These data demonstrate that nucleoside analog resistance arises from mutations in multiple domains. Many of the mutations are located far from the dNTP binding sites. These changes inhibit drug function by altering the conformation of the target protein in a manner analogous to those conformational changes that may be induced by an allosteric inhibitor. 136 232/116

Table 5 summarizes the mutations that alter the function of non-nucleoside inhibitor drugs

Table 5

Location and postulated mechanism of amino acid substitutions which confer resistance to non-nucleoside analog inhibitors.

137 232/116

It is evident from these examples that the substitutions which inhibit drug functions are distributed across several domains. Different inhibitory mechanisms have been postulated in domains throughout the protein, based on the three-dimensional structure of the protein. Most involve conformational disruption of the protein secondary and tertiary structure.

Thyrotropin receptor Naturally occurring antibodies against the thyrotropin receptor can cause activation of thyroid function (Grave's disease) or inhibition of thyroid function (Hashimoto's disease). The sites within the thyrotropin receptor that are targeted by these natural antibodies have been mapped in detail and have been tested with monoclonal antibodies. Most of the inhibitory antibodies do not interfere with binding of thyrotropin to its receptor, and thus, are allosteric rather than competitive inhibitors. Several independent classes of inhibitory antibodies have been identified that bind to epitopes within different domains of the receptor. At least one of these epitopes is in a domain that is entirely unimportant for receptor activity and can be deleted by site-directed mutagenesis without disrupting the function of the receptor. These experiments provide an explicit precedent for achieving allosteric inhibitory effects from ligands that target widely dispersed sequences within the protein.

Thermus aquaticus DNA polymerase The inhibitory activity of 24 monoclonal antibodies to Thermus aquaticus DNA polymerase has been investigated. The antibodies recognized 13 non-overlapping epitopes. Antibody binding to eight epitopes was inhibitory. Inhibitory antibodies mapped to several distinct domains, including the 5' nuclease domain, the polymerase domain and the boundary region between the 5' nuclease and polymerase domains. Some antibodies recognized epitopes overlapping the DNA binding groove of the polymerase. Significantly, the inhibitory antibodies recognized epitopes constituting as much as 50% of the Taq polymerase surface, and the non-inhibitory antibodies a further -25%. 138 232/116 -lactamase The β-lactamases are a diverse family of enzymes which catalyze the hydrolysis of the β-lactam ring of penicillin and cephalosporin antibiotics. Interactions of these proteins with various small molecule drugs have been characterized in detail as the pharmaceutical industry has worked to develop chemically modified penicillins and cephalosporins to elude inactivation by β- lactamases. In addition, a β-lactamase inhibitor (clavulanic acid) has also been introduced into clinical use.

As each new drug has been introduced into wide use, mutant β-lactamases have emerged that are resistant to the drug. Over 190 β-lactamases have been described with differential specificity for the various penicillins and cephalosporins. Many of these differ by only a few amino acids. Many different amino acid substitutions at various sites within the protein can change the substrate specificity of the enzyme.

kat G (Isoniazid resistance) The kat G protein of M. tuberculosis encodes a catalase-peroxidase enzyme that is one of two mycobacterial genes frequently altered in isoniazid resistant strains (the other is inhA). There are a wide variety of amino acid substitutions in katG associated with drug resistance distributed evenly across the 740 amino acids of the protein. The mechanism by which some of these substitutions inhibit katG function can be inferred from the structure of the homologous yeast and E. coli enzymes and knowledge of the catalytic function of the enzyme. For example, insertion of an He between positions 125 and 126 affects a conserved interhelical loop near the active site residues; substitutions at amino acid 275 and 315 are likely to affect the ligand access channel; substitutions at amino acid 463 may affect a N-terminal substrate binding site. Other substitutions occur in regions that are not directly related to the functional sites of the protein.

The examples described above demonstrate that small molecules can discriminate in activity between polypeptides or proteins which have one a single amino acid 139 232/116 difference in sequence, i.e., a single amino acid sequence variance.

The application of small molecule inhibitor identification is specifically discussed in Example 39 below in connection with the methylguanine methyltransferase gene.

C. Antibody Inhibition.

Once an essential gene is identified and is determined to exist in two or more allelic forms which encode different proteins, antibodies can be raised against both allelic forms of the protein. The techniques for using a specific protein or an oligopeptide as an antigen to elicit antibodies which specifically recognize epitopes on the peptide or protein are well known. Preferably monoclonal antibodies (MABs) are used.

In one embodiment, the DNA sequence of the desired allelic form of the target gene can be cloned by insertion into an appropriate expression vector and translated into protein in a prokaryotic or eukaryotic host cell. The protein can be recovered and used as an antigen to elicit the production of specific antibodies. In another embodiment, the DNA of the desired allelic form of the target gene is amplified by PCR technology and is subsequently translated in vitro into protein to be used as the antigen to elicit the production of specific antibodies. A third embodiment is to use the DNA sequence of the alternative alleles as a basis for the generation of synthetic peptides representing the amino acid sequence of the alleles for use as antigen to elicit the production of specific antibodies.

Antibodies can be generated either by standard monoclonal antibody techniques or generated through recombinant based expression systems. See generally, Abbas, Lichtman, and Pober, Cellular and Molecular Immunology. W.B. Saunders Co. (1991). The term "antibodies" is meant to include intact antibody molecules of the 140 232/116

IgD isotype as well as antibody fragments or derivatives, such as Fab and F(ab')2, which are capable of specifically binding to antigen. The antibodies so produced will preferentially bind only the protein produced in the allelic form which was used as an antigen to create the antibody. If the targeted protein is expressed on the cell surface, the antibody or antibody derivative can be tested as a therapeutic.

Antibody inhibitors are most effective when they are directed against cell surface proteins or receptors. If the essential protein produced by the targeted allele is not a cell surface protein or receptor, the development of antibody inhibitors may also require the use of a special antibody-delivery system to facilitate entry of the antibody into the tumor cells. The plasma membrane that surrounds all cells is designed to limit the entrance of most compounds. Entry is generally restricted to small, non-charged molecules (absence of charge allows them to slip through the fatty membrane) or to those factors that can penetrate the cell using existing, specialized import mechanisms. The introduction into cells of much larger molecules, such as specific antibodies, other proteins, or peptides, requires appropriate delivery systems such as are known in the art. Alternatively, the structure of the variable region of allele specific antibodies can be used as the basis for design of smaller allele specific inhibitory molecules.

D. Oligopeptides Oligopeptides can be demonstrated to have a very high degree of specificity in their interaction with functional polypeptides such as cellular enzymes, receptors or other polypeptides essential for cell viability. Methods for screening peptide sequences which have high specificity for binding to, and functional inhibition of, a specific polypeptide target have been well described previously. Scott, J.K. and Smith G.P., "Searching for Peptide Ligands with an Epitope Library," Science 249:386-390 (1990). These methods include the screening of Ml 3 libraries by "phage display" of polypeptide sequences as well as direct screening of peptides or mixtures of synthetic peptides for binding to or inhibition of the target functional polypeptide. 141 232/116

The oligopeptides of this invention can be synthesized chemically or through an appropriate gene expression system. Synthetic peptides can include both naturally occurring amino acids and laboratory synthesized, modified amino acids.

Also provided herein are functional derivatives of a polypeptide or protein. By "functional derivative" is meant a "chemical derivative," "fragment," "variant," "chimera," or "hybrid" of the polypeptide or protein, which terms are defined below. A functional derivative retains at least a portion of the function of the protein, for example reactivity with a specific antibody, enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention.

A "chemical derivative" of the complex contains additional chemical moieties not normally a part of the protein. Such moieties may improve the molecule's solubility, absoφtion, biological half life, and the like. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art. Covalent modifications of the protein or peptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, as described below.

Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N- 142 232/116 alkylmaleimides, 3-nitro-2 -pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloro- mercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3- diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect or reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing primary amine containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with gly oxy late.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high p , of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine alpha-amino group.

Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. 143 232/116

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction carbodiimide (R'-N-C-N-R') such as l-cyclohexyl-3-(2-moφholinyl(4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residue are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful, for example, for cross-linking component peptides to each other or the complex to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, for example, l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobi- functional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maleimido-l,8-octane. Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T.E., Proteins:

Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 144 232/116

(1983)), acetylation of the Nterminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.

Such derivatized moieties may improve the stability, solubility, absoφtion, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein complex and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences. 18th ed., Mack Publishing Co., Easton, PA (1990).

The term "fragment" is used to indicate a polypeptide derived from the amino acid sequence of the protein or polypeptide having a length less than the full-length polypeptide from which it has been derived. Such a fragment may, for example, be produced by proteolytic cleavage of the full-length protein. Preferably, the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence.

Another functional derivative intended to be within the scope of the present invention is a "variant" polypeptide which either lack one or more amino acids or contain additional or substituted amino acids relative to the native polypeptide. The variant may be derived from a naturally occurring polypeptide by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N- terminus, and/or within the native sequence.

A functional derivative of a protein or polypeptide with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well- known to those of ordinary skill in the art. For example, the modified components of the functional derivatives may be produced using site-directed mutagenesis 145 232/116 techniques (as exemplified by Adelman et al., 1983, DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is modified, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above. Alternatively, components of functional derivatives of complexes with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.

E. Complementary Oligonucleotides and Ribozymes

Oligonucleotides or oligonucleotide analogs which interact with complementary sequences of cellular target DNA or RNA can be synthesized and used to inhibit or control gene expression at the levels of transcription or translation. The oligonucleotides of this invention can be either oligodeoxyribonucleotides or oligoribonucleotides, or derivatives thereof, which are complementary to the allelic forms of the targeted essential gene or they can act enzymatically, such as ribozymes. Both antisense RNA and DNA can be used in this capacity as chemotherapeutic agents for inhibiting gene transcription or translation. Trojan, J., et al, "Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin-like growth factor I RNA," Science 259:94-97 (1993). Inhibitory complementary oligonucleotides may be used as inhibitors for cancer therapeutics because of their high specificity and lack of toxicity.

Included in the scope of the invention are oligoribonucleotides, including antisense RNA and DNA molecules and ribozymes that function to inhibit expression of an essential gene in an allele specific manner. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation or directing RNase mediated degradation of the mRNA. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of the relevant 146 232/116 nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific interaction of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead, haiφin, and other motif ribozyme molecules that catalyze sequence specific endonucleolytic cleavage of RNA sequences encoding a gene product essential for cell survival, growth, or vitality.

Specific ribozyme cleavage sites within any potential RNA target can initially be identified by scanning the target molecule for ribozyme cleavage sites, such as sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays. See, for example, Draper PCT WO 93/23569. For the present invention, the target site will generally include a sequence variance site as described above.

Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA and DNA molecules. See, for example, Draper, supra, hereby incoφorated by reference herein. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the 147 232/116 antisense or ribozyme RNA molecule. Such DNA sequences may be incoφorated into a wide variety of vectors which incoφorate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense or ribozyme cDNA constructs that synthesize antisense or ribozymes RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or methyl phosphonate rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. Modifications may also be made on the nucleotidic sugar or purine or pyrimidine base, such as 2'-O-alkyl (e.g., 2'-O- methyl), 2'-O-allyl, 2'-amino, or 2'-halo (e.g., 2'-F). A variety of other substitutions are also known in the art and may be used in the present invention. More than one type of nucleotide modification may be used in a single modified oligonucleotide.

A specific application of generating inhibitors which are either complementary oligonucleotides or inhibitory oligopeptides is described in Holzmayer, Pestov, and Roninson, "Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments," Nucleic Acids Research 20:711-717 (1992). In this study, genetic suppressor elements (GSEs) are identified by random DNA fragmentation and cloning in expression plasmids.

Preferred oligonucleotide inhibitors include oligonucleotide analogues which are resistant to degradation or hydrolysis by nucleases. These analogues include neutral, or nonionic, methylphosphonate analogues, which retain the ability to 148 232/116 interact strongly with complementary nucleic acids. Miller and Ts'O, Anti-Cancer Drug Des. 2:11-128 (1987). Further oligonucleotide analogues include those containing a sulfur atom in place of the 3'-oxygen in the phosphate backbone, and oligonucleotides having one or more nucleotides which have modified bases and/or modified sugars. Particularly useful modifications include phosphorothioate linkages and 2'-modification (e.g., 2'-O-methyl, 2'-F, 2'-amino).

F. Gene Therapy

Nucleic acid molecules encoding oligonucleotide or polypeptide inhibitors will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, (1992). Miller indicates that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. An in vivo model of gene therapy for human severe combined immunodeficiency is described in Ferrari, et al., Science 251 :1363-1366, (1991). The basic science of gene therapy is described in Mulligan, Science 260:926-931, (1993).

Some methods of delivery that may be used include: a. complexation with lipids, b. transduction by retro viral vectors, c. localization to nuclear compartment utilizing nuclear targeting site found on most nuclear proteins, d. transfection of cells ex vivo with subsequent reimplantation or administration of the transfected cells, e. a DNA transporter system.

A nucleic acid sequence encoding an inhibitor may be administered utilizing an ex vivo approach whereby cells are removed from an animal, transduced with the nucleic acid sequence and reimplanted into the animal. The liver can be accessed by an ex vivo 149 232/116 approach by removing hepatocytes from an animal, transducing the hepatocytes in vitro with the nucleic acid sequence and reimplanting them into the animal (e.g., as described for rabbits by Chowdhury et al, Science 254: 1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3: 179-222, 1992) incoφorated herein by reference.

Many nonviral techniques for the delivery of a nucleic acid sequence encoding an inhibitor into a cell can be used, including direct naked DNA uptake (e.g., Wolff et al., Science 247: 1465-1468, 1990), receptor-mediated DNA uptake, e.g., using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu et al., J Biol. Chem. 266: 14338-14342, 1991), and liposome-mediated delivery (e.g., Kaneda et al., Expt. Cell Res. 173: 56-69, 1987; Kaneda et al., Science 243: 375- 378, 1989; Zhu et al., Science 261: 209-211, 1993). Many of these physical methods can be combined with one another and with viral techniques; enhancement of receptor-mediated DNA uptake can be effected, for example, by combining its use with adenovirus (Curiel et al., Proc. Natl. Acad. Sci. USA 88: 8850-8854, 1991; Cristiano et al, Proc. Natl. Acad. Sci. USA 90: 2122-2126, 1993).

In one preferred embodiment, an expression vector containing a sequence encoding a ribozyme or an antisense oligonucleotideis inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.

The gene therapy may involve the use of an adenovirus containing a sequence encoding a ribozyme or an antisense oligonucleotide targeted to a tumor.

Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, heφes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences into the targeted 150 232/116 cell population (e.g., tumor cells). Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences. See, for example, the techniques described in Maniatis et. al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, N.Y. (1989), and in Ausubel et. al., Current Protocols in Molecular Biology. Greene Publishing

Associates and Wiley Interscience, N.Y. (1989). Alternatively, recombinant nucleic acid molecules encoding protein sequences can be used as naked DNA or in reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (See e.g., Feigner et. al., Nature 337:387-8, 1989). Several other methods for the direct transfer of plasmid DNA into cells exist for use in human gene therapy and involve targeting the DNA to receptors on cells by complexing the plasmid DNA to proteins. See, Miller, supra.

In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA (e.g., a plasmid vector encoding an inhibitor) into the nucleus of a cell, through a process of microinjection. Capecchi MR, Cell 22:479-88 (1980). The DNA can be part of a formulation which protects the DNA from degradation or prolongs the bioavailability or the DNA, for example by complexing the DNA with a compound such as polyvinylpyrrolidone. Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been used for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with CaPO₄ and taken into cells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52 (1987)); electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu G. et al., Nucleic Acids Res., 15:1311-26 (1987)); lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles which fuse with a target cell (Feigner PL., et al., Proc. Natl. Acad. Sci. USA_± 84:7413-7 (1987)); and particle bombardment using DNA bound to small 151 232/116 projectiles (Yang NS. et al., Proc. Natl. Acad. Sci. 87:9568-72 (1990)). Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins.

It has also been shown that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene. Curiel DT et al, Am. J. Respir. Cell. Mol. Biol, 6:247-52 (1992).

As used herein "gene transfer" means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene. The product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.

As used herein "gene therapy" is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient. 152 232/116

In another preferred embodiment, a vector having nucleic acid sequences encoding an allele specific inhibitor is provided in which the nucleic acid sequence is expressed only in specific tissue. Examples or methods of achieving tissue-specific gene expression are described in International Publication No. WO 93/09236, published May 13, 1993.

VII. Utility of allele-specific inhibitors of essential genes A. Conditions susceptible to therapy.

The fraction of all cancers could be treated with allele specific inhibitors directed against allele specific essential gene targets is a function of the frequency of the target allele and the frequency of LOH. The ideal target would be deleted in 100% of all major cancers and would exist in two allelic forms, each with an allele frequency of 0.5 so that half the population would be heterozygous. An inhibitor of one allele of such an ideal target would be a useful agent for 25% of all cancer patients. An inhibitor of the other allele of the same ideal target would be therapeutic for an additional 25% of all patients, making 50% of all patients treatable. The ideal target has so far not been identified, but we have identified many essential gene sequence variance targets which are deleted in 30-70% of several major cancers, and which are heterozygous in 25-50% of North Americans. Allele specific inhibitors of both alleles of such targets would be expected to address 0.4 x 0.5 = 0.2 or 20% of the relevant cancer population. The relevant cancer population often includes breast, colon and lung cancer, which sum to -500,000 new cases per year in the United States. Thus a total available market of 100,000 patients is not unusual, and many targets would be expected to address markets of at least 50,000 patients.

The targets of this invention are suitable for treatment of many different cancers, which includes cancers of different types, as well as non-malignant proliferative 153 232/116 disorders, as well as being suitable for use in other applications involving targeting alternative allelic forms of a gene. The classification and nomenclature for a variety of benign and malignant tumors relevant to the present invention is shown in the following table (Table 6-1 from Robbins et al., Pathologic Basis of Disease. 3rd ed. (1984), however, the invention is not limited to these cancers or classifications.

Table 6

Tissue of Origin Benign Malignant

I. Composed of one parenchymal cell type Sarcomas

A. Tumors of mesenchymal origin

(1) Connective tissue and derivatives fibrous tissue fibroma fibrosarcoma myxomatous tissue myxoma myxocarmo fatty tisssue lipoma liposarcoma cartilage chondroma chondrasarcoma bone osteoma osteosarcoma osteogenic sarcoma

(2) Endothelial & related tissues blood vessels hemangioma angiosarcoma capillary cavernous sclerosing hemangioendothelioma endotheliosarcoma, Kaposi's sarcoma lymph vessels lymphoangioma lymphangiosarcoma synovia synovioma (synoviosarcoma) mesothelium mesothelioma (mesotheliosarcoma) brain coverings meningioma glomus glomus tumor 154 232/116

?endothelial or Ewing's tumor mesenchymal cells

(3) Blood cells & related cells hematopoietic cells myelogenous leukemia monocytic leukemia lymphoid tissue malignant lymphomas lymphocytic leukemia plastocytoma (multiple myeloma) monocyte-macrophage histiocytosis X Langerhans' cells ?histiocytic lymphoma (4) Muscle ?Hodgkin's disease smooth muscle leiomyoma leiomyosarcoma striated muscle rhabdomyoma rhabdomyosarcoma

B. Tumors of epithelial origin Carcinomas stratified squamous squamous cell squamous cell or papilloma epidermoid carcinoma basal cells of skin or adnexia basal cell carcinoma skin adnexal glands sweat glands sweat gland adenoma sweat gland carcinoma sebaceous gland sebaceous gland sebaceous gland adenoma carcinoma epithelial lining glands or ducts -well adenoma adenocarcinoma differentiated papillary adenoma papillary group cystadenoma adenocarcinoma cystadenocarcinoma poorly differentiated group medullary carcinoma undifferentiated carcinoma (simplex) respiratory tract bronckogenic carcinoma bronchial "adenoma" neuroectoderm nevus melanoma (melanocarcinoma) renal epithelium renal tubular adenoma renal cell carcinoma (hypernephroma) 155 232/116

liver cells liver cell adenoma hepatocellular carcinoma bile duct bile duct adenoma bile duct carcinoma

(cholangiocarcinoma) urinary tract epithelium transitional cell papillary carcinoma (transitional) papilloma transitional cell carcinoma squamous cell carcinoma placental eptithelium hydatiform mole choriocarcinoma testicular epithelium (germ seminoma cells) embryonal carcinoma

II. More than one neoplastic cell type— mixed tumors — usually derived from mixed tumor of salivary malignant mixed tumor one germ layer gland origin of salivary gland origin salivary glands (pleiomoφhic adenoma) renal anlage Wilms' tumor

III. More than one neoplastic cell type derived from more than one germ layer — teratogenous totipotential cells in gonads or teratoma, dermoid cyst malignant teratoma and in teratocarcinoma embryonic rests

Allele specific therapy can be targeted to essential genes which undergo LOH in many different tumor types, including the tumors and tumor types described in the tables above, and in Figure 3.

For the treatment of patients suffering from a tumor using an allele specific inhibitor, 156 232/116 the preferred method of preparation or administration will generally vary depending on the type of inhibitor to be used. Thus, those skilled in the art will understand that administration methods as known in the art will also be appropriate for the inhibitors of this invention.

B. Pharmaceutical Formulations and Modes of Administration

The particular compound, antibody, antisense or ribozyme molecule that exhibits allele specific inhibitor activity can be administered to a patient either by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of a agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating plasma concentration range that 157 232/116 includes the IC₅₀ as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et. al., in The Pharmacological Basis of Therapeutics. 1975, Ch. 1 p.l). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences. 18th ed., Mack Publishing Co., Easton, PA (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, 158 232/116 penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incoφorated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended puφose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical 159 232/116 compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable 160 232/116 coatings. For this puφose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Factors specific for the delivery of antisense and ribozyme nucleic acids are known in the art, for example as discussed in Couture et al., WO 94/02595, which is hereby incoφorated herein by reference. This reference also describes the synthesis of nucleic acid molecules having a variety of 2' modified nucleotides.

The references cited herein are incoφorated by reference to the same extent as if each had been individually incoφorated by reference. The invention is illustrated further by the following examples, which are not to be taken as limiting in any way. The examples, individually, and together, further demonstrate that one skilled in the art would be able to practice each of the steps in developing useful pharmaceutical products as described in the invention. Generally, the development of such a product involves the following steps: 1. Select candidate target gene essential for cell survival or proliferation. 2. Determine chromosome location and LOH frequency. 3. Identify common variance in the normal population. 161 232/116

4. Demonstrate antiproliferative effects from inhibition of candidate gene.

5. Design variance-specific inhibitor.

6. Achieve variance-specific antiproliferative effects in cancer cells.

EXAMPLES

Example 1. Genes required for Cell Proliferation

Many genes are involved in the process of cell proliferation and are potential targets for anti-proliferative drugs in this invention. Dividing cells progress through a repeating cycle of four stages, each of which is critical to the proliferation process. During the first phase, GI, cells ready the proteins they need to replicate their DNA, which occurs during S phase. Following S phase, cells enter G2, in which they prepare to divide into two daughter cells, each of which will contain the DNA content of the original cell. The final stage of the cell cycle is M phase, in which cells undergo mitosis. During mitosis, the cell nucleus disappears and the two sets of replicated chromosomes are separated to opposite sides of the cell. The cell then divides into two cells, the nucleus reforms in each new cell, and the cycle begins again. Cell proliferation is exceedingly complex and requires the precise coordination of many processes, including DNA synthesis, chromosome condensation and separation, and cell fission. In eukaryotic cells such as yeast, many of the proteins involved in cell division are encoded by essential genes, including those contributing to the duplication of the nucleus and the functions of microtubules, spindle pole bodies the centromere and the kinetochore.

A number of proteins are essential for cell proliferation. Proteins that are critical to this process can be divided into two classes: (i) proteins that regulate cell division; (ii) proteins that form structures involved in cell division. Proteins that regulate cell division include, but are not limited to, proteins involved in the regulation of particular 162 232/116 steps in the division process, such as nuclear breakdown and the transition between the different stages of mitosis, as well as proteins regulating the initiation of mitosis, such as the cylins, cyclin-dependent kinases (CDKs), and the kinases and phosphatases that regulate CDKs. Cyclin B, the cyclin-dependent kinase cdc2, and the cdc25C phosphatase are examples of proteins that regulate the initiation of mitosis. Deletion of yeast homologs of these genes is lethal, verifying their critical role in regulating the entry into mitosis. (It has been established that many human genes which encode proteins involved in highly conserved cellular processes can substitute for their yeast counteφarts, and vice versa. For example such conservation has been demonstrated for components of the transcriptional apparatus, as well as components of the translational apparatus.)

Proteins that form structures involved in cell division include, but are not limited to, those involved in the processes of chromosome condensation and separation. Examples are tubulin and kinesin, which participate in the separation of chromosomes, and KIAA0165 and CDC37, involved in the spindle pole. Deletion of the yeast homolog of CDC37 is lethal.

Inhibiting the ability of a cell to divide induces, by definition, a cytostatic response, often followed by cell death. Colchicine and nocodazole are examples of drugs that inhibit microtubule function in vitro, thereby preventing chromosome separation and leading to cell cycle arrest during mitosis. Vinblastine and vincristine, which also inhibit microtubule function and therefore cell proliferation, have been used widely in the treatment of cancer.

Examples of genes that are involved in the process of cell proliferation, and are thus essential for cell survival or proliferation are shown in the accompanying table. Each of these genes has been disrupted in Saccharomyces cerevisiae and the mutant yeast shown to be nonviable. 163 232/116

Table: Genes Essential for Cell Proliferation in Yeast

164 232/116

It will be evident to one skilled in the art that many genes that express essential metabolic and homeostatic functions of the cell will also be essential for cell proliferation.

Example 2. Genes required to maintain inorganic ions at levels compatible with cell growth or survival.

Inorganic Ions are Essential for Cellular Life

Inorganic ions are required for virtually all cellular processes: they are important for maintenance of cell shape and osmolality; they are prosthetic groups of a wide variety of enzymes; they are required for ATP production coupled to ion diffusion; they mediate signal transduction both from intracellular and extracellular signals. Hence maintenance of inorganic ions at physiological concentrations is essential for cell 165 232/116 proliferation and cell survival. The importance of maintaining physiological ion concentrations is further demonstrated by the observation that deviation from normal levels leads to cytostatic or cytotoxic effects, as demonstrated by the effects of selectively poisoning ion channels or placing cells in hypotonic or hypertonic extracellular fluid.

Inorganic Ions Must be Transported Across Membranes

Maintenance of ion concentrations at optimal concentrations within cells is complicated by the presence of membranes which, because of their hydrophobic interior, form a highly impermeable barrier to most polar molecules, including inorganic ions. Important cell membranes include the plasma membrane as well as the nuclear membrane, mitochondrial membranes, the endoplasmic reticulum and Golgi apparatus, lysosomes and vesicles of various types, all of which are essential for cell proliferation or survival. Therefore maintaining the concentration of essential polar molecules, including both organic and inorganic ions, at levels compatible with cell growth or survival requires specialized mechanisms for moving such ions across the plasma membrane and the various intracellular membrane bound compartments.

Vital components of the apparatus for maintaining ion concentrations at levels essential for cell survival include regulatory molecules that sense the concentration of ions in different cellular compartments and produce signals to increase or decrease the concentration of said ions to levels compatible with cell survival; proteins that actively or passively transport ions across membranes; and proteins that modify ions so they can be transported across membranes.

Membrane transport proteins can be divided into several categories depending on whether they require energy (provided either by ATP hydrolysis or by co-transport of ions such as sodium or protons down their electrochemical gradients), produce energy 166 232/116

(ATP synthetases, which are usually coupled to proton diffusion) or are energy neutral. Other categories of transporters include those that transport one or more solutes (one or more of which may be ions), gated vs. non-gated - i.e. open only transiently (ligand gated and voltage gated channels) or open continuously, allowing ions to move down their concentration and electrochemical gradients. Specific types of essential membrane transporters include uniports, which simply transport one solute from one side of the membrane to the other, and cotransports, in which the transport of one solute is dependent on the simultaneous or sequential transport of a second solute in the same direction (symport) or in the opposite direction (antiport).

Other inorganic ions, such as iron, are transported bound to carrier proteins (transferrin in the case of iron). Transport of the iron carrier protein involves a complex cycle that begins with binding of iron to transferrin, binding of the iron-transferrin complex to transferrin receptor, formation of coated pits, endocytosis of the transferrin-iron complex via the coated pits, release of iron from transferrin in endosomes upon acidification to pH 5, and then recycling of the transferrin receptor-apotransferrin complex to the surface of the cell where, at neutral pH, the apotransferrin is released from transferrin receptor into the extracellular fluid to bind more iron and participate in another cycle. Thus in the case of transferrin-mediated iron transfer there are a variety of specialized proteins which must interact in a coordinated manner for transport to occur effectively.

Some of the specific inorganic ions which must be transported across the both the plasma membrane and intracellular membranes are sodium, potassium, chloride, calcium, hydrogen, magnesium, manganese, phosphate, selenium, molybdenum, iron, copper, zinc, fluorine, iodine, chromium, silicon, tin and arsenic. Specific transporters have been identified for many of these solutes including sodium, potassium, chloride, protons, copper and iron among others. 167 232/116

Regulation of ion concentrations at appropriate levels is often an energy-dependent process; intracellular and extracellular concentrations may differ by 10 fold or more (see Table).

Ion Concetrations Inside and Outside a Typical Mammalian Cell

Cations

Anions

Cl - 4 110

Inhibitors of Ion Transporting Proteins are Cytostatic or Cytotoxic

Blocking import of essential cell nutrients, including inorganic ions, prevents cell growth and can lead to cell death. A well studied example is blockade of iron transport by inhibition of transferrin receptor. Dividing cells require iron, and transferrin receptor-mediated uptake of iron-transferrin complexes is the principal route for iron aquisition. Iron uptake requires multiple steps, including receptor binding, endocytosis via coated pits, acidification of endosomes and consequent release of iron from transferrin, followed by recycling of transferrin receptor-apotransferrin to the cell surface for another round of binding. Each step requires the coordinated function of a variety of proteins. Anti-transferrin receptor antibodies arrest cell growth by blocking iron uptake; antitumor effects have been demonstrated in vitro and in vivo with such antibodies.

Ion pumps are another class of proteins for which cytotoxic inhibitors have been 168 232/116 identified. All animal cells contain a Na⁺, K⁺ pump which operates as an antiport, actively pumping Na⁺ out of the cell and K⁺ in against their concentration gradients. In coupling the hydrolysis of ATP to the active transport of 3 Na⁺ out and 2 K⁺ into the cell the pump is electrogenic. The electrochemical gradients generated and maintained by the Na⁺,K⁺ pump are essential for regulation of cell volume and for the secondary, sodium-coupled active transport of a variety of organic and inorganic molecules including glucose, amino acids and CaA Hence the sodium potassium pump plays an essential role in cellular physiology. More than one third of a typical animal cells energy requirement is expended in fueling this pump. (Alberts et al. Molecular Biology of the Cell, Garland Publishing, New York, 1983, p.291.) Ouabain is an inhibitor of the Na⁺, K⁺ ATPase. It binds to the catalytic alpha 1 subunit of sodium potassium ATPase and is a potent cytotoxic drug. Cells treated with ouabain swell and eventually burst as they are unable to maintain a balance of osmotic forces because they can no longer pump out Na+. See Example 11 for a more detailed description of the essential properties of the Na⁺, K⁺ ATPase. Amiloride is another cytotoxic drug; it blocks the sodium-proton antiporter. Thus inhibition of proteins essential for maintaining physiologial levels of inorganic ions is toxic to cells.

Ion Transporting Proteins are Evolutionary Conserved and Essential in Other Species

Many of the proteins required to maintain inorganic ions at physiologic levels are widely conserved in eukaryotes, reflecting an ancient and vital role. A number of gene disruption experiments in non-human cells demonstrate the importance of ion transponting proteins for cell growth and survival. For example in the yeast

Saccharomyces Cerevisiae the gene encoding CDC1 protein, involved in maintaining ion homeostasis, has been disrupted resulting in non-viable yeast. Another essential yeast gene is PMA1, which encodes a H+ transporting P-type ATPase of the plasma membrane; activity of the encoded protien is rate limiting for growth at low pH. 169 232/116

As a result of the essential functions provided by proteins required for maintenance of inorganic ions at levels required for cell growth or survival, those genes which undergo LOH in a neoplastic disorder and which have sequence variants (nucleic acid or amino acid sequences) in a population as described above, are appropriate potential targets for allele specific inhibition, and thus can be used in the methods for identifying allele specific inhibitors and in other aspects of this invention. The provision of the exemplary ion transport genes, including sodium-potassium ATPase alpha 1 subunit as well as the other genes listed in the Target Genes Table, indicates that other genes within this category or related subcategories will also be appropriate potential targets. Such a gene can be identified as an essential gene by reference to the art, or by the essential gene identification methods known in the art, examples of which are referenced herein. The LOH and sequence variance characteristics can then be readily determined by the described methods, thereby demonstrating that the gene is an appropriate potential target gene for allele specific inhibition.

Example 3. Genes required to maintain organic compounds at levels compatible with cell growth or survival.

Organic Compounds are Essential for Cellular Life

Organic compounds include the amino acids, carbohydrates, lipids, nucleosides and nucleotides, ions such as bicarbonate, vitamins such as ascorbic acid, pantothenic acid, riboflavin, nicotinamide, thiamine, vitamin B6, vitamin B12, and folate, essential nutrients such as linoleic acid and a wide variety of metabolic intermediates. Organic compounds are required for virtually all vital cellular processes: they are the building blocks of all cellular macromolecules including larger organic comounds such as proteins, starches, polynucleotides and complex lipids as well as glycolipids, 170 232/116 glycoproteins, lipoproteins, etc.; they are constituents of all cell structural molecules including proteins and membranes; they constitute all the metabolic intermediates in such vital cell processes as glycolysis, the Krebs cycle, oxidative phosphorylation, gluconeogenesis, the urea cycle, nucleotide biosynthesis, amino acid biosynthesis, etc. Maintaining organic compounds at levels compatible with cell growth or survival constitutes a large fraction of the work of the cell. Deviation from normal levels of organic compounds will generally have cytotoxic or cytostatic effects on cells (if the appropriate homeostatic cellular machinery for maintaining organic compounds at levels compatible with cell growth or survival is not operating to bring levels back to normal), as demonstrated by the effects of preventing transport of organic ions such as essential amino acids, vitamins or ions such as bicarbonate or blocking such processes as glycolysis or amino acid biosynthesis or transport of proteins into mitochondria, or required post-translational processing of proteins, lipids or carbohydrates.

Maintaining Organic Compounds at Levels Compatible with Cell Growth or Survival

Requires Membrane Transport, Biosynthesis, Energy Extraction, Energy Production, Degradation and Excretion Pathways

Maintenance of organic compounds at optimal concentrations within cells is complicated by the presence of membranes which, because of their hydrophobic interior, form a highly impermeable barrier to most polar or charged molecules or molecules over 100 Daltons, including many organic compounds. Important cell membranes include the plasma membrane as well as the nuclear membrane, mitochondrial membranes, the endoplasmic reticulum and Golgi apparatus, lysosomes and vesicles of various types, all of which are essential for cell proliferation or survival. Therefore maintaining the concentration of essential organic compounds at levels compatible with cell growth or survival requires specialized mechanisms for moving such compounds across the plasma membrane and the various intracellular membrane bound compartments. 171 232/116

Vital components of the apparatus for maintaining organic compounds concentrations at levels essential for cell survival include regulatory molecules that sense the concentration of ions in different cellular compartments and produce signals to increase or decrease the concentration of said compounds to levels compatible with cell survival; proteins that actively or passively transport organic compounds across membranes; and proteins that modify or bind to organic compounds so they can be transported across membranes.

Some of the specific inorganic ions which must be transported across the both the plasma membrane and intracellular membranes are sodium, potassium, chloride, calcium, hydrogen, magnesium, manganese, phosphate, selenium, molybdenum, iron, copper, zinc, fluorine, iodine, chromium, silicon, tin and arsenic. Specific transporters have been identified for many of these solutes including sodium, potassium, chloride, protons, copper and iron among others.

The number of essential membrane proteins is not known. A crude estimate can be derived by adding up the proteins which perform essential functions enumerated above. There are many presently known organic compounds which must be transported across the cell membrane, including small molecules such as essential amino acids, lipids, sugars, the vitamins pantothenic acid, folic acid, riboflavin, nicotinamide, thiamine, vitamin Bg, vitamin B γ^an^ ascorbic acid as well as larger molecules such as proteins. (It is important to note that some essential functions are performed by families of transporters with overlapping tissue expression. In such cases it may be that no one protein is essential despite the fact that the protein family collectively carries out an essential cell function. Conversely, there are likely to be a number of essential membrane proteins not yet identified.)

Examples of Genes Essential to Maintain Organic Compounds at Levels Compatible with Cell Growth or Survival, From Yeast 172 232/116

The yeast Saccharomyces Cerevisiae is a eukaryote which shares many genes in common with humans. Approximately 70% of the essential genes in yeast have human homologs. Many human genes can be exchanged with their yeast counteφarts with minimal effects on growth in yeast or human cells. The study of essential genes in yeast is much further advanced than in mammalian systems: over half of the -6,000 genes of Saccharomyces Cerevisiae have been disrupted and the phenotype of the resulting strains tested on minimal growth media. Over 20% of disrupted yeast genes are essential, and a significant fraction of their human counteφarts are likely to be essential for cell survival. Among the yeast genes disrupted are a variety of genes that encode proteins required to maintain organic compounds at levels compatible with cell growth or survival. Many of these genes are essential for cell survival. Many of the disrupted essential yeast proteins have closely related human homologs, and it is likely that the human homologs are also essential. Specific examples of yeast genes that are essential are listed below. (This is a partial list; see the web site .proteome.com for an up to date list.)

The yeast ACC1 gene encodes acetyl co-A carboxylase and, like the human enzyme, is the first and rate limiting step in fatty acid biosynthesis.

The yeast DYS1 gene encodes deoxyhypusine synthase which catalyzes the first step in biosynthesis of the polyamine deoxyhypusine.

The yeast FBA gene encodes fructose-bisphosphate aldolase II, the sixth step in glycolysis, while the essential yeast genes GND1, ENO2, GPM1 and PYK1 encode 6- phosphogluconate dehydrogenase, enolase 2, phosphoglycerate mutase and pyruvate kinase (the last step of glycolysis).

The yeast ERG 10 gene encodes acetyl-CoA-acetyltranferase, the first step in the mevalonate/sterol pathway. The essential ERG1 gene encodes squalene 173 232/116 monooxygenase, an later enzyme of the sterol biosynthesis pathway. ERG7, ERG8, ERG9, ERGl 1, ERG20, ERG24 and ERG25 encode enzymes on the same or related pathways.

The yeast ALG1 and ALG2 genes encode mannosyltransferases requried for N- glycosylation, and the ALG7, DPMI and NMT1 genes encode transferases for UDP-N- acetyl-glucosamine-1-P, mannose and myristate, respectively. RAM2 encodes a protein that is a subunit of both farnesyltransferases and (with BET2) geranylgeranyltransferases .

The yeast LCB1 gene encodes serine C-palmitoyltransferase which catalyzes the first step in the pathway for synthesis of the long chain base component of shingolipids, while the yeast AURl gene encodes a phosphoinositol transferase also essential for shingolipid synthesis.

The yeast PRO1 and PRO2 genes encode the three enzymes of proline biosynthesis. THR1 catalyzes the first step of threonine biosynthesis.

Example 4. Genes required to maintain cellular proteins at levels compatible with cell growth or survival.

Proteins carry out a host of essential enzymatic and structural functions required for cell proliferation and cell survival. Consequently, complete inhibition of protein synthesis is eventually lethal to all cells. The requirement of dividing cells (including cancer cells) for high level protein synthesis makes them more sensitive than quiescent cells to the cytostatic and cytotoxic effects of protein synthesis inhibitors. Because the basic scheme of protein synthesis remains the same in all living organisms there are many attractive schemes for screening human targets in heterologous organisms. 174 232/116

Polypeptide Synthesis Occurs in Several Steps and Requires Over 100 Proteins

The machinery of polypeptide synthesis includes:

Aminoacyl tRNA synthetases, which covalently couple amino acids to their cognate tRNAs. Eukaryotic cells have two sets of tRNA synthetases, one for aminoacylation of cytoplasmic tRNAs and one for aminoacylation of mitochondrial tRNAs. Both types of tRNA synthetases are encoded in the nuclear genome. Ribosomes, which translate mRNA into protein and integrate the action of the other components of the polypeptide polymerization machinery.

Initiation factors, which mediate the steps before the first peptide bond is formed, including formation of an initiation complex consisting of a ribosome, an mRNA and the first aminoacyl tRNA. Initiation is generally the rate limiting step in polypeptide synthesis. Elongation factors, which function in all the reactions between synthesis of the first peptide bond and addition of the last amino acid. Termination factors, which perform the reactions required to release completed polypeptide chains from ribosomes. Polypeptide chaperonins and other folding factors such as isomerases, which are necessary for the proper folding (and hence function) of proteins.

Polypeptide degradation machinery, including the ubiquitin system for tagging proteins for degradation and the proteasome and its constituents for cleaving proteins targeted for degradation. As cells grow and respond to changing circumstances there is a continual need to new protein synthesis. However, without some mechanism for eliminating existing unneeded or damaged proteins cells are not able to survive or proliferate.

There are approximately 20 cytoplasmic and 20 mitochondrial tRNA synthetases, approximately 80 ribosomal proteins, and over 20 protein constituents of initiation 175 232/116 factors, elongation factors and termination factors. The available evidence suggests that virtually all of these proteins are encoded by single copy genes. Thus at least 150 genes and their encoded proteins are potential candidates for allele specific targeting. (Conversely, the RNA constituents of the translational apparatus - transfer RNAs and ribosomal RNAs - are encoded by multicopy genes and do not constitute targets for allele specific targeting).

Inhibitors Have Been Identified for Most Steps of Peptide polymerization and processing

Well over 100 protein synthesis inhibitors with a wide variety of structures and mechanisms of action of have been characterized in both prokaryotes and eukaryotes.

Specific inhibitors have been identified for each step of translation described above.

See Table from Vasquez (ref. 1) for a summary of translation inhibitors. Inhibition of aminoacyl tRNA synthetases has been accomplished by at least three different mechanisms: amino acid analogs such as borrelidin and histidinol result in arrest of cell division by competing with natural amino acids for aminoacylation by tRNA synthetases. Inhibition of prokaryotic cell growth has also been demonstrated with RNA minihelices which mimic the acceptor stems of tRNAs. The minihelices compete with authentic tRNAs for aminoacylation by cognate tRNA synthetases. A third class of synthetase inhibitor is represented by pseudomonic acid A, a species specific inhibitor of gram positive isoleucyl tRNA synthetase produced by a gram negative organism. Pseudomonic acid A does not mimic amino acids or tRNAs, but binds to isoleucyl tRNA synthetase to inhibit peptide polymerization and processing.

Peptide polymerization and processing inhibitors that act on ribosomes include agents which bind the protein components and agents which bind or cleave the RNA components of ribosomes. An example of the former is the small 176 232/116 molecule drug emetine, which binds to ribosomal protein S14 and inhibits peptide polymerization and processing.

Peptide polymerization and processing Inhibitors are Cytostatic or Cytotoxic Drugs

Some of the most potent cytotoxic agents known are protein synthesis inhibitors. For example, a single molecule of ricin or diphtheria toxin is sufficient to kill a cell.

The largest class of protein synthesis inhibitors act on the elongation step of translation, with many inhibitors known for both prokaryotes and eukaryotes. Among the best studied prokaryotic elongation inhibitors are molecules belonging to the major antibiotic groups: the tetracyclines, streptomycin and other aminoglycosides, lincomycin and related compounds, erythromycin and related macrolide antibiotics and puromycin. Among the better characterized eukaryotic elongation inhibitors are toxins such as ricin and diphtheria toxin.

Cancer Chemotherapy by Inhibition of Peptide polymerization and processing

The best studied chemotherapeutic agent that acts solely by inhibiting protein synthesis is the enzyme L-asparaginase, used frequently in the treatment of acute lymphoblastic leukemia and occasionally in the treatment of other cancers. The therapeutic effect of L-asparaginase treatment is hydrolysis of serum L-asparagine to L-aspartate, with a rapidly ensuing drop in serum asparagine levels. While asparagine is not an essential amino acid, leukemia cells generally do not express asparagine synthase and are therefore reliant on importation of asparagine from serum via amino acid transporters in the plasma membrane. The effect of sudden asparagine starvation on rapidly dividing leukemia cells is to induce apoptotic death. Subsequent retreatment with L- asparaginase is generally not as effective as the initial treatment because the leukemia cells which survived the initial treatment have had time to induce expression of 177 232/116 asparagine synthase and are no longer dependent on external asparagine.

Examples of Genes Essential to Maintain Cellular Proteins at Levels Compatible with Cell Growth or Survival, From Yeast

The yeast Saccharomyces Cerevisiae is a eukaryote which shares many genes in common with humans. Approximately 70% of the essential genes in yeast have human homologs. Many human genes can be exchanged with their yeast counteφarts with minimal effects on growth in yeast or human cells. The study of essential genes in yeast is much further advanced than in mammalian systems: over half of the -6,000 genes of Saccharomyces Cerevisiae have been disrupted and the phenotype of the resulting strains tested on minimal growth media. Over 20% of disrupted yeast genes are essential, and a significant fraction of their human counteφarts are likely to be essential for cell survival. Among the yeast genes disrupted are a variety of genes that encode proteins required to maintain proteins at levels compatible with cell growth or survival. Many of these genes are essential for cell survival. Many of the disrupted essential yeast genes have closely related human homologs, and it is likely that the human homologs are also essential. Specific examples of yeast genes that are essential are listed below. All of these genes have human homologs. (This is a partial list because the Saccharomyces gene disruption project is only halfway done; see the web site http//quest7.proteome.com for an up to date list.)

GRC5, NHP2, NIP1, RPL1, RPL25, RPL27, RPL32, RPL35, RPL7, and URP2 are yeast ribosomal proteins that have been disrupted and found to be essential.

CDC33, GCD1, GCD10, GCD11, GCD2, GCD6, GCD7, PRT1, SIS1, SUI1, SUI2, SUI3, TIF 11, TIF34, and TIF5 are essential translation factors, mostly translation initiation factors that initiate translation at ATG. 178 232/116

EFBl and YEF3 are translation elongation factors that have been disrupted and found essential.

SUP35 and SUP45 are essential translation termination factors.

ALA1, HTS1, DED81, THS1, VAS1, WRS1 and KRSl are essential yeast cytoplasmic tRNA synthetases.

References 1. Vazquez, D. ( 1979) Molecular Biology and Biophysics, vol.30. Inhibitors of Protein

Synthesis. Springer- Verlag, Berlin.

2. Lim-Sylianco, C.Y. (1990) Toxins that alter the expression of genetic information: genotoxins and inhibitors of RNA or protein synthesis, pp. 338-421 in Shier, W.T. and D. Mebs, eds., Handbook of Toxinology. Marcel Dekker, New York.

Example 5. Genes required to maintain cellular nucleic acids at levels compatible with cell growth or survival.

Cellular nucleic acids including deoxyribonucleic acids and ribonucleic acids are essential elements for cell survival and proliferation. Many different genes are involved in maintaining these constituents at levels required for cell growth and proliferation including genes encoding enzymes for nucleotide synthesis, nucleotide degradation and salvage, polymerization of DNA (replication), polymerization of RNA (transcription), modifications of DNA including methylation, modifications of RNA including polyadenylation and capping, and processing or DNA and RNA. Many of these genes and their gene products are targets for conventional antiproliferative drugs.

RNA and DNA precursor Biosynthesis is Essential for Cell Proliferation 179 232/116

Nucleotides, the building blocks for both RNA and DNA, are essential for cell survival. Eukaryotic cells have several pathways for the production of nucleotides: de novo purine and pyrimidine biosynthesis, salvage pathways and membrane transport.

Over 50 Proteins Participate in RNA and DNA precursor Biosynthesis The principal enzyme groups involved in RNA and DNA precursor biosynthesis are the 14 enzymes of de novo purine biosynthesis, 5 enzymes of de novo pyrimidine biosynthesis (encoded in two polypeptides) and the enzymes of the nucleotide salvage pathways, which number at least 10.

Inhibitors of RNA and DNA precursor Biosynthesis are Cytostatic or Cytotoxic Drugs Useful in Cancer Chemotherapy Many of the most clinically effective antineoplastic agents block steps in RNA and DNA precursor biosynthesis. Examples include agents which block enzymes of de novo purine and pyrimidine biosynthesis or interfere with salvage pathways. For example, hydroxyurea blocks production of deoxyribonucleotides by ribonucleotide diphosphate reductase.

Purine Biosynthesis is essential for cell proliferation Pharmacologic inhibitors of purine biosynthesis are cytotoxic. These include drugs like azaserine and 6-diazo-5-oxo-L-norleucine (DON), glutamine analogs which inhibit three steps in purine synthesis, the most important being inhibition of the enzyme formylglycinamide ribonucleotide amidotransferase. 8-azaguanine and mycophenolic acid interfere with guanylate biosynthesis. (See Kornberg, A., DNA Replication, W.H. Freeman and Company, San Francisco, 1980, for a review of drugs that inhibit purine and pyrimidine biosynthesis.) There is also evidence of the essentiality of purine biosynthesis from yeast. For example, the saccharomyces cerevisiae PUR5 gene encodes inosine 5 '-monophosphate dehydrogenase, which converts inosine 5'- phosphate and NAD to xanthosine 5 '-phosphate and NADH, the first reaction unique 180 232/116 to GMP biosynthesis. Disruption of PUR5 is lethal.

Pyrimidine Biosynthesis is essential for cell proliferation

Pharmacologic inhibitors of pyrimidine biosynthesis are cytotoxic. These include drugs like phosphonacetyl-L-aspartate (PALA) which inhibits aspartate transcarbamylase, a key enzyme in de nove pyrimidine synthesis. Also, there is evidence of the essentiality of pyrimidine biosynthesis from yeast. For example, the saccharomyces cerevisiae CDC8 gene encodes thymidylate kinase, required for synthesis of dTTP. Disruption of CDC8 is lethal.

DNA synthesis and polymerization.

Cell division clearly requires DNA polymerization to replicate the chromosomes so that each daughter cell has the same genetic makeup as the parent cell. Much of the basic machinery of DNA replication is conserved in prokaryotic and eukaryotic cells

(1). Disruption of genes that encode proteins of DNA replication in yeast - including Polymerases I and III (the counteφarts of human polymerases a and d), and accessory factors such as Replication Protein A and Replication Factor C - is lethal in S. cerevisiae (2). Nucleotide analogs that are incoφorated into DNA are cytotoxic drugs. Examples of such analogs are the antineoplastic drug 6-mercaptopurine and arabinosyl

NTPs, which interfere with DNA polymerization. Since inability to replicate DNA is lethal for growing cells, mutants in DNA replication must be obtained as conditional lethals in both prokaryotes and eukaryotes.

Second strand DNA polymerization on takes place in three main steps, each requiring different protein machinery: (1) At the start of replication an initiation complex is formed at chromosome structures called origins of replication. The parental DNA strands are transiently separated, a replication fork is formed and DNA synthesis is primed. (2) The elongation phase of replication is thought to take place in two 181 232/116 complexes, one moving forward on the leading strand and the other moving iteratively in the opposite direction to form the lagging strand. Elongation, then, requires replicative DNA polymerases and associated factors for unwinding and transiently stabilizing single stranded DNA, proofreading the newly synthesized template and, on the lagging strand, removing RNA primers and covalently linking adjacent newly synthesized lagging strands (Okazaki fragments). (3) During the final phase of DNA synthesis replication is terminated and the newly synthesized strands are separated.

Origin recognition complexes are formed by at least 6 origin recognition complex proteins (ORC 1 through 6) along with other factors, including "licensing" proteins such as the MCM family as well as "regulating" factors.

The two principal nuclear replicative polymerases are DNA polymerase a, which is responsible for priming synthesis and for synthesis of the lagging strand, and DNA Polymerase d, which synthesizes the leading strand. Both are multisubunit proteins, which function in multiprotein assemblies that include Replication Protein A, Replication Factor C, Proliferating Cell Nuclear Antigen and other proteins.

DNA Polmerases b and e are believed to principally carry out nuclear repair synthesis, while Polymerase g is the mitochondrial replicative enzyme. These polymerases are also multiprotein complexes. Proteins such as topoisomerases I and II and other DNA helicases are also required during replication to maintain DNA topology. The biochemistry of replication termination is not well characterized however the proteins which carry out this final step of replication are likely to be essential.

Inhibitors Have Been Identified for Several Steps of DNA Replication

In addition to lethal disruptions of genes encoding proteins required for replication, a variety of cytotoxic inhibitors of DNA replication have been identified. They include 182 232/116 agents which act on production of DNA precursors as well as inhibitors of DNA polymerases.

DNA Replication Inhibitors are Cytostatic or Cytotoxic Drugs

There are several chemotherapy drugs that arrest DNA replication and poison cells by inhibiting production of deoxynucleotides, the precursors of DNA. These drugs include hydroxyurea, which inhibits ribonucleotide reductase, and 5-fluorouracil, which inhibits thymidylate synthase. Other inhibitors of replication appear to act, at least in part, by blocking DNA polymerases. These include nucleotide analogs that block DNA polymerases, such as 2',3' dideoxy NTPs and 3' deoxy ATP (cordycepin) as well as the chemotherapy drugs cytarabine (cytosine arabinoside), fludarabine phosphate and 2-chlorodeoxyadenosine. Cytarabine, after metabolism to the di- and trinucleotide phosphate forms, is incoφorated into DNA and inhibits chain elongation leading to cell death, apparently by inducing apoptosis. Fludarabine, after metabolism to the triphosphate derivative, inhibits DNA polymerase, DNA primase and ribonucleotide reductase and is incoφorated into DNA and RNA (3).

DNA polymerization is essential for cell proliferation

The essentiality of the function of DNA polymerization is clear, as such polymerization is needed for cell division, and therefore for tissue or tumor growth. As indicated for other categories, confirmation of the essentiality of a particular gene and the presence of a single locus, along with the determination of appropriate LOH and sequence variance heterozygosity characteristics identifies or confirms a gene in this category as an appropriate gene for potential allele specific targeting.

References 183 232/116

1. O'Donnell, M., Onrust, R., Dean, F.B., Chen, M. and J. Hurwitz (1993) Homology in accessory proteins of replicative polymerases - E. coli to humans. Nucleic Acids Research 21 :1-3.

2. Stillman, B. (1996) Cell Cycle Control of DNA Replication. Science 274: 1659- 1664.

3. Gandhi, V., Huang, P. and W. Plunkett (1994) Fludarabine inhibits DNA replication: a rationale for its use in the treatment of acute leukemias. Leukemia and Lymphoma 14 Suppl. 2: 3-9.

Maintaining RNA at levels required for cell growth or survival

Gene transcription is necessary for the production of messenger RNAs, the precursors of all cellular proteins. Transcription is also required for the production of ribosomal RNA, essential to formation of ribosomes, and for the production of transfer RNA, required for formation of aminoacyl tRNAs, the building blocks of protein synthesis. Turning off transcription - which can be accomplished with drugs that act on DNA templates or RNA polymerase - leads to rapid arrest of cell growth and subsequent cell death. Beyond gene transcription lie a series of essential RNA processing steps, including, but not necessarily limited to, mRNA splicing, capping, polyadenylation and export to the cytoplasm. Interference with any of these steps prevents the production of mature mRNA competent for translation, and therefore has the same cytotoxic effects as blocking transcription.

Gene Transcription and RNA Processing Require Many Proteins Transcription of eukaryotic genes is carried out by three different RNA polymerases, each of which works with a different set of accessory factors. RNA Polymerase I is responsible for transcription of ribosomal RNAs, RNA Polymerase II transcribes protein coding genes and RNA Polymerase III transcribes transfer RNAs and other small RNAs. All three polymerases are multiprotein complexes. Several protein subunits are common components of all three polymerases, but each polymerase also 184 232/116 has unique subunits and accessory factors, not all of which have yet been identified or characterized. Some of the key proteins identified so far are:

RNA Polymerase I subunits and accessory factors including UBF1 and SL1. (SL1 has been shown to consist of TATA binding protein and three TATA associated factors.)

RNA Polymerase III subunits and accessory factors including TFIIIA, TFIIIB and TFIIIC.

RNA Polymerase II and its accessory factors are by far the most extensively characterized and most complex system. The large multisubunit protein complex that transcribes protein coding genes has recently come to be called the RNA Pol II holoenzyme (reviewed by Berk, ref. 1). The holoenzyme consists of more than 50 proteins, among which are:

RNA polymerase, the catalytic complex at the core of the holoenzyme. It consists of 14 subunits, many of which can complement their yeast counteφarts in vivo.

The general transcription factors. These are proteins which either make direct contact with DNA, like TATA binding protein and associated factors, or interact with other transcription factors and/or transcriptional regulators. The general transcription factors, including TFII A, TFII B, TFII D, TFII E, TFII F, TFII H and TFII I, are multimeric protein complexes with >30 protein constituents (2,3). For example, there are 8-13 proteins which associate with TATA binding protein (called TATA associated factors, or TAFs) to collectively make up TFII D. Some of these factors (e.g. TFII250) have already been proven essential for cell proliferation. Accessory proteins such as elongation factors, termination factors, activator and mediator proteins, srb (suppressor of RNA Polymerase B; see ref. 1 and references therein) proteins, RNA methylases and a variety of other processing factors. RNA helicases, which are required for proper folding of RNAs, 185 232/116

Once transcribed, genes are spliced by multiprotein assemblies termed spliceosomes (4), which are made up of pre-mRNA, small nuclear ribonucleoproteins including (snRNPs) Ul, U2, U4/6 and U5 and other proteins including SF2/ASF, U2AF and SC35. Recently progress has been made in cloning cDNAs for several splicing factors, however many of the proteins which process mRNAs have not yet been well characterized. After splicing, mRNAs are polyadenylated and exported to the cytoplasm (5). Several of the proteins of polyadenylation have been purified and cloned. The export of mRNAs is less well studied but is clearly a specific process requiring protein machinery. Several essential yeast genes required for mRNA transport have been identified.

Inhibitors Have Been Identified for Several Steps of Gene Transcription

The best studied inhibitors of gene transcription are small molecules that inhibit RNA polymerase or interact with DNA to block transcription. Inhibitors of RNA polymerase include actinomycin D, which intercalates into double stranded DNA and blocks the movement of RNA polymerase and rifampicin, an antibiotic which binds the b subunit of E. Coli RNA polymerase and blocks initiation of transcription. The best studied specific inhibitor of eukaryotic RNA Polymerase II is the potent mushroom toxin a-amanitin, a cyclic octapeptide which binds to the polymerase with high affinity (Kd -10-9 M). Several mutations conferring resistance to alpha-amanitin have been characterized and they all map to the RNA Polymerase II protein coding sequence.

Examples of essential yeast genes (disruption shown to be lethal) required to maintain cellular nucleic acids at levels compatible with cell growth or survival

A number of yeast genes involved in DNA (including nuclear DNA and mt DNA) and RNA (including mRNA, tRNA and rRNA) metabolism have been disrupted and shown 186 232/116 essential for yeast cell viability. Many of these genes are conserved in all eukaryotes. Human homologs of these yeast genes are likely to be essential for human cell growth or survival. Specific examples:

The yeast DNA2 gene encodes a DNA helicase required for DNA replication. DNA2 is essential to the function of TOP2 (topoisomerase) which is also an essential gene.

POL1, POL2, POL3 and POL12 encode DNA polymerases. The disruption of any one of these genes is lethal. Knockout of polymerase associated genes DBP2 and POB3 is also lethal. These genes are essential for the synthesis of DNA.

ORC1, ORC2, ORC3, ORC4, ORC5, ORC6, CDC7, CDC46 and CDC54 are essential in yeast. These genes encode origin recognition complex proteins responsible for the initiation of DNA synthesis. There are direct human homologs of the ORC genes.

General replication factors RFA1, RFA2, RFA3, RFC1, RFC2, RFC3, RFC4 and RFC5 are all essential yeast genes. These genes encode replication protein A and replication factor C which are essential for DNA replication and have direct human homologs.

TBF1, TEL2 and CDC 13 are essential yeast genes that encode proteins that responsible for the synthesis and maintenance of telomeres.

RNR1 (Ribonucleotide Reductase 1), RNR2 (Ribonucleotide Reductase 2) CDC8 (Thymidylate Kinase) and PUR5 (Inosine-5'-monophosphate dehydrogenase) are essential yeast genes involve in the purine/pyrimidine biosynthesis pathways and in the conversion of ribonucleotides to deoxyribonucleotides.

ROX3, RPA135, RPA190, RPA43, RPB10, RPB11, RPB2, RPB3, RPB5, RPB6, 187 232/116

RPB7, RPB8, RPCIO, RPC128, RPC19, RPC25, RPC31, RPC34, RPC40, RPC53, and RPC82 are subunits of RNA polymerases I, II and III. These genes have been disrupted and shown to be essential. RNA polymerase I, II, and II are responsible for the synthesis of rRNA, mRNA, and tRNA respectively and have human homologs.

BRR2, DBP5, DBP6, DED1, HCA4, MAK5, and ROK1 are RNA helicases that are essential for processes such as pre-mRNA splicing and ribosomal RNA splicing.

Yeast TATA binding proteins TAF145, TAF17, TAF19, TAF25, TAF40, TAF47, TAF47, TAF60, TAF61 , TAF67, and TAF90 are required for mRNA transcription by the RNA Polymerase II holoenzyme.

Transcription elongation factors RPO21 and RPO31 are essential.

General transcription factors SPT15, SSL1, SSL2, SUA7, TFA1, TFA2, TFB1, TFB2,

TFB4, TFC2, TFC3, TFC4, TFC5, TFG1, TFG2, TOA1, and TOA2 have been disrupted and proven to be essential. These genes encode proteins that constitute the general machinery of RNA transcription.

Specific transcription factors BBP1, BRF1, BUR6, CDC39, HSF1, KIN28, MET30,

RAP1, and REB1 are essential yeast genes. These genes encode proteins that are involved in the transcription of specific genes.

CUS1, GIN10, MSL5, PRP19, PRP31, SLU7, SME1, SNP2, USS1, and YHC1 are essential genes responsible for normal RNA splicing.

ESS1, FIP1, NAB2, NAB3, NAB4, PAP1, RNA14, RNA15, and YTH1 are essential genes required for RNA modification. The encoded proteins perform functions such as cleavage and polyadenylation of 3' ends of RNAs to produce mature mRNA 188 232/116 molecules.

References

1. Berk, A.J. (1995) Biochemistry meets genetics in the holoenzyme. Proc. Natl. Acad.

Sci. U.S.A. 92: 11952-11954.

2. Buratowski, S.(1994) The basics of basal transcription by RNA polymerase II. Cell 77: 1-3.

3. Tjian, R. and T. Maniatis (1994) Transcriptional activation: a complex puzzle with few easy pieces. Cell 77: 5-8.

4. Green, M.R. (1991) Annual Review of Cell Biology 7: 559.

5. Proudfoot, N. (1991) Poly(A) signals. Cell 64: 671-674.

Example 6. Genes required to maintain integrity and function of cellular and subcellular structures compatible with cell growth or survival.

In order to survive and grow cells must be able to maintain their shape and internal architecture, including the structural integrity of a wide variety of subcellular organelles including the nucleus, mitochondria, endoplasmic reticulum and Golgi vesicles and a variety of lysosomes, peroxisomes vesicles and vacuoles. These structures perform essential functions such as:

(i) Movement of proteins and other macromolecules across membranes to maintain their concentration at levels compatible with cell growth or survival. Newly synthesized proteins are transported to the endoplasmic reticulum by specialized transport apparatus which assists in protein folding and posttranslational modification. From the ER, proteins may be transported to distant cellular sites via vesicles which are comprised of specialized proteins. Some proteins synthesized in the cytoplasm must be transported into the mitochondia for proper mitochondrial function. There 189 232/116 also exist specialized apparatus for transport of mRNA from the nucleus, (ii) Fusion or fission of various membrane bound cytoplasmic or nuclear organelles requires the specialized function of molecules that affect membrane properties to allow joining or separating and that provide a scaffold for moving membrane bound structures together or apart. The relationship of the ER and Golgi vesicles involves a continuous process of fission, while various classes of vacuoles or vesicles may fuse, (iii) There must be effective coordination of the function of all cellular compartments. Coordination is accomplished by the transmission of signals from membrane to nucleus, from cytosol to mitochondria, from nucleus to cytoplasm, etc. Signals are transmitted by enzymes such as adenylate cyclases, protein kinases and protein phosphatases.

(iv) Maintenance of the integrity of cellular and subcellular structures also requires processes and structures for eliminating, transforming, sequestering or otherwise regulating levels of endogenous cellular toxins or waste substances. This may be accomplished by transfer of waste molecules to organelles such as vacuoles, lysosomes or peroxisomes, by inactivation of toxic byproducts of oxygen metabolism such as free radicals or by export of molecules that have reached excessive levels in the cell, (v) The structure of the cytoplasm is maintained by the cytoskeleton, while different organelles in some cases are made up of specialized structural molecules. For example, the nucleus, bound by a double layered nuclear envelope, contains the nuclear matrix, consisting of over 100 unique proteins, as well as the histones and other proteins which form chromatin and the proteins which form subnuclear structures such as nucleoli, nuclear pores and the protein structures which convey mRNA out of the nucleus. (Darnell, J. et al., Molecular Cell Biology, Scientific American Books, 1990.)

The fibrous proteins of the cytoplasm are collectively referred to as the cytoskeleton. Among the important cytoskeletal proteins are microfilaments made up of actin molecules, microtubules made up of tubulin molecules, and intermediate filaments, made up of one of a variety of subunit types. The cytoskeleton is important not only 190 232/116 for maintenance of cell shape, strength and rigidity but also for providing a frame for movement of other structures. Microtubules, for example, are critical for chromosome movement during cell division, while actin microfilaments and intermediate filaments affect the organization and mobility of surface membrane proteins. Actins and other cytoskeletal proteins are vital for processes such as endocytosis, which is the only route of essential nutrients such as transferrin-bound iron. Cells also contain a variety of proteins essential for anchoring organelles to the cytoskeleton, or anchoring the plasma membrane to adjacent structures such as basement membranes and adjacent cells.

A variety of yeast structural proteins required to maintain integrity and function of cellular and subcellular structures have been disrupted and shown essential for cell survival. Since most structural proteins are highly conserved in eukaryotes it is likely that the human counteφarts of these yeast genes are also essential. Specific examples:

The genes encoding yeast nuclear pore proteins (nucleoporin) NIC96, NSP1 , NUP49,

NUP57, NUP82, NUP145 and NUP159 are lethal when disrupted, as is the pore trafficking protein GSP1. NNF1 is an essential protein of the nuclear envelope required for proper nuclear moφhology.

The yeast nucleolar protein NOP2, homologous to human proliferation associated nucleolar antigen pl20, is essential. NOP4 encodes another essential yeast nucleolar protein.

Knockout of the yeast ACT1 gene, which encodes actin, is lethal, as is knockout of the actin related proteins ARP100, ARP2, ARP3 and ARP4. The actin binding and severing protein cofilin, encoded by the yeast COF1 gene, is also essential, as is profilin (PFY1), which can complex with actin monomers and prevent actin polymerization. PAN1 is an essential protein involved in normal regulation of the actin cytoskeleton. 191 232/116

The RET1, RET2, RET3, SEC1, SEC4, SEC5, SEC6, SEC7, SEC8, SEC10, SEC11, SEC 14, SEC 16, SEC 17, SEC 18, SEC20, SEC26, SEC27, SEC31, SEC61, SEC62, SEC63, SFT1, SLY1, BET1, BET3, UFE1, USO1, VTI1, TIP20, KAR2 and BOS1 genes are all essential in yeast. These genes encode proteins which are vital for the function of the endoplasmic reticulum and Golgi vesicles, including processes such as protein transport across the ER, membrane fusion and formation of vesicles.

The essential yeast histone-like protein CSE4 is required for chromosome segregation. STH1, RSC6 and RSC8 are components of the essential abundant chromatin remodeling complex, while SPT5 and SPT6 influence gene expression through effects on chromatin structure.

The essential yeast intermediate filament protein MDM1 is involved in organelle inheritance and mitochondrial moφhology.

The essential yeast mitochondrial proteins MGEl and SSCl participate in folding of proteins during mitochondrial import. TIM 17, TIM22 and TIM23 are essential mitochondrial inner membrane proteins involved in import and translocation of proteins. ATM1 is an ATP binding mitochondrial inner membrane protein.

The RAT1, MTR2 and MTR3 genes encode proteins essential for mRNA transport from the nucleus to the cytoplasm.

DBF8 is an essential yeast protein involved in protein transport.

APS2 is an essential clathrin associated protein, involved in membrane transport.

The yeast PKC1 gene encodes the essential protein kinase C, which regulates the MAP kinase cascade; CDC 15 is an essential component of the MAP kinase kinase kinase 192 232/116 family of signaling proteins.

CYR1 is an essential adenylate cyclase which generates cAMP in response to signals including ras activation. GDI1 is an essential GDP dissociation inhibitor.

Example 8: Validation of Target Gene Essentiality

To investigate whether specific target genes are essential for cell proliferation and/or survival, a method was developed to use antisense oligonucleotides to inhibit gene expression. Phosphorothioate antisense oligonucleotides targeting polymoφhic sites were transfected into human cell lines, and mRNA down-regulation was assessed by northern blotting. mRNA down-regulation was achieved for 19 of the 35 polymoφhisms targeted (54.2%). Oligonucleotides targeting each polymoφhic allele were (separately) transfected to assess the allele-specificity of the mRNA down- regulation. In 15 of the 19 sites accessible to oligos, the oligonucleotide targeting the allele found in the cell down-regulated mRNA to a level significantly lower than did the mismatched oligonucleotide. In 6 of these 15 cases, striking allele-specificity was observed.

The consequence of down-regulating the mRNA of an essential gene should be cell death. Allele-specific cell death was indeed observed in these experiments, both upon transfecting cells daily for three days with the phosphorothioate oligos described above (followed by a recovery period during which control-treated cells continued to divide while essential gene inhibition prevented division) or upon extended (5-10 days) daily transfections with less toxic oligonucleotide chemistries. In an experiment targeting either RNA polymerase II or the glutamyl-prolyl tRNA synthetase (EPRS), cells were transfected for five consecutive days with oligos either targeting the allele found in the cell (match) or carrying a 1 bp mismatch, targeting the other allele (mismatch). One day after the fifth transfection, cells remaining on the plate were quantitated by staining 193 232/116 with sulforhodamine B. The matched oligonucleotide was significantly more cytotoxic than the mismatched oligonucleotide.

Example 9: Aminoacyl tRNA Synthetases are Essential for Cell Survival

Each aminoacyl-tRNA synthetase performs an analogous role in protein synthesis, and each represents a target for the present invention.

Aminoacyl-tRNA synthetases perform a basic cell function

Aminoacyl-tRNA synthetases are present in all living cells (1). (A recent paper entitled "A minimal gene set for cellular life derived by comparison of complete bacterial genomes" [ref. 2] concludes that as few as 256 genes may be required for prokaryotic cell life; all 20 tRNA synthetases are included in this minimal gene set.) Each tRNA synthetase catalyzes ATP dependent covalent attachment of a specific amino acid to its cognate tRNA. It is the specificity of each synthetase for a single amino acid and transfer RNA that establishes the universal rules of the genetic code. The aminoacyl- tRNAs produced by tRNA synthetases constitute the precursors for protein assembly by ribosomes - thus tRNA synthetases are vital for peptide polymerization and processing. Blockade of peptide polymerization and processing at any one of multiple different steps (see above) results in arrest of cell growth and eventually cell death in a variety of organisms and cell types.

Aminoacyl-tRNA synthetases have been shown essential in all tested organisms

It has been demonstrated by mutagenesis experiments that tRNA synthetases are essential for prokaryotic, yeast and mammalian cell survival (ref. 1-5). The most relevant data concerns mammalian cells: mutagenesis of Chinese hamster ovary (CHO) 194 232/116 and Chinese hamster lung cells followed by "suicide" selection at 39oC for temperature sensitive (ts), conditionally lethal protein synthesis mutants has led to isolation of cell lines with mutant tRNA synthetases (reviewed in ref. 5). (The "suicide" of dividing cells is accomplished by adding thialysine or tritiated [3H] amino acids to cell growth media. Only cells that incoφorate these amino acid analogs into protein die - thus cells that are protein synthesis deficient at 39oC survive the selection.) The fraction of cells surviving a single round of suicide selection ranges from one in 105 to one in 108. Biochemical and genetic characterization of surviving cells has led to identification of specific ts aminoacyl-tRNA synthetase mutants. Cell lines with mutant leucyl- or asparaginyl-tRNA synthetases have been isolated repeatedly because the genes for leu and asn tRNA synthetases are haploid in the CHO cell line used for selection, and therefore require only one mutation. Less frequently, mutant alanyl-, arginyl-, glutaminyl-, histidyl-, lysyl-, methionyl-, tryptophanyl- and valyl-tRNA synthetases have been isolated. The properties of these mutant cell lines are similar: when shifted to 39oC, the non-permissive temperature, the rate of protein synthesis drops, in some cases to almost undetectable levels. Soon thereafter the cells stop replicating DNA and within a few days cell death ensues. These experiments constitute proof of the essential role of tRNA synthetases in mammalian cells. Arrest of protein synthesis and consequent cell death can be prevented in some cases by supplementing cell media with the amino acid substrate of the defective tRNA synthetase (thereby driving the aminoacylation reaction), or by fusing the mutant cell line with a normal cell line, or a cell line mutant for a different tRNA synthetase (thereby complementing the mutant synthetase). The cell fusion experiments show that the aminoacyl-tRNA synthetase mutations are recessive at the cellular level. The chromosomal map positions of a number of human tRNA synthetases were first determined by analysis of (human) X (ts mutant CHO cell) hybrids. Human chromosomes are progressively lost in such hybrids, but one human chromosome - the one which contains the human synthetase complementary to the mutant hamster synthetase - is consistently retained. Such experiments provided the first evidence that 195 232/116 human tRNA synthetases are single copy genes in man (or at least confined to a single chromosome; refs. 6, 7). Subsequently Southern blotting and fluorescence in situ hybridization analyses have confirmed and extended these observations for thirteen synthetases (8-14). These Southern blotting and in situ hybridization mapping studies established beyond doubt that each of the human tRNA synthetase genes investigated is encoded at a single locus. The table below summarizes the chromosomal location of tRNA synthetases mapped to date.

Chromosome Location of tRNA Synthetases

tRNA synthase Chromosome tRNA Synthetase Chromosome

Ala 16q22 Tφ 14q21-32

Arg, Leu, His, Thr 5 Asp 2

Asn 18 Gin 3p

Cys l lplS.5 Gly 7

Glu/Pro lq32-42 He (mitochondrial) 2

Gly 7pl5 Lys 16q21

He 9q21 Ser lpl2

Lys 16q23-24 Tyr lp31

Met 12 Val 6p21.3 9

Classification of tRNA synthetases

The twenty tRNA synthetases are divided into two groups based on structural features and functional properties that are conserved throughout evolution. There are ten class I synthetases, all of which contain two short conserved amino acid segments which fold together to form an ATP binding pocket called the Rossman fold, in the amino 196 232/116 terminal half of the proteins. The C-terminal end of the Class I synthetases contains the tRNA binding fold. Class II synthetases, of which there are also ten, share up to three short conserved amino acid motifs.

References

1. Schimmel, P., Giege, R., Moras, D. and S. Yokoyama (1993) An operational RNA code for amino acids and possible relationship to genetic code. Proc. Natl. Acad. Sci. U.S.A. 90:8763-8768. 2. Mushegian, A.R. and EN. Koonin (1996) A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc. Natl. Acad. Sci. U.S.A. 93:10268-10273.

3. Schmitt, E. and P. Schimmel (1994) Dominant lethality by expression of a catalytically inactive class I tRΝA synthetase. Proc. Natl. Acad. Sci. U.S.A. 90:6919- 6923.

4. Ripmaster, T.L., Shiba, K. and P. Schimmel (1995) Wide cross-species aminoacyl- tRΝA synthetase replacement in vivo: Yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proc. Natl. Acad. Sci. U.S.A. 92:4932-4936.

5. Wasmuth, J.J., Chinese Hamster Cell Protein Synthesis Mutants, pp. 375-421 in Gottesman, M., editor, Molecular Cell Genetics, John Wiley & Sons, New York, 1985.

6. Kunze, N., Bittler, E., Fett, R., Schray, B., Hameister, H., Wiedorn, K.-H. and R. Knippers (1990) The human QARS locus: assignment of the human gene for glutaminyl-tRNA synthetase to chromosome lq32-42. Human Genetics 85:527-530. (This gene was later shown to be glutamyl-prolyl tRNA synthetase [abbreviated EPRS]).

12. Cirullo, R.E., Arredondo-Vega, F.X., Smith, M. and JJ. Wasmuth (1983) Isolation and characterization of interspecific heat-resistant hybrids between a temperature- sensitive Chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: assignment of human asnS gene to chromosome 18. Somatic Cell Genetics 197 232/116

9:215-233.

13. Nichols, R.C., Pai, S.I., Ge, Q., Targoff, I.N., Plotz, P.H. and P. Liu (1995) Localization of two human autoantigen genes by PCR screening and in situ hybridization - glycyl tRNA synthetase locates to 7pl5 and alanyl-tRNA synthetase locates to 16q22. Genomics 30:131-132.

14. Nichols, R.C., Blinder, J., Pai, S.I. et al. (1996) Assignment of two human autoantigen genes: isoleucyl tRNA synthetase locates to 9q21 and lysysl-tRNA synthetase locates to 16q23-24. Genomics: 210-213.

Example 11: Sodium Potassium ATPase, 1 subunit (ATP1A1) - Target Gene VARIA125

Sodium Potassium ATPase is essential for cell survival

The plasma membranes of virtually all eukaryotic cells contain a Na+, K+ pump that operates as an antiport, pumping Na+ out of the cell and K+ in against their concentration gradients. In coupling the hydrolysis of ATP to the active transport of 3 Na+ out and 2 K+ into the cell the pump is electrogenic. The electrochemical gradients generated and maintained by the Na+,K+ pump are essential for the regulation of cell volume, and for the secondary, sodium-coupled active transport of a variety of organic and inorganic molecules including glucose, amino acids and Ca++. Hence the sodium potassium pump plays an essential role in cellular physiology (1).

Sodium Potassium ATPase is a heterodimer composed of a -100 kDa catalytic subunit and a -55 kDa glycoprotein subunit of unknown function. Biochemical studies and gene cloning have demonstrated the existence of three isoforms and two -like isoforms of the catalytic subunit, each encoded by a separate gene and with a characteristic expression pattern (reviewed in refs. 2 and 3). Of these, only the 1 gene 198 232/116

(ATP1A1) is ubiquitously expressed; the other subunits have restricted tissue distribution.

Sodium Potassium ATPase is the target of the cardiac glycoside drugs, including digoxin and the poison ouabain. Ouabain binds to the extracellular face of the 1 subunit and inhibits Na+,K+ exchange, leading to cell death. The 1 subunit from primates is sensitive to nanomolar concentrations of ouabain while the rodent 1 subunit is resistant to -1000 fold higher concentrations, enabling precise definition of the ouabain binding site. Study of human-rat chimeric 1 subunits combined with site directed mutagenesis has localized the ouabain interacting domain in the amino- terminal portion of the 1 subunit (4,5). Other structure-function studies have contributed to an understanding of 1 subunit cation binding and ATPase functions, while electron microscopy and low resolution (20-30 ) diffraction analyses of membrane preparations have elucidated the geometry of the protein in the membrane (1).

The I subunit of Sodium Potassium ATPase has sequence variants

The cDNA sequence of the human 1 subunit of sodium-potassium ATPase has been published by four groups (6-9). We undertook a systematic search for DNA sequence variance by analyzing the 1 cDNA from 36 unrelated individuals using the single strand conformation polymoφhism technique. Primers were designed using the sequence of Kawakami et al. (GENBANK accession D00099; see ref. 6). SSCP analysis revealed 7 sequence variances, and subsequent DNA sequence analysis confirmed that nucleotides 1059 (A vs. C), 1428 (G vs. A), 2538 (T vs. C), 3324 (C vs.

T), 3375 (G vs. A), 3397 (G vs. A) and 3408 (C vs. A) vary as shown in the Target Summary Table. The first five sequence variances are in the coding sequence while the latter two are in the 3' untranslated region. 199 232/116

The frequency of heterozygotes for the seven sequence variants ranged from 3-11% among the 36 individuals tested. Some of the sequence variances appear to occur more commonly in certain racial or ethnic groups. For example, heterozygotes for four sequence variances (at nucleotides 1059, 1428, 3324 and 3375) were detected solely or predominantly in North American Blacks, with heterozygote frequencies of 1/4 or

2/4. The nucleotide 2538 variance was detected solely in North American Whites (4/16) and results in an amino acid exchange (see below). The nucleotide 3397 sequence variance was detected solely in one Japanese individual (of four tested). The nucleotide 2538 sequence variant results in an aspartic acid vs. glutamic acid substitution at amino acid 740 of the 1024 amino acid protein. This residue lies in the cytoplasmic loop of the 1 subunit.

The alphal subunit of Sodium Potassium ATPase maps to chromosome lplS-pll

The gene for the 1 subunit of sodium-potassium ATPase has been mapped to chromosome band lpl3-pl 1 by several techniques. Yang-Feng et al. (10) assigned the ATP1 Al gene to lp21-cen by Southern analysis of DNA from panels of rodent/human somatic cell hybrid lines. This localization was confirmed and refined by Chehab et al., who showed that the gene for the ATP1A1 subunit is on lpl3-pll using hybridization to flow-sorted chromosomes and in situ hybridization (9).

Chromosome band lpl -pll is a site of frequent loss of heterozygosity

The short arm of chromosome 1 is comparatively well investigated for allele loss, especially in breast and colon cancers, however most of these studies are principally concerned with the lp36 region, and there is comparatively little data on lpl3-pl l. The best studies of proximal lp allele loss are in breast and testicular cancers. These studies show LOH occurs in approximately 15-35% of breast cancers (11,12) and 15- 25% of testicular cancers (13). Data from more distal loci on lp show >25% LOH in 200 232/116 glioma, colon cancer, stomach cancer, ovarian cancer, and liver cancer (14). The LOH observed in this region indicates that other essential genes mapping to the lp chromosomal arm, and especially to the lpl 1 region, which have LOH and for which sequence variances, and therefore heterozygotes for a sequence variance, exist in normal somatic cells of individuals in a population are potential target genes

References

1. Jorgensen, P.L. Na, K-ATPase, structure and transport mechanism. In De Pont, ed. Molecular Aspects of Transport Proteins. Elsevier Science Publishers, The

Netherlands, 1992, pp. 1-26..

2. Sweadner, K.J. (1989) Biochimica et Biophysica Acta 1154: 185-220.

3. Lingrel, J.B., Orlowski, J., Shull, M.M. and E.M. Price (1989) Prog. Nucleic Acid Research and Mol. Biol. 38: 37-89. 4. Price, E.M. and J.B. Lingrel (1988) Structure-function relationships in the Na, K-

ATPase a subunit: site directed mutagenesis of glutamine-111 to arginine and asparagine 122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27: 8400-8408.

5. Emanuel, J.R., Graw, S., Housman, D. and R. Levenson (1989) Identification of a region within the Na, K-ATPase a subunit that contributes to differential ouabain sensitivity. Molecular and Cellular Biology 9: 3744-3749.

6. Kawakami, K., Ohta, T., Nojima, H., Nagano, K. (1986) Primary structure of the alpha-subunit of human Na,K- ATPase deduced from cDNA sequence. J. Biochem. 100: 389-397. 7. Ovchinnikov, Y. A., Monastyrskaya, G. S., Broude, N. E., et al. (1987) The family of human Na+,K+- ATPase genes: a partial nucleotide sequence related to the alpha- subunit. FEBSLett. 213: 73-80.

8. Shull, M. M. and J.B. Lingrel (1987) Multiple genes encode the human Na+,K+- ATPase catalytic subunit. Proc. Nat. Acad. Sci. U.S.A. 84: 4039-4043. 201 232/116

9. Chehab, F. F., Kan, Y. W., Law, M. L., Hartz, J., Kao, F.-T. and R. Blostein (1987) Human placental Na+,K+-ATPase alpha subunit: cDNA cloning, tissue expression, DNA polymoφhism, and chromosomal localization. Proc. Nat. Acad. Sci. U.S.A. 84: 7901-7905. 10. Yang-Feng, T.L., Schneider, J.W., Lindgren, V., Shull, M.M., Benz, E.J., Jr.,

Lingrel, J.B. and U. Francke (1988) Chromosomal localization of human Na+,K+- ATPase alpha- and beta-subunit genes. Genomics 2: 128-138.

11. Bieche, I., Champeme, M.H., Matifas, F., Cropp, C.S., Callahan, R. and R. Lidereau (1993) Two distinct regions involved in lp deletion in human primary breast cancer. Cancer Res. 53:1990-4.

12. Nagai H, Negrini M, Carter SL, et al. (1995) Detection and cloning of a common region of loss of heterozygosity at chromosome lp in breast cancer. Cancer Res. 55:1752-7.

13. Mathew S., Murty V.V., Bosl G.J., Chaganti R.S.K. (1994) Loss of heterozygosity identifies multiple sites of allelic deletions on chromosome 1 in human male germ cell tumors. Cancer Res. 54:6265-9.

14. Yeh S.H., Chen P.J., Chen H.L., Lai M.Y., Wang CC. and D.S. Chen (1994) Frequent genetic alterations at the distal region of chromosome lp in human hepatocellular carcinomas. Cancer Res. 54:4188-92.

Example 12: Ribonucleotide Reductase, Ml subunit (RRM1) - Target Gene VARIA200

Ribonucleotide Reductase is essential for cell growth

Human ribonucleotide reductase (also called ribonucleoside diphosphate reductase) is essential in dividing cells for the production of deoxyribonucleotides prior to DNA synthesis in S phase. Ribonucleotide reductase catalyzes the reduction of all four 202 232/116 ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates by replacing the 2' hydroxyl moiety of ribose with a hydride ion to form deoxyribose; these reactions constitute the first committed steps in the creation of DNA precursors (deoxyribonucleotides), and are therefore tightly regulated by allosteric nucleotide binding sites on the Ml subunit (2,3). The enzyme is an 2 2 tetramer apparently conserved in all prokaryotes and eukaryotes (1). The two subunits, Ml and M2, are both required for enzyme activity. The RRM2 subunit contains the catalytic site, while the RRM1 subunit provides an indispensable allosteric function. (See pages 758-763 of Biochemistry by C.K. Mathews and K.E. van Holde, Benjamin/Cummings Publishing Biochemistry, Company, Redwood City, 1990 for a fuller account of ribonucleotide reductase function.)

Both ribonucleotide reductase subunits are expressed in all proliferating cells but are generally nondetectable in quiescent cells. Ribonucleotide reductase subunit M2 is the target of several antineoplastic compounds, including hydroxyurea. Hydroxyurea is used in the chemotherapy of a variety of myeloproliferative disorders (4). It acts by reversibly destroying a tyrosyl free radical in the catalytic site of the M2 subunit (3). Hydroxyurea and other ribonucleotide reductase poisons are specific for the S phase of the cell cycle, resulting in growth arrest at the Gl-S boundary and apoptotic death in tumor cells (5). Exposure of cell cultures to hydroxyurea results in selection of cells expressing high levels ribonucleotide reductase, demonstrating that ribonucleotide reductase is required for these cells to grow (6).

The human ribonucleotide reductase gene has sequence variances

The cDNA sequence of the human ribonucleotide reductase Ml subunit has been published by two groups (7,8). We undertook a systematic search for DNA sequence variance in the cDNA of the Ml subunit by analysing 36 unrelated individuals using the single strand conformation polymoφhism technique. Primers were designed using 203 232/116 the sequence of Parker et al. (GENBANK accession X59543; see ref. 7). SSCP analysis revealed 4 sequence variances, and subsequent DNA sequence analysis confirmed that nucleotides 1037 (C vs. A), 2410 (A vs. G), 2419 (A vs. G) and 2717 (T vs. A) vary as shown in the Target Summary Table. (The sequence variance at nt 1037 was previously noted by Parker et al., ref. 7.) Also, DNA sequencing revealed an insertion/deletion sequence variance: the 9 consecutive T nucleotides between positions 2724 and 2732 (numbering from ref. 7) were augmented in some cDNAs by a tenth T. (This sequence variance is designated T9 vs. T10 in the Target Summary Table.)

Both alleles at nt 1037 were detected in North American Whites, Hispanics, Chinese, Japanese, Arabs and Indians. Similarly, both alleles of the sequence variance at nt 2410 were detected in virtually all tested populations: North American White, North American Black, Hispanic, Chinese, Arab and Indian. In contrast, the sequence variances at nt 2419 and 2717 were prevalent in North American Blacks, Hispanics,

Chinese, and Japanese, but not North American Whites. The insertion/deletion sequence variance at nt 2724 was only studied in four individuals so no firm conclusions can be drawn regarding population distribution, however it appears to be in linkage disequilibrium with the 2419 and 2724 sequence variances.

The human ribonucleotide reductase gene maps to chromosome lip 15.5

The gene for human ribonucleotide reductase has been mapped to band 1 lpl5.5 by several techniques. Initially the gene was localized by Southern hybridization analysis of human X rodent somatic cell hybrids and by chromosomal in situ hybridization (9).

Subsequently RRM1 has been placed on a yeast artificial chromosome (YAC) physical map of chromosome 1 lpl5 (10). The precise physical localization of the RRM1 gene facilitates inteφretation of LOH results at adjacent polymoφhic markers (see below). 204 232/116

Chromosome band lip 15.5 is a site of frequent loss of heterozygosity

The short arm of chromosome 11 is the site of several tumor suppressor genes, including the WT1 gene and the Beckwith-Weidemann syndrome gene. As a result there are many studies of LOH in l lpl5.5, particularly focusing on breast, cervix, kidney, liver, lung, ovarian, stomach and testicular cancers. These studies show that the 1 lpl5.5 band of chromosome 11 is frequently reduced to one copy (11-28). For example, LOH occurs in approximately 13-33% of breast cancers (11-13), 14-42% of cervical cancers (14), 0-50% of liver cancers (16), 0-80% of lung cancers (17-19), 18- 54% of ovarian cancers (20,21), 0-71% of stomach cancers (22) and 0-50% of testicular cancers (23,24). Other studies show that 1 lpl5.5 LOH may also be frequent in bladder cancer (25), esophageal cancer (26), some leukemias (27) and sarcomas (28). Many deletions in the l lpl5.5 region span relatively short chromosomal segments (2 - 10 megabases; see ref. 17).

References

1. Caras, I.W., Levinson, B.B., Fabry, M., et al. (1985) Cloned mouse ribonucleotide reductase subunit Ml cDNA reveals amino acid sequence homology with Escherichia Coli and heφesvirus ribonucleotide reductases. J. Biol. Chem. 260: 7015-7022.

2. Thelander, L., and P. Reichard, (1979) Reduction of Ribonucleotides. Annu. Rev. Biochem. 48:133-158.

3. Reichard, P. and A Ehrenberg (1983) Ribonucleotide reductase: a radical enzyme. Science 221: 514-9. 4. Donehower, R.C. (1992) An Overview of the clinical experience with hydroxyurea. Seminars in Oncology 19:11-19, 1992.

5. Wright, P.S., Cross-Doersen, D., ThOng, J.P., et al. (1996) A ribonucleotide reductase inhibitor, MDL 101,731, induces apoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts. Experimental Cell Research 22: 54-60. 205 232/116

6. Cocking, J.M., Tonin, P.N., Stokoe, et al. (1987) Gene for Ml subunit of ribonucleotide reductase is amplified in hydroxyurea-resistant hamster cells. Somat. Cell. Mol Genet. 13:221-33.

7. Parker, N.J., Begley, C.G. and R.M. Fox. (1991) The Human Ml Subunit of Ribonucleotide Reductase: cDNA Sequence and Expression in Stimulated

Lymphocytes. Nucleic Acids research 9:3741.

8. Pavloff, N., Rivard, D., Masson, S., Shen, S.H. and A.M. Mes-Masson. (1992) Sequence Analysis of the Large and Small Subunits of Human Ribonucleotide Reductase. DNA Sequence 2:227. 9. Brissenden, J.E., Caras, I., Thelander, L. and Francke, U. (1988) The structural gene for the Ml subunit of ribonucleotide reductase maps to chromosome 11, band pl5, in human and to chromosome 7 in mouse. Exp. Cell. Res. 174:302-8.

10. See: http://shows.med.buffalo.edu/home.html

11. Ali, I., Lidereau, R., Theilley, C. and R. Callahan (1987) Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia. Science

238:185-8.

12. Winqvist, R., Mannermaa, A., Alavaikko, et al. (1993) Refinement of regional loss of heterozygosity for chromosome 1 lpl 5.5 in human breast tumors. Cancer Research 53: 4486-4488. 13. Carter, S.L., Negrini, M., Baffa, R., et al. (1994) Loss of heterozygosity at 1 lq22- q23 in breast cancer. Cancer Research 54:6270-4.

14. Mitra, A.B., Murty, V.V.N.S., Li, R.G., et al. (1994) Allelotype analysis of cervical carcinoma. Cancer Research 54:4481.

15. Fujimori, M., Tokino, T., Hino, O., et al. (1991) Allelotype study of primary heptocellular carcinoma. Cancer Research 51 : 89-93.

16. Wang, H.P. & C.E. Rogler (1988) Deletions in human chromosomes 1 lp and 13q in primary hepatocellular carcinomas. Cytogenetics and Cell Genetics 48:72-78.

17. Bepler, G. and Garcia-Blanco, M.A. (1994) Three Tumor Suppressor Regions on Chromosome l ip Identified by High Resolution Deletion Mapping in Human Νon- 206 232/116

Small Cell Lung Cancer. Proc. Natl. Acad. Sci. U.S.A. 91:5513-7.

18. Iizuka, M., Sugiyama, Y.,Shiraishi, M., et al. (1995) Allelic losses in human chromosome 11 in lung cancers. Genes, Chromosomes & Cancer 13:40-46.

19. Weston, A., Willey, J.C., Modali, R., et al. (1989) Differential DNA sequence deletions from chromosomes 3, 11, 13 and 17 in squamous cell carcinoma , large-cell carcinoma and adenocarcinoma of the human lung. Proc. Natl. Acad. Sci. U.S.A. 86:5099-5103.

20. Kiechle-Schwartz, M., Bauknecht, T., Wienker, T., et al. (1993) Loss of Constitutional Heterozygosity on Chromosome l ip in Human Ovarian Cancer. Cancer 72:2423-32.

21. Viel, A., Giannini, F., Tumiotti, L., Sopracordevole, F., Visentin, M.C. and M. Boiocchi (1992) Chromosomal localization of two putative l ip oncosuppressor genes involved in human ovarian tumors British Journal of Cancer 66: 1030-1036.

22. Baffa, R., Negrini, M., Mandes, B., et al. (1996) Loss of heterozygosity for chromosome 11 in adenocarcinoma of the stomach. Cancer Research 56: 268-72.

23. Lothe, R.A., Hastie, N., Heimdal, K., et al. (1993) Frequent loss of lpl3 and 1 lpl5 loci in male germ cell tumors. Genes, Chromosomes & Cancer 7:96-101.

24 Smith, R.C, and Rukstalis, D.B. (1995) Frequent Loss of Heterozygosity at 1 lp Loci in Testicular Cancer. The Journal of Urology 153:1684-7. 25. Shaw, M.E. and Knowles, M.A. (1995) Deletion Mapping of Chromosome 11 in

Carcinoma of the Bladder. Genes, Chromosomes & Cancer 13:1-8.

26. Shibagaki, I., Shimada, Y., Wagata, T., et al. (1994) Allelotype analysis of esophageal squamous cell carcinoma. Cancer Research 54: 2996-3000.

27. Ahuja, H.G., Foti, A., Zhou, D.J. and M.J. Cline (1990) Analysis of proto- oncogenes in acute myeloid leukemia: loss of heterozygosity for the Ha-ras gene.

28. Yamaguchi, T., Toguchida, J., Yamamuro, T., et al. (1992) Allelotype analysis in osteosarcoma: frequent allele loss on 3q, 13q, 17p and 18q. Cancer Res. 52: 2419. 207 232/116

Example 13: Thymidylate Synthase (TS) - Target Gene VARIA250

Thymidylate Synthase is essential for cell growth

Human thymidylate synthase (TS) catalyzes the formation of thymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP) by transfer of a methyl group from N5,N10-methylenetetrahydrofolate to carbon 5 of dUMP. This is the sole de novo pathway to dTMP, an essential precursor for DNA synthesis. TS also plays an important role in balancing the four nucleotide precursors for DNA polymer synthesis (1). Thus TS is an attractive target for antiproliferative drugs. (See

Biochemistry by CK. Mathews and K.E. van Holde, Benjamin/Cummings Publishing Company, Redwood City, 1990, pages 763-768, for a fuller account of thymidylate synthase function.)

Like some other growth associated genes involved in DNA synthesis, thymidylate synthase is expressed in proliferating cells at 20-40 fold higher levels than in quiescent cells. Increased expression occurs at the Gl-S transition of the cell cycle when quiescent cells are stimulated with serum. Levels of thymidylate synthase are finely controlled by autoregulatory feedback loops wherein TS protein regulates the transcription, stability and translational efficiency of TS mRNA (2). Transcription increases by only 2-4 fold, so posttranscriptional events constitute the predominant regulatory mechanisms (3). One mechanism of 5-FU resistance is increased expression ofTS Mrna.

Thymidylate synthase is the target of 5-fluorouracil (5-FU), a potent antineoplastic compound. Once inside cells 5-FU is ribosylated and phosphorylated to 5-fluoro-2'- deoxyuridine 5'-monophosphate (F-dUMP), which acts as an inhibitory transition state analog of TS when bound in the presence of the enzyme's second substrate, N5,N10- methylenetetrahydrofolate. (5-FU is also incoφorated into both DNA and RNA, 208 232/116 augmenting its toxicity.) 5-FU induces partial responses in 10-30% of patients with a variety of cancers, including metastatic breast and gastrointestinal tract cancers (4). While 5-FU is a potent antiproliferative agent in tissue culture cells, as with most antineoplastic drugs, its clinical utility is limited by lack of discrimination between normal cells and tumor cells: common toxic effects include stomatitis, diarrhea, bone marrow suppression, hair loss and occasionally cardiac and neurologic symptoms.

The human thymidylate synthase gene has sequence variances

The sequence of a human thymidylate synthase cDNA was determined by Takeishi et al. (5), who later determined the genomic sequence as well (6). We undertook a systematic search for DNA sequence variance by analysing 36 unrelated individuals using the single strand conformation polymoφhism. Primers were designed using the sequence of Takeishi et al. (5). SSCP analysis revealed 3 DNA fragments having sequence variances, and subsequent DNA sequence analysis showed that nucleotides

1066 (C vs. T), 1136 (A vs. G) and 1497 (A vs. T) vary among normal individuals as shown in the Target Summary Table. All three sequence variances are in the 3' untranslated region of the gene. The nucleotide 1066 and 1497 sequence variances are in complete linkage disequilibrium in the 36 individuals examined. Both alleles of all three sequence variances were detected in North American Whites, North American

Blacks, Chinese, Japanese, Arabs and Indians.

Another TS sequence variance has been described by Berger and colleagues (7-9). They detected a T to C change at nucleotide 276 of the TS gene, resulting in the substitution of histidine for an evolutionarily conserved tyrosine at residue 33 of TS protein. So far the histidine allele has been detected in only one cell line, HCTl 16 (7). The rare his-33 form of the protein is 3-4 fold more resistant to FdUrd than the tyr-33 form, due to an 8 fold lower catalytic efficiency (kcat), suggesting that histidine at residue 33 perturbs the structure of the TS active site (9) 209 232/116

The human thymidylate synthase gene maps to chromosome 18p 11.32

The gene for human thymidylate synthase was initially mapped to the long arm of chromosome 18 (18q21.31-qter) by somatic cell hybrid analysis (10), however two subsequent reports place the gene in band 18p 11.32 using fluorescence in situ hybridization (11,12).

Chromosome band 18p 11.32 is a site of loss of heterozygosity

The long arm of chromosome 18 contains the DCC (deleted in colon cancer) candidate tumor suppressor gene and has been well studied in a variety of tumors. The short arm (18p), where TS apparently resides, has not been studied as extensively. The available data suggests there is LOH in approximately 45% of colon cancers (13) and 25-30% of cervical (14), head and neck (15), lung (16) and ovarian (17) cancers and sarcomas. LOH has also been described in breast, brain, esophagus, kidney and prostate cancers

(0-15%). 18p has not been studied for allele loss in several other major cancers, including bladder, leukemia, lymphoma, liver, pancreas, stomach and testicular cancers.

References

1. Chu, E., Koeller, D.M., Casey, J.L., et al. (1991) Autoregulation of human thymidylate synthase messenger RNA translation by thymidyate synthase. Proc. Natl. Acad. Sci. U.S.A. 88: 8977-81. 2. Seno, T., Ayusawa, D., Shimizu, K., et al. (1985) in de Serres, FJ. (ed.) Genetic

Consequences of Nucleotide Pool Imbalance. Plenum Publishing Company, New York, pp. 241-263.

3. Johnson, L.F. (1994) Posttranscriptional regulation of thymidylate synthase gene expression. Journal of Cellular Biochemistry 54: 387-392. 210 232/116

4. Calabresi, P. and B. Chabner (1996) in Hardman, J.G., Limbird, L.E., et al. (eds.) Goodman and Gilman's The Pharmacological Basis of Therapeutics. McGraw Hill, New York, pp. 1247-1251.

5. Takeishi, K., Kaneda, S., Aysawa, D., Shimizu, K., Gotoh, O. and T. Seno (1985) Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic

Acids Research 13: 2035-2043.

6. Kaneda, S., Nalbantoglu, K., Takeishi, K., et al. (1990) Structural and functional analysis of the human thymidylate synthase gene. Journal of Biological Chemistry 265: 20277-84. 7. Barbour, K.W., Berger, S.H. and S.G. Berger (1990) Single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase. Molecular Pharmacology 37: 515-518.

8. Barbour, K.W., Hoganson, D.K., Berger, S.H. and F.G. Berger (1992) A naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxoyuridine in mammalian and bacterial cells. Molecular Pharmacology 42: 242-248

9. Hughey, C.T., Barbour, K.W., Berger, F.G. and S.H. Berger (1993) Functional effects of a naturally occurring amino acid substitution in human thymidylate synthase. Molecular Pharmacology AA: 316-323. 10. Nussbaum, R.L., McCarrick-Walmsley, R., Lesko, J.G., et al. (1985) Thymidylate synthase deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression. American Journal of Human Genetics 37: 1192-1205.

11. Hori, T., Takahashi, E., Ayusawa, D., et al. (1990) Regional assignment of the human thymidylate synthase gene to chromosome band 18pl 1.32 by nonisotopic in situ hybridization. Human Genetics 85: 576-580.

12. Silverman, G.A., Kuo, W.-L., Taillon-Miller, P. and J.W. Gray (1993) Chromosomal reassignment: YACs containing both YES1 and thymidylate synthase map to the short arm of chromosome 18. Genomics 15: 442-445. 211 232/116

13. Vogelstein, B., Fearon, E.R., Kern, S.E., et al. (1989) Allelotype of colorectal carcinomas. Science 244: 207-211.

14. Mullokandov, M.R., Kholodilov, N.G., Atkin, N.B., et al. (1996) Genomic Alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status. Cancer Research 56: 197-205.

15. Nawroz, H., van der Riet, P., Hruban, R.H., et al. (1994) Allelotype of head and neck squamous cell carcinoma. Cancer Research 54: 1152-55.

16. Allelotype of non-small cell lung carcinoma - comparison between loss of heterozy-gosity in squamous cell carcinoma and adenocarcinoma. Cancer Research: 52: 2478-81.

17. Abeln, E.C.A., Kuipers-Dijkshoorn, N.J., Berns, E.M.J.J., et al. (1995) Molecular genetic evidence for unifocal origin of advanced epithelial ovarian cancer and for minor clonal divergence. British Journal of Cancer 72: 1330-1336.

Example 14: Cytidine Triphosphate Synthetase (CTPS) - Target Gene VARIA260

Cytidine Triphosphate Synthetase is essential for cell growth

Human cytidine triphosphate synthetase catalyzes the glutamination of UTP to form

CTP. The reaction is: UTP + ATP + glutamine --> CTP + ADP + Vi + glutamate. This is the rate limiting step in the synthesis of cytidine nucleotides from both the de novo and uridine salvage synthesis routes (see ref. 1 and references therein). CTPS also plays a vital regulatory function in balancing nucleotide pools for DNA polymer synthesis; it is allosterically regulated by CTP (negatively) and GTP (positively).

There is compelling evidence that CTPS is essential for cell survival:

CTPS is evolutionarily conserved in yeast and bacteria, with a high degree of amino acid identity in regions mediating allosteric regulation and catalysis (1- 212 232/116

3). (Another example: the human and hamster enzymes are identical in length and 98% amino acid identical over 591 amino acids.) Mutant hamster cells lacking functional CTPS need exogenous cytidine to survive (3). There is no known human deficiency disease of CTPS.

CTPS function is increased in proliferating cells (4).

Thus CTPS is an attractive target for antiproliferative drugs. Cyclopentyl cytosine (CPE-C) is a synthetic cytidine analog in which a cyclopentyl group replaces the furan ring of the ribose sugar. CPE-C has antineoplastic and antiviral effects in animal models (5). The drug is kinased intracellularly to the triphosphorylated nucleotide form (CPE-CTP). Exposure of cells to CPE-C leads to rapid depletion of CTP pools, as a result of inhibition of CTPS by CPE-CTP (6,7). Upregulation of CTP synthetase, or loss of negative allosteric modulation by CTP is associated with resistance to the cancer chemotherapy drugs arabinosyl cytosine (ara-C), 5-fluorouracil and other cytotoxic nucleoside analogs as well as alkylating agents (3).

The human cytidine triphosphate synthetase gene has sequence variances

The sequence of a human cytidine triphosphate synthetase cDNA was determined by

Yamauchi et al. (1), who later determined the genomic sequence as well (2). We undertook a systematic search for DNA sequence variance by analysing 36 unrelated individuals using the single strand conformation polymoφhism technique. Primers were designed using the sequence of Yamauchi et al. (1). SSCP analysis revealed 3 DNA fragments having sequence variances, and subsequent DNA sequence analysis showed that nucleotides 576 (A vs. G), 2093 (C v-s.T) and 2135 (G vs. A) vary among normal individuals as shown in the Target Summary Table. The nucleotide 576 sequence variance is a silent substitution in the coding region, while the latter two sequence variances are in the 3' untranslated region of the cDNA. All three sequence 213 232/116 variances were detected at low frequency in the panel of 36 individuals (3-8%), however all but one of the heterozygotes is Asian, and it seems likely that a larger survey of Asian populations would show higher allele frequencies in Chinese and other groups. For example among the four Chinese in the panel two (50%) are heterozygous for the residue 2135 sequence variance, and one (25%) is heterozygous for the nt 576 sequence variance. Also, the one Cambodian in the panel is heterozygous for both the 2093 and 2135 sequence variances.

The human cytidine triphosphate synthetase gene maps to chromosome lp34.1

The gene for human cytidine triphosphate synthetase has been mapped to lp34.1 by somatic cell hybrid analysis (2).

Chromosome band lp34.1 is a site of frequent loss of heterozygosity

The short arm of chromosome 1 is comparatively well investigated for allele loss, especially in breast and colon cancers. The lp35-32 and lp22-13 regions flank lp34.1 and are the best available markers for LOH on lp. Studies of these regions show 30- 50% LOH frequency in breast cancer (8-12), 41-75% in glioma (a brain cancer subtype) (13), 20-40% in colon cancer (14,15), -50% in stomach cancer (16), -20% in lung cancer (17) and 20-30% in ovarian cancer (18). High frequency LOH has been detected in several uncommon cancers such as pheochromocytoma (50-86%) and neuroblastoma (-50%). Most other common cancers have not been adequately investigated to assess LOH frequency in this region.

References

1. Yamauchi, M., Yamauchi, N. and M. Meuth (1990) Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase 214 232/116 chromosomes. EMBO Journal 9: 2095-2099.

2. Yamauchi, M., Yamauchi, N., Phear, G., et al. (1991) Genomic organization and chromo-somal localization of the human CTP synthetase gene(CTPS). Genomics 11: 1088-96. 3. Whelan, J., Phear, G., Yamauchi, M. and M. Meuth (1993) Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells. Nature Genetics 3: 317- 322.

4. van den Berg, A., van Lenthe, H., Busch, S., et al. (1993) Evidence for transformation related increase in CTP synthetase activity in situ in human lymphoblastic leukemia. European Journal of Biochemistry 216: 161-167.

5. Marquez, V.E., Lim, M.-L, Treanor, S.P., et al. (1988) Cyclopentylcytosine: a carbocyclic nucleoside with antitumor and antiviral properties. Journal of Medical Chemistry 31 : 1687-94.

6. Kang, G.J., Cooney, D.A., Moyer, J.D., et al. (1989) Cyclopentenyl triphosphate: formation and inhibition of CTP synthetase. Journal of Biological Chemistry 264:

713-718.

7. Glazer, R.I., Knode, M.C. Lim, M.-L, and V.E. Marquez (1985) Cyclopentyl cytidine analogue: an inhibitor of cytidine triphosphate synthesis in human colon carcinoma cells. Biochemical Pharmacology 34: 2535-2539. 8. Bieche I, Champeme MH, Matifas F, Cropp CS, Callahan R, Lidereau R. (1993)

Two distinct regions involved in lp deletion in human primary breast cancer. Cancer Res. 53:1990-4.

9. Borg A, Zhang QX, Olsson H, Wenngren E. (1992) Chromosome 1 alterations in breast cancer: allelic loss on lp and lq is related to lymphogenic metastases and poor prognosis. Genes Chromosomes & Cancer. 5:311-20.

10. Sato T, Tanigami A, Yamakawa K, et al. (1990) Allelotype of breast cancer: cumulative allele losses promote tumor progression in primary breast cancer. Cancer Res. 50:7184-9.

1 1. Devilee P, van Vliet M, Bardoel A, et al. (1991) Frequent somatic imbalance of 215 232/116 marker alleles for chromosome 1 in human primary breast carcinoma. Cancer Res. 51:1020-5.

12. Loupart ML, Armour J, Walker R, Adams S, Brammar W, Varley J. (1995) Allelic imbalance on chromosome 1 in human breast cancer. I. Minisatellite and RFLP analysis. Genes Chromosomes & Cancer. 12:16-23.

13. Reifenberger, J., Reifenberger, G., Liu, L., et al. (1994) Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and lp. American Journal of Pathology 145: 1175-1190.

14 Meling GI, Lothe RA, Borresen AL, et al. (1991) Genetic alterations within the retinoblastoma locus in colorectal carcinomas. Relation to DNA ploidy pattern studied by flow cytometric analysis. Br J Cancer. 64:475-80.

15. Lothe RA, Nakamura Y, Woodward S, Gedde DT, Jr., White R. (1988) VNTR

(variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas. Cytogenet Cell Genet. 48:167-9. 16. Ezaki, T., Yanagisawa, A., Ohta, K., et al. ((1996) Deletion mapping on chromosome lp in well-differentiated gastric cancer. British Journal of Cancer 73:

424-428.

17. Hiyama K, Ishioka S, Shirotani Y, et al. (1995) Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb. Oncogene. 10:937-44.

18. Yang-Feng TL, Han H, Chen KC, et al. (1993) Allelic loss in ovarian cancer. Int J Cancer. 54:546-51.

Example 15: Cysteinyl tRNA Synthetase (CARS) - Target Gene VARIA301

The human cysteinyl tRNA synthetase gene is essential for cell survival

Cysteinyl-tRNA synthetase (CARS) catalyzes ATP dependent covalent attachment of 216 232/116 cysteine to its cognate tRNA to form cysteinyl-tRNA. In the absence of cysteinyl- tRNA, protein synthesis is blocked. Since Cysteinyl-tRNA synthetase is a single copy gene in man, inhibition of its function is expected to be cell lethal. This has been shown for other tRNA synthetases (summarized above).

The human cysteinyl-tRNA synthetase gene and mRNA have sequences variances

A human cDNA encoding cysteinyl tRNA synthetase (CARS) was cloned based on the similarity of a human expressed sequence tag (EST) to E. coli cysteinyl tRNA synthetase (1). The published human CARS cDNA is 2048 nucleotides in length and includes a 30 nucleotide 5' untranslated region followed by an open reading frame of 1914 nucleotides and a 3' untranslated region of 134 nucleotides (1). An EMBL/ GENBANK submission (accession # L06845) by the authors of ref. 1 includes a 3' untranslated region 423 nucleotides longer than the published sequence, but lacks 19 consecutive A nucleotides after position 2029 (making a net increase of: 423 - 19 =

404 nucleotides, and a composite cDNA of: 2048 + 404 = 2452 nucleotides in length. We have confirmed the existence of 2452 nt transcripts by PCR amplification of reverse transcribed mRNA.) We designed primers as shown on the annotated cDNA sequence and screened the composite 2452 nt cDNA for sequence variance in 36 unrelated individuals by the single strand conformation polymoφhism (SSCP) technique. Two sequence variances were identified. One of the sequence variances, located in the 5' untranslated region, was below the desired level of 20% heterozygosity. The other sequence variance is a C vs. T transition near the 3' end of the coding sequence at nucleotide 1739 (see annotated sequence).

The human cysteinyl tRNA synthetase protein has sequence variances The deduced amino acid sequence of the human CARS gene encodes a protein of 638 amino acids which probably functions as a monomer, by analogy to related synthetases. The deduced protein contains two sequence motifs, HIGH (residues 64- 217 232/116

67) and KMSKS (residues 406-410), which define Class I synthetases (see ref. 2 for background information on tRNA synthetases). These two conserved motifs form an ATP binding fold (the Rossman fold) in the amino terminal half of the protein. Cytosine at nucleotide 1739 encodes proline at residue 622 of the protein, while thymine at nucleotide 1739 encodes leucine. The pro/leu amino acid sequence variance is a mere 16 residues from the C terminus of the protein. The C-terminal portion of CARS, by analogy to other class I synthetases, contains the tRNA binding site.

Frequency of CARS heterozygotes

The frequency of heterozygotes for the nucleotide 1739 sequence variance is -45-50% in all major racial groups surveyed (see accompanying table), including North American Whites (8/15=53%), North American Blacks (2/4=50%), Chinese (2/4=50%), Swedish (127/344=37%) and Japanese (1/4=25%). The wide population distribution of both alleles suggests that other population groups will also have a high frequency of heterozygotes.

Gene Mapping of CARS to llpl5.5

Human CARS cDNA has been mapped to chromosome 1 lp 15.5 by screening human X Chinese hamster somatic cell hybrids informative for all human chromosomes, and by fluorescence in situ hybridization (3). Both mapping techniques were conclusive in showing only one locus for human CARS. Detailed physical maps of 1 lpl5.5 have subsequently allowed precise localization of the CARS gene relative to other DNA markers (4).

LOH at llpl5.5 is well documented in many cancer types

The short arm of chromosome 11, and particularly the 1 lpl5.5 region, is deleted in a 218 232/116 variety of human cancers, including (but not limited to) ovarian (18 - 50% LOH), non- small cell lung (22 - 71%), breast (12 - 33%), bladder (40 -50%), esophageal (18 - 40%) and testicular cancers (18 - 66%) (refs. 5-12). Many deletions in the 1 lpl5.5 region span relatively short chromosomal segments (2 - 10 megabases; see ref. 8). Using the specific variances identified in the CARS gene as markers for heterozygosity, we have determined that LOH occurs in 10/20 ovarian cancers (50%) and 10/52 non-small cell lung cancers (19%).

Assays for human CARS inhibitors

There is no published work on the protein encoded by the putative human CARS cDNA, nor on any other eukaryotic CARS protein, however the extensive characterization of other Class I synthetases from both prokaryotes and eukaryotes provides a template for modeling the structure of human CARS. (For an example of how this can be done see ref. 14, in which the three dimensional structure of human alanyl-tRNA synthetase has been modeled up to amino 249 by neural net software and multiple alignments of partial and complete human AARS sequences with heterologous prokaryotic class II synthetases for which crystal structures exist.) With respect to the C-terminal location of the variant amino acid residue in human CARS, it is worth noting that single amino acid substitutions in the C-terminal region of alanyl tRNA synthetase can have greater than 100 fold effects on catalytic activity (15).

References

1. Wasmuth, JJ. Cruzen, M. E. and S.M. Arfϊn (1994) Nucleotide and deduced amino acid sequence of human cysteinyl-tRNA synthetase. DNA Sequence 4:243-248.

2. Moras, D. (1992) Structural and functional relationships between aminoacyl-tRNA synthetases. Trends in Biochemical Sciences 17: 159-164.

3. Cruzen, M.E., Bengtsson, U., McMahon, J., Wasmuth, JJ. and S.M. Arfin (1993) 219 232/116

Assignment of the cysteinyl-tRNA synthetase gene (CARS) to 1 lp 15.5. Genomics 15: 692-693.

5. Winqvist, R., Mannermaa, A., Alavaikko, M., Blanco, G., Taskinen, P J., Kiviniemi, H., Newsham, I. and W. Cavenee (1993) Refinement of regional loss of heterozygosity for chromosome 1 lpl5.5 in human breast tumors. Cancer Research

53: 4486-4488.

6. Kiechle-Schwartz, M., Bauknecht, T., Wienker, T., et al. (1993) Loss of Constitutional Heterozygosity on Chromosome l ip in Human Ovarian Cancer. Cancer 72:2423-32. 7. Viel, A., Giannini, F., Tumiotti, L., Sopracordevole, F., Visentin, M.C. and M.

Boiocchi (1992) Chromosomal localisation of two putative 1 lp oncosuppressor genes involved in human ovarian tumors British Journal of Cancer 66: 1030-1036.

8. Bepler, G. and Garcia-Blanco, M.A. (1994) Three Tumor Suppressor Regions on Chromosome l ip Identified by High Resolution Deletion Mapping in Human Non- Small Cell Lung Cancer. Proc. Natl. Acad. Sci. U.S.A. 91:5513-7.

9. Iizuka, M., Sugiyama, Y.,Shiraishi, M., Jones, C and T. Sekiya (1995) Allelic losses in human chromosome 11 in lung cancers. Genes, Chromosomes & Cancer 13:40-46.

10. Shaw, M.E. and Knowles, M.A. (1995) Deletion Mapping of Chromosome 11 in Carcinoma of the Bladder. Genes, Chromosomes & Cancer 13:1-8. 11. Smith, R.C., and Rukstalis, D.B. Frequent Loss of Heterozygosity at 1 lp Loci in

Testicular Cancer. The Journal of Urology 153:1684-7, 1995. 12. Shibagaki, I., Shimada, Y., Wagata, T., Ikenaga, M., Imamura, M. and K. Ishizaki (1994) Allelotype analysis of esophageal squamous cell carcinoma. Cancer Research 54: 2996-3000. 13. Shiba, K., Suzuki, N., Shigesada, K., Namba, Y., Schimmel, P. and T. Noda (1994)

Human cytoplasmic isoleudyl-tRNA synthetase: selective divergence of the anticodon- binding domain and acquisition of a new structural unit. Proc. Natl. Acad. Sci. U.S.A. 91 :7435-7439. 14. Shiba, K., Ripmaster, T., Suzuki, N., Nichols, R., Plotz, P., Noda, T. and P. 220 232/116

Schimmel (1995) Human alanyl-tRNA synthetase: conservation in evolution of catalytic core and microhelix recognition. Biochemistry 34: 10340-10349. 15. Wu, M.-X., Filley, S.J., Xiong, J., Lee, JJ. and K.A.W. Hill (1994) A cysteine in the C-terminal region of alanyl-tRNA synthetase is important for aminoacylation activity. Biochemistry 33: 12260-12266.

Example 16: Glutamyl-Prolyl tRNA Synthetase (EPRS): - Target Gene VARIA300

The human glutamyl -proly I tRNA synthetase gene is essential for cell survival

Glutamyl-prolyl-tRNA synthetase (EPRS) catalyzes ATP dependent covalent attachment of glutamine and proline to their cognate tRNAs to form glutamyl-tRNA and prolyl-tRNA. In the absence of glutamyl-tRNA or prolyl-tRNA, protein synthesis is blocked. Since glutamyl-prolyl-tRNA synthetase is a single copy gene in man, inhibition of its function is expected to be cell lethal. This has been shown for other tRNA synthetases (summarized above).

The human glutamyl-prolyl tRNA synthetase gene, mRNA and protein have sequence variances

A human cDNA encoding glutamyl-prolyl tRNA synthetase (EPRS) was initially misidentified as glutaminyl-tRNA synthetase (1) based on misleading sequence alignments with bacterial and yeast glutaminyl-tRNA synthetase (2). Subsequently, biochemical studies of the protein encoded by a D. melanogaster gene -70% identical to the human gene demonstrated glutamyl (not glutaminyl) tRNA synthetase activity, and also showed that a single gene encodes both glutamyl- and prolyl-tRNA synthetases in the fly (3). These observations eventually led to the realization that 221 232/116 human EPRS is also a single polypeptide containing two synthetases (2). The aminoacyl tRNA synthetases are divided into two classes (see Background on tRNA Synthetases, above). Glutamyl-tRNA synthetase belongs to Class I while Prolyl-tRNA synthetase belongs to class II. Thus the two halves of EPRS evolved independently and likely represent an evolutionarily recent fusion. The published human EPRS cDNA is 4,586 nt long and includes a 5' untranslated region of 58 nt followed by an open reading frame of 4320 nt and a 3' untranslated sequence of 208 nt (1). The gene encodes a polypeptide of 1440 amino acids. The glutamyl-tRNA synthetase activity is encoded by an imprecisely defined segment at 5' end of the gene probably spanning at least amino acids 105-426, while the prolyl-tRNA synthetase activity is encoded by a segment likely including residues 942-1369 at the 3' end of the gene (2). The two synthetase moieties are connected by a central domain of unknown function. It has been speculated that the central domain may attach the enzyme to the cytoskeleton or to other aminoacyl-tRNA synthetases in a multienzyme complex (2, 3).

The human glutamyl-prolyl-tRNA synthetase gene and mRNA have sequence variances We designed primers and screened the 4586 nt cDNA for sequence variance in 36 unrelated individuals by the single strand conformation polymoφhism technique. Seven sequence variances were identified, four located in the coding sequence and three located in the 3' untranslated region. As shown on the Annotated Glutamyl-

Prolyl tRNA Synthetase cDNA Sequence and in the Target Summary Page, the sequence variance nucleotides are 2520 (C vs. A), 2944 (G vs. A), 2963 (C vs. T), 2969 (A vs. G), 3247 (A vs. G), 4459 (G vs. A) and 4506 (G vs. A). The sequences flanking the alternate allelic forms and their frequencies of occurrence are shown on the Target Summary Page. Less than 10% of individuals surveyed are heterozygous for sequence variances at 2520, 2944 and 2963. Heterozygotes for the other 4 sequence variances occur more frequently and appear to be widely distributed in the surveyed populations (see below). 222 232/116

The human glutamyl-prolyl tRNA synthetase protein has sequence variances Three nucleotide sequence variances, at 2520, 2963 and 2969, alter the amino acid coding sequence of EPRS at residues 821 (pro/his), 969 (his/tyr) and 971 (ile/val). The residue 821 his and 969 tyr alleles are relatively rare, with fewer than 10% heterozygotes in the surveyed populations. The more common residue 971 sequence variance lies in the PRS domain of the protein, near one of the widely conserved defining motifs for class II tRNA synthetases.

EPRS heterozygotes are frequent in non-Asian populations. While the overall frequency of residue 971 heterozygotes is 8/36 (24%), the frequency of heterozygotes varies among different populations. For example, there are no heterozygotes among 10 Asians surveyed (Chinese, Japanese, Filipino and Korean), while 8/26 (31%) of non-Asians, including North American Whites, Blacks and Hispanics, are heterozygotes.

The EPRS Gene Maps to Iq41-q42

Human EPRS cDNA has been mapped to chromosome lq41-42 by screening human X Chinese hamster somatic cell hybrids informative for all human chromosomes, and by fluorescence in situ hybridization (3). Both mapping techniques were conclusive in showing only one locus for human EPRS.

Loss of heterozygosity at lq41-42 is documented in several cancer types. 17-25% of breast cancers have allele loss in the Iq41-q42 region (4, 5), 29-46% of colon cancers (6, 7) and 17-26% of cervical cancers (8). One report describes 27% LOH in stomach cancer (9). One or two studies of brain, esophageal, kidney, liver and ovarian cancers also report LOH. No studies of LOH in the Iq41-q42 region have been reported in bladder, endocrine, head and neck, lung, or pancreas cancers or in leukemia or lymphoma. 223 232/116

Antisense considerations The sequence variances at 2963 and 2969 are close enough that a 20-mer antisense oligonucleotide could easily span them. Such an oligonucleotide should afford greater allele discrimination than is possible with a single nucleotide difference. However, the 2963 sequence variance is fairly rare (<10% heterozygotes) and not in linkage disequilibrium with the 2963 sequence variance, so there are more than two haplotypes in the populations tested.

References

1. Fett, R. and R. Knippers (1991) The primary structure of human glutaminyl tRNA synthetase. Journal of Biological Chemistry 266: 1448-1455. 2. Cerini, C, Kerjan,P., Astier, M., Gratecos, D., Mirande, M. and M. Semeriva (1991) A component of the multisynthetase complex is a multifunctional aminoacyl- tRNA synthetase. The EMBO Journal 10: 4267-4277. 3. Kaiser, E., Hu, B., Becher, S., Eberhard, D., et al. (1994) The human EPRS locus

(formerly the QARS locus): a gene encoding a class I and a class II aminoacyl-tRNA synthetase. Genomics 19: 280-290.

4. Journal of The National Cancer Institute 84: 506.

5. Cancer Research 51: 1020. 6. International Journal of Cancer 53: 382.

7. Genes, Chromosomes & Cancer 12: 16.

8. Cancer Research 56: 197.

9. Cancer Research 52: 3099.

10. Shiba, K., Ripmaster, T., Suzuki, N., Nichols, R., Plotz, P., Noda, T. and P. Schimmel (1995) Human alanyl-tRNA synthetase: conservation in evolution of catalytic core and microhelix recognition. Biochemistry 34: 10340-10349.

Example 17: Alanyl-tRNA Synthetase (AARS) - Target Gene VARIA304 224 232/116

The human glutamyl-prolyl tRNA synthetase gene is essential for cell survival

Alanyl-tRNA synthetase (AARS) catalyzes ATP dependent covalent attachment of alanine to its cognate tRNA to form alanyl-tRNA. In the absence of alanyl-tRNA, protein synthesis is blocked. Since alanyl-tRNA synthetase is a single copy gene in man (see below) inhibition of its function is expected to be cell lethal. This has been shown for other tRNA synthetases (summarized above).

The human alanyl-tRNA synthetase gene and mRNA have sequence variances

A human cDNA encoding alanyl tRNA synthetase (AARS) was cloned by Shiba et al. (1) using cross species PCR: AARS sequences from four evolutionarily distant species were compared and primers were designed to conserved regions specific to AARS. The cloned human cDNA is 3344 nt in length and includes a 110 nt 5' untranslated region, an open reading frame of 2904 nt encoding a 968 residue polypeptide, and a 3' untranslated region of 330 nt (ref. 1; Genbank accession D32050).

We designed primers. The 3344 nt cDNA was screened for sequence variance in 36 unrelated individuals by the single strand conformation polymoφhism (SSCP) technique. One sequence variance was identified, a C vs. T transition at nucleotide

1013, within the coding sequence. The published nucleotide at position 1013 is T (1).

The frequency of AARS heterozygotes is 25-50% in all populations surveyed. The frequency of heterozygotes for the nucleotide 1013 sequence variance is 57% in the 36 individuals tested. Both alleles are present in all major racial groups surveyed (see

Target Gene Summary Table), including North American Whites (9/15=60% heterozygotes), North American Blacks (3/4=75%), Chinese (2/4=50%), Japanese (1/4=25%) and Hispanic (1/2). The wide population distribution of both alleles suggests that other population groups will also have a high frequency of heterozygotes. 225 232/116

The AARS gene maps to 16q22

The human AARS cDNA has been mapped to chromosome 16q22 by us and by Nichols et al. (ref. 2). We designed primers to the 3' untranslated region of AARS and used PCR to analyze the National Institute of General Medical Sciences (NIGMS)

Human/Rodent Somatic Cell Hybrid Mapping Panel #2 (see page 704 of the NIGMS 1994/1995 Catalog of Cell Lines, available from the Coriell Cell Repository, Camden, NJ). The panel consists of 24 hybrid cell lines, each monochromosomal for one human chromosome. The AARS PCR product mapped to the hybrid containing human chromosome 16. Subsequently we screened the Radiation Hybrid Mapping Panel created at Stanford University (rhserver@shgc.stanford.edu) and distributed by Research Genetics (RH01). The AARS PCR product mapped near D16S496 with a lod score>10. D16S496 is a polymoφhic DNA marker at 16q22. The AARS PCR product mapped near D16S496 with a LOD score >10. DH16S496 is a polymoφhic DNA marker at 16q22. (See, ref. 29 for a full explanation of modification hybrid mapping.) Similar results were obtained by Nichols et al., who mapped AARS by analysis of the same NIGMS hybrid mapping panel, by PCR mapping in a chromosome 16 regional mapping panel and by fluorescence in situ hybridization to metaphase chromosomes. All mapping techniques were conclusive in showing only one locus for human AARS.

LOH at 16q22 is well documented in many cancer types. Loss of heterozygosity studies of chromosome 16q have principally focused on breast and liver cancers. In six detailed studies of breast cancer in the 16q22 region LOH frequencies of 40-60% have been reported (refs 3-8). 16q22 LOH has ben reported in 25-90% of liver cancers

(9-13), with the average around 45%. Less extensive studies of other cancer types report 16q22 LOH in 19% of bladder cancers, 20% of colon cancers (14), 19-27% of esophageal cancers (15), 25% of small cell lung cancers (16), 16-37% of ovarian cancers (17-19) and 22% of uterine cancers (20), and 31-50% of prostate cancers (21- 226 232/116

22).

References

1. Shiba, K., Ripmaster, T., Suzuki, N., Nichols, R., Plotz, P., Noda, T. and P. Schimmel (1995) Human alanyl-tRNA synthetase: conservation in evolution of catalytic core and microhelix recognition. Biochemistry 34: 10340-10349.

2. Nichols, R.C., Pai, S.I., Ge, Q., Targoff, I.N., Plotz, P.H. and P. Liu (1995) Localization of two human autoantigen genes by PCR screening and in situ hybridization - glycyl tRNA synthetase locates to 7pl5 and alanyl-tRNA synthetase locates to 16q22. Genomics 30:131-132.

3. Cleton-Jansen AM, Moerland EW, Kuipers-DijkshoornNJ, et al. (1994) At least two different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Genes Chromosom Cancer. 9:101-7.

4. Dorion-Bonnet F, Mautalen S, Hostein I, Longy M. (1995) Allelic imbalance study of 16q in human primary breast carcinomas using microsatellite markers. Genes

Chromosomes Cancer. 14:171-81.

5. Kashiwaba M, Tamura G, Suzuki Y, et al. (1995) Epithelial-cadherin gene is not mutated in ductal carcinomas of the breast. Jpn J Cancer Res. 86:1054-9.

6. 0'Connell P, Pekkel V, Fuqua S, Osborne CK, Allred DC. (1994) Molecular genetic studies of early breast cancer evolution. Breast Cancer Res Treat. 32:5-12.

7. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51:5794-9.

8. Tsuda H, Callen DF, Fukutomi T, Nakamura Y, Hirohashi S. (1994) Allele loss on chromosome 16q24.2-qter occurs frequently in breast cancers irrespectively of differences in phenotype and extent of spread. Cancer Res. 54:513-7.

9. Fujimori M, Tokino T, Hino O, et al. (1991) Allelotype study of primary hepatocellular carcinoma. Cancer Res. 51:89-93.

10. Fujimoto Y, Hampton LL, Wirth PJ, Wang NJ, Xie JP, Thorgeirsson SS. (1994) Alterations of tumor suppressor genes and allelic losses in human hepatocellular 227 232/116 carcinomas in China [see comments]. Cancer Res. 54:281-5. 11. Tsuda H, Zhang WD, Shimosato Y, et al. (1990) Allele loss on chromosome 16 associated with progression of human hepatocellular carcinoma. Proc Natl Acad Sci USA. 87:6791-4. 12. Tsuda H, Oda T, Sakamoto M, Hirohashi S. (1992) Different pattern of chromosomal allele loss in multiple hepatocellular carcinomas as evidence of their multifocal origin. Cancer Res. 52:1504-9.

13. Zhang WD, Hirohashi S, Tsuda H, et al. (1990) Frequent loss of heterozygosity on chromosomes 16 and 4 in human hepatocellular carcinoma. JpnJ Cancer Res. 81:108- 11.

14. Ookawa K, Sakamoto M, Hirohashi S, et al. (1993) Concordant p53 and DCC alterations and allelic losses on chromosomes 13q and 14q associated with liver metastases of colorectal carcinoma. Int J Cancer. 53:382-7.

15. Genes, Chromosomes & Cancer 10: 177. 16. Yokota J, Wada M, Shimosato Y, Terada M, Sugimura T. (1987) Loss of heterozygosity on chromosomes 3, 13, and 17 in small-cell carcinoma and on chromosome 3 in adenocarcinoma of the lung. Proc Natl Acad Sci USA. 84:9252-6.

17. Cancer Research 51: 5118.

18. Osborne RJ, Leech V. (1994) Polymerase chain reaction allelotyping of human ovarian cancer. Br J Cancer. 69:429-38.

19. Yang-Feng TL, Han H, Chen KC, et al. (1993) Allelic loss in ovarian cancer. Int J Cancer. 54:546-51.

20. Okamoto A, Sameshima Y, Yamada Y, et al. (1991) Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus. Cancer Res. 51:5632-5.

21. Carter, B.S., Ewing, CM., Ward, S.W., et al. (1990) Allelic loss of chromosomes 16q and lOq in human prostate cancer. Proc Natl A cadSci USA. 87: 8751-8755.

22. Bergerheim, U.S.R., Kunimi, K., Collins, V.P. and P. Ekman (1991) Deletion mapping of chromosomes 8, 10, and 16 in human prostatic carcinoma. Genes, 228 232/116

Chromosomes & Cancer 3: 215-220.

23. Boehnke, M., Lange, K. and D.R. Cox (1991) Statistical methods for multipoint radiation hybrid mapping. Am. J. Hum. Genet. 49: 1174-88.

Example 18: Threonyl-tRNA Synthetase (TARS) - Target Gene VARIA302

The human threonyl-tRNA synthetase gene is essential for cell survival

Threonyl-tRNA synthetase (TARS) catalyzes ATP dependent covalent attachment of threonine to its cognate tRNA to form threonyl-tRNA. In the absence of threonyl- tRNA, protein synthesis is blocked. Threonyl-tRNA synthetase is a single copy gene in man (see below) and inhibition of TARS is cell lethal. This has been shown using the specific TARS inhibitor borrelidin, a threonine analog. Borrelidin resistant CHO cell lines have been isolated; the most resistant lines contain -60-100 fold more immunologically reactive protein and 10-20 fold higher TARS activity than non- selected CHO cells (1-3).

The human TARS enzyme is a homodimeric member of the class II tRNA synthetases. The human protein is 53% amino acid identical to S. cerevisiae cytoplasmic TARS,

40% amino acid identical to E. coli TARS and 39% amino acid identical to yeast mitochondrial TARS. The degree of evolutionary conservation is 52-64% when conservative substitutions are allowed.

The human Threonyl-tRNA synthetase gene and mRNA have sequence variances. A human cDNA encoding threonyl tRNA synthetase was cloned by Cruzen and Arfin (GENBANK accession M63180; ref. 2) using anti-TARS antibodies to screen a lgtl 1 expression library. The cDNA is 2644 nt in length and includes a 138 nt 5' untranslated region, an open reading frame of 2136 nt encoding a 712 residue polypeptide, and a 3' 229 232/116 untranslated region of 370 nt.

We designed primers for amplification. The 2644 nt cDNA was screened for sequence variance in 36 unrelated individuals by the single strand conformation polymoφhism (SSCP) technique. Three sequence variances were identified: G vs. A transitions at nucleotides 1608 and 1755 within the coding sequence, and a C vs. T transition at nucleotide 2395 of the 3' untranslated region. None of the sequence variances alters the sense of the coding strand. The published sequence shows G, G and T at the three sequence variance sites

The frequency of TARS heterozygotes is 25-45% in all populations surveyed. The nucleotide 1608 sequence variance was genotyped only in North American Whites, 45% of whom were heterozygotes. The nucleotide 1608 and 1755 sequence variances were both genotyped in 36 individuals, with overall heterozygosity rates of 31% and 25%, respectively. Both sequence variances were detected in North American Whites,

North American Blacks, Hispanics and Chinese. Of 14 North American Whites genotyped at all 3 sequence variance nucleotides, 11 (79%) were heterozygous for a least one polymor-phism (see threonyl tRNA synthetase summary table).

The TARS gene maps to 5pl3-CEN The human TARS cDNA has been mapped to chromosome 5pl3-CEN by analysis of TARS isoelectric focusing patterns in human/Chinese hamster hybrids (). The mapping studies were consistent with one human TARS locus.

LOH at 5pl3-CEN is documented in several cancer types. The best data on 5p LOH is in cervical cancer where 9 markers have been tested in 3 different studies. The frequency of LOH ranges from 12-57%, averaging -45%. Other cancers that have been studied are breast (10-24% LOH), head and neck (20% LOH), adenocarcinoma of the lung (40% LOH, but only 5 cancers were studied), melanoma (40%) and ovary (15- 230 232/116

21%).

Assays for human TARS inhibitors. Human TARS protein is a homodimeric class II synthetase. Antibodies to rat TARS were used to clone the human protein. The high degree of amino acid conservation throughout the protein suggests that it may be possible to create yeast and/or bacterial strains with human CARS.

References

1. Gantt, J.S., Bennett, CA. and S.M. Arfin (1981) Increased levels of threonyl tRNA synthetase in a borrelidin-resistant Chinese hamster ovary cell line. Proc. Natl. Acad.

Sci. U. S. A. 92: 5367-5370.

2. Gerken, S.C. and S.M. Arfin (1984) Chinese hamster ovary cells resistant to borrelidin oveφroduce threonyl-tRNA synthetase. The Journal of Biological Chemistry 259: 9202-9206. 3. Kontis, KJ. and S.M. Arfin (1989) Isolation of a cDNA clone for human threonyl tRNA synthetase: amplification of the structural gene in borrelidin resistant cell lines. Molecular and Cellular Biology 9: 1832-1838.

4. Cruzen, M.E. and S.M. Arfin (1991) Nucleotide and deduced amino acid sequence of human threonyl-tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes. The Journal of Biological Chemistry 266: 9919-9923.

5. Gerken, S.C, Wasmuth, JJ. and S.M. Arfin (1986) Threonyl-tRNA synthesis gene maps close to leucyl-tRNA synthetase gene on human chromosome 5. Somatic Cell and Molecular Genetics 12: 519-522.

6. Mitra AB, Murty VV, Singh V, et al. (1995) Genetic alterations at 5pl 5: a potential marker for progression of precancerous lesions of the uterine cervix. J Natl Cancer

Inst. 87:742-5.

7. Mitra AB, Murty VV, Li RG, Pratap M, Luthra UK, Chaganti RS. (1994) Allelotype analysis of cervical carcinoma. Cancer Res. 54:4481-7.

8. Mullokandov MR, Kholodilov NG, Atkin NB, Burk RD, Johnson AB, Klinger HP. 231 232/116

(1996) Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status. Cancer Res. 56:197-205. 9. Larsson C, Bystrom C, Skoog L, Rotstein S, Nordenskjold M. (1990) Genomic alterations in human breast carcinomas. Genes Chromosomes Cancer. 2:191-7. 10. Cancer Research 54: 1152

11. Wieland I, Bohm M, Arden KC, et al. (1996) Allelic deletion mapping on chromosome 5 in human carcinomas. Oncogene. 12:97-102.

12. Dracopoli NC, Houghton AN, Old LJ. (1985) Loss of polymoφhic restriction fragments in malignant melanoma: implications for tumor heterogeneity. Proc Natl AcadSci USA. S2:\A70-A.

13. Osborne RJ, Leech V. (1994) Polymerase chain reaction allelotyping of human ovarian cancer. Br J Cancer. 69:429-38.

Example 19: Glutaminyl-tRNA Synthetase (QARS) - Target Gene VARIA305

The human glutaminyl-tRNA synthetase gene is essential for cell survival

Glutaminyl-tRNA synthetase (QARS) catalyzes ATP dependent covalent attachment of glutamine to its cognate tRNA to form glutaminyl-tRNA. In the absence of glutaminyl-tRNA, protein synthesis is blocked in eucaryotic cells. Glutaminyl-tRNA synthetase is a single copy gene in man . Inhibition of its function is expected to be cell lethal, as shown for other tRNA synthetases (summarized above).

The human Glutaminyl-tRNA synthetase gene and mRNA have sequence variances.

A human cDNA encoding glutaminyl tRNA synthetase (QARS) was cloned by Lamour et al. (1) who expressed the cDNA in E. coli and demonstrated glutaminyl tRNA synthetase activity in bacterial extracts. The cloned human cDNA 232 232/116

(Genbank/EMBL accession number X76013) is 2437 nt in length and includes a 5' untranslated region of 5 nucleotides, an open reading frame of 2325 nucleotides encoding a 775 amino acid polypeptide, and a 3' untranslated region of 107 nt including 8 terminal nt of poly A.

We designed primers for amplification. The QARS cDNA was screened for sequence variance in 36 unrelated individuals using the single strand conformation polymoφhism (SSCP) technique. One sequence variance was identified, a C vs. T transition at nucleotide 404, within the coding sequence. The published nucleotide at position 404 is C The sequence variance does not affect the protein encoded.

The frequency of heterozygotes for the nucleotide 404 sequence variance is 11% in the 36 individuals tested (4/36). However three of 16 North American Whites are heterozygotes (19%), and one of four Japanese (25%) (see Target Gene Summary Table).

The QARS gene maps to 3p

The human QARS cDNA has been mapped to chromosome 3 by hybridization of a QARS probe to a panel of 25 human/rodent somatic cell hybrids (1). One somatic cell hybrid, not known to contain human chromosome 3, was positive for both the QARS probe and an ACY1 probe. ACY1 maps to human 3p21, suggesting QARS may also map in this area. We independently mapped QARS to chromosome 3 using primers from the 3' untranslated region to analyze the National Institute of General Medical Sciences (NIGMS) Human/Rodent Somatic Cell Hybrid Mapping Panel #2 by PCR

(see page 704 of the NIGMS 1994/1995 Catalog of Cell Lines, available from the Coriell Cell Repository, Camden, NJ). The panel consists of 24 hybrid cell lines, each monochromosomal for one human chromosome. The QARS PCR product mapped to the hybrid containing human chromosome 3. All mapping techniques were conclusive 233 232/116 in showing only one locus for human QARS.

Chromosome band 3p21 is a site of frequent loss of heterozygosity. The short arm of chromosome 3 has been well studied in breast, cervical, esophageal, kidney, and lung cancers. These studies report frequent allele loss at 3p21, varying up to 100% in some studies of small cell lung cancer. Among other cancers LOH occurs in approximately 20-30% of breast cancers (2,3), 30-60% of cervical cancers (4,5), 10-40% of esophageal cancers (6,7), 45-80% of kidney cancers (8-10), 50-100% of nasopharyngeal cancers (11), 0-75% of squamous cell head and neck cancers (12), 30- 60% of melanomas (13), 30-100% of non-small cell lung cancers (14-16) and 80-

100% in small cell lung cancer (17-19). Other for which there are reports of LOH in at least 20% of cases include leukemia, pancreas cancer, sarcoma, testis cancer and ovarian cancer. Other cancer types, including bladder and lymphoma, have not been studied for LOH at 3p21.

References

1. Nomura, N., Nagase, T., Miyajima, N., et al. (1994) Prediction of the coding sequences of unidentified human genes. II. The coding sequences of 40 new genes (KIAA0041-KIAA0080) deduced by analysis of cDNA clones from human cell line KG- 1. DNA Research 1 :225-229.

2. Nichols, R.C., Blinder, J., Pai, S.I. et al. (1996) Assignment of two human autoantigen genes: isoleucyl tRNA synthetase locates to 9q21 and lysysl-tRNA synthetase locates to 16q23-24. Genomics: 210-213.

3. Cleton-Jansen AM, Moerland EW, Kuipers-Dijkshoorn NJ, et al. (1994) At least two different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Genes Chromosom Cancer. 9:101-7.

4. Dorion-Bonnet F, Mautalen S, Hostein I, Longy M. (1995) Allelic imbalance study of 16q in human primary breast carcinomas using microsatellite markers. Genes Chromosomes Cancer. 14:171-81. 234 232/116

5. Kashiwaba M, Tamura G, Suzuki Y, et al. (1995) Epithelial-cadherin gene is not mutated in ductal carcinomas of the breast. Jpn J Cancer Res. 86:1054-9. 6. 0'Connell P, Pekkel V, Fuqua S, Osborne CK, Allred DC. (1994) Molecular genetic studies of early breast cancer evolution. Breast Cancer Res Treat. 32:5-12. 7. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51:5794-9. 8. Tsuda H, Callen DF, Fukutomi T, Nakamura Y, Hirohashi S. (1994) Allele loss on chromosome 16q24.2-qter occurs frequently in breast cancers irrespectively of differences in phenotype and extent of spread. Cancer Res. 54:513-7. 9. Fujimori M, Tokino T, Hino O, et al. (1991) Allelotype study of primary hepatocellular carcinoma. Cancer Res. 51 :89-93.

10. Fujimoto Y, Hampton LL, Wirth PJ, Wang NJ, Xie JP, Thorgeirsson SS. (1994) Alterations of tumor suppressor genes and allelic losses in human hepatocellular carcinomas in China [see comments]. Cancer Res. 54:281-5. 11. Tsuda H, Zhang WD, et al. (1990) Allele loss on chromosome 16 associated with progression of human hepatocellula carcinoma. Proc Natl Acad Sci USA. 87:6791-4. 12. Tsuda H, Oda T, Sakamoto M, Hirohashi S. (1992) Different pattern of chromosomal allele loss in multiple hepatocellular carcinomas as evidence of their multifocal origin. Cancer Res. 52:1504-9. 13. Zhang WD, Hirohashi S, Tsuda H, et al. (1990) Frequent loss of heterozygosity on chromosomes 16 and 4 in human hepatocellular carcinoma. Jpn J Cancer Res. 81:108- 11.

14. Ookawa K, Sakamoto M, Hirohashi S, et al. (1993) Concordant p53 and DCC alterations and allelic losses on chromosomes 13q and 14q associated with liver metastases of colorectal carcinoma. Int J Cancer. 53 :382-7.

15. Genes, Chromosomes & Cancer 10: 177.

16. Cancer Research 54: 2996.

17. Gallion H.H., Powell D.E., Morrow J.K., et al. (1992) Molecular genetic changes in human epithelial ovarian malignancies [see comments]. Gynecol Oncol. 47:137-42. 235 232/116

20. British Journal of Urology 73: 390.

21. Okamoto A, Sameshima Y, Yamada Y, et al. (1991) Allelic loss on chromosome 17p and p53 mutations in endometrial carcinoma of the uterus. Cancer Res. 51 :5632-5.

Example 20: Lysyl-tRNA Synthetase (KARS) - Target Gene VARIA303

Human Lysyl t-RNA synthase gene is essential

Lysyl-tRNA synthetase (KARS) catalyzes ATP dependent covalent attachment of lysine to its cognate tRNA to form lysyl-tRNA. In the absence of lysyl-tRNA, protein synthesis is blocked. Since lysyl-tRNA synthetase is a single copy gene in man, inhibition of its function is expected to be cell lethal. This has been shown for other tRNA synthetases (summarized above).

The human Lysyl-tRNA synthetase gene and mRNA have sequence variances

A human cDNA encoding a sequence similar to bacterial lysyl tRNA synthetases was cloned by Nomura et al. (GenBank/DDBJ submission D31890; see ref. 1) while sequencing random cDNAs. No biochemical studies of the protein encoded by this sequence have been reported. The 5' end of the sequence apparently begins in the coding region and the open reading frame continues for 1805 nucleotides, encoding 601 residues of a polypeptide (the full length of which has not been established), followed by a 3' untranslated region of 165 nucleotides. 236 232/116

We designed primers for amplification. The reported partial cDNA was screened for sequence variance in 36 unrelated individuals using the single strand conformation polymoφhism (SSCP) technique as described in the methods section. Two sequence variances were identified, an A vs. G transition at nucleotide 89 and a G vs. C transversion at nucleotide 1798, both within the coding sequence. The published nucleotides are A and G, respectively. The nucleotide 1798 sequence variance alters the sense of the 599th codon (the third codon from the end of the coding sequence) to serine vs. threonine.

The frequency of KARS heterozygotes varies among the populations surveyed. The frequency of heterozygotes for the nucleotide 89 sequence variance is 19% in the 36 individuals tested. However all heterozygous individuals were either North American Whites (4/16; 25% heterozygotes), North American Blacks (1/4; 25%), or Hispanics (1/3; 33% heterozygotes). The frequency of heterozygotes for the nucleotide 1798 sequence variance is 6% in the 36 individuals tested. However all heterozygous individuals were North American Blacks (2/4; 50%) (see Target Gene Summary Table). Further study of these and other population groups will better establish the frequency of heterozygotes for these two sequence variances.

The KARS gene maps to 16q23-q24

The human KARS cDNA has been mapped to chromosome 16q22 by Nichols et al. (ref. 2) and by us. We designed primers to the 3' untranslated region of KARS and used PCR to analyze the National Institute of General Medical Sciences (NIGMS) Human/Rodent Somatic Cell Hybrid Mapping Panel #2 (see page 704 of the NIGMS

1994/1995 Catalog of Cell Lines, available from the Coriell Cell Repository, Camden, NJ). The panel consists of 24 hybrid cell lines, each monochromosomal for one human chromosome. The KARS PCR product mapped to the hybrid containing human chromosome 16. Similar results were obtained by Nichols et al., who mapped KARS 237 232/116 by analysis of the same NIGMS hybrid mapping panel, by PCR mapping in a chromosome 16 regional mapping panel and by fluorescence in situ hybridization to metaphase chromosomes. The in situ hybridization showed KARS maps to 16q23- q24. All mapping techniques were conclusive in showing only one locus for human KARS.

Loss of heterozygosity occurs frequently at 16q23-q24 in many cancer types. Loss of heterozygosity studies of chromosome 16q have principally focused on breast and liver cancers. In six detailed studies of breast cancer in the 16q23-q24 region LOH frequencies of 30-60% have been reported (refs 3-8). 16q22 LOH has ben reported in

35-65% of liver cancers (9-13), with the average around 45%. Studies of other cancer types report 16q22 LOH in 19% of colon cancers (14), 17-27% of esophageal cancers (15,16), 37% of ovarian cancers (new ref) (17-19), 18% of prostate cancers (20) and 23% of uterine cancers (21). Cancer types not yet investigated for LOH include kidney, leukemia and lymphoma, lung, melanoma, neuroblastoma, stomach and testis.

References

1. Nomura, N., Nagase, T., Miyajima, N., et al. (1994) Prediction of the coding sequences of unidentified human genes. II. The coding sequences of 40 new genes

(KIAA0041-KIAA0080) deduced by analysis of cDNA clones from human cell line KG-1. DNA Research 1:225-229.

4. Dorion-Bonnet F, Mautalen S, Hostein I, Longy M. (1995) Allelic imbalance study 238 232/116 of 16q in human primary breast carcinomas using microsatellite markers. Genes Chromosomes Cancer. 14:171-81.

5. Kashiwaba M, Tamura G, Suzuki Y, et al. (1995) Epithelial-cadherin gene is not mutated in ductal carcinomas of the breast. Jpn J Cancer Res. 86:1054-9. 6. 0'Connell P, Pekkel V, Fuqua S, Osborne CK, Allred DC. (1994) Molecular genetic studies of early breast cancer evolution. Breast Cancer Res Treat. 32:5-12.

10. Fujimoto Y, Hampton LL, Wirth PJ, Wang NJ, Xie JP, Thorgeirsson SS. (1994) Alterations of tumor suppressor genes and allelic losses in human hepatocellular carcinomas in China [see comments]. Cancer Res. 54:281-5.

11. Tsuda H, Zhang WD, Shimosato Y, et al. (1990) Allele loss on chromosome 16 associated with progression of human hepatocellular carcinoma. Proc Natl Acad Sci USA. 87:6791-4. 12. Tsuda H, Oda T, Sakamoto M, Hirohashi S. (1992) Different pattern of chromosomal allele loss in multiple hepatocellular carcinomas as evidence of their multifocal origin. Cancer Res. 52:1504-9.

13. Zhang WD, Hirohashi S, Tsuda H, et al. (1990) Frequent loss of heterozygosity on chromosomes 16 and 4 in human hepatocellular carcinoma. Jpn J Cancer Res. 81:108- 11.

15. Genes, Chromosomes & Cancer 10: 177- 239 232/116

16. Cancer Research 54: 2996-

17. Gallion HH, Powell DE, Morrow JK, et al. (1992) Molecular genetic changes in human epithelial ovarian malignancies [see comments]. Gynecol Oncol. Al:\2>l-A2.

20. British Journal of Urology 73: 390-.

21. Okamoto A, Sameshima Y, Yamada Y, et al. (1991) Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus.Cαwcer

Res.51:5632-5.

Example 21: Ribosomal Protein S14 (RPS14) - Target Gene VARIA326

Ribosomal protein SI 4 is essential for cell growth

Human ribosomal protein S14 (RPS14) is one of -80 unique protein constituents of the mammalian ribosome. Many of the protein subunits of ribosomes, the protein making machines of all cells, are highly conserved throughout prokaryotic and eukaryotic evolution (1). For example, human RPS14 protein is 100% amino acid identical to hamster S14 protein, 72% identical to yeast φ59 protein and 43% identical to E. Coli ribosomal protein SI 1 (2,3). Mammalian S14 and yeast φ59 are components of the 40S ribosomal subunit while E. coli Sl l is part of the corresponding bacterial S30 subunit. Thus human RPS 14 is a ribosomal component fixed early in evolution.

There are many antibiotics and eukaryotic cell poisons that act by inhibiting ribosome function (reviewed in ref. 4). One such drug is emetine, which inhibits protein translation by interacting with the eukaryotic RPS 14 subunit to prevent elongation 240 232/116 factor dependent translocation of peptidyl-tRNAs bound to eukaryotic ribosomes in vitro (4).

Chinese hamster ovary (CHO) cell lines resistant to emetine have been shown to contain mutant RPS 14 loci (also referred to as the EMTB locus) (5). Such lines have been used to investigate the effects of mutant RPS 14 on ribosome function (5-8). Human-CHO cell hybrids are emetine-sensitive, indicating that the EMTB/RPS14 mutation is recessive in CHO cells. This is apparently because arrest of protein synthesis in half of ribosomes blocks translation of all polysomic mRNAs by blocking any functional ribosomes upstream of frozen mutant ribosomes. RPS 14 appears to contribute to the structural integrity of the 40S subunit: 40S subunits containing mutant S14 protein are more easily dissociable in high ionic strength wash buffers (9). Ribosomal subunit genes are coordinately expressed in all cells and ribosomal proteins constitute a large fraction of the cell mass in all cell types.

The human RPS 14 gene has sequence variances

Rhoads et al. reported the sequence of the human RPS 14 gene and cDNA (3). The cDNA contains a 33 nucleotide 5' untranslated region, a 453 nt coding region and a 60 nt 3' untranslated region (including 12 nt of poly A). We undertook a systematic search for DNA sequence variance in the cDNA of RPS 14 by analysing 36 unrelated individuals using the single strand conformation polymoφhism technique. Primers were designed using the sequence of Rhoads et al. (GENBANK accession Ml 3934, M13641; see ref. 3). SSCP analysis revealed 1 sequence variance, and subsequent DNA sequence analysis confirmed an A vs. G transition at nucleotide 183 of the coding sequence. (This change was noted as a difference between the cDNA and genomic sequences in ref. 3.)

As shown in the Target Summary Table, both alleles were detected in all major 241 232/116 populations surveyed, including North American Whites, North American Blacks, Hispanics, Chinese and Japanese.

The human RPS 14 gene maps to chromosome 5q23-q33

Dana and Wasmuth (11) used Chinese hamster/human somatic cell hybrids to map the RPS14 gene (designated EMTB) to 5q23-5q35. Later Nakamichi et al. (12) placed the RPS 14 gene on the segment 5q23-q33 using similar techniques.

Chromosome band 5q23-q33 is a site of frequent loss of heterozygosity. There have been many studies of LOH on 5q, particularly the 5q21-q22 region where the Adenomatous Polyposis Coli (APC) tumor suppressor gene lies. The most extensively studied cancers are those of the gastrointestinal tract, lung and ovary. The available data on the 5q23-q33 region just distal to APC (where RPS14 lies), suggests that LOH occurs in this region at a frequency of -30% in cervical cancer (13), 20-40% in colon cancer (14,15), 30-50% in ovarian cancer (16,17), 38% in stomach cancer (18) and 23% in testicular cancer (19). There is also evidence for LOH in head and neck, lung, and liver cancers.

References

1. Chambliss, G., Craven, G.R., Davies, J., et al., editors, Ribosomes: Structure. Function and Genetics. University Park Press, Baltimore, 1980.

2. Chen, I.-T., Dixit, A., |Rhoads, D.D. and D.J. Roufa (1986) Homologous ribosomal proteins in bacteria, yeast and humans. Proc. Natl. Acad. Sci. U.S.A. 83: 6907-6911.

3. Rhoads, D. D.; Dixit, A.; Roufa, D. J. (1986) Primary structure of human ribosomal protein S14 and the gene that encodes it. Molec. Cell Biol. 6: 2774-2783.

4. Vazquez, D. (1979) Molecular Biology and Biophysics, vol.30. Inhibitors of Protein Synthesis. Springer- Verlag, Berlin. 242 232/116

5. Wasmuth, J.J. (1985) Chinese hamster cell protein synthesis mutants. In Gottesman, M., ed. Molecular Cell Genetics, pp. 397-421.

6. Rhoads, D.D. and D J. Roufa (1985) Emetine resistance in Chinese hamster cells: structures of wild-type and mutant ribosomal protein ASM mRNAs. Mol. Cell Biol. 5: 1655-1659.

7. Madjar, J.J., Nielsen-Smith, K., Frahm, M. and D. Roufa (1982) Emetine resistance in Chinese hamster ovary cells is associated with an altered ribosomal protein S14 mRNA. Proc. Natl. Acad. Sci. U.S.A. 79: 1003-1007.

8. Dana, S. L., Chang, S. and JJ. Wasmuth (1985) Synthesis and incoφoration of human ribosomal protein S 14 into functional ribosomes in human-Chinese hamster cell hybrids containing human chromosome 5: human RPS 14 gene is the structural gene for ribosomal protein S 14. Somat. Cell Molec. Genet. 11: 625-631.

9. Madjar, J.-J., Frahm, M., McGill, S. and D J. Roufa (1983) Molec. Cell. Biol. 3: 190- 197. 10. Mount, S. (1982) A catalogue of splice junction sequences. Nucleic Acids Research

19: 459-472.

11. Dana, S. and J.J. Wasmuth (1982) Selective linkage disruption in human-Chinese hamster cell hybrids: deletion mapping of the leuS, hexB, emtB, and chr genes on human chromosome 5. Molec. Cell. Biol. 2: 1220-1228. 12. Nakamichi, N. N.; Kao, F.-T.; Wasmuth, J.; Roufa, D. J. (1986) Ribosomal protein gene sequences map to human chromosomes 5, 8 and 17. Somat. Cell. Molec. Genet.

12: 225-236.

13. Mitra AB, Murty VV, Li RG, Pratap M, Luthra UK, Chaganti RS. (1994)

Allelotype analysis of cervical carcinoma. Cancer Res. 54:4481-7. 14. Japanese Journal of Cancer Research 82: 1003.

15. Cunningham C, Dunlop MG, Wyllie AH, Bird CC. (1993) Deletion mapping in colorectal cancer of a putative tumor suppressor gene in 8p22-p21.3. Oncogene. 8:1391-6.

16. British Journal of Cancer 69: 429. 243 232/116

17. Weitzel J.N., Patel J., Smith D.M., Goodman A., Safaii H., Ball H.G. (1994) Molecular genetic changes associated with ovarian cancer. Gynecol Oncol. 55:245-52.

18. Genes, Chromosomes and Cancer 3: 468

19. Murty VV, Bosl GJ, Houldsworth J, et al. (1994) Allelic loss and somatic differentiation in human male germ cell tumors. Oncogene. 9:2245-51.

Example 22: Eukaryotic Initiation Factor 5A (eIF-5A) - Target Gene VARIA351

Initiation Factor 5 A is essential for cell growth

Human Initiation Factor 5 A (eIF-5 A), formerly named Initiation Factor 4D, is an 18- kD protein which promotes formation of the first peptide bond in in vitro translation systems - hence the name 'initiation factor' (1,2); however, the full physiological role of eIF-5A is not understood. Inhibition of elF 5 A formation blocks proliferation in all tested cell types (3); the presence of functional elF 5 A has been shown to correlate with the onset of DNA replication (4) - perhaps due to elF 5 A dependent translation of mRNAs encoding proteins necessary for DNA replication (3), and eIF-5A is an essential co-factor for HIV-1 Rev protein (5).

elF 5 A is an unusual protein: one of its lysine residues (amino acid 50) is modified by transfer and hydroxylation of the butylamino-group from the polyamine spermidine to form hypusine, a post translational modification unique to elF 5A. All of the biological activities of elF 5A are abrogated in the absence of the hypusine modification, as demonstrated by pharmacological inhibition of hypusine formation in human cell lines (3) and by site directed mutagenesis of the modified lysine residue in the yeast enzyme (6). There are two enzymes responsible for hypusine formation, one of which, deoxyhypusyl hydroxylase, can be inhibited with the drug mimosine (3), providing a convenient pharmacological inhibitor of eFI 5 A formation. 244 232/116

The genome of the yeast Saccharomyces cerevisiae encodes two elF 5A genes. Disruption of one (form A) slows growth, disruption of the other (form B) arrests growth and strains with both forms disrupted are non-viable (6). The yeast A form substitutes for human elF 5A in the mammalian methionyl-puromycin synthesis assay (6), while the human gene complements elF 5A disrupted yeast (7). elF 5A is a highly conserved protein, with counteφarts in archeae, bacteria and eukaryotes. The yeast proteins are -63% identical to the human protein (6).

The human elF 5 A gene and mRNA have sequence variances

Smit-McBride, et al. reported the sequence of a human cDNA encoding eIF-5A (8) and Koettnitz et al. (8) later reported the sequence of the active elF 5A gene, which contains three introns (GenBank accession U 17969). A composite sequence made from the cDNA and genomic versions is 1309 nucleotides long and contains a 5' untranslated region of 145 nucleotides, a 462 nt coding region and a 702 nt 3' untranslated region (see annotated sequence). We undertook a systematic search for DNA sequence variance in the cDNA of elF 5 A by analysing 36 unrelated individuals using the single strand conformation polymoφhism technique. Primers were designed for amplification. SSCP analysis revealed 2 sequence variances, and subsequent DNA sequence analysis confirmed an A vs. G transition at nucleotide 623 and a T vs. C transition at nucleotide 1012, both in the 3' untranslated sequence.

Neither sequence variance affects the protein coding sequence, however nucleotide 623 is one nucleotide away from a splice acceptor site at position 622, and could therefore be targeted by an oligonucleotide intended to abrogate splicing in an allele specific manner. The second exonic nucleotide (+2 position) of a splice acceptor site is not highly conserved, nonetheless the A vs. G transition at nucleotide 623 may affect the mechanics of splicing. 245 232/116

As shown in the Target Summary Table, both alleles were detected in all major populations surveyed, including North American Whites, North American Blacks, Hispanics, Arabs, Indians and Japanese, except only the nucleotide 1012 variance was detected in the four Chinese surveyed. The overall frequency of heterozygotes was 37% for the nucleotide 623 sequence variance and 52% for the nucleotide 1012 sequence variance.

The human elF 5 A gene maps to chromosome 17pl3-pl2

Steinkasserer et al. (1995) mapped the elF 5A gene to 17pl3-pl2 by fluorescence in situ hybridization (9). Three elF 5 A pseudogenes were mapped to 10q23, 17q25 and 19ql3.

Chromosome band 17pl3-pl2 is a site of frequent loss of heterozygosity. There have been many studies of LOH on 17p, particularly the 17pl3 region where the p53 tumor suppressor gene maps. Virtually all cancer types have been surveyed for LOH in this area, with particularly extensive studies of breast, colon, ovarian, and stomach cancers. These studies report LOH in approximately 40-60% of breast cancers (10-18), 50-70% of colon cancers (19-25), 25-75% of ovarian cancers (26-30), 20-60% of stomach cancers (31 -34), 20-50% of brain cancers (35,36), 45-70% of esophageal cancers (37),

35-65% of non-small cell lung cancers (38,39) and 100% of small cell lung cancers, 15-50% of cervical cancers, 30-80% of head and neck cancers, 20-60% of liver cancers, over 50% of sarcomas and 10-30% of a variety of other cancer types.

References

1. Wolff, E.C., Park, M.H. and J.E. Folk (1990) Journal of Biological Chemistry 265: 4793-4799.

2. Park, M.H., Wolff, E.C and J.E. Folk (1993) Hypusine: its post-translational formation in eukaryotic translation factor 5A and its potential role in cellular 246 232/116 regulation. Biofactors A 95-104.

3. Hanauske-Abel, H.M., Park, M.-H., Hanauske, A.-R., et al. (1994) Inhibition of the Gl-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation. Biochimica et Biophysica Acta 1221 : 115- 124. 4. Hanauske-Abel, H.M., Slowinska, B., Zagulska, S., et al. (1995) Detection of a subset of polysomal mRNAs associated with modulation of hypusine formation at the Gl-S boundary. Proposal of a role for elF 5 A in onset of DNA replication. FEBSLett. 366: 92-98.

5. Ruhl, M., Himmelspach, M., Bahr, G.M., et al. (1993) Eukaryotic initiation factor 5 A is a cellular target of the HIV-1 Rev activation domain mediating trans-activation.

J. Cell Biol. 123:1309-1320.

6. Schnier, J., Schwelberger, H.G., Smit-McBride, Z, et al. (1991) Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces Cerevisiae. Molecular and Cellular Biology 11 : 3105-3114. 7. Koettnitz, K., Wohl, T., Kappel, B., Lottspeich, F., Hauber, J. and D. Bevec (1995)

Identification of a new member of the human eIF-5A gene family. Gene 159: 283-284.

8. Smit-McBride, Z., Dever, T.E., Hershey, J.W.B., et al. (1989) Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine containing protein. Journal of Biological Chemistry 264: 1578-1583.

9. Steinkasserer, A.; Jones, T.; Sheer, D.; Koettnitz, K.; Hauber, J. and D. Bevec (1995) The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17pl2-pl3: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19ql3.2. Genomics 25: 749-752. 10. Cornells RS, van Vliet M, Vos CB, et al. ( 1994) Evidence for a gene on 17p 13.3 , distal to TP53, as a target for allele loss in breast tumors without p53 mutations. Cancer Res. 54:4200-6.

11. Lindblom A, Skoog L, Rotstein S, Werelius B, Larsson C, Nordenskjold M. (1993) Loss of heterozygosity in familial breast carcinomas. Cancer Res. 53:4356-61. 247 232/116

12. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51 :5794-9.

13. Singh S, Simon M, Meybohm I, et al. (1993) Human breast cancer: frequent p53 allele loss and protein overexpression. Hum Genet. 90:635-40. 14. Thorlacius S, Borresen AL, et al. (1993) Somatic p53 mutations in human breast carcinomas in an Icelandic population: a prognostic factor. Cancer Res. 53:1637-41. 15. Tsuda H, Hirohashi S. (1994) Association among p53 gene mutation, nuclear accumulation of the p53 protein and aggressive phenotypes in breast cancer. Int J Cancer. 57:498-503. 16. Watatani M, Nagayama K, Imanishi Y, et al. (1993) Genetic alterations on chromosome 17 in human breast cancer: relationships to clinical features and DNA ploidy. Breast Cancer Res Treat. 28:231-9.

17. Chen LC, Neubauer A, Kurisu W, et al. (1991) Loss of heterozygosity on the short arm of chromosome 17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer. Proc Natl Acad Sci U S A. 88:3847-51.

18. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51 :5794-9.

19. Burmer GC, Rabinovitch PS, Haggitt RC, et al. (1992) Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of a p53 allele [see comments]. Gastroenterology. 103:1602-10.

20. Cunningham C, Dunlop MG, Wyllie AH, Bird CC. (1993) Deletion mapping in colorectal cancer of a putative tumor suppressor gene in 8p22-p21. Oncogene. 8:1391- 6

21. Kikuchi-Yanoshita R, Konishi M, Ito S, et al. (1992) Genetic changes of both p53 alleles associated with the conversion from colorectal adenoma to early carcinoma in familial adenomatous polyposis and non-familial adenomatous polyposis patients. Cancer Res. 52:3965-71.

22. Yin J, Haφaz N, Tong Y, et al. (1993) p53 point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology. 104:1633-9. 248 232/116

23. lacopetta B, DiGrandi S, Dix B, et al. (1994) Loss of heterozygosity of tumor suppressor gene loci in human colorectal carcinoma. Eur J Cancer. 5:664-70.

24. Law DJ, Olschwang S, Monpezat JP, et al. (1988) Concerted nonsyntenic allelic loss in human colorectal carcinoma. Science. 241:961-5. 25. Lothe RA, Nakamura Y, Woodward S, Gedde DT, Jr., White R. (1988) VNTR

(variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas. Cytogenet Cell Genet. 48:167-9. 26. Foulkes WD, Stamp GW, Afzal S, et al. (1995) MDM2 overexpression is rare in ovarian carcinoma irrespective of TP53 mutation status. Br J Cancer. 72:883-8. 27. Phillips NJ, Ziegler MR, Radford DM, et al. (1996) Allelic deletion on chromosome 17pl3.3 in early ovarian cancer. Cancer Res. 56:606-11. 28. Foulkes WD, Black DM, Stamp GW, Solomon E, Trowsdale J. (1993) Very frequent loss of heterozygosity throughout chromosome 17 in sporadic ovarian carcinoma. Int J Cancer. 54:220-5. 29. Gallion HH, Powell DE, Morrow JK, et al. (1992) Molecular genetic changes in human epithelial ovarian malignancies [see comments]. Gynecol Oncol. 47:137-42.

30. Phillips N, Ziegler M, Saha B, Xynos F. (1993) Allelic loss on chromosome 17 in human ovarian cancer. Int J Cancer. 54:85-91.

31. Seruca R, David L, Castedo S, Veiga I, Borresen AL, Sobrinho-Simoes M. (1994) p53 alterations in gastric carcinoma: a study of 56 primary tumors and 204 nodal metastases. Cancer Genet Cytogenet. 75:45-50.

32. Kim CJ, Kim WH, Kim CW, Lee JB, Lee CK, Kim YL. (1995) Detection of 17p loss in gastric carcinoma using polymerase chain reaction. Lab Invest. 72:232-6.

33. Ranzani GN, Renault B, PellegataNS, et al. (1993) Loss of heterozygosity and K- ras gene mutations in gastric cancer. Hum Genet. 92:244-9.

34. Sano T, Tsujino T, Yoshida K, et al. (1991) Frequent loss of heterozygosity on chromosomes lq, 5q, and 17p in human gastric carcinomas. Cancer Res. 51 :2926-31.

35. Frankel RH, Bayona W, Koslow M, Newcomb EW. (1992) p53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation 249 232/116 frequency. Cancer Res. 52:1427-33.

36. Hermanson M, Funa K, Koopmann J, et al. (1996) Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor alpha receptor expression in human malignant gliomas. Cancer Res. 56:164-71. 37. Aoki T, Mori T, Du X, Nisihira T, Matsubara T, Nakamura Y. (1994) Allelotype study of esophageal carcinoma. Genes Chromosomes Cancer. 10:177-82.

38. Tsuchiya E, Nakamura Y, Weng SY, et al. (1992) Allelotype of non-small cell lung carcinoma— comparison between loss of heterozygosity in squamous cell carcinoma and adenocarcinoma. Cancer Res. 52:2478-81. 39. Hiyama K, Ishioka S, Shirotani Y, et al. (1995) Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb.

Oncogene. 10:937-44.

Example 23: Replication Protein A, 32 kDa Subunit (RPA32) - Target Gene

VARIA402

The human RPA32 gene encodes a protein essential for cell survival

Replication Protein A (RPA; also known as Replication Factor A, Activator 1, Single

Strand Binding Protein or SSB) is a heterotrimeric protein which participates in DNA replication, homologous recombination and nucleotide excision repair (1-3). The evidence that RPA is an essential protein comes from in vitro and in vivo data.

DNA replication is essential for cell proliferation, as discussed above for RPA70.

The best studied function of RPA32 is in DNA replication. Because of the complexity of DNA replication in higher eukaryotic genomes, the small genome of the papovavirus SV40 has been used as a model system to study DNA replication in human cell extracts. In the 1980s several research groups 250 232/116 developed cell free systems to study DNA replication using SV40 chromosomes as templates (4-8). An effort to identify the minimal set of factors required for DNA replication led to the discovery of RPA. Subsequent work proved that each of the three subunits of RPA is essential for DNA replication (9,10). This was proved in several ways, including by using antibodies to various constituents of the replication complex. Anti-RPA32 antibodies inhibit DNA replication, providing clear in vitro evidence for the essential function of this subunit of RPA in human DNA replication (10). The yeast S. cerevisiae has a trimeric replication protein A which is structurally and functionally homologous to the human protein. It consists of three subunits similar in size to the human subunits. All three yeast subunits have been disrupted and each disruption produces non-viable yeast (9).

77-e human RPA32 gene and mRNA are polymorphic.

The published cDNA for the 32 kD subunit of Replication Protein A is 1512 nucleotides long and includes a 5' untranslated segment of 77 nucleotides, followed by a protein coding region of 810 nucleotides and a 3' untranslated region of 625 nucleotides (10). We undertook a systematic search for DNA polymoφhism by analysing the RPA32 cDNA from 36 unrelated individuals using the single strand conformation polymoφhism technique (described in the methods section). Primers were designed using the sequence of Erdile et al. (GenBank accession J05249; see ref. 10). SSCP analysis revealed 2 variances, one of which was sequenced. Sequencing revealed a G vs. A transition at nucleotide 40 of the 5' untranslated region. Four of 36 individuals were heterozygotes, all of them Caucasians. Thus the allele frequency is

25% (4/16) in North American Whites, while no heterozygosity was detected in other populations (see Target Summary sheet).

The RPA32 gene maps to chromosome lp35 251 232/116

The gene for RPA32 was mapped to chromosome band lp35 by in situ hybridization, somatic cell hybrid analysis and yeast artificial chromosome mapping (11,12). Only one locus was detected by all methods.

Chromosome band lp35 is a site of frequent loss of heterozygosity. The short arm of chromosome 1 is comparatively well investigated for allele loss, especially in breast and colon cancers. Studies of the lp35 region show LOH in 15-40% of breast cancers (13,14), -50% of gliomas (a brain cancer subtype) (15), 20-70% of colon cancers (16,17), -50% of stomach cancers (18), -20% of lung cancers (19) and 10-30% of ovarian cancers. High frequency LOH has been detected in several uncommon cancers such as pheochromocytoma (50-80%) and neuroblastoma (-50%).

References

1. Erdile, L. F., et al. Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication, [published erratum appears in J. Biol. Chem. 1993 Jan 25;268(3):2268]. J. Biol. Chem. 266.18 (1991): 12090-8. 2. Jones, K. A., et al. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell 48.1 (1987): 79-89.

3. He, Z., et al. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature 374.6522 (1995): 566-9.

4. Challberg, M. D., and T. J. Kelly. Eukaryotic DNA replication: viral and plasmid model systems. Annu Rev Biochem 51 (1982): 901-34.

5. Wold, M. S., et al. Identification of cellular proteins required for simian virus 40 DNA replication. Journal Biological Chemistry 264.5 (1989): 2801-9.

6. Kelly, T. J. DNA replication in mammalian cells: insights from the SV40 model system. Harvey Lecture 85 (1989): 173-88. 252 232/116

7. Hurwitz, J., Dean, F.B., Kwong, A.D and S.-H. Lee (1990) Journal of Biological Chemistry 265: 18043-18046.

8. Stillman, B. (1992) Initiation of chromosome replication in eukaryotic cells. Harvey Lecture 88: 115-40. 9. Brill, S.J. and B. Stillman (1991) Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes and Development 5: 589-1600.

10. Erdile, L. F., M. S. Wold, and T. J. Kelly. The primary structure of the 32-kDa subunit of human replication protein A. J Biol Chem 265.6 (1990): 3177-82. 11. Ozawa, K., Dean, F., et al. (1993) Mapping of the 70 kDa 34kDa and 1 lkDa subunit genes of the human multimeric single-stranded DNA binding protein (hSSB/RPA) to chromosome bands 17pl3, Ip35-p36.1 and 7p21-p22. Cell Struct Funct 18: 221-230.

12. Umbricht, C. B., et al. High-resolution genomic mapping of the three human replication protein A genes (RPA1, RPA2, and RPA3). Genomics 20.2 (1994): 249-

57.

13. Bieche I, Champeme MH, Matifas F, Cropp CS, et al. (1993) Two distinct regions involved in lp deletion in human primary breast cancer. Cancer Res. 53:1990-4.

14. Borg A, Zhang QX, Olsson H, Wenngren E. (1992) Chromosome 1 alterations in breast cancer: allelic loss on lp and lq is related to lymphogenic metastases and poor prognosis. Genes Chromosomes & Cancer. 5:311-20.

15. Reifenberger, J., Reifenberger, G., Liu, L., et al. (1994) Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and lp. American Journal of Pathology 145: 1175-1190. 16. (1990) Cancer Research 50: 7232.

17.Lothe RA, Nakamura Y, Woodward S, Gedde DT, Jr., White R. (1988) VNTR (variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas. Cytogenet Cell Genet. 48:167-9. 18. Ezaki, T., Yanagisawa, A., Ohta, K., et al. (1996) Deletion mapping on 253 232/116 chromosome lp in well-differentiated gastric cancer. British Journal of Cancer 73:

424-428.

19. Hiyama K, Ishioka S, et al. (1995) Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb. Oncogene. 10:937-44.

Example 24: Replication Protein A, 70 kD subunit (RPA70) - Target Gene VARIA401

The human RPA 70 gene encodes a protein essential for cell survival

Replication Protein A (also known as Replication Factor A, Activator or Single Strand Binding protein [SSB]) is a heterotrimeric protein which participates in DNA replication, homologous recombination and nucleotide excision repair (1-3). The evidence that RPA is an essential protein comes from in vitro, in vivo and evolutionary data.

DNA replication is essential for cell proliferation, and a variety of antiproliferative drugs act, at least in part, by inhibiting DNA replication. Such drugs include nucleotide analogs that block DNA polymerases, such as 2',3' dideoxy NTPs and 3' deoxy ATP (cordycepin); inhibitors that bind to or modify DNA such as intercalating agents, DNA crosslinking drugs or alkylating agents, and inhibitors that bind to polymerases and replication proteins such as topoisomerase inhibitors like the epipodophyllotoxins, which prevent DNA unwinding necessary for replication (and transcription) and antibiotics which bind to polymerases such as arylhydrazino-pyrimidines.

The best studied function of RPA70 is in DNA replication. Because of the complexity of DNA replication in higher eukaryotic genomes, the small genome of the papovavirus SV40 has been used as a model system to study DNA replication in human cell extracts. In the 1980s several research groups 254 232/116 developed cell free systems to study DNA replication using SV40 chromosomes as templates (4-8). These studies, in seeking to identify the minimal set of factors required for DNA replication, led to the discovery of replication protein A. Subsequent work proved that each of the three subunits of RPA is essential for DNA replications. This was proved in several ways, including by using antibodies to various constituents of the replication complex. These antibodies are effectively inhibitors of RPA70. Anti-RPA70 antibody mediated abrogation of DNA replication provides clear in vitro evidence for the essential function of RPA70 in human DNA replication (10). The yeast S. cerevisiae has a trimeric replication protein A which is structurally and functionally homologous to the human protein. It consists of three subunits similar in size to the human subunits. The yeast 70 kDa subunit is 31% identical and 75% similar (including conserved amino acids) to its human counteφart (1). All three yeast subunits have been disrupted and each disruption produces non- viable yeast. The yeast 70 kD protein is also a single stranded DNA binding protein.

Single stranded DNA binding proteins (SSBs) are required for DNA replication in a wide variety of organisms, including bacteriophage, bacteria and some DNA viruses of higher eukaryotes. Recently the crystal structure of the DNA binding domain of human RPA was solved and found to be remarkably similar in three dimensional shape to the bacteriophage single stranded DNA binding proteins PC and gene V from fl phage.

The human RPA70 gene, mRNA and protein have sequence variances

The published cDNA for the 70 kD subunit of Replication Protein A is 2393 nucleotides long and includes a 5' untranslated segment of 69 nucleotides, followed by a protein coding region of 1848 nucleotides and a 3' untranslated region of 476 nucleotides (1). We undertook a systematic search for DNA polymoφhism by 255 232/116 analyzing the RPA70 cDNA from 36 unrelated individuals using the single strand conformation polymoφhism technique (described in the methods section). Primers were designed using the sequence of Erdile et al. (GenBank accession M63488; see ref. 1). SSCP analysis revealed 5 variances, and subsequent DNA sequence analysis of those variances led to identification of four additional variances. SSCP revealed the variances at nucleotides 81 (G vs. A), 1120 (A vs. G), 1674 (T vs. C), 2050 (T vs. C) and 2297, where an insertion/deletion variance of one C nucleotide was observed (8 vs. 9 C's in a row). In the course of sequencing around the nucleotide 2297 polymoφhism an additional variance was detected at nucleotide 2341 (A vs. G). Also, while sequencing additional Swedish individuals around nucleotide 1120 two new variances were observed at nucleotides 1124 and 125 (both C vs. T). Finally, in three individuals sequenced for the 2050 variance we noted a difference from the published sequence at nucleotide 2046: we detect 3 T's while the published clone shows just two. This difference may represent another insertion deletion polymoφhism. Five of the nine detected variances are in the coding sequence while four are in the 3' untranslated region.

The frequency of heterozygotes for the five SSCP positive variances ranged from 25- 42% among the 36 individuals tested. The small number of individuals genotyped for the other four variances precludes definitive assessment of heterozygosity rates. Some of the polymoφhisms appear to occur more commonly in certain racial or ethnic groups (see Target Summary sheet for details). For example, only one of the variances (nt 1674) was detected in Japanese individuals. In general, higher levels of polymoφhism were detected in North American Whites than in other groups. The nucleotide 1120 polymoφhism, for instance, was heterozygous in 9/36 individuals overall (25%), but in 8/16 North American Whites (50%).

The RPA70 cDNA encodes a 616 amino acid protein. The nucleotide 1120 and 1124 variances result in amino acid substitutions at residues 351 and 352, the former an alanine-threonine exchange (approximately 50% of Caucasians are heterozygotes) and 256 232/116 the latter a serine-phenylalanine exchange (rare in the populations tested). In the recently published crystal structure of the DNA binding segment of RPA70 (amino acids 181 -422) it is possible to place residue 351 in the second of two tandemly arrayed DNA binding domains (domain B; see ref. 10). Domain B extends from residue 1305 to N402, thus the variant residue 351 is in the middle. The published structure is a co- crystal of RPA70 amino acids 181-422 complexed to octadeoxy cytosine. Several RPA70 residues contact the oligonucleotide (Figure 4 of ref. 11), including amino acids K343 and T359, which lie 8 residues away from the polymoφhism in either direction. Modeling the two variant forms of the protein using the atomic coordinates deposited in the Protein Data Bank (1 JMC) should clarify the structural consequences of the alanine-threonine variance. Residue 351 lies in the center of a 50 amino acid segment of the protein that is relatively poorly conserved between yeast and man: 11 of the 50 residues are identical and 25 more are conservative substitutions. Towards the C terminus there is strong conservation: starting 25 residues C-terminal of the polymoφhism, 27 of the next 37 residues are identical between yeast and man.

Towards the N terminus there is -30% conservation. Both yeast and human 70 kD RPA subunits contain putative C4-type zinc finger motifs at positions -480-500.

The RPA 70 gene maps to chromosome 17pl3.3

The gene for RPA70 has been mapped to chromosome band 17pl3.3 by in situ hybridization (12). Only one locus was detected.

Chromosome band 17pl3.3 is a site of frequent loss of heterozygosity. RPA70 lies just telomeric to the TP53 tumor suppressor gene which is located in cytogenetic band

17pl3.1. This region of chromosome 17 is extremely well investigated for allele loss. In general, studies report LOH in approximately 40-60% of breast cancers (13-21), 50- 70% of colon cancers (22-28), 25-75% of ovarian cancers (29-33), 20-60% of stomach cancers (34-37), 20-50% of brain cancers (38,39), 45-70% of esophageal cancers (40), 257 232/116

35-65% of non-small cell lung cancers (41,42) and 100% of small cell lung cancers, 15-50% of cervical cancers, 30-80% of head and neck cancers, 20-60% of liver cancers, over 50% of sarcomas and 10-30% of a variety of other cancer types.

Assays developed for RPA: Protein and DNA contacts

Human cDNAs encoding all 3 subunits (70, 34 and 11 kD) of RPA have been cloned and expressed in E. coli and in insect cells via baculovirus vectors. The bacterially expressed 70 kDa protein is indistinguishable from its purified human counteφart immunologically and in several functional assays (see Table below). There is good evidence that the 70 kD subunit of RPA interacts with a number of different molecules. A partial list would include the 34 and 11 kD subunits of RPA, DNA, the xeroderma pigmentosum damage recognition and endonuclease proteins XPA and XPG, and DNA polymerase a-primase. These experimentally proven contacts (and almost certainly others) may constrain the topology of the protein in ways that have implications for inhibitor design. In summary a broad array of assays exists to screen for small molecule inhibitors of RPA (possibly including modified nucleotides), that act via competitive, allosteric or protein-protein blocking mechanisms. Table 4

Assays and reagents available for RPA inhibitor screening

RPA 70 kD, Assay Systems

Purified Purified Bacterial or

Human Protein Baculovirus Protein

ASSAY

Immunoreactivity

Single stranded DNA binding

DNA Polymerase alpha primase

258 232/116

DNA strand exchange Nucleotide excision repair Support SV40 Replication

References

1. Erdile, L. F., et al. Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication, [published erratum appears in J. Biol. Chem. 1993 Jan 25;268(3):2268]. J. Biol. Chem. 266.18 (1991): 12090-8.

2. Jones, K. A., et al. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell 48.1 (1987): 79-89.

3. He, Z., et al. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature 374.6522 (1995): 566-9. 4. Challberg, M. D., and T. J. Kelly. Eukaryotic DNA replication: viral and plasmid model systems. Annu Rev Biochem 51 (1982): 901-34.

6. Kelly, T. J. DNA replication in mammalian cells: insights from the SV40 model system. Harvey lectwre 85 (1989): 173-88.

8. Stillman, B. (1992) Initiation of chromosome replication in eukaryotic cells. Harvey Lecture d: 115-40. 9. Heyer, W. D., et al. An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A. EMBO Journal 9.7 (1990): 2321-9.

10. Erdile, L. F., M. S. Wold, and T. J. Kelly. The primary structure of the 32-kDa subunit of human replication protein A. J Biol Chem 265.6 (1990): 3177-82. 11. Bochkarev, A., Pfuetzner, R. A., Edwards, A.M. and L. Frappier ( 1997) Structure 259 232/116 of the single stranded DNA binding domain of replication protein A bound to DNA. Nature 385: 176-181.

12. Umbricht, C B., et al. High-resolution genomic mapping of the three human replication protein A genes (RPA1, RPA2, and RPA3). Genomics 20.2 (1994): 249- 57.

13. Cornelis RS, van Vliet M, Vos CB, et al. (1994) Evidence for a gene on 17pl3.3, distal to TP53, as a target for allele loss in breast tumors without p53 mutations. Cancer Res. 54:4200-6.

14. Lindblom A, Skoog L, Rotstein S, Werelius B, Larsson C, Nordenskjold M. (1993) Loss of heterozygosity in familial breast carcinomas. Cancer Res. 53:4356-61.

15. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51 :5794-9.

16. Singh S, Simon M, Meybohm I, et al. (1993) Human breast cancer: frequent p53 allele loss and protein over expression. Hum Genet. 90:635-40. 17. Thorlacius S, Borresen AL, et al. (1993) Somatic p53 mutations in human breast carcinomas in an Icelandic population: a prognostic factor. Cancer Res. 53: 1637-41. 18. Tsuda H, Hirohashi S. (1994) Association among p53 gene mutation, nuclear accumulation of the p53 protein and aggressive phenotypes in breast cancer. Int J Cancer. 57:498-503. 19. Watatani M, Nagayama K, Imanishi Y, et al. (1993) Genetic alterations on chromosome 17 in human breast cancer: relationships to clinical features and DNA ploidy. Breast Cancer Res Treat. 28:231-9.

20. Chen LC, Neubauer A, Kurisu W, et al. (1991) Loss of heterozygosity on the short arm of chromosome 17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer. Proc Natl Acad Sci USA. 88:3847-51.

21. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51 :5794-9.

22. Burmer GC, Rabinovitch PS, Haggitt RC, et al. (1992) Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of a p53 allele [see comments]. 260 232/116

Gastroenterology. 103:1602-10.

23. Cunningham C, Dunlop MG, Wyllie AH, Bird CC. (1993) Deletion mapping in colorectal cancer of a putative tumor suppressor gene in 8p22-p21 .0ncogene. : 1391 - 6 24. Kikuchi-Yanoshita R, Konishi M, Ito S, et al. (1992) Genetic changes of both p53 alleles associated with the conversion from colorectal adenoma to early carcinoma in familial adenomatous polyposis and non-familial adenomatous polyposis patients. Cancer Res. 52:3965-71.

25. Yin J, Haφaz N, Tong Y, et al. (1993) p53 point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology. 104:1633-9.

26. lacopetta B, DiGrandi S, Dix B, et al. (1994) Loss of heterozygosity of tumour suppressor gene loci in human colorectal carcinoma. Eur J Cancer. 5:664-70.

27. Law DJ, Olschwang S, Monpezat JP, et al. (1988) Concerted nonsyntenic allelic loss in human colorectal carcinoma. Science. 241 :961-5. 28. Lothe RA, Nakamura Y, Woodward S, Gedde DT, Jr., White R. (1988) VNTR

(variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas. Cytogenet Cell Genet. 48:167-9. 29. Foulkes WD, Stamp GW, Afzal S, et al. (1995) MDM2 over expression is rare in ovarian carcinoma irrespective of TP53 mutation status. Br J Cancer. 72:883-8. 30. Phillips NJ, Ziegler MR, Radford DM, et al. (1996) Allelic deletion on chromosome 17pl3.3 in early ovarian cancer. Cancer Res. 56:606-11. 31. Foulkes WD, Black DM, Stamp GW, Solomon E, Trowsdale J. (1993) Very frequent loss of heterozygosity throughout chromosome 17 in sporadic ovarian carcinoma. Int J Cancer. 54:220-5. 32. Gallion HH, Powell DE, Morrow JK, et al. (1992) Molecular genetic changes in human epithelial ovarian malignancies [see comments]. Gynecol Oncol. 47:137-42.

33. Phillips N, Ziegler M, Saha B, Xynos F. (1993) Allelic loss on chromosome 17 in human ovarian cancer. Int J Cancer. 54:85-91.

34. Seruca R, David L, Castedo S, Veiga I, Borresen AL, Sobrinho-Simoes M. (1994) 261 232/116 p53 alterations in gastric carcinoma: a study of 56 primary tumors and 204 nodal metastases. Cancer Genet Cytogenet. 75:45-50.

35. Kim CJ, Kim WH, Kim CW, Lee JB, Lee CK, Kim YL. (1995) Detection of 17p loss in gastric carcinoma using polymerase chain reaction. Lab Invest. 72:232-6. 36. Ranzani GN, Renault B, Pellegata NS, et al. (1993) Loss of heterozygosity and K- ras gene mutations in gastric cancer. Hum Genet. 92:244-9.

37. Sano T, Tsujino T, Yoshida K, et al. (1991) Frequent loss of heterozygosity on chromosomes lq, 5q, and 17p in human gastric carcinomas. Cancer Res. 51 :2926-31.

38. Frankel RH, Bayona W, Koslow M, Newcomb EW. (1992) p53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency. Cancer Res. 52:1427-33.

39. Hermanson M, Funa K, Koopmann J, et al. (1996) Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor alpha receptor expression in human malignant gliomas. Cancer Res. 56:164-71. 40. Aoki T, Mori T, Du X, Nisihira T, Matsubara T, Nakamura Y. (1994) Allelotype study of esophageal carcinoma. Genes Chromosomes Cancer. 10:177-82. 41. Tsuchiya E, Nakamura Y, Weng SY, et al. (1992) Allelotype of non-small cell lung carcinoma—comparison between loss of heterozygosity in squamous cell carcinoma and adenocarcinoma. Cancer Res. 52:2478-81. 42. Hiyama K, Ishioka S, et al. (1995) Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb. Oncogene. 10:937-44.

Example 25: RNA Polymerase II, 220-kD subunit (RPOL2A) - Target Gene VARIA500

The human RPOL2A gene encodes a protein essential for cell survival

DNA-dependent RNA polymerase II (also known as RPB1 or POLR2A), a complex 262 232/116 multisubunit enzyme, is responsible for the transcription of mRNA from all protein coding genes.

RNA polymerases are found in all cellular organisms. The subunit structure of RNA polymerases is highly conserved in eukaryotes. RNA polymerase acts in concert with as many as 50 other proteins in gene transcription (reviewed in ref. 1). See refs. 2 and 3 for a review of basal transcription by RNA polymerase II and recent progress in identifying and purifying transcription factors and cloning the genes that encode them. Several subunits of S. cerevisiae RPOL2A have been disrupted, always resulting in non-viable yeast.

A variety of inhibitors of RNA polymerase are cytotoxic drugs, such as actinomycin D, which intercalates into double stranded DNA and blocks the movement of RNA polymerase; rifampicin binds the b subunit of E. coli RNA polymerase and blocks initiation of transcription. The best studied specific inhibitor of eukaryotic RPOL2A, however, is the potent mushroom toxin - amanitin, a cyclic octapeptide which binds with high affinity (Kd -10-9 M) to RPOL2A. Several mutations conferring resistance to a-amanitin have been characterized and they all map to the RPOL2A protein coding sequence. Recently a-amanitin binding has been shown to trigger specific degradation of

RPOL2A (4).

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA pol II, but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on PolB Il-transcribed genes is incompletely understood.

The human RPOL2A gene and mRNA have sequence variances

Wintzerith et al. and later Mita et al. cloned and sequenced the complete human gene 263 232/116 for RPOL2A (5, 6); the deduced amino acid sequences are identical. The RPOL2A gene contains 29 exons and spans about 32 kb of DNA. The cDNA sequence we evaluated is 6732 nucleotides long (see Annotated RPOL2A Sequence) and contains a 5' untranslated region of 386 nucleotides, a 5910 nucleotide coding region specifying 1970 amino acids, and a 436 nucleotide 3' untranslated region (see annotated sequence). We undertook a systematic search for DNA sequence variance in the cDNA of RPOL2A by analyzing 36 unrelated individuals using the single strand conformation polymoφhism technique. Primers were designed for amplification. SSCP analysis revealed 10 sequence variances, and subsequent DNA sequence analysis confirmed a G vs. A transition at nucleotide 857, a C vs. T transition at nucleotide

1260, a C vs. T transition at nucleotide 1346, a C vs. T transition at nucleotide 1544, a C vs. T transition at nucleotide 1847, a C vs. T transition at nucleotide 2678, a C vs. T transition at nucleotide 3059, a C vs. T transition at nucleotide 3827, a T vs. C transition at nucleotide 6466 and a T vs. C transition at nucleotide 6557. The former seven sequence variances are in coding sequence and the latter two are in the 3' untranslated sequence. Only one of the ten sequence variances alters the protein coding sequence: the nucleotide 1260 alleles encode arginine (common) or cysteine (rare) at amino acid 292. Only 2/36 individuals surveyed are heterozygotes (6%), however both are North American Whites (2/16 = 12.5%) so further investigation of this population is required. The prevalence of heterozygotes for the other sequence variances varies from 3% to 50%, with 6 sequence variances above 22% (see RPOL2A Target Summary Sheet). The 6 common sequence variances are widely prevalent among all or nearly all the tested populations.

The human RPOL2A gene maps to chromosome 17pl 3.105

The human RPOL2A gene was initially assigned to the distal portion of the short arm of chromosome 17 (17pter-pl2) by in situ hybridization and Southern analysis of DNA from human/rodent somatic cell hybrids (7, 8). Subsequent somatic cell hybrid studies narrowed the assignment to 17pl3.105-ρl2 [vanTuinen and Ledbetter (1987)], which 264 232/116 was later confirmed by in situ hybridization to 17pl3 (9).

Chromosome band 17 p 13.1 is a site of frequent loss of heterozygosity There have been many studies of LOH on 17p, particularly the 17p 13.1 region where the p53 tumor suppressor gene maps. Virtually all cancer types have been surveyed for LOH in this area, with particularly extensive studies of breast, colon, ovarian, and stomach cancers. These studies report LOH in approximately 40-60% of breast cancers (10-18), 50-70% of colon cancers (19-25), 25-75% of ovarian cancers (26-30), 20-60% of stomach cancers (31-34), 20-50% of brain cancers (35,36), 45-70% of esophageal cancers (37), 35-65% of non-small cell lung cancers (38,39) and 100% of small cell lung cancers, 15-50% of cervical cancers, 30-80% of head and neck cancers, 20-60% of liver cancers, over 50% of sarcomas and 10-30% of a variety of other cancer types.

References

1. Acker, J.; Mattei, M.-G.; Wintzerith, M.; Roeckel, N.; Depetris, D.; Vigneron, M.;

Kedinger, C. (1994) Chromosomal localization of human RNA polymerase II subunit genes. Genomics 20: 496-499. 4. Buratowski, S. (1994) The basics of basal transcription by RNA polymerase II. Cell

77:1-3.

5. Cannizzaro, L. A., Emanuel, B. S., Cho, K. W. Y. and R. Weinmann (1986) The gene encoding the large subunit of human RNA polymerase II is located on the short arm of chromosome 17. Am. J. Hum. Genet. 38: 812-818. 8. Mita, K.; Tsuji, H.; Morimyo, M.; Takahashi, E.; Nenoi, M.; Ichimura, S.;

Yamauchi, M.; Hongo, E., Hayashi, A. (1995) The human gene encoding the largest subunit of RNA polymerase II. Gene 159: 285-286.

9. Pravtcheva, D.; Rabin, M.; Bartolomei, M.; Corden, J.; Ruddle, F. H. (1986)

Chromosomal assignment of gene encoding the largest subunit of RNA polymerase II 265 232/116 in the mouse. Somat. Cell Molec. Genet. 12: 523-528.

13. Wintzerith, M., Acker, J., Vicaire, S., Vigneron, M. and C Kedinger (1992)

Complete sequence of the human RNA polymerase II largest subunit. Nucleic Acids

Res. 20: 910. 10. Cornells RS, van Vliet M, Vos CB, et al. (1994) Evidence for a gene on 17pl3.3, distal to TP53, as a target for allele loss in breast tumors without p53 mutations.

Cancer Res. 54:4200-6.

11. Lindblom A, Skoog L, Rotstein S, Werelius B, Larsson C, Nordenskjold M. (1993)

Loss of heterozygosity in familial breast carcinomas. Cancer Res. 53:4356-61. 12. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51 :5794-9.

13. Singh S, Simon M, Meybohm I, et al. (1993) Human breast cancer: frequent p53 allele loss and protein over expression. Hum Genet. 90:635-40.

14. Thorlacius S, Borresen AL, et al. (1993) Somatic p53 mutations in human breast carcinomas in an Icelandic population: a prognostic factor. Cancer Res. 53 : 1637-41.

15. Tsuda H, Hirohashi S. (1994) Association among p53 gene mutation, nuclear accumulation of the p53 protein and aggressive phenotypes in breast cancer. Int J Cancer. 57:498-503.

16. Watatani M, Nagayama K, Imanishi Y, et al. (1993) Genetic alterations on chromosome 17 in human breast cancer: relationships to clinical features and DNA ploidy. Breast Cancer Res Treat. 28:231-9.

17. Chen LC, Neubauer A, Kurisu W, et al. (1991) Loss of heterozygosity on the short arm of chromosome 17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer. Proc Natl Acad Sci USA. 88:3847-51. 18. Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y. (1991) Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res. 51 :5794-9. 19. Burmer GC, Rabinovitch PS, Haggitt RC, et al. (1992) Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of a p53 allele [see comments]. Gastroenterology. 103:1602-10. 266 232/116

20. Cunningham C, Dunlop MG, Wyllie AH, Bird CC. (1993) Deletion mapping in colorectal cancer of a putative tumour suppressor gene in 8p22-p21.3. Oncogene. 8:1391-6

22. Yin J, Haφaz N, Tong Y, et al. (1993) p53 point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology. 104:1633-9. 23. lacopetta B, DiGrandi S, Dix B, et al. (1994) Loss of heterozygosity of tumour suppressor gene loci in human colorectal carcinoma. Eur J Cancer. 5:664-70.

24. Law DJ, Olschwang S, Monpezat JP, et al. (1988) Concerted nonsyntenic allelic loss in human colorectal carcinoma. Science. 241:961-5.

25. Lothe RA, Nakamura Y, Woodward S, Gedde DT, Jr., White R. (1988) VNTR (variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas. Cytogenet Cell Genet. 48:167-9.

26. Foulkes WD, Stamp GW, Afzal S, et al. (1995) MDM2 over expression is rare in ovarian carcinoma irrespective of TP53 mutation status. Br J Cancer. 72:883-8.

27. Phillips NJ, Ziegler MR, Radford DM, et al. (1996) Allelic deletion on chromosome 17pl3.3 in early ovarian cancer. Cancer Res. 56:606-11.

28. Foulkes WD, Black DM, Stamp GW, Solomon E, Trowsdale J. (1993) Very frequent loss of heterozygosity throughout chromosome 17 in sporadic ovarian carcinoma. Int J Cancer. 54:220-5.

29. Gallion HH, Powell DE, Morrow JK, et al. (1992) Molecular genetic changes in human epithelial ovarian malignancies [see comments]. Gynecol Oncol. 47:137-42.

31. Seruca R, David L, Castedo S, Veiga I, Borresen AL, Sobrinho-Simoes M. (1994) p53 alterations in gastric carcinoma: a study of 56 primary tumors and 204 nodal 267 232/116 metastases. Cancer Genet Cytogenet. 75:45-50.

33. Ranzani GN, Renault B, Pellegata NS, et al. (1993) Loss of heterozygosity and K- ras gene mutations in gastric cancer. Hum Genet. 92:244-9.

34. Sano T, Tsujino T, Yoshida K, et al. (1991) Frequent loss of heterozygosity on chromosomes lq, 5q, and 17p in human gastric carcinomas. Cancer Res. 51:2926-31.

35. Frankel RH, Bayona W, Koslow M, Newcomb EW. (1992) p53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency. Cancer Res. 52:1427-33.

36. Hermanson M, Funa K, Koopmann J, et al. (1996) Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor alpha receptor expression in human malignant gliomas. Cancer Res. 56:164-71.

37. Aoki T, Mori T, Du X, Nisihira T, Matsubara T, Nakamura Y. (1994) Allelotype study of esophageal carcinoma. Genes Chromosomes Cancer. 10:177-82.

38. Tsuchiya E, Nakamura Y, Weng SY, et al. (1992) Allelotype of non-small cell lung carcinoma—comparison between loss of heterozygosity in squamous cell carcinoma and adenocarcinoma. Cancer Res. 52:2478-81.

39. Hiyama K, Ishioka S, Shirotani Y, et al. (1995) Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb.

Oncogene. 10:937-44.

Example 26: TATA Associated Factor 30 kD subunit (TAF2H) - Target Gene VARIA 520

The human TAF2H gene encodes a component of the transcriptional apparatus

Transcription initiation by RNA polymerase II requires the assembly of a complex of 268 232/116 basic transcription factors which include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIG/TFIIJ and TFIIH/BTF2 into a preinitiation complex (1,2). TFIID is the first factor to contact the promotor, and subsequent assembly of the transcription complex is dependent on TFIID binding. TFIID is a 700-750 kD multiprotein complex which includes TATA binding protein (TBP) and between eight and 13 TBP-associated factors (TAFs) ranging from 250 to 17 kDa. The TAFs have been shown necessary to reconstitute activation of transcription in vitro, leading to the hypothesis that some TAFs link transcription activation domains to the basal transcription complex. The TFIID complex also supports transcription from TATA-less promoters, while TBP fails to do so. Therefore TAFs may also contribute to formation of stable initiation complexes by interacting directly with DNA (2). Conditional temperature sensitive Chinese hamster mutants of another TAF, TAFII250, were detected because, at the non-permissive temperature, DNA synthesis was inhibited leading to arrest of cell division at the GI phase (3,4). Transfection of a human TAFII250 gene relieved the block at the non-permissive temperature. Thus an essential role has been proven for

TAFs in mammalian cells.

A gene (TAF2H) encoding the 30 kDa human TAF protein (TAFII30) was cloned and its functional properties examined by Jacq, et al. (5). The protein was shown to be present in a subset of TFIID complexes and to mediate transcriptional activation by a specific region of the estrogen receptor. Estrogen mediated transcriptional activation could be abrogated by adding an antibody against TAFII30. TAFII30 was not required for basal transcription or for transcription activation by VP-16. It is likely that TAFII30 is required for transcriptional activation by a variety of other transactivating proteins, and is therefore essential for cell proliferation or cell survival.

The human TAF2H gene and mRNA have sequence variants

A human TAF2H cDNA has been cloned and sequenced (5). It encodes a cDNA of 756 nucleotides including a 5' untranslated region of 17 nucleotides, a 657 nucleotide 269 232/116 coding region specifying 218 amino acids, and an 82 nucleotide 3' untranslated region (GenBank accession U13991; see annotated TAF2H cDNA sequence). (Note that the numbering of the sequence in ref. 5 differs slightly from that in the GenBank accession.) We undertook a systematic search for DNA variance in the cDNA of TAF2H by analysing 36 unrelated individuals using the single strand conformation polymoφhism technique Primers were designed for amplification. SSCP analysis revealed 1 polymoφhism, and subsequent DNA sequence analysis confirmed a G vs. A transition at nucleotide 554 (nt 556 of the sequence in ref. 3) of the coding sequence. This variance does not alter the protein coding sequence. Eight of 36 individuals surveyed are heterozygotes (22%). The variance occurs in North American Whites

(3/16 = 19%), North American Blacks (2/4) and Hispanics (3/3).

The human TAF2H gene maps to chromosome Ilpl5.5-pl5.2 The human TAF2H cDNA has been mapped to I lpl5.5-pl5.2 by fluorescent in situ hybridization (6). There appears to be a single TAF2H locus. Chromosome band Ilpl5-pl4 is a site of frequent loss of heterozygosity

There have been many studies of LOH on l ip, particularly the l lpl5 and l lpl3 segments where the Beckwith-Weidemann syndrome and WT1 genes reside. As a result there are many studies of LOH in l lpl5.5, particularly focusing on breast, cervix, kidney, liver, lung, ovarian, stomach and testicular cancers. These studies show that the 1 lpl5.5 band of chromosome 11 is frequently reduced to one copy (7-24). For example, LOH occurs in approximately 13-33% of breast cancers (7-9), 14-42% of cervical cancers (10), 0-50% of liver cancers (11,12), 0-80% of lung cancers (13-15), 18-54% of ovarian cancers (14,15), 0-71% of stomach cancers (18) and 0-50% of testicular cancers (19,20). Other studies show that 1 lpl5.5 LOH may also be frequent in bladder cancer (21), esophageal cancer (22), some leukemias (23) and sarcomas (24). Many deletions in the l lpl5.5 region span relatively short chromosomal segments (2 - 10 megabases; see ref. 13). 270 232/116

References

1. Buratowski, S.(1994) The basics of basal transcription by RNA polymerase II. Cell

77: 1-3. 2. Tjian, R. and T. Maniatis (1994) Transcriptional activation: a complex puzzle with few easy pieces. Cell 77: 5-8.

3. Sekiguchi, T., Miyata, T. and T. Nishimoto (1988) Molecular cloning of the cDNA of human X chromosomal gene (CCGl) which complements the temperature sensitive

G(l) mutants, tsBN462 and tsl3, of the BHK cell line. EMBO Journal 7: 1683-1687. 4. Hisatake, K., Hasegawa, S., Takada, R., et al. (1993) The p250 subunit of native

TATA box-binding factor TFIID is the cell -cycle regulatory protein CCGl . Nature

362: 172-181.

5. Jacq, X., Brou, C, Lutz, Y., Davidson, I., Chambon, P. and L. Tora (1994) Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor. Cell 79: 107-117.

6. Scheer, E., Mattei, M.G., Jacq, X., Chambon, P. and L. Tora (1995) Organization and chromosomal localization of the gene (TAF2H) encoding the human TBP- associated factor II 30 (TAFII30). Genomics 29: 269-272.

7. AH, I., Lidereau, R., Theilley, C. and R. Callahan (1987) Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia. Science

238:185-8.

8. Winqvist, R., Mannermaa, A., Alavaikko, M., Blanco, G., Taskinen, P.J., Kiviniemi, H., Newsham, I. and W. Cavenee (1993) Refinement of regional loss of heterozygosity for chromosome 1 lpl5.5 in human breast tumors. Cancer Research 53: 4486-4488.

9. Carter, S.L., Negrini, M., Baffa, R., et al. (1994) Loss of heterozygosity at 1 lq22- q23 in breast cancer. Cancer Research 54:6270-4.

10. Mitra, A.B., Murty, V.N.V.S., Li, R.G., et al. (1994) Allelotype analysis of cervical carcinoma. Cancer Research 54:4481. 271 232/116

11. Fujimori, M., Tokino, T., Hino, O., et al. (1991) Allelotype study of primary heptocellular carcinoma. Cancer Research 51 : 89-93.

12. Wang, H.P. and C.E. Rogler (1988) Deletions in human chromosome arms 1 lp and 13q in primary hepatocellular carcinomas. Cytogenetics and Cell Genetics 48:72- 78.

13. Bepler, G. and Garcia-Blanco, MA. (1994) Three Tumor Suppressor Regions on Chromosome l ip Identified by High Resolution Deletion Mapping in Human Non- Small Cell Lung Cancer. Proc. Natl. Acad. Sci. U.S.A. 91 :5513-7.

14. Iizuka, M., Sugiyama, Y.,Shiraishi, M., Jones, C and T. Sekiya (1995) Allelic losses in human chromosome 11 in lung cancers. Genes, Chromosomes & Cancer

13:40-46.

15. Weston, A., Willey, J.C, Modali, R., et al. (1989) Differential DNA sequence deletions from chromosomes 3, 11, 13 and 17 in squamous cell carcinoma , large-cell carcinoma and adenocarcinoma of the human lung. Proc. Natl. Acad. Sci. U.S.A. 86:5099-5103.

16. Kiechle-Schwartz, M., Bauknecht, T., Wienker, T., et al. (1993) Loss of Constitutional Heterozygosity on Chromosome l ip in Human Ovarian Cancer. Cancer 72:2423-32.

17. Viel, A., Giannini, F., Tumiotti, L., Sopracordevole, F., Visentin, M.C. and M. Boiocchi (1992) Chromosomal localization of two putative 1 lp oncosuppressor genes involved in human ovarian tumors British Journal oj Cancer 66: 1030-1036.

18. Baffa, R., Negrini, M., Mandes, B., et al. (1996) Loss of heterozygosity for chromosome 11 in adenocarcinoma of the stomach. Cancer Research 56: 268-72.

19. Lothe, R.A., Hastie, N., Heimdal, K., et al. (1993) Frequent loss of lpl3 and 1 lpl5 loci in male germ cell tumors. Genes, Chromosomes & Cancer 7: 96-101.

20. Smith, R.C., and Rukstalis, D.B. (1995) Frequent Loss of Heterozygosity at 1 lp Loci in Testicular Cancer. The Journal of Urology 153: 1684-7.

21. Shaw, M.E. and Knowles, MA. (1995) Deletion Mapping of Chromosome 11 in Carcinoma of the Bladder. Genes, Chromosomes & Cancer 13: 1-8. 272 232/116

22. Shibagaki, I., Shimada, Y., Wagata, T., Ikenaga, M., Imamura, M. and K. Ishizaki (1994) Allelotype analysis of esophageal squamous cell carcinoma. Cancer Research 54: 2996-3000.

23. Ahuja, H.G., Foti, A., Zhou, D.J. and M.J. Cline (1990) Analysis of proto- oncogenes in acute myeloid leukemia: loss of heterozygosity for the Ha-ras gene. Blood 75: 819-822.

Example 27 - cDNA synthesis

In order to analyze an essential gene for sequence variances, it is generally useful to have a cDNA(s) containing the coding sequence for further sequencing or amplification puφoses. cDNAs for some genes are available, however, in some cases it is useful to synthesize the cDNA de novo. Methods for obtaining cDNA are known to those skilled in the art, as are methods for sequencing or amplifying the cDNA or portions thereof. An example of a useful cDNA production protocol is provided below, however, as recognized by those skilled in the art, other specific protocols can also be used.

cDNA Production

** Make sure that all tubes and pipette tips are RNase-free. (Bake them overnight at 1 OOoC in the vacuum oven to make them RNase-free.)

1 Add the following to a RNase-free 0.2 ml micro-amp tube and mix gently: 24 ul water (DEPC treated)

12 ul RNA (lug/ul)

12 ul random hexamers(50 ng/ul)

2 Heat the mixture to 70oC for ten minutes.

3 Incubate on ice for 1 minute. 273 232/116

Add the following:

16 ul 5 X Synthesis Buffer

8 ul 0.1 M DTT

4 ul 10 mM dNTP mix ( 10 mM each dNTP)

4 ul Superscript RT II enzyme Pipette gently to mix.

5 Incubate at 42oC for 50 minutes.

6 Heat to 70oC for ten minutes to kill the enzyme, then place it on ice.

7 Add 160 ul of water to the reaction so that the final volume is 240 ul.

8 Use PCR to check the quality of the cDNA. Use primer pairs that will give a -800 base pair long piece. See "PCR Optimization" for the PCR protocol.

The following chart shows the reagent amounts for a 20 ul reaction, a 80 ul reaction, and a batch of 39 (which makes enough mix for 36) reactions:

Example 28 - Variance detection by SSCP 274 232/116

This example describes the SSCP technique as used for the identification of sequence variances of the exemplary genes, which were then sequenced to confirm the specific base variances. One common technique currently employed in the identification of such single nucleotide differences is the single strand conformation polymoφhism (SSCP) method, (originally described in Orita, et al, "Rapid and Sensitive Detection of Point Mutations and DNA Polymoφhisms Using the Polymerase Chain Reaction, Genomics, 5:874-879 (1989)) Also employed are restriction fragment length polymoφhism (RFLP), heteroduplex analysis, ligase chain reaction (LCR), denaturing gradient gel electrophoresis (DGGE) (Myers, Maniatis, and Lerman, Methods Enzymol, 155:501-527 (1987)) or direct nucleotide sequencing. A review of polymoφhism detection techniques, including SSCP, is provided in Grompe, 1993, Nature Genetics 5:111-117, which includes a comparison of the commonly used methods.

The SSCP method reveals the presence of sequence variation between individuals as shifts in electrophoretic mobility, but does not show the sequence itself. Direct sequencing of DNAs with altered mobility in the SSCP assay identifies the precise nucleic acid sequence differences among the various alleles. From the nucleic acid sequence data, the amino acid sequence can be determined. One example of the use of this technique is in Pelletier et al., Cell, 67:437-447 (1991). The single strand conformation polymoφhism methodology is effective for scanning essential genes for sequence variants. It remains the standard technique in human genetics for variance detection, with numerous studies of its efficacy (>90%) and schemes for improved throughput. The SSCP method has been shown to be quite sensitive in the detection of single base changes, for example as shown in Ravnik-Glava et al., 1994, Human

Mol. Genet. 3:801-807 (human cystic fibrosis gene) and Glava & Dean, 1993, Human Mutation 2:404-414 (mouse -globin gene).

A flow chart of the SSCP method as used to identify essential gene sequence variants is shown in Fig. 2 (SSCP OVERVIEW). The method involves the steps of 1) PCR 275 232/116 amplifying a portion of an essential gene cDNA of known sequence (labeled products), 2) selecting restriction enzymes which will produce fragments approximately 100-400 bases in length for 3 independent digestions of the PCR products, 3) heat denaturing the digestion products, 4) running single strand digestion products on non-denaturing gels, 5) identifying bands having different mobilities when compared between individuals, thereby identifying potential sequence variants, 6) sequence at least the region around the potential sequence variance, that region being identified by comparison of the expected fragment sizes resulting from the digestions, 7) record the specific location and base identity of the confirmed sequence variant, 8) calculate the percent occurrence of each sequence variance for the gene as found for the sample of the population. The method is further described in Example 2.

Single strand conformation polymoφhism screening is a widely used technique for identifying an discriminating DNA fragments which differ from each other by as little as a single nucleotide. As originally developed by Orita (supra), the technique was used on genomic DNA, however the same group showed that the technique works very well on PCR amplified DNA as well. In the last 8 years the technique has been used in hundreds of published papers, and the modifications of the technique have been described in dozens of papers. The enduring popularity of the technique is due to (1) a high degree of sensitivity to single base differences (>90%) (2) a high degree of selectivity, measured as a low frequency of false positives, and (3) technical ease. SSCP is almost always used together with DNA sequencing because SSCP does not directly provide the sequence basis of differential fragment mobility. The basic steps of the SSCP procedure are described below and summarized in Fig. 2 in flow chart form.

Because the intent of our SSCP screening was to identify as many target gene variances as practically possible, we developed a protocol designed to look at a relatively large number of individuals (36) with a high degree of redundancy, so as to minimize both the false negative and false positive rates. 276 232/116

The 36 individuals examined are reasonably representative of most of the worlds major populations. The racial or geographic origin of the 36 cell lines is detailed in the Target Summary Tables (Figure 5). All cell lines are EBV immortalized lyphoblastoid cells obtained from the Coriell Cell Repository (Camden, NJ), which includes the racial/ethnic/geographic background of cell line donors in its catalog. The cell lines were also selected for their rapid growth rates. In several cases a panel of cDNAs isolated from French Canadians was used instead, or in addition to, the Coriell panel.

SSCP was used to analyze cDNAs (rather than genomic DNAs) because in many cases the full genomic sequence of the target gene is not available, however, the technique is also applicable to genomic sequences. To produce cDNA requires RNA. Therefore each of the 36 cell lines was grown to mass culture and RNA was isolated using the acid phenol protocol, sold in kit form as TRIAZOL™ by Life Technologies (Gaithersberg, MD). The unfractionated RNA was used to produce cDNA by the action of a modified Maloney Murine Leukemia Virus Reverse Transcriptase, purchased in kit form from Life Technologies (SUPERSCRIPT II™ kit). The reverse transcriptase was primed with random hexamer primers to initiate cDNA synthesis along the whole length of the RNAs. This proved useful later in obtaining good PCR products from the 5' ends of some genes.

Material for SSCP analysis was prepared by PCR amplification of the cDNA in the presence of one³²P labeled dNTP (usually³²P dCTP). Usually the concentration of nonradioactive dCTP was dropped from 200 uM (the standard concentration for all four dNTPs) to about 100 uM, and³²P dCTP was added to a concentration of about 0.1 -0.3 uM. This involved adding a 0.3- 1 ul (3-10 uCi) of³²P cCTP to a 10 ul PCR reaction. All radioactivity was purchased from DuPont/New England Nuclear.

The customary practice is to amplify about 200 base pair PCR products for SSCP, however, we found that it was preferable to amplify about 0.8-1.4 kb fragments and 277 232/116 then use several cocktails of restriction endonucleases to digest those into smaller fragments of about 0.1-0.4kb, aiming to have as many fragments as possible between .15 and .3 kb. The digestion strategy had the advantage that less PCR was required, reducing both time and costs. Also, we routinely performed three different digests on each sample (for all 36 cDNAs), and then ran each of the digests separately on SSCP gels. This had the effect of increasing the redundancy of our method, lessening both the false negative and false positive rates. For example: a site of variance might lie within 2 bases of the end of a fragment in one digest, and as a result not affect the conformation of that strand; the same variance, in a second or third digest, would likely lie in a location more prone to affect strand folding, and therefore be detected by SSCP.

After digestion, the radiolabeled PCR products were diluted 1 :5 by adding formamide load buffer (80% formamide, IX SSCP gel buffer) and then denatured by heating to 90%C for 10 minutes, and then allowed to renature by quickly chilling on ice. This procedure (both the dilution and the quick chilling) promotes intra- (rather than inter-) strand association and secondary structure formation. The secondary structure of the single strands influences their mobility on nondenaturing gels, presumably by influencing the number of collisions between the molecule and the gel matrix (i.e., gel sieving). Even single base differences consistently produce changes in intrastrand folding sufficient to register as mobility differences on SSCP.

The single strands were then resolved on two gels, one a 5.5% acrylamide, 0.5X TBE gel, the other an 8% acrylamide, 10% glycerol, IX TTE gel. The use of two gels provides a greater opportunity to recognize mobility differences. Both glycerol and acrylamide concentration have been shown to influence SSCP performance. The gel apparatus we use (from Owl Scientific, MA) allows 108 samples to be loaded per gel. Since all 36 samples are routinely digested with three different endonuclease mixes there are 108 samples to be analyzed for each PCR product. By routinely analyzing three different digests under two gel conditions (effectively 6 conditions), and by 278 232/116 looking at both strands under all 6 conditions, we achieve a 12-fold sampling of each base pair of cDNA.

All of the sequence variances described in this disclosure were determined by DNA cycle sequencing of³²P labeled PCR products using the femtomole DNA cycle sequencing kit from Promega (WI) and the instructions provided with the kit. Fragments were selected for DNA sequencing based on their behavior in the SSCP assay.

Example 29 - Variance detection by using T4 endonuclease VII mismatch cleavage method

The enzyme T4 endonuclease VII is derived from the bacteriophage T4. T4 endonuclease VII is used by the bacteriophage to cleave branched DNA intermediates which form during replication so the DNA can be processed and packaged. T4 endonuclease can also recognize and cleave heteroduplex DNA containing single base mismatches as well as deletions and insertions. This activity of the T4 endonuclease VII enzyme can be exploited to detect sequence variances present in the general population.

The following are the major steps involved in identifying sequence variations in a candidate gene by T4 endonuclease VII mismatch cleavage:

1. Amplification by the polymerase chain reaction (PCR) of 400-600 bp regions of the candidate gene from a panel of DNA samples The DNA samples can either be cDNA or genomic DNA and will represent some cross section of the world population.

2. Mixing of a fluorescently labeled probe DNA with the sample DNA. Heating 279 232/116 and cooling the mixtures causing heteroduplex formation between the probe DNA and the sample DNA.

3. Addition of T4 endonuclease VII to the heteroduplex DNA samples. T4 endonuclease will recognize and cleave at sequence variance mismatches formed in the heteroduplex DNA.

4. Electrophoresis of the cleaved fragments on an ABI sequencer to determine the site of cleavage.

5. Sequencing of a subset of PCR fragments identified by T4 endonuclease VI to contain variances to establish the specific base variation at that location.

A more detailed description of the procedure is as follows:

A candidate gene sequence is downloaded from an appropriate database. Primers for PCR amplification are designed which will result in the target sequence being divided into amplification products of between 400 and 600 bp. There will be a minimum of a 50 bp of overlap not including the primer sequences between the 5' and 3' ends of adjacent fragments to ensure the detection of variances which are located close to one of the primers.

Optimal PCR conditions for each of the primer pairs is determined experimentally.

Parameters including but not limited to annealing temperature, pH, MgCl₂ concentration, and KC1 concentration will be varied until conditions for optimal PCR amplification are established. The PCR conditions derived for each primer pair is then used to amplify a panel of DNA samples (cDNA or genomic DNA) which is chosen to best represent the various ethnic backgrounds of the world population or some designated subset of that population.

One of the DNA samples is chosen to be used as a probe. The same PCR conditions used to amplify the panel are used to amplify the probe DNA. However, a 280 232/116 flourescently labeled nucleotide is included in the deoxy-nucleotide mix so that a percentage of the incoφorated nucleotides will be fluorescently labeled.

The labeled probe is mixed with the corresponding PCR products from each of the DNA samples and then heated and cooled rapidly. This allows the formation of heteroduplexes between the probe and the PCR fragments from each of the DNA samples. T4 endonuclease VII is added directly to these reactions and allowed to incubate for 30 min. at 37 C 10 ul of the Formamide loading buffer is added directly to each of the samples and then denatured by heating and cooling. A portion of each of these samples is electrophoresed on an ABI 377 sequencer. If there is a sequence variance between the probe DNA and the sample DNA a mismatch will be present in the heteroduplex fragment formed. The enzyme T4 endonuclease VII will recognize the mismatch and cleave at the site of the mismatch. This will result in the appearance of two peaks corresponding to the two cleavage products when run on the ABI 377 sequencer.

Fragments identified as containing sequencing variances are subsequently sequenced using conventional methods to establish the exact location and sequence variance.

Example 30 - Identification of Sequence Variances by Informatics-based analysis of gene-sequence databases

In addition to and/or in conjunction with the molecular biology based approaches for identifying sequence variances in genes, particularly in essential genes, such sequence variances can be identified by analysis of public and/or private genetic sequence databases. Such information can be either genomic or cDNA sequence information.

The data base analysis process includes the following major steps: 281 232/116

1.

1. capture of homologous sequences of a particular gene from data bases. It is preferable to obtain a large number of independent sequences of a particular gene 2. analysis of collected sequences of a particular gene to identify authentic sequence variances. This step involves the discrimination of authentic sequence variances, which are sequence variances which actually exist in the population, from sequencing errors and artifacts. It is expected that about 0.1- 0.3% of the bases will occur as true variances, while the frequency of sequencing artifacts is expected to be 1-3%. This discrimination utilizes the expected frequencies of occurrence of specific types of nucleotide sequence changes. Such information includes the characteristic frequency of specific transitions and transversions and of the characteristic frequency of deletions and insertions in authentic variations. It uses the frequency of occurrence of known types of sequencing artifacts such as single base insertions or deletions adjacent to repeated C or G nucleotides. Additional information for such discrimination is provided if particular putative authentic variations are observed in multiple independently derived sequences of the gene.

An implementation of this sequence variance identification process utilizes a reference sequence of an essential gene. Preferably, the reference sequence is a high quality sequence, meaning that there is a low frequency of occurrence of sequencing errors or artifacts. The second step is the retrieval of allelic sequences of that essential gene from available databases such as the BLAST server, the UNIGENE database, or other such sequence database. Such allelic sequences need not be complete, but are preferably long enough to ensure that they are in fact allelic sequences. The third step involves alignment analysis to identify and tabulate sequence differences between the different available sequences. An algorithm for such analysis is the Smith- Waterman local alignment algorithm. Use of an algorithm of this type involves a series of pair- 282 232/116 wise alignments of each retrieved sequence with the reference sequence. The fourth step involves analysis of the observed sequence differences and assignment of a probability that each sequence difference represents an authentic variance. This analysis utilizes program filters which are combined in a weighted fashion to determine a final probability . Such program filters include comparison of the observed difference with common mutational changes and sequencing errors, a weighting of the reliability of a particular retrieved sequence based on the total number of differences observed, a weighting based on the location within a retrieved sequence where a change was observed and a significant weighting based on the observance of a particular difference in multiple independently derived retrieved sequences.

Using such an implementation, a database analysis with respect to a particular reference sequence produces a list of putative authentic sequence variances and a probability for each of those variances that the sequence difference is an authentic variance. As described above, the probability is obtained through the use of a series of weighted program filters and thus these filters are modified to produce optimal authentic variance discrimination.

Example 31 - Antiproliferative effects of variance specific inhibition of RPA70

This example describes experiments showing the practicality and utility of variance- specific inhibition of essential genes for cancer therapy. Specifically, this example describes in vitro experiments showing the design and production of variance-specific oligonucleotides for antisense inhibition of variant alleles of the essential Replication Protein A, 70 kDa subunit (RPA70) for inhibition of RPA70 mRNA, and the use of these oligonucleotides to inhibit cell proliferation and to reduce the number of cells in a variance-specific manner.

Variance-specific inhibition and cell killing with antisense oligonucleotides against 283 232/116

RPA70

These experiments with RPA70 illustrate the feasibility of each of the steps for development of a variance specific inhibitor:

Select candidate target gene essential for cell survival or proliferation. As described above, RPA is essential for replication in prokaryotic and eukaryotic cells, mitochondria, phage, viruses and in in vitro (SV40) replication systems. The protein is a heterotrimer required for loading DNA polymerase onto the DNA template during cell replication. The 70 kDa subunit, RPA70, is a single strand binding protein that mediates the interaction of RPA with DNA. Without this protein, the replication complex does not associate with DNA and the replication of DNA does not occur.

Confirm chromosome location and LOH frequency. RPA70 is encoded by a single gene locus on chromosome 17pl3.3, immediately adjacent to the p53 gene at 17pl3.1. LOH involving chromosome band 17pl3.3 has been documented in 50-70% of colon, lung, breast, and ovarian cancers. LOH at this locus also occurs in other cancers. The inventor as confirmed LOH involving RPA 70 in breast, colon, lung and other cancers.

Identify common variances in the normal population. We have identified five common variances in the RPA70 gene (Figure 8). The most common occurs in 42% of the normal population. One variance alters the amino acid sequence and is present in 25% of the normal population (44% of Caucasians). This variance occurs within the active DNA binding domain (discussed below). These variances are described in the description above and in Fig. 1.

Demonstrate antiproliferative effects due to inhibition of candidate gene. The inventor has shown that inhibition of RPA70 in T24 bladder carcinoma cells with an antisense oligonucleotide reduces cell number. This effect is comparable to treatment of these cells with antisense oligonucleotide against ras, previously shown to have antitumor 284 232/116 effects in vitro and in vivo (Figure 9).

Design variance-specific inhibitor. Variance specific antisense oligonucleotides were designed to differentially inhibit the two variant forms of RPA70. Experiments were performed using tumor cell lines that are homozygous for each form of the target gene.

Figure 10 shows inhibition of mRNA levels in Mia Paca II cells by the 13085 oligonucleotide which matches the variance in these cells. In contrast, in T24 cells (and A549 cells, see below) the 12781 oligonucleotide matches the target gene and inhibits mRNA levels. In both cell lines neither the control oligonucleotide differing by one base (13085 in T24 cells and 12781 in Mia Paca II cells) nor a random- sequence oligonucleotide control (13706) inhibit mRNA levels to the same extent as the matched oligonucleotide.

Figure 10 demonstrates that the RPA 70 mRNA can be specifically down regulated in an allele-specific manner. However, the 13085 oligomer used also has a small effect on the level of the unmatched RNA. In order to increase the discrimination we altered the structure of the targeting oligomer, 13085. The results are shown in Figure 11. By shortening the oligomer we retain its ability to down-regulate its matched target RNA

(Mia Paca II cells, right half of Figure 11). Strikingly, however, this alteration dramatically altered the ability of this oligomer to down-regulate the mismatched variant RNA T24 cells, left half of Figure 11. The reciprocal regulation by oligomer

12781 was augmented by altering transfection conditions. These data suggest that even simple changes to the rudimentary "first generation" chemistry and transfection techniques can have significant effects in enhancing the ability of the oligomers to recognize and down regulate specific mRNAs.

Achieve variance-specific antiproliferative effects in cancer cells. Cell proliferation in each cell line, determined by BrdU incorporation, was suppressed to a greater degree by the matched oligonucleotide than by the controls differing by one base (Figure 12). 285 232/116

Cell proliferation in A549 cells was inhibited by oligomer 12781 to a greater degree than by oligomer 13085. Cell proliferation in Mia Paca 11 cells was inhibited more by oligomer 13085.

Additional studies were performed to characterize the antiproliferative effect in A549 cells (12781 genotype). A dose response curve demonstrates inhibition of BrdU incorporation by the matched oligonucleotide (12781) at concentrations 8-fold lower than the oligonucleotide with one base mismatch (13085) (Figure 13).

Cell survival was measured by staining cells with Sulforhodamine B dye 72 hours after treatment with oligonucleotides. Dose dependent reductions in cell number were observed in cells treated with the matched oligonucleotide (12781) but not with an oligonucleotide containing the one base mismatch (13085) (Figure 14). In contrast, in Mia Paca II cells, more cell killing was observed with the 13085 oligonucleotide than with the 12781 oligonucleotide (Figure 15). The oligonucleotides used in these studies have not been optimized for achieving allele-specific effects. Oligonucleotides using advanced chemistries can be utilized to optimize the potency and provide greater discrimination between variant targets at lower levels.

Example 32 - variance specific inhibition of essential genes

This example describes experiments showing the practicality and utility of variance- specific inhibition of essential genes for cancer therapy including RNA Pol II, and ribonucleotide reductase. Specifically, this example describes in vitro experiments showing the design and production of variance-specific oligonucleotides for antisense inhibition of variant alleles of the essential Ribonuclotide Reductase (RR), the design and production of variance-specific oligonucleotides against RR, and the use of these oligonucleotides to inhibit RR mRNA in a variance-specific manner. 286 232/116

Variance-specific inhibition of Ribonucleotide Reductase.

Ribonucleotide Reductase (RR) is an essential gene of nucleoside metabolism. Inhibitors of this function are known to be cell lethal. Two variances were discovered at position 2410 and 2419. Oligonucleotides were synthesized to a sequence spanning these two variations. In one case the oligomer targeted the GnnnnnnnnA variation (oligomer Varia 2410GA or RR2410GA) and in the other case the oligomer targeted the AnnnnnnnnG variant (oligomer Varia 2410AG or RR2410AG). In Mia Paca II cells which contain the GnnnnnnnnA variance, the RR2410GA antisense oligomer dramatically knocked down the level of RR mRNA. However, the oligomer targeting the other variance, oligomer Varia 2410AG, had little to no effect on the level of mRNA (Figure 16). The reciprocal regulation was demonstrated in MDA-MB 468 cells which express the other variance, AnnnnnnnnG (Figure 17). In these cells Varia 2410AG dramatically lowered the level of RR mRNA. In contrast, Varia 2410GA had no effect on the level of mRNA. These data taken together, are another example of allele-specific targeting of gene expression. We are also determining the effect of down regulating RR gene expression on cellular growth.

Example 33 - variance specific inhibition of essential genes using advanced oligonucleotide chemistries.

This example describes experiments showing the practicality and utility of variance- specific inhibition of essential genes for cancer therapy. Specifically, this example describes in vitro experiments showing the design and production of variance-specific oligonucleotides for antisense inhibition of variant alleles of the essential Glutamyl/prolyl tRNA Synthetase (EPRS), the design and production of variance- specific oligonucleotides against EPRS, and the use of these oligonucleotides to inhibit EPRS mRNA in a variance-specific manner. 287 232/116

Glutamyl-prolyl-tRNA synthetase (EPRS) is an essential gene, required for the synthesis of both glutamic acid tRNA and proline tRNA. Without EPRS protein synthesis is blocked. Two variances were discovered in this gene at positions 2963 and 2969 in the cDNA. We have demonstrated variance-specific inhibition of this gene with antisense oligonucleotides exploiting several different types of chemistry.

The experiments described above with RPA70 and RR utilized phophorothioate chemistry. This chemistry was developed to achieve greater stability in vivo, and this compound ha been used in several successful clinical trials. Phosphorothioates, however have low affinity for the RNA target, and, consequently, relatively lower specificity. We have achieved improved variance-specific inhibition using alternative chemistries. Specifically, we have synthesized hybrid oligonucleotides that contain both phosphorothioate and nucleotides with higher affinities. These hybrids contain "wings" consisting of six nucleotides with a 2' sugar modification (ethoxy-methoxy radical at the 2' position) and either a phosphorothioate or phosphodiester backbone.

Between the "wings" is a 8 nucleotide sequence of phosphorothioates that overlaps the variance. (In these constructs the 5' position of cytosine has been methylated.) As shown in Figure 18, variance specific inhibition is observed with the conventional phoφhorothioates. Greater inhibition of target mRNA is observed using the hybrid chemistries at lower doses. Inhibition by the matched hybrid oligomer, 14977, occurs at approximately 50-100 nM. The effect is extremely oligomer-specific. The mismatched oligomer, 14971, has no effect on mRNA levels at concentrations as high as 400 nM (Figure 19).

Example 34 - in vivo cancer therapy using oligonucleotides

This example describes reported in vitro and in vivo data on the treatment of cancer in animal models using antisense oligonucleotides against c-raf, showing the expected 288 232/116 correlation between in vitro suppression of mRNA and cell proliferation with oligonucleotides, and in vivo anticancer activity.

In vitro evidence for inhibition of mRNA by antisense oligonucleotides and inhibition of cell proliferation is commonly used to predict in vivo effects on tumors. This is exemplified by the publication by Monia et al (Nature Medicine, Volume 2 Number

6, June 1996) who demonstrated anticancer effects using oligonucleotides against C-raf kinase. In vitro treatment of human tumor cells with appropriate phosphorothioate antisense oligomers led to specific inhibition of C-raf kinase gene expression and subsequent decrease in cellular proliferation, IC50=50- 1 OOnM. Administration of C- raf antisense oligomers to nude mice having a tumor burden derived from these cells significantly inhibited tumor growth in vivo, IC50= 0.06-0.6 mg/kg. Remarkably, the investigators were able to show that the anti-C-raf oligomers down-regulated the level of C-raf kinase mRNA in vivo by assaying mRNA levels in cells removed from the tumor.

Example 35 - in vivo cancer therapy by oligonucleotide inhibition of ras

This example describes reported in vivo data showing an anticancer effect using an allele-specific inhibitor for suppression of mutant H-ras. Schwab et al (Proc. Nat. Acad. Sci. USA 91:10460-464, Oct 1994) demonstrated antitumor effects of an antisense oligonucleotide specific for the mutant ras in animal models. In these experiments HBL100 cells were transformed with the RAS oncogene. In vitro studies demonstrated that the RAS mRNA could be specifically down-regulated by a nanoparticle conjugated phosphodiester antisense oligomer. Only the transforming RAS mRNA was targeted by the oligomer. The normal cellular RAS rnRNA, differing by a single base, was not affected by the antisense oligomer. The decrease in RAS expression was associated with a decrease in the growth rate of the cells. The 289 232/116 transformed HBLIOO cells were injected into nude mice to form tumors; following subcutaneous injection of nanoparticle-conjugated phosphodiester antisense oligomers, Schwab et al measured both a decrease in targeted tumor weight and volume. Specificity for tumor cell growth correlated well with the in vitro data having a 5 -fold differential between antisense and control groups.

The authors of this paper are proceeding with clinical trial of these oligonucleotides for the treatment of cancer, demonstrating the potential clinical utility of these methods.

Example 36. Variance detection by DGGE

This example describes denaturing gradient gel electrophoresis (DGGE), a technique used for the identification of DNA sequence variances in genomic DNA, cDNA or in PCR products amplified from genomic DNA or cDNA. The DGGE method was originally described by Fischer and Lerman (Two Dimensional Electrophoretic Separation of Restriction Enzyme Fragments of DNA. Methods in Enzymology, vol. 68: 183-191, 1979; DNA Fragments Differing by Single Base-Pair Substitutions are Separated in Denaturing Gradient Gels: Correspondence with Melting Theory. Proc. Natl. Acad. Sci. U.S.A. 80:1579, 1983) and has been improved since then by many investigators. See, for example: Myers, et al., Mutation Detection by PCR, GC- Clamps, and Denaturing Gradient Gel Electrophoresis, pp. 71-88 in Erlich, H.A., editor: PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, 1989; Myers, et al., Detecting Changes in DNA: Ribonuclease Cleavage and Denaturing Gradient Gel Electrophoresis, in Davies, K.E., editor: Genomic Analysis: A Practical Approach. IRL Press Ltd., Oxford, 1988, pp. 95- 139; E.S. Abrams and V.P. Stanton Jr., Use of Denaturing Gradient Gel Electrophoresis, pp. 71-104 in Lilley, D.M.J. and Dahlberg, J.E., editors: DNA Structures. Part B: Chemical and Electrophoretic Analysis of DNA. Methods in 290 232/116

Enzymology, volume 212, Academic Press, 1992; .) Descriptions of current applications of the technique can be found in

The basic principal of DGGE involves the creation of a gradient of denaturant in a gel, which is then used to resolve double stranded DNA (or RNA) fragments on the basis of conformational differences associated with strand melting. The denaturant can be chemical (as in DGGE, where a gradient of formamide and urea is typically used) or thermal (as in a related technique called thermal gradient gel electrophoresis, or TGGE, where a gradient of heat is used). To obtain conditions where double stranded DNA is close to melting, DGGE gels are immersed in a heated bath of electrophoresis buffer, while TGGE gels have a fixed concentration of chemical denaturant.

As a double stranded DNA molecule migrates through a DGGE gel from a low concetration of denaturant at the origin to higher concentrations of denaturant toward the end of the gel it eventually reaches a level of denaturant that will cause partial melting. (Some design of DNA molecules is often necessary to assure that the partial melting will occur as desired; see below.) The concentration of denaturant required to melt a given DNA segment is highly sensitive to sequence differences in the DNA, including changes as subtle as a single nucleotide substitution. Partially melted DNA fragments move through gels at a much slower rates than their fully duplex counteφarts. Thus two DNA fragments differing at a single nucleotide can be distinguished on the basis of their gel position after an appropriate period of electrophoresis: the fragment with the more stable structure (resulting from, for example, a G:C base pair in place of an A:T pair) will travel further in the gel than its less stable counteφart, because it will encounter the concentration of gradient required to melt it (and consequently dramatically retard or nearly stop its movement) at a point further along in the gel.

The DGGE method reveals the presence of sequence variation between individuals as 291 232/116 shifts in electrophoretic mobility, but does not show the sequence itself. Direct sequencing of DNA fragments (from different individuals) with altered mobility in the DGGE assay will reveal the precise sequence differences among them (see example 37, Variance Detection by DNA Sequencing). From the nucleic acid sequence data, the amino acid sequence can be determined and any amino acid differences can be identified.

The DGGE method is suitable for analysis of restriction enzyme digested genomic DNAs, as initially described by Lerman and co-workers (supra) and later extended (Gray, M. Detection of DNA Sequence Polymoφhisms in Human Genomic DNA by

Denaturing Gradient Blots, American Journal of Human Genetics, 50: 331-346, 1992). DGGE is equally suitable for analysis of cloned DNA fragments or DNA fragments produced by PCR. The analysis of cloned fragments or PCR fragments has the advantage that non-natural sequences, rich in G and C nucleotides can easily be added to the 5' ends (either flanking the cloning site or at the 5' ends of PCR primers). Such

DNA fragments have very stable double stranded segments, called GC clamps, at one or both ends. The GC clamps alter the melting properties of the fragments, and can be designed so as to insure melting of the inter-primer segment of the PCR product at a lower temperature than the clamps, thereby optimizing the detection of sequence differences (see Myers et alia, supra and Myers et alia, Nearly All Single Base

Substitutions in DNA Fragments Joined to a GC Clamp Can be Detected by Denaturing Gradient Gel Electrophoresis. Nucleic Acids Research 13: 3131, 1985). GC clamps can be rationally designed for any specific DNA fragment of known sequence by use of a computer program (MELT87, written by L. Lerman) that accurately predicts melting behavior based on analysis of primary sequence. When GC clamps are used correctly, the DGGE method is highly efficient at detecting DNA sequence differences. Not only are nearly 100% of differences detected, but the false positive rate is essentially zero. (Abrams, E.S., et alia, Comprehensive Detection of Single Base Changes in Human Genomic DNA Using Denaturing Gradient Gel 292 232/116

Electrophoresis and a GC Clamp. Genomics 7: 463-475, 1990.) Recently methods for increasing the throughput of DGGE have been developed, based on multiplex PCR.

The steps in carrying out DGGE with GC clamps are:

1. Design DNA fragments with optimal melting behavior. Select oligonucleotide primers, using GC clamps as necessary, to produce a single melting domain over the length of the sequence to be analyzed. (It may be necessary to divide the sequence into overlapping fragments to achieve this goal.) Design of primers and simulated analysis of fragments can be performed with the computer program described by Lerman.

(Lerman, L.S. and Silverstein, K. Computational Simulation of DNA Melting and its Application to Denaturing Gradient Gel Electrophoresis. Methods in Enzymology 155: 482-501, 1987.) The output of the program is the melting map of the fragment, from which it will also be possible to determine the optimal range of denaturant in the gradient and the approximate electrophoresis time for fragments to reach the point of melting in the gradient.

2. Amplify the fragment by PCR. Procedures for optimizing PCR are briefly described in other examples and are well known in the art. Template DNA samples can either be cDNA or genomic DNA and will typically be drawn from a panel of unrelated individuals.

3. Pour a denaturing gradient gel. Briefly, make up two gel solutions containing the desired beginning and end concentrations of denaturant. The gel solutions are generally made up by mixing "0%" and "100%" denaturant stock solutions, where the 0% stock consists of 7% acrylamide in Tris-acetate EDTA (TAE) electrophoresis buffer, and the 100% stock is also 7% acrylamide in TAE, plus 40% formamide by volume and 7 molar urea. Equal volumes of the two solutions (e.g. twelve milliliters of each solution) are poured into the two chambers of a gradient maker (usually between 20 and 40% denaturant in the upstream chamber and 60 to 80% in the lower 293 232/116 one) immediately after addition of ammonium persulfate and TEMED for acrylamide polymerization. Open the stopcock of the gradient maker and pour the gradient gel. Usually gels are .75 to 1 mm in thickness, and gel combs that form 10-30 wells are used. With commercially available apparatus multiple gradient gels can be poured simultaneously. Suitable apparatus is sold by several vendors, including the BioRad

(Hercules, CA) Dcode system and the C.B.S. Scientific DGGE system.

4. Place the gel in a heated bath of electrophoresis buffer. Gels are electrophoresed at elevated temperature which, together with the denaturant, brings the DNA fragments to their melting point. Gels are often run at 60°C in IX TAE buffer, with constant recirculation of buffer to the upper buffer chamber. Once the gel has been placed in the heated tank and allowed to equilibrate it can be loaded. Multiple gels can be run simultaneously in the same tank with the apparatus listed above.

5. Load and run gel. Usually enough PCR product from each sample is loaded on the gel so that samples can be detected by a simple DNA staining procedure; use of radioactivity, dyes or hybridization procedures can thereby be avoided. At least 100 mg of each sample should be loaded, but preferably over 200 ng. Gel running conditions can be estimated from the output of the MELT87 program, however empirical adjustment will often be necessary. Usually a voltage of ~ 80 to 200V is applied for periods of 5-20 hours, depending on the characteristics of the fragments being analyzed.

6. Stain and analyze gel. After electrophoresis gels are stained with ethidium bromide, SYBR Green, silver or some other procedure. The location of PCR products produced with the same primer pairs should be compared. Altered location, and usually the appearance of two or more bands instead of one, signify the presence of DNA sequence differences. (The reason for more than two bands from a diploid sample is that during the terminal cycle of heating and cooling of the PCR 294 232/116 step heteroduplexes are formed between the maternally and paternally inherited alleles. If those alleles differ in sequence, the heteroduplexes will have mispaired nucleotides at the sites of difference. As a result the heteroduplexes will be less stable than either of the homoduplex species, and will consequently melt and be retarded in the gel at a lower concentration of denaturant. Altogether one may see four bands in such samples: two reciprocol heteroduplexes and two homoduplexes.) The specific pattern of fragments in each lane constitutes a signature for a specific nucleotide change.

7. Sequence DNA fragments with altered mobility. Examples of all different signatures should next be analyzed by DNA sequencing to identify the base difference(s) accounting for altered mobility in the gradient gel. See example 37 for a description of this procedure and the subsequent steps of recording the sequence variances and analyzing their frequency and structural and functional consequences.

Example 37: Variance detection by sequencing.

Sequencing by the Sanger dideoxy method or the Maxim Gilbert chemical cleavage method is widely used to determine the nucleotide sequence of genes. Presently, a worldwide effort is being put forward to sequence the entire human genome. The Human Genome Project as it is called has already resulted in the identification and sequencing of many new human genes. Sequencing can not only be used to identify new genes, but can also be used to identify variations between individuals in the sequence of those genes.

The following are the major steps involved in identifying sequence variations in a candidate gene by sequencing: 295 232/116

1. Amplification by the polymerase chain reaction (PCR) of 400-700 bp regions of the candidate gene from a panel of DNA samples The DNA samples can either be cDNA or genomic DNA and will represent some cross section of the world population. 2. Sequencing of the resulting PCR fragments using the Sanger dideoxy method.

Sequencing reactions are performed using flourescently labeled dideoxy terminators and electrophoresedon an ABI 377 sequencer or its equivalent.

3. Analysis of the resulting data from the ABI 377 sequencer using software programs designed to identify sequence variations between the different samples analyzed.

A more detailed description of the procedure is as follows:

A candidate gene sequence is downloaded from an appropriate database. Primers for PCR amplification are designed which will result in the target sequence being divided into amplification products of between 400 and 700 bp. There will be a minimum of a 50 bp of overlap not including the primer sequences between the 5' and 3' ends of adjacent fragments to ensure the detection of variances which are located close to one of the primers.

Optimal PCR conditions for each of the primer pairs is determined experimentally. Parameters including but not limited to annealing temperature, pH, MgCl₂ concentration, and KCl concentration will be varied until conditions for optimal PCR amplification are established. The PCR conditions derived for each primer pair is then used to amplify a panel of DNA samples (cDNA or genomic DNA) which is chosen to best represent the various ethnic backgrounds of the world population or some designated subset of that population.

PCR reactions are purified using the QIAquick 8 PCR purification kit (Qiagen cat# 296 232/116

28142) to remove nucleotides, proteins and buffers. The PCR reactions are mixed with 5 volumes of Buffer PB and applied to the wells of the QIAquick strips. The liquid is pulled through the strips by applying a vacuum. The wells are then washed two times with 1 ml of buffer PE and allowed to dry for 5 minutes under vacuum. The PCR products are eluted from the strips using 60 ul of elution buffer.

The purified PCR fragments are sequenced in both directions using the Perkin Elmer ABI Prism™ Big Dye™ terminator Cycle Sequencing Ready Reaction Kit (Cat# 4303150). The following sequencing reaction is set up: 8.0 ul Terminator Ready Reaction Mix, 6.0 ul of purified PCR fragment, 20 picomoles of primer, deionized water to 20 ul. The reactions are run through the following cycles 25 times: 96°C for 10 second, annealing temperature for that particular PCR product for 5 seconds, 60°C for 4 minutes.

The above sequencing reactions are ethanol precipitated directly in the PCR plate, washed with 70% ethanol, and brought up in a volume of 6 ul of formamide dye. The reactions are heated to 90°C for 2 minutes and then quickly cooled to 4°C 1 ul of each sequencing reaction is then loaded and run on an ABI 377 sequencer.

The output for the ABI sequencer appears as a series of peaks where each of the different nucleotides, A, C, G, and T appear as a different color. The nucleotide at each position in the sequence is determined by the most prominent peak at each location. Comparison of each of the sequencing outputs for each sample can be examined using software programs to determine the presence of a variance in the sequence. One example of heterozygote detection using sequencing with dye labeled terminators is described in Pui- Yan Kwok et. al. (Pui-Yan Kwok, Christopher Carlson, Thomas D. Yager, Wendy Ankener, and Deborah A. Nickerson, Genomics 23, 138- 144 (1994)). The software compares each of the normalized peaks between all the samples base by base and looks for a 40% decrease in peak height and the concomitant 297 232/116 appearance of a new peak underneath. Possible variances flagged by the software are further analyzed visually to confirm their validity

Example 38. Loss of heterozygosity.

Loss of chromosomes or segments of chromosomes in disease cells results in loss of alleles in the disease cells compared to normal diploid cells. Such allele losses are a common occurrence in cancer, where they have been documented in over 1,500 publications in the past 14 years. More recent work has documented the occurrence of allele loss in other proliferative diseases. Several cytogenetic and molecular techniques have been developed to measure chromosome losses. The molecular techniques are preferable for identification of allele loss because they also show which allele is lost, and are therefore best suited to provide the information needed to implement the present invention.

In order to measure chromosome loss using molecular techniques it is necessary to be able to distinguish the paternally and maternally inherited copies of a given chromosome. DNA variances allow the two copies of a given chromosome to be distinguished because different alleles can be resolved electrophoretically. The standard method for analyzing allele loss in cancer is to compare tumor cell DNA with normal cell DNA, either in a Southern blot or using PCR based techniques. A patient's tumor DNA is said to be "informative" for allele loss only at loci where the patient's normal cells are heterozygous. When such heterozygous loci are examined in tumor cells often only one allele is detected. Such tumor cells have lost the heterozygous state which characterizes all normal somatic cells of the patient, hence the term loss of heterozygosity (LOH). 298 232/116

Several effective molecular procedures have been developed to measure LOH. These procedures have been applied most extensively to cancer tissues, however the same methods are effective in the study of nonmalignant diseases such as atherosclerotic plaques and endometriosis. The main steps are:

1. Identify DNA variances at or near the locus to be investigated for LOH.

LOH usually affects large segments of DNA, ranging from several megabases to an entire chromosome. As a result, accurate estimation of LOH at a specific locus can be obtained by measuring the frequency of LOH at neighboring polymoφhic markers on the same chromosome, or more preferably on the same chromosome arm, or most preferably within several 10-20 megabases of the locus. However, to precisely measure LOH at a specific locus requires a variance at the locus. Different types of variances have been used to study LOH, including single nucleotide polymophisms (SNPs), specifically SNPs that alter restriction endonuclease cleavage sites, called

RFLPs. (For details of this approach see Vogelstein, B., et al., Allelotype of colorectal carcinomas. Science 244: 207-211, 1989). Also short tandem repeat polymoφhisms (STRPs), including di-, tri- and tetranucleotide repeat polymoφhisms have been used to measure LOH. (For details of this procedure see Jones and Nakamura, Deletion Mapping of Chromosome 3p in Female Genital Tract Malignancies Using

Microsatellite Polymoφhisms. Oncogene 7: 1631-1634, 1992.) Procedures for identifying variances are described in Examples 28, 29, 30 and 36.

2. Prepare DNA from paired normal and disease tissue samples from patients being studied.

Before preparing genomic DNA from tumor tissue it is important to assess tumor cell purity and viability, using microscopic examination of frozen sections if necessary. If embedded pathological specimens are being analyzed tumor cell purity can be 299 232/116 assessed by examining histologic sections before selecting areas for cell isolation and DNA purification. (See Johnson, et al., Direct Molecular Analysis of Archival Tumor Tissue for Loss of Heterozygosity, BioTechniques 19:190-191, 1995, and references therein for description of techniques for purifying tumor cell DNA from archival pathology samples.) Areas of necrosis and extensive admixture of normal and tumor tissue should be avoided. For Southern blotting -5-10 ug of genomic DNA is required for each sample being analyzed. For PCR based methods as little as 5 to 10 ng of genomic DNA is sufficient; much less will suffice if two successive rounds of PCR amplification are used. 3. Determine genotype in the normal and disease tissues using a quantitative or semi- quantitative procedure that allows the amount of each allele to be measured. Compare the ratio of alleles in the normal tissue to the ratio in the tumor tissue

In order to show LOH at a given locus it is necessary to establish that the patient is constitutionally heterozygous at the locus. Thus DNA from normal tissue must be tested, either before or in parallel with tumor tissue DNA. A variety of methods can be used for quantitation of signal from the two alleles. If the alleles are compared on a Southern blot then signal in the bands corresponding to the two alleles can be counted by radioactive or nonradioactive techniques (see Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons). One method employs phosphor technology using a Molecular Dynamics Phosphorlmager with ImageQuant software to measure signals. If the alleles are compared after PCR amplification then DNA sequencing can provide accurate quantitation of allele ratios. See, for example,

Goldsborough and Kornberg, AUele-Specific Quantification of Drosophila Engrailed and Invected Transcripts, Proc. Natl. Acad. Sci. U.S.A. 91 : 12696-12700, 1994.

Using highly variable markers distributed across the genome a comprehensive map of LOH can be assembled for a specific cancer type. Such data sets have been termed allelotypes. Separate studies are necessary for different cancer (or other disease) types 300 232/116 as the patterns of LOH differ significantly in different diseases.

Other techniques that have been used to detect allele loss in cancer include Comparative Genomic Hybridization (CGH) and Representation Difference Analysis (RDA) however these methods are more complex than the Southern blot or PCR based techniques. Chromosome loss can also be detected cytogenetically. Mitelman (Catalog of Chromosome Aberrations in Cancer. Wiley-Liss, New York, 1995.) has compiled a catalog of over 10,000 published karyotypes of cancer cells which documents chromosome deletions as well as other changes.

Example 39. Small molecule inhibitors of variant sequences: Methylguanine Methyltransferase (MGMT) Gene VARIA 1534

The methylguanine methyltransferase gene is essential for cell growth or survival in the presence of alkylating agents

Methylguanine methyltransferase (MGMT) is a nuclear protein that repairs alkylating agent damage, specifically alkylation of the O6 position of guanine bases in genomic DNA. MGMT acts as a suicide protein in removing methyl or alkyl groups from guanine and covalently binding them to cysteine 145 of MGMT. The protein is subsequently degraded; it does not act as an enzyme. O6-benzylguanine is an inhibitor of MGMT that mimics the natural substrate, alkylated DNA; transfer of the benzyl group to cysteine 145 of MGMT inactivates the protein. Concurrent administration of O6-benzylguanine and an alkylating agent such as carmustine (BCNU) or lomustine (CCNU) renders tumor cells more sensitive to the toxic effects of the nitrosoureas by inactivataing MGMT and thereby inhibiting the tumor cells ability to repair alkylated 301 232/116

DNA. MGMT is thus a conditionally essential gene in the presence of nitrosoureas and other alkylating agents. The conditional essentiality of MGMT has been demonstrated in mice. Animals homozygous for disrupted MGMT genes are more than ten times as sensitive to alkylating agents as normal mice. The relative sensitivity has been measured as the LD50, the dose required to kill 50% of treated animals. (Tsuzuki, T., et al. Targeted disruption of the DNA repair methyltransferase gene renders mice hypersensitive to alkylating agent. Carcinogenesis 17: 1215-1220, 1996.) 06- benzylguanine is being developed as a chemosensitizing agent (with alkylating agents) for treatment of human cancer. This treatment regimen is not specific for cancer cells.

In a cancer patient with two alternative functional MGMT alleles in normal tissues and LOH at 10q23 resulting in only one copy of MGMT in cancer cells, an allele specific inhibitor of MGMT could be used to specifically sensitize cancer cells to the action of alkylating agents. Treatment would consist of the administration of the appropriate allele specific inhibitor (directed to the one allele remaining in cancer cells) plus an alkylating agent. The tumor cells would be unable to effectively repair the alkylating agent induced DNA damage, while the uninhibited allele in normal cells would be able to function. Thus normal cells, including sensitive normal cell populations such as bone marrow stem cells, would be able to tolerate higher doses of alkylating agents than cancer cells.

The MGMT gene and encoded protein are polymorphic

Four variances in human MGMT have been discovered by the inventors or reported in the literature, including three variances that affect the protein sequence. There is a C/T variance at nucleotide 255 (11% heterozygotes among 36 individuals surveyed) which does not affect the encoded protein. There is a second C/T variance at nt. 346 which results in a L84F amino acid variance (5% heterozygotes among 36 individuals surveyed). There is an A/G variance at nt. 523 which results in a 1143V amino acid 302 232/116 variance (24% heterozygotes among 36 individuals surveyed). This variance occurs only two residues from the active site cysteine at 145. A fourth variance, G/A has been reported in the Japanese population at codon 160, GGA vs. AGA, resulting in a glycine vs. arginine amino acid variance. Fifteen percent of 40 Japanese individuals studied were heterozygotes for this variance. (Imai, Y., et al. A polymoφhism at codon 160 of human O6-methylguanine-DNA methyltransferase gene in young patients with adult type cancers and functional assay. Carcinogenesis [London] 16:2441-24445, 1995.)

Allele specific inhibitors of MGMT

Two of the amino acid variances in MGMT, at residues 143 and 160, are near the active site of the protein. Substantial work has already been done to characterize the functional consequences of the residue 160 glycine/arginine variance. Studies of MGMT kinetics and activity have shown that the 160arginine allele is at least 20 fold more resistant to O6 benzylguanine inactivation, measured as an increase in the ED50 and or as a reduction in the production of guanine from O6-benzyl[8-3H] guanine. The 160gly and 160arg forms of MGMT were nearly equal in alkyltransferase activity in an assay that measured repair of O6-methylguanine in methylated DNA. These results demonstrate variance-specific effects of a small molecule, O6-benzylguanine, on normal (non-mutant) alleles of the conditionally essential MGMT gene. (Edara, S., et al. Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine. Cancer Research 56: 5571-5575, 1996)

Administration of O6-benzylguanine to patients who are heterozygous for the variance in their normal cells, and contain only the alternative form of the gene with a glycine residue at position 160 in their cancer cells, together with methylating or chloroethylating agents, will specifically sensitize cancer cells to the cytotoxic effects of the alkylating agents without increasing toxicity to normal cells which, since they 303 232/116 contain the O6-benzylguanine resistant 160arginine form of the protein, will continue to repair alkylated DNA.

There is no published data concerning the residue 143 variance, however the proximity of this variance to the active site - both in the primary sequence and upon inspection of the three dimensional structure of the bacterial AGT protein, a functional and structural homolog of human MGMT - suggests that allele specific drugs could be discovered for this variance.

The structural difference between 143isoleucine and 143valine is a hydrophobic methyl group. It is well known that most small molecule protein inhibitors interact via hydrophobic interactions. Favorable Van der Waals distances between hydrophobic groups of a substrate and a ligand are vital for high affinity interaction. One possible mechanism of allele specific inhibition would be to exploit the greater bulk of the isoleucine by finding a small molecule that fits into the active site pocket of the valine allele but has a very unfavorable Van der Waals interaction the methyl group of the isoleucine. Other schemes based on the different size and geometry of isoleucine and valine could also be effective.

One approach to identification of such inhibitors would be to make small molecule libraries in which various positions of guanine are substituted with moities of appropriate size and structure. Such libraries could then be tested in various screens of MGMT activity. The two alleles (143 isoleucine and 143valine, or any of the other allele pairs of MGMT described above) would be assayed in parallel. Identification of molecules with allele specific inhibitory activity could be the basis for synthesis of additional libraries in which the moities that are best correlated with differential activity are further varied. Methods for the iterative design of high affinity or highly discriminating small molecule inhibitors are known in the art. 304 232/116

Libraries of restricted size can be screened for allele specific inhibitors using a combinatorial strategy based on known inhibitors of MGMT such as O6-benzyl- guanine. A library or libraries can be constructed in which substitutions are indroduced at positions C6 and N9 which have previouly been found to affect inactivation of MGMT, or at positions C2 and N8 which can be easily substituted.

For example a series of 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogs in which 4(6)-benzyloxy groups were replaced with (2-, 3-, or 4 fluorobenzyl)oxy or (2-, 3-, or 4-pyridylmethyl)oxy groups have been synthesized and tested for MGMT inhibition. (Terashima I., and K. Kohda. Inhibition of human O6-alkylguanine-DNA alkyltransferase and potentiation of the cytotoxicity of chloroethylnitrosourea by 4(6)-(Benzyloxy)- 2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues. JMed Chem 41 : 503-508, 1998.) Substitutions at N7 have been found to be detrimental in general (Moschel, R.C. et al & Pegg, A. E., J. Med. Chem. 35: 4486-4491, 1992).

Combinatorial libraries can be constructed according to a published procedure (Norman, T. C et al., A Structure-Based Library Approach to Kinase Inhibitors. J. Am. Chem.Soc. 118: 7430-7431, 1996) where guanine based libraries were made by anchoring a chemically modified guanine (at C6, C2, or C8) to solid supports at C2 via a glycinamide linkage or at N9 via a hydroxyethyl linkage. Chemical reactions can be carried out to introduce a library of hydrophobic substituents of different size at positions C6, C2, or C8. Hydrophobic substituents of various bulkiness and orientation can be indroduced through derivatives of O6-benzyl and O6-phenyl groups, O6-alkyl groups, N9-alkyl groups, and C2-amino-alkyl groups.

Libraries constructed as above can be screened for MGMT activity in several types of assays. Methods for bacterial expression and purification of human MGMT protein have been described (see Edara, et al., cited above). Both allelic forms of MGMT could be screened for repair of alkylated or methylated DNA by measuring transfer of tritium from a tritium labelled (methylated) DNA substrate in the 305 232/116 presence of various concentrations of library compounds for various times. Alternatively, library compounds could be tritiated and MGMT proteins could be screened for the rate at which they interact with (either via association or cleavage of a moiety from the compound). Other assays for MGMT activity are known in the art.

Example 41. Clinical use of variance specific inhibitors for treating cancer

Inhibitors that are the object of the present invention are designed to be administered to patients who are heterozygous for the target gene, meaning that their cells normally contain two alternative copies of the gene, one that is sensitive to inhibition by said inhibitors, and one that is not sensitive to said inhibitors. It is apparent that several such inhibitors may be developed according to this invention targeted to alternative alleles of a single target gene or to several different target genes. The inventors propose that a series of such inhibitors will be developed according to this invention.

The clinical use of this invention involves the steps of: (a) testing normal cells from a patient to identify target genes that are heterozygous, present in two alternative forms.

(b) testing biopsy tissue from a tumor or proliferative lesion to determine whether one of the two alternative forms is eliminated due to LOH.

(c) selecting a drug for inhibition based on the presence of the sensitive allele in the tumor and the presence of an insensitive allele in normal cells

(d) administering said drug to the patient in an appropriate dose to inhibit the essential function in the cancer cell.

Testing of normal cells to identify heterozygosity of the target gene is performed 306 232/116 using conventional diagnostic methods that are known in the art. Normal cells are commonly derived from a blood sample, hair sample, or buccal smear. Alternatively normal cells may be obtained by cultivating primary cells such as lymphoblasts or fibroblasts in vitro. The presence of two alternative alleles may be determined by methods including allele-specific hybridization with oligonucleotides containing the variant sequences and a number of non- variant nucleotides to allow differential binding to the alternative forms of the gene or other methods known in the art using purified DNA or RNA or amplified DNA or cDNA sequences. Testing of biopsy tissue is performed by separating tumor cells or cells of the proliferative lesion to isolate a sample of cells characteristic of the proliferative lesion for analysis. This is performed by a variety of methods known in the art including manual dissection or laser assisted methods for eliminating normal cells or selecting abnormal cells. Samples of abnormal tissue, and samples of normal tissue as a control, are analyzed to identify the presence or absence of alternative forms of the target gene. The presence of two altrnative alleles may be determined by methods including allele-specific hybridization with oligonucleotides containing the variant sequences and a number of non- variant nucleotides to allow differential binding to the alternative forms of the gene or other methods known in the art using purified DNA or RNA or amplified DNA or cDNA sequences.

Selection of a drug for administration will be based on clinical trial data indicating that the drug is effective in eliminating abnormally proliferating cells and causing an improvement in the patient's clinical condition for patients who have the sensitive allele of the target gene in their pathological lesion. In one aspect of this invention, the product label will describe that the drug is indicated in patients who have only a specific allele of the target gene in their lesion and an alternative allele in their normal cells. Any such drug will be indicated only for a fraction of patients having two alternative alleles of the target gene in their normal cells and LOH. The fraction of patients who may be treated with any one drug may be determined by 307 232/116 multiplying the number of patients with a given cancer times the fraction of tumors exhibiting LOH of the target gene locus times the fraction of patients who will be heterozygous. For a target gene exhibiting 50% heterozygosity in the population and a 70% fraction of LOH in a specific cancer (several such examples are shown), a single inhibitor will treat -17% of such cancers. A second compound directed against the alternative allele would treat another 17% of said cancer. In the preferred use of this invention, a panel of such drugs will be available enabling therapy with at least one such drug in most patients.

Administration of the drug to the patient ration to the patient will involve conventional means such as parenteral, oral, or intratumoral administration. The route of administration will be determined separately for each inhibitor and will be based on the bioavailability of the compound to the lesion. The compound may be administered in one or more doses as a single agent or in combination with other allele specific agents or conventional antiproliferative drugs or agents commonly used for the treatment of cancer or support of cancer patients.

Example 42.Cell Division Cycle 25C (CDC25C) - Gene VARIA10

Cdc25C is essential for cell growth

A vital regulator of cell proliferation is the protein kinase Cdc2, whose activation at the end of G2 of the cell cycle initiates mitosis. Gene disruption experiments in yeast confirm the importance of this protein, as cells lacking Cdc2 fail to progress through the cell cycle. As would be expected for such an important protein, Cdc2 activity is tightly regulated. Its activity depends on complex formation with Cyclin B, a protein that accumulates through the cell cycle and is then abruptly degraded during mitosis. Phosphorylation of Cdc2 on Tyr-15 and Thr-14 by the Weel/Mikl 308 232/116 kinases maintains the Cdc2/Cyclin B complex in an inactive state until the end of G2. The dual-specificity phosphatase Cdc25C is then stimulated to dephosphorylate Cdc2 on both residues, resulting in activation of the complex. Just as Cdc2 is essential for cell growth, the regulation of its activity is essential. The best evidence for this is that the individual disruption of cdc2, cyclin B, wee 1 and cdc25 in the yeast S. pombe are lethal events. When cdc25 is deleted from these cells they display a phenotype consistent with their function; they grow without dividing, becoming dramatically elongated.

The human CDC25C gene and protein have variances

The CDC25C cDNA was cloned by Sadhu et al. (1) (Genbank accession number M34065, GI number 181075). To determine whether CDC25 is polymoφhic, VARIAGENICS scanned cDNA from 32 unrelated individuals using the T4 Endonuclease VII method, which involves the cleavage of DNA heteroduplexes followed by DNA sequencing of polymoφhic regions (see description of method in examples). A transversion at nucleotide 1099 (G or C) was identified (nucleotide numbering is from reference 1). This results in an amino acid difference at residue 297, with G encoding glycine and C encoding arginine. Overall, 9.4% of individuals analyzed are heterozygous. The rate of heterozygosity increases to

33.3% in Caucasians.

The human CDC 25 C gene maps to chromosome 5q31, a site of frequent loss of heterozygosity

Sartor et al. (2) mapped the human CDC25 gene to 5q31 by fluorescence in situ hybridization using the cDNA cloned by Sadhu et al. This mapping location was confirmed by Taviaux and Demaille (3), also using fluorescence in situ hybridization. There have been many studies of LOH on 5q, particularly the 5q21- 309 232/116 q22 region where the Adenomatous Polyposis Coli (APC) tumor suppressor gene lies. The most extensively studied cancers are those of the gastrointestinal tract, lung and ovary. There have been fewer studies of the 5q23-q33 region just distal to APC (where CDC25C lies), however the available data suggests that LOH occurs in this region at a frequency of -30% in cervical cancer (4), 20-40% in colon cancer

(5,6), 30-50% in ovarian cancer (7,8), up to 38% in stomach cancer (9), and 23% in testicular cancer (10). There is also evidence for LOH in head and neck, lung and liver cancers. In most of these studies only one or two markers were used. Definitive assessment of LOH frequency at the CDC25C locus will require direct analysis of the polymoφhisms identified in various tumor types.

References

1) Sadhu, K., Reed, S.I., Richardson, H., Russell, P. (1990) Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G(2). Proc. Natl. Acad. Sci. U.S.A. 87: 5139-5143.

2) Sartor, H., Ehlert, F., Grzeschik, K.-H., Muller, R., Adolph, S. (1992) Assignment of two human cell cycle genes, CDC25C and CCNB1, to 5q31 and 5q 12, respectively. Genomics 13: 911-912.

3) Taviaux, S.A., Demaille, J.G. (1993) Localization of human cell cycle regulatory genes CDC25C to 5q31 and WEE1 to 1 lpl 5.3-1 lpl 5.1 by fluorescence in situ hybridization. Genomics 15: 194-196.

4) Mitra, A.B., Murty, V.V., Li, R.G., Pratap, M., Luthra, U.K., Chaganti, R.S. (1994) Allelotype analysis of cervical carcinoma. Cancer Res. 54: 4481-7.

5) Japanese Journal of Cancer Research 82: 1003. 6) Cunningham, C, Dunlop, M.G., Wyllie, A.H., Bird, CC (1993) Deletion mapping in colorectal cancer of a putative tumour suppressor gene in 8p22-p21.3. Oncogene 8: 1391-6.

7) British Journal of Cancer 69: 429.

8) Weitzel, J.N., Patel, J., Smith, D.M., Goodman, A., Safaii, H., Ball, H.G. (1994) 310 232/116

Molecular genetic changes associated with ovarian cancer. Gynecol Oncol. 55: 245-52.

9) Genes, Chromosomes and Cancer 3: 468.

10) Murty, V.V., Bosl, G.J., Houldsworth, J., et al. (1994) Allelic loss and somatic differentiation in human male germ cell tumors. Oncogene 9: 2245-51.

Example 43. Dihydropyrimidine Dehydrogenase (DPD)

DPD is conditionally essential

Dihydropyrimidine Dehydrogenase is essential for cell survival in the presence of pyrimidine nucleotide analogs such as 5-FU and fluorodeoxyuridine. 5-fluorouracil (5-FU) and related compounds are antineoplastic drugs used in the treatment of breast, gastrointestinal, head and neck and other cancers. These drugs have widely varying clinical effects in cancer patients, ranging from induction of complete response (tumor disappearance) in some patients to severe toxicity in others. There is currently no reliable basis for predicting individual patient responses, and therefore patients receiving 5-FU must be monitored carefully for toxic reactions.

There are a variety of anabolic and catabolic pathways that affect the action of 5-FU (reviewed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, 8th edition). For example, in order to exert its antiproliferative effects the pyrimidine analog 5-FU must be converted enzymatically to the nucleotide level (fluorodeoxyuridine) by phosphorylation and ribosylation; fluorodeoxyuridine is sometimes given directly because it bypasses most of these steps, and simply requires phosphorylation by thymidine kinase. The 5-fluoronucleotide is an irreversible inhibitor of thymidylate synthase, the enzyme which converts dUMP to dTMP and is required for de novo synthesis of thymidine, and hence for DNA 311 232/116 synthesis.

There is a three step pathway for catabolism of pyrimidines (thymine and uracil) to beta alanine. Pyrimidine analogs such as 5-FU are catabolized by the same pathway. The first and rate limiting step in this pathway is catalyzed by dihydropyrimidine dehyrogenase (DPD). DPD accounts for catabolism of as much as 90% of a 5-FU dose in normal individuals, and the half life of 5-FU in normals is -8-20 minutes. Patients homozygous for mutant DPD alleles have been identified, a condition variously called DPD Deficiency, Hereditary Thymine-Uraciluria or Familial Pyrimidinemia. In such patients -90% of 5-FU is excreted unchanged in the urine, and the drug has a half life longer that 2.5 hours. As a result of the drastically reduced catabolism of 5-FU the toxic effects of the drug are magnified and patients are subject to severe toxic reactions. There are reports of deaths in patients with DPD deficiency after treatment with 5-FU. Thus cell (and organism) survival in the presence of 5-FU depends on presence of functional DPD protein to transform 5-FU to the inactive dihydroxy metabolite.

This principal has also been demonstrated in cancer cells both in vitro and in vivo: cancer cells with lower DPD levels are more susceptible to the toxic effects of 5-FU. It has been suggested that measuring DPD levels would be useful for calibration of 5-FU dosage.

The DPD gene exhibits variances

We have identified four common sites of variance in DPD mRNA by screening cDNA from 36 unrelated individuals. The variant nucleotides are 166, 577, 3925 and 3937 (see DPD Variance Table; numbering is from Yokota, et al. cDNA Cloning and Chromosome Mapping of Human Dihydropyrimidine Dehydrogenase, an Enzyme Associated with 5-fluorouracil Toxicity and Congenital Thymine 312 232/116

Uraciluria. Journal of Biological Chemistry. 269: 23192-23196, 1994). Two of the variances in nucleotide sequence alter the amino acid coding sequence: amino acid 29 is usually cysteine but arginine alleles were also detected; cys/arg heterozygotes were found at a frequency of 11%. Residue 166 of DPD is reported to be methionine but valine is present at 166 in some alleles; 9% of the population surveyed are met/val heterozygotes. One double heterozygote was identified out of 36 patients. Both these amino acid polymoφhisms are located in the N-terminal NAD/FAD binding domain of DPD. Residue 166 is located in a highly conserved domain of DPD. Two other polymoφhisms are located in the 3' untranslated region of DPD, only 11 nucleotides apart.

The DPD gene maps to chromosome lp22, a region frequently subject to LOH in different cancers

The DPD gene has been mapped to chromosome lp22 by fluorescense in situ hybridization. LOH at lp22 has been reported in colon, breast, and other cancers.

Allele specific inhibition of DPD to potentiate 5-FU action in cancer cells with LOH at the DPD locus

The DPD gene is polymoφhic and conditionally essential in the presence of 5-FU.

These properties can be exploited in a therapeutic strategy for cancer patients with LOH at the DPD locus. Specifically, in a patient with two alternative alleles for DPD in normal cells and one allele in cancer cells due to LOH, an allele specific drug can be used to sensitize cancer cells to the action of 5-FU by inhibiting its catabolism. Cancer cells (but not normal cells) would be poisoned by high levels of

5-FU due to low clearance. Normal cells, containing an uninhibited allele, would be able to catabolize DPD at close to normal levels.

Alternatively, patients heterozygous for functional and defective copies of DPD, 313 232/116 and in whom LOH resulted in loss of the functional allele, could be treated by 5-FU without the necessity for an allele specific inhibitor. Identification of such patients would require a test for heterozygosity at DPD and a test for LOH which could show which allele is deleted in cancer cells. Such an approach would be expected to identify patients likely to respond well to 5-FU even though they might have cancers not traditionally treated with pyrimidine analogs.

Example 44. Fanconi Anemia genes A, B, C, D, E, F, G and H (FAA, FAB, FAC, FAD, FAE, FAF, FAG, FAH)

The Fanconi Anemia genes are conditionally essential.

The Fanconi Anemia genes are essential for cell growth or survival in the presence of DNA cross linking agents. In order for cells to survive or proliferate in an abnormal environment characterized by the presence of DNA cross linking molecules such as Mitomycin C and diepoxybutane it is necessary that the cells are capable of efficiently repairing damage caused by these agents. Cells contain proteins necessary for such repair. One way such repair proteins can be identified is by absence of function in specific patients who, as a consequence, are particularly susceptible to the toxic effects of cross linking agents.

Fanconi Anemia (FA) is a hereditary disease, autosomal recessive in transmission, characterized by progressive bone marrow failure, birth defects and predisposition to malignancies. FA patients are hypersensitive to the toxicity of DNA cross linking agents. This hypersensitivity can be measured in cultured FA cells, which is one method used to establish the diagnosis of FA.

Patients heterozygous for defective FA genes are generally not hypersensitive to 314 232/116

DNA crosslinking agents in contrast to those that are homozygous. This suggests that treating heterozygous cancer patients with an inhibitor specific for one allele of the FA gene (and thereby reducing levels of FA protein function by up to 50% in normal cells) would be well tolerated. Inhibition of the FA allele present in cancer cells but not the alternative form present only in normal cells would make cancer cells selectively sensitive to crosslinking agents, leading to a cytotoxic antiproliferative effect. Normal cells would be able to repair damage caused by such agents, by analogy to the clinical data from patients heterozygous for defective FA genes.

The FA genes and gene products are polymorphic

Seven FA genes have been identified by complementation studies. The genes for FAA and FAC have been cloned. DNA variances have been reported in both genes. For example, Savino et al. report three variances in FAA, all of which alter the protein coding sequence. (Savino, M., et al. Mutations in the Fanconi Anemia Group A Gene (FAA) in Italian Patients. American Journal of Human Genetics 61:1246-1253, 1997.) The location of these variances is shown in the Table below, reproduced from the paper by Savino.

Variances in the FAA Gene

FA genes map to chromosomes that are frequently subject to LOH in different cancers

The FAC gene maps to chromosome 9q22.3, (as do three other FA complementation 315 232/116 groups according to Strathdee, C.A., et al. Evidence for at least four Fanconi anaemia genes including FACC on chromosome 9. Nature Genetics 1: 196-198, 1992). The FAA gene maps to chromosome 16q24.3. FAD maps to 3p26-p22. All FA genes mapped so far lie in regions subject to frequent LOH. LOH affecting chromosome 9 is well documented in many cancers. For example, loss of the 9q arm is well documented in cancers such as bladder, esophagus, ovary, testis and uterus. LOH frequencies in these cancers range from 20% to 62%. LOH affecting chromosome arm 16q, particularly the 16q24 region is well documented, particularly in breast, prostate and liver cancers. For example, in six detailed studies of breast cancer in the 16q22-q24 region LOH frequencies of 40-60% have been reported. Further, 16q22

LOH has been reported in 25-90% of liver cancers, with the average around 45%. Less extensive studies of other cancer types report 16q22 LOH in 19% of bladder cancers, 20% of colon cancers, 19-27% of esophageal cancers, 25% of small cell lung cancers, 16-37% of ovarian cancers 22% of uterine cancers, and 31-50% of prostate cancers. Loss of chromosome 3p26-21 is common in lung cancer, kidney cancer, head and neck cancer and breast cancer among other cancers. Reports of >50% LOH are common in these cancer types.

Other genes conditionally essential for response to DNA cross linking agents

In a related aspect, other genes which, when defective, sensitize cells to toxic effects of DNA crosslinking agents would be amenable to the therapeutic strategy outlined above for the FA genes. Specifically, in a patient with two alternative alleles for such a gene and LOH at the relevant locus, an allele specific drug could be used to sensitize cancer cells to the action of cross linking agents. Such drugs could then be used to treat cancer patients constitutionally heterozygous for two normal alleles at the relevant locus, in whom LOH had rendered cancer cells hemizygous or homozygous for one allele. Treatment would consist in the administration of the appropriate allele specific inhibitor plus a cross linking agent or treatment to induce damage in all cells. Cancer 316 232/116 cells (but not normal cells) would be rendered unable to respond by inhibition of expression of the relevant repair gene. Examples of such genes are the excision repair cross complementing (ERCC) genes, twelve of which have been identified (see Target Gene Table). Defects in these genes are associated with Xeroderma Pigmentosum and Cockayne Syndrome. (Scriver, C R. et al., The Metabolic and Molecular Bases of

Inherited Disease, 7th edition, McGraw Hill, New York, 1995.)

Alternatively, patients heterozygous for functional and defective copies of such genes, and in whom LOH resulted in loss of the functional allele, could be treated by a cross- link inducing procedure without the necessity for an allele specific inhibitor.

Identification of such patients would require a test for heterozygosity at the target locus and a test for LOH which could show which allele is deleted in cancer cells. Such an approach would be expected to identify patients likely to respond well to cross linking agents or procedures even though they might have cancers not traditionally treated with such agents.

Example 45. Asparagine Synthetase (AS). Variagenics Target Gene

Asparagine Synthase is conditionally essential

Cells require a continuous supply of amino acids for protein biosynthesis. Cells can import amino acids from serum via amino acid transporters (the only source besides protein catabolism for the ten essential amino acids), or amino acids cells can be synthesized de novo by cells (only an option for the ten nonessential amino acids). The essential amino acids are isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and histidine. Alterations in the nutritional environment of growing cells that result in a decreased extracellular concentration of essential amino 317 232/116 acids cause arrested cell growth and may result in cell death.

Even a nonessential amino acid can become essential in a cell where (i) at least one enzyme required for its biosynthesis is not expressed (perhaps due to downregulation in response to an abundant extracellular supply of the amino acid), or (ii) the biosythetic pathway is blocked by an inhibitor.

Asparagine is a nonessential amino acid which is, however, essential for survival of rapidly dividing cells that are not expressing asparagine synthetase, the terminal enzyme in asparagine biosynthesis. Asparagine synthetase, considered to be a housekeeping gene, catalyzes the ATP dependent conversion of aspartic acid to asparagine in mammalian cells. A number of different cancer types do not usually express asparagine synthetase, including childhood acute leukemias. One common therapeutic used in the treatment of childhood acute lymphocytic leukemia is the enzyme L-asparaginase (purified from E. coli or Erwinia carotovora) which, upon injection, rapidly depletes serum asparagine (by hydrolysis to aspartate), thereby lowering blood levels of asparagine to undetectable levels within hours of injection. (Ohnuma, T. et al. Biochemical and Pharmacological Studies with L-Asparaginase in Man. Cancer Research 30: 2297-2305, 1970.) Leukemic cells have high rates of protein synthesis but do not express asparagine synthetase and are therefore highly vulnerable to the rapid loss of asparagine and consequent shutdown of protein synthesis. Cell death after L-asparaginase induced asparagine starvation has been shown to be apoptotic. (Bussolati, O. Characterization of Apoptotic Phenomena Induced by Treatment with L-Asparaginase in NIH3T3 Cells. Experimental Cell Research 220: 283-291, 1995.) After one or more doses leukemic cells often become resistant to L-asparaginase due to induction of asparagine synthetase activity and consequent autonomy for asparagine.

In a patient with two alternative alleles for asparagine synthetase and LOH at 7q, an 318 232/116 allele specific drug could be used to sensitize cancer cells to the action of L- asparaginase. Such drugs could then be used to treat cancer patients constitutionally heterozygous for two normal alleles at the asparagine synthetase locus, in whom LOH had rendered cancer cells hemizygous or homozygous for one allele. Treatment would consist in the administration of the appropriate allele specific inhibitor plus L- asparaginase to deplete the concentration of this amino acid in serum while rendering cancer cells (but not normal cells) unable to respond by upregulating asparagine synthetase.

The Asparagine Synthetase gene maps to chromosome 7q21.3, a region frequently subject to LOH in different cancers

The asparagine synthetase gene has been mapped to chromosome 7q21.3 by fluorescence in situ hybridization, following localization to 7q by analysis of somatic cell hybrids. The q21 region of chromosome 7 is subject to frequent LOH, particularly in colon, breast and prostate cancers. 7q21.3 LOH is detected in up to 50% of colon cancers, up to 37% of prostate cancers (83% of prostate cancers have LOH in the adjacent chromosome band, 7q31) and in 10-55% of breast cancers, where again, there is even more frequent LOH in 7q31. LOH at 7q21 has also been reported in uterine cancer and head and neck cancer. Several other cancer types have not yet been well studied for LOH affecting this region.

Example 46. Methionine Synthase (MS). Variagenics Target Gene

Methionine Synthase is conditionally essential in dividing cells

Cells require a continuous supply of amino acids for protein biosynthesis. L- 319 232/116 methionine is one of ten essential amino acids. Consequently dividing cells must obtain their methionine from serum via amino acid transporter (the only source besides protein catabolism for the ten essential amino acids). Alterations in the nutritional environment of growing cells that result in a decreased extracellular concentration of essential amino acids such as methionine cause arrested cell growth and may result in cell death. Cancer cells are particularly sensitive to methionine deprivation. (Tan, Y., et al., Anticancer Efficacy of Methioninase in vivo. Anticancer Research 16: 3931- 3936.)

The cellular requirement for methionine can be bypassed: if L-homocysteine is provided to cells it can be methylated to form methionine by the enzyme methionine synthase (MS). In this reaction the methyl group is provided by 5- methyltetrahydrofolate and MS-bound methylcobalamin serves as an intermediate methyl carrier. A second enzyme may be required for reductive activation of methionine synthase, based on complementation studies.

It occured to the inventors that the apparent antineoplastic effects of methionine deprivation could be enhanced and made tumor cell specific by preventing cells from converting endogenous homocysteine to methionine by allele specific inhibition of methionine synthase (or other enzymes required for the conversion of homocysteine to methionine; see: Scriver, C, et al., editors, The Metabolic and Molecular Basis of Inherited Disease. McGraw Hill, New York, pp. 3111-3128 and 3129-3149). This strategy would be useful in cancer patients that are heterozygous for methionine synthase (or another enzyme required for conversion of homocysteine to methionine) and who have LOH at the methionine synthase (or other) gene locus. In such patients an allele specific inhibitor of MS directed to the sole allele present in cancer cells, coupled with methionine starvation or methioninase treatment, would selectively prevent tumor cells from responding to methionine deprivation. The provision of supplemental homocysteine, which could only be converted to methionine by the 320 232/116 normal cells, would provide a way to amplify the differential toxicity to cancer cells. Also, the methionine analog ethionine has been shown to potentiate the effects of methionine starvation. (Poirson-Bichat, F., et al., Growth of methionine-dependent human prostate cancer (PC-3) is inhibited by ethionine combined with methionine starvation. Br. J. Cancer 75: 1605-1612.) Ethionine or similar agents could be used in conjunction with an allele specific inhibitor of methionine synthesis.

An alternative approach to allele specific therapy of cancer cells with LOH would be to target the amino acid transport system for methionine in patients heterozygous for this protein and in whom only one allele is present in cancer tissue as a result of LOH.

This would result in selective methionine starvation for cancer cells. Allele specific transport inhibition could be combined with methionine starvation or methioninase treatment to enhance the cytotoxic effect.

The Methionine Synthase gene maps to chromosome lq43, a region subject to LOH in several cancers

The MS gene has been mapped to chromosome lq43 by fluoresence in situ hybridization. The q43 region of chromosome 1 is subject to frequent LOH particularly in colon, head and neck, ovarian and liver cancers, where LOH frequencies vary from 11 to 39%. LOH at lq43 has also been reported in cervix, pancreas, stomach and testis cancers. Several other cancer types have not yet been well studied for LOH in this region.

Other amino acid biosynthetic enzymes are candidates for allele specific inhibition

It will be evident to one skilled in the art that strategies similar to those described above for asparagine (an essential amino acid) and methionine (a non-essential amino acid) could be undertaken for other amino acid biosynthetic enzymes. For example, 321 232/116

L-glutaminase has also been shown to have antiproliferative effects on mammalian cell growth. Allele specific blockade of glutamine synthesis in heterozygous patients with LOH for genes essential for glutamine synthesis could be the basis of a cancer specific therapy.

Example 47. Methylthioadenosine phosphorylase (MTAP). Variagenics Target Gene

Methylthioadenosine phosphorylase can convert methylthioadenosine to methionine, an essential amino acid

Cells require a continuous supply of amino acids for protein biosynthesis. L- methionine is one of ten essential amino acids. Consequently dividing cells must obtain methionine from serum via amino acid transporter (the only source besides protein catabolism or conversion of L-homocysteine). Alterations in the nutritional environment of growing cells that result in a decreased extracellular concentration of essential amino acids such as methionine cause arrested cell growth and may result in cell death. Cancer cells are particularly sensitive to methionine deprivation. (Tan, Y., et al., Anticancer Efficacy of Methioninase in vivo. Anticancer Research 16: 3931- 3936.)

The cellular requirement for methionine can be bypassed by conversion of L- homocysteine to methionine as discussed above. An alternative pathway for methionine synthesis is conversion of 5 '-methylthioadenosine (5'-MTA) via the action of 5'-MTA phosphorylase (MTAP). (Tisdale, M J., Methionine Synthesis from 5'- methylthioadenosine by Tumor Cells. Biochemical Pharmacology 32: 2915-2920.) In tissue culture experiments low concentrations of 5'-MTA can substitute for 322 232/116 methionine in some cell lines. Thus 5'-MTA can rescue cells from methionine deprivation.

It occured to the inventors that allele specific inhibition of MTAP in cancer patients heterozygous for MTAP and whose cancer cells have only one allele of MTAP as a consequence of LOH, in combination with methionine deprivation (methionine starvation or L-methioninase treatment) and dietary supplementation with 5'- methylthioadenosine would provide a source of convertible methionine substrate selectively useful to normal cells. Tumor cells would have no source of methionine, being unable to convert the 5 '-methylthioadenosine, and hence would be selectively poisoned. This therapeutic strategy would not necessarily require an allele specific inhibitor as all copies of MTAP are deleted in some cancers. Such cancers should be differentially poisoned vis a vis normal cells by methionine deprivation in the presence of 5 '-methylthioadenosine.

The MTAP gene maps to 9p21, a region frequently subject to LOH in many cancers

The MTAP gene has been mapped to chromosome 9p21 by physical techniques (pulsed field gel electrophoresis and yeast artificial chromosome mapping). The gene lies near the cyclin dependent kinase inhibitors pl6 and pl5 which are frequently reduced to one or zero copies in cancer cells. (Nobori, et al., Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers. Proc. Natl. Acad. Sci. U.S.A. 93: 6203-6208.) The p21 region of chromosome 9 is subject to frequent LOH particularly in cancers of the bladder, breast, esophagus, head and neck, kidney, lung, melanoma and ovary. The frequency of LOH in these cancers ranges from 20% to nearly 100%. 323 232/116

Example 48. DNA dependent protein kinase (DNA-PK) and associated factors. Variagenics Target Genes

DNA dependent protein kinase is conditionally essential

Cells exposed to ionizing radiation, such as gamma radiation, are damaged by base modifications and DNA strand breaks. Double strand DNA breaks are among the most lethal form of radiation damage; one such break, if unrepaired, can be cell lethal. Four complementation groups of mammalian cell mutants that are defective in repair of double strand (ds) breaks have been identified. All four complementation groups are hypersensitive to ionizing radiation. The loci for three of these groups have been shown to encode components of DNA-dependent protein kinase (DNA-PK). The fourth group is deficient in the gene encoding XRCC4, a factor that associates with and stimulates DNA Ligase IV. Ligation of ds breaks by DNA ligase IV in a cell free system in increased 7-8 fold by co-expression of XRCC4.

DNA-PK is a multiprotein complex with a DNA binding regulatory subunit, the Ku heterodimer [Ku70 (XRCC6) and Ku80, also referred to as Ku86 (XRCC5)], and a catalytic subunit, DNA-PKcs (probably XRCC7), that is activated by the regulatory subunit upon binding to DNA ds ends, with consequent expression of serine/threonine kinase activity resulting in phosphorylation of a variety of DNA binding proteins. A fourth protein called KARP-1 is expressed from the Ku80/86 locus and is also implicated in DNA-PK function.

Cells lacking any of the components of DNA-PK are exquisitely sensitive to gamma irradation. This has been demonstrated directly in mice with targeted disruption of the Ku80/86 and DNA-PKcs genes. The Ku80/86 deficient mice were also sensitive to methyl methane sulfonate, a DNA alkylating agent that induces single strand breaks and to etoposide, a topoisomerase II inhibitor. Thus the components of DNA-PK can 324 232/116 also be important for repair of a variety of chemically induced DNA lesions as well as ionizing radiation.

In a cancer patient with two alternative alleles for a component of DNA-PK and LOH at the heterozygous locus, an allele specific inhibitory drug could be used to sensitize cancer cells to the action of ds break inducing treatments. Such a drug could be used to treat cancer patients constitutionally heterozygous for two normal alleles at any of the DNA-PK loci in whom LOH had rendered cancer cells hemizygous or homozygous for one allele. Treatment would consist in the administration of the appropriate allele specific inhibitor plus a ds break inducing agent or procedure. The tumor cells would be unable to effectively repair ds breaks, while the uninhibited allele in normal cells would be able to function. Alternatively, patients heterozygous for functional and defective copies of genes required for repair of strand breaks, and in whom LOH resulted in loss of the functional allele, could be treated by a strand break inducing procedure without the necessity for an allele specific inhibitor. Identification of such patients would require a test for heterozygosity at the target locus and a test for LOH which could show which allele is deleted in cancer cells. Such an approach would be expected to identify patients likely to respond well to strand breaking agents or procedures (exposure to ionizing radiation) even though they might have cancers not traditionally treated with such measures.

77je genes encoding constituents of DNA-PK map to chromosomes frequently subject to LOH in different cancers

The DNA-PKcs gene has been mapped to 8ql 1, the Ku80/86 gene to 2ql l-ql3 and the

Ku70 gene to 22ql l-ql3. All three regions are subject to LOH in different cancers. LOH on 2q has been reported in lung ovary and cervical cancers at frequencies ranging from 11% to 39%. LOH for 8q has been reported in cervix, head and neck, kidney, lung, ovary, prostate and testis cancers at frequencies ranging from 20% to 50% of 325 232/116 cancers. LOH on 22q has been reported in brain, breast colon, head and neck, lung, ovary, pediatric and stomach cancers at frequencies ranging from 10 to 76%. Several other cancer types have not yet been well studied for LOH affecting either region.

Other proteins required for repair of DNA strand breaks are also candidates for allele specific therapy of cancer

It will be evident to one skilled in the art that strategies similar to those described above for DNA-PK could be undertaken for other proteins required for repair of DNA strand breaks. For a recent review of such proteins see: Zdzienicka, M.Z., Mammalian mutants defective in the response to ionizing radiation-induced DNA damage. Mutation Research 336: 203-213, 1995; Thompson, L.H. and P.A. Jeggo, Nomenclature of human genes involved in ionizing radiation sensitivity. Mutation Research 337: 131-134, 1995; Thacker, J. and R.E. Wilkinson, The gentic basis of cellular recovery from radiation damage: response of the radiosensitive irs lines to low- dose rate irradiation. Radiation Research 144: 294-300, 1995. Two other syndromes with hypersensitivity to X-rays are Diamond-Blackfan anemia and aplastic anemia (Diemen, P.C, X-ray-sensitivity of lymphocytes of aplastic- and Diamond-Blackfan- anemia patients as detected by conventional cytogentic and chromosome painting techniques. Mutation Resarch 373: 225-235, 1997). Recently evidence of several other genes responsible for DNA double strand break repair has been described. (Nicolas, N., Finnie, N.J., et al., Eur. J. Immunol. 26:1118-1122, 1996.) The above genes which, when defective, sensitize cells to toxic effects of DNA strand breaking agents would be amenable to the therapeutic strategy outlined above for the DNA-PK genes. Specifically, in a patient with two alternative alleles for such a gene and LOH at the relevant locus, an allele specific drug could be used to sensitize cancer cells to the action of strand breaking agents. Such drugs could then be used to treat cancer patients constitutionally heterozygous for two normal alleles at the relevant locus, in whom LOH had rendered cancer cells hemizygous or homozygous for one allele. 326 232/116

Treatment would consist in the administration of the appropriate allele specific inhibitor plus a strand breaking agent or treatment to induce damage in all cells. Cancer cells (but not normal cells) would be rendered unable to respond by inhibition of expression of the relevant repair gene.

Alternatively, patients heterozygous for functional and defective copies of genes required for repair of strand breaks, and in whom LOH resulted in loss of the functional allele, could be treated by a strand break inducing procedure without the necessity for an allele specific inhibitor. Identification of such patients would require a test for heterozygosity at the target locus and a test for LOH which could show which allele is deleted in cancer cells. Such an approach would be expected to identify patients likely to respond well to strand breaking agents or procedures (exposure to ionizing radiation) even though they might have cancers not traditionally treated with such measures.

Example 49. Ataxia Telangiectasia Mutated (ATM) and c-Abl Variagenics Target Gene

The Ataxia Telangiectasia gene is essential for cell growth or survival in the presence of ionizing radiation or DNA damaging molecules

In order for cells to survive or proliferate in the presence of ionizing radiation (IR) or radiomimetic chemicals it is necessary that they are capable of efficiently repairing IR induced damage. Cells contain proteins necessary for such repair. One way such proteins can be identified is by their absence in specific patients who are particularly susceptible to the toxic effects of IR. 327 232/116

Ataxia Telangiectasia (AT) is a genetically transmitted autosomal recessive disorder characterized by variable degrees of immunodeficiency, telagiectasia (small blood vessels growing near the surface of the skin or eye), cerebellar ataxia (loss of balance due to abnormal development of the cerebellum) and increased sensitivity to both ionizing radiation and radiomimetic drugs, including bleomycin; AT cells are killed by lower doses of ionizing radiation or radiomimetic drugs than normal cells. Further, heterozygotes for mutant and normal AT alleles have radiation sensitivity close to that of homozygous normals. Therefore cancer cells from individuals heterozygous for null alleles of the AT gene (called ATM) should be highly susceptible to radiation therapy when only the deficient AT allele remains in cancer cells due to LOH, compared to normal cells from the same patients. Such patients could be treated by a DNA damage inducing procedure without the necessity for an allele specific inhibitor. Identification of such patients would require a test for heterozygosity at the target locus and a test for LOH which could show which allele is deleted in cancer cells. Such an approach would be expected to identify patients likely to respond well to strand breaking agents or procedures (such as exposure to ionizing radiation) even though they might have cancers not traditionally treated with such measures. In a related aspect, this approach is applicable to heterozygotes for other genes associated with ATM-mediated radiosensitivity. One such protein is the c-Abl protein tyrosine kinase, which binds to the ATM protein and regulates its function. c-Abl is known to be important in the stress response to ionizing radiation. One of its functions is activation of stress activated protein kinases (SAPKs) after irradiation or exposure to alkylating agents such as cw-platinum or mitomycin C, a response that is defective in ATM cells. Correction of the SAPK activation defect in ATM cells by non-mutant ATM cDNA suggests that the ATM - c-Abl interaction is necesary for the DNA damage response.

(Kharbanda, S., et al. Nature 376: 785-788, 1995.)

In a cancer patient with two alternative functional alleles for a component of ATM and LOH at the ATM locus, an allele specific inhibitory drug could be used to sensitize 328 232/116 cancer cells to the action of DNA damage inducing treatments such as ionizing radiation or radiomimetic drugs. Such an allele specific drug could be used to treat cancer patients constitutionally heterozygous for two normal ATM alleles in whom LOH had rendered cancer cells hemizygous or homozygous for one allele. Treatment would consist of the administration of the appropriate allele specific inhibitor plus a

DNA damage inducing treatment or procedure. The tumor cells would be unable to effectively the DNA damage, while the uninhibited allele in normal cells would be able to function. A similar approach could be taken to

The A TM gene is polymorphic

The ATM cDNA is 9.58 kb. Several likely polymoφhisms have been identified, although population studies have not yet been performed to determine allele frequencies. One of the reported polymoφhisms, an ATG to ATA change in codon 847, results in a methionine vs. isoleucine difference. Thus ATM is potentially targetable at the DNA, RNA and protein levels. It is likely that additional variances will be identified with broader population surveys and computational variance detection.

The ATM gene maps to chromosome llq23 and the c-Abl gene maps to 9q34.1, two regions of high frequency LOH in different cancer types

Chromosome 9q34 is lost in a high fraction of bladder, esophagus, ovary, head & neck and testis cancers (17 - 76%) and in a lesser fraction of breast, liver and prostate cancers and leukemias. Chromosome 1 lq23 is lost in brain, cervix, esophagus, breast, kidney, colon, stomach, head & neck and lung cancers at frequencies ranging from

16% to 100%.

Other proteins required for repair of DNA damage are also candidates for allele specific therapy of cancer 329 232/116

It will be evident to one skilled in the art that strategies similar to those described above for ATM and c-Abl could be undertaken for other proteins required for the stress response to DNA damaging agents, such as other stress activated protein kinases or downstream effector proteins.

330 232/116

Methylguanine Methyltransferase (MGMT) Gene VARIA 1534

Methylguanine methyltransferase (MGMT) is a suicide protein that repairs alkylating agent damage, specifically alkylation of the⁶O position of guanine. Alkyl groups are covalently bound to an active site cysteine (residue 145) of MGMT, thereby irreversibly inactivating the protein.⁶O-benzylguanine is an analog inhibitor of

MGMT that, by inactivating MGMT, renders tumor cells more sensitive to the toxic effects of methylating and chloroethylating agents. MGMT is thus a conditionally essential gene in the presence of such drugs.⁶O-benzylguanine is being developed as a chemosensitizing agent.

In a cancer patient with two alternative functional MGMT alleles an allele specific inhibitory drug could be used to sensitize cancer cells to the action of alkylating agents. Such an allele specific drug could be used to treat cancer patients constitutionally heterozygous for two normal MGMT alleles in whom LOH had rendered cancer cells hemizygous or homozygous for one allele. Treatment would consist of the administration of the appropriate allele specific inhibitor plus an alkylating agent. The tumor cells would be unable to effectively repair the alkylating agent induced DNA damage, while the uninhibited allele in normal cells would be able to function.

The MGMT gene is polymorphic

Several variances have been reported in human MGMT, or discovered by Variagenics, including three protein polymoφhisms. There is a silent C/T variance at position 255 (11% heterozygotes among 36 individuals surveyed), another C/T variance at nt. 346 331 232/116 which results in a L84F amino acid variance (5% heterozygotes), an A G variance at nt. 523 which results in a 1143 V amino acid variance (24% heterozygotes). A variance has been reported in Japanese at codon 160, GGA vs. AGA, converting glycine to arginine. 15% of the population studied were heterozygotes.

The alteration of glycine 160 to arginine reduced the inactivation by O6-benzylguanine with an approximately 20 fold increase in the IC50 concentration. These results demonstrate variance-specific effects of a small molecule, O6-benzylguanine, on normal (non-mutant) alleles of the conditionally essential MGMT gene.

Administration of 06 benzylguanine to patients who are heterozygous for the residue 160 gly/arg variance in their normal cells, and contain only the form of the gene with a glycine residue at position 160 in their cancer cells, together with methylating or chloroethylating agents for chemotherapy, will be specifically toxic to cancer cells without increasing toxicity to normal cells.

References

1. Imai, Y, Carcinogenesis (1995), 16:2441-24445

2. Edara, S. (1996) Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine. Cancer

Research 56, 5571-5575.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incoφorated by reference to the same extent as if each reference had been incoφorated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well 332 232/116 adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The groups of genes and the particular genes described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, those skilled in the art will readily recognize that the methods and inhibitors can utilize a variety of different target genes within the groups described. Thus, such additional embodiments are within the scope of the present invention and the following claims.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. 333 232/116

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

Thus, additional embodiments are within the scope of the invention and within the following claims.

Claims

334 232/116

What we claim is:

L A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:

(a) determining at least two alleles of a said gene, wherein said gene encodes a product required for cell proliferation;

(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles; wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

2. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:

(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival; (b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles; wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of 335 232/116 said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

3. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:

(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival;

4. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:

(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival;

(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles; wherein inhibition of expression of at least one but less than all of said alleles 336 232/116 or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

5. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:

(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival;

6. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:

(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures;

(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles; 337 232/116 wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

7. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of: (a) determining at least two alleles of a said gene, wherein said gene is located on a high frequency LOH chromosomal region;

8. The method of claim 7, wherein said gene is located on a chromosomal arm which has a frequency of allele loss of at least 15% in a cancer.

9. The method of claim 7, wherein said gene is located in proximity to a chromosomal marker which undergoes LOH at a frequency of at least 10% in a cancer.

10. The method of claim 7, wherein said gene is located in proximity to a tumor suppressor gene which undergoes LOH at a frequency of at least 10% in a cancer. 338 232/116

11. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of: (a) determining at least two alleles of a said gene, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene;

12. The method of claim 11, wherein said gene is located on a high frequency LOH chromosomal region.

13. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene encodes a product required for cell proliferation, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

14. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, said gene has at least two alternative 339 232/116 alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

15. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

16. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

17. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles. 340 232/116

18. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

19. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene is located on a high frequency LOH chromosomal arm region, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

20. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

21. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required for cell proliferation; and a pharmaceutically acceptable carrier or excipient. 341 232/116

22. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival; and a pharmaceutically acceptable carrier or excipient.

23. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival; and a pharmaceutically acceptable carrier or excipient.

24. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival; and a pharmaceutically acceptable earner or excipient.

25. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival; and a pharmaceutically acceptable canier or excipient. 342 232/116

26. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures; and a pharmaceutically acceptable carrier or excipient.

27. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene is located on a high frequency LOH chromosomal arm region; and a pharmaceutically acceptable carrier or excipient.

28. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene; and a pharmaceutically acceptable carrier or excipient.

29. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:

(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required for cell proliferation; 343 232/116

(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and

(c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

30. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:

(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival;

31. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:

(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival; 344 232/116

32. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:

(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival; (b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and

33. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of: (a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival;

(b) screening to identify an inhibitor which inhibits said at least one but less 345 232/116 than all of said at least two alternative alleles; and

34. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of: (a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures;

35. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:

(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene is located on a high frequency LOH chromosomal arm region;

(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and 346 232/116

36. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:

(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene;

37. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required for cell proliferation; and 347 232/116 wherein cells of said precancerous condition have undergone LOH of said first gene.

38. The method of claim 37, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

39. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

40. The method of claim 39, wherein the cells of said precancerous condition are 348 232/116 not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

41. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

42. The method of claim 41 , wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in 349 232/116 cells of said precancerous condition.

43. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

44. The method of claim 43, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

45. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are 350 232/116 heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

46. The method of claim 45, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

47. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain the integrity and function of cellular and subcellular structures; and wherein cells of said precancerous condition have undergone LOH of said first gene. 351 232/116

48. The method of claim 47, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

49. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene is located on a high frequency LOH chromosomal arm region; and wherein cells of said precancerous condition have undergone LOH of said first gene.

50. The method of claim 49, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for 352 232/116 each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

51. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene; and wherein cells of said precancerous condition have undergone LOH of said first gene.

52. The method of claim 51 , wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

53. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at 353 232/116 least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required for cell proliferation, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

54. The method of claim 53, further comprising the steps of:

(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or

(b) determining whether cancerous cells of said patient have only one allele of said particular gene; or

(c) both (a) and (b).

55. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: aώninistering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

56. The method of claim 55, further comprising the steps of:

(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).

57. A method for treating a patient suffering from a cancer, wherein said patient 354 232/116 is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

58. The method of claim 57, further comprising the steps of: (a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or

(c) both (a) and (b).

59. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

60. The method of claim 59, further comprising the steps of:

(b) determining whether cancerous cells of said patient have only one allele of said particular gene; or 355 232/116

(c) both (a) and (b).

61. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

62. The method of claim 61, further comprising the steps of:

(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determimng whether cancerous cells of said patient have only one allele of said particular gene; or

(c) both (a) and (b).

63. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

64. The method of claim 63, further comprising the steps of:

(a) determimng whether non-cancerous cells of said patient are 356 232/116 heterozygous for a particular gene essential for cell growth or viability; or

(c) both (a) and (b).

65. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene is located on a high frequency LOH chromosomal arm region, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

66. The method of claim 65, further comprising the steps of:

(b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).

67. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of: administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said 357 232/116 patient.

68. The method of claim 67, further comprising the steps of:

(c) both (a) and (b).

69. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required for cell proliferation, and wherein said inhibitor is less active on at least one other allele of said gene.

70. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.

71. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene. 358 232/116

72. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.

73. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.

74. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, and wherein said inhibitor is less active on at least one other allele of said gene.

75. A method of inhibiting growth of a cell comprising the step of: administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene is located on a high frequency LOH chromosomal arm region, and wherein said inhibitor is less active on at least one other allele of said gene.

76. A method of inhibiting growth of a cell comprising the step of: 359 232/116 administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, and wherein said inhibitor is less active on at least one other allele of said gene.

77. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: identifying a patient heterozygous for a said gene encoding a product required for cell proliferation, wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

78. The method of claim 77, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

79. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required for cell proliferation, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment. 360 232/116

80. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: identifying a patient heterozygous for a said gene encoding a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

81. The method of claim 80, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

82. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

83. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of: 361 232/116 identifying a patient heterozygous for a said gene encoding a product required to maintain organic compounds at levels compatible with cell growth or survival; wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

84. The method of claim 83, further comprising the step of determimng whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

85. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain organic compounds at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

86. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of: identifying a patient heterozygous for a said gene encoding a product required to maintain cellular proteins at levels compatible with cell growth or survival ; wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment. 362 232/116

87. The method of claim 86, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

88. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain cellular proteins at levels compatible with cell growth or survival , wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

89. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of: identifying a patient heterozygous for a said gene encoding a product required to maintain cellular nucleotides at levels compatible with cell growth or survival ; wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

90. The method of claim 89, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment. 363 232/116

91. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

92. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of: identifying a patient heterozygous for a said gene encoding a product required to maintain the integrity and function of cellular and subcellular structures ; wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

93. The method of claim 91 , further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

94. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a 364 232/116 said gene encoding a product required to maintain the integrity and function of cellular and subcellular structures, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

95. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of: identifying a patient heterozygous for a said gene located on a high frequency

LOH chromosomal arm region ; wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

96. The method of claim 95, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

97. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene located on a high frequency LOH chromosomal arm region, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

98. A method of identifying a potential patient for treatment with an inhibitor 365 232/116 active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of: identifying a patient heterozygous for a said gene which has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene; wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

99. The method of claim 98, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

100. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene which has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

101. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required for cell proliferation, wherein said portion comprises a sequence variance site, and wherein said probe 366 232/116 hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

102. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a conesponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

103. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a conesponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

104. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain cellular 367 232/116 proteins at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a conesponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

105. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

106. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

107. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or 368 232/116 viability, wherein said gene is located on a high frequency LOH chromosomal arm region, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

108. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

109. The method .inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 1, 13, 21, 29, 37, 53, 69, 77, and 101, wherein said gene is selected from the group consisting of 14-3-3 Protein TAU, CCNA(G2/Mitotic-Specific Cyclin

A ), CCNBl(G2/Mitotic-Specific Cyclin Bl), CCND1(G1/S-Specific Cyclin Dl), CCND2(G1/S-Specific Cyclin D2), CCND3(G1/S-Specific Cyclin D3), Cell division control protein 16, Cell division cycle 2, GI to S and G2 to M, Cell division cycle 25 A, Cell division cycle 25B, Cell division cycle 25C, Cell division cycle 27, Cell division- associated protein BIMB, Cyclin Al(G2/Mitotic-Specific Cyclin Al), Cyclin C

(Gl/S-Specific Cyclin C), Cyclin Gl(G2/Mitotic-Specific Cyclin G), Cyclin G2 (G2/Mitotic-Specific Cyclin G), Cyclin H, Cyclin H Assembly, GSPT1(G1 to S phase transition 1), Mitotic MAD2 Protein, MRNP7, RANBP1(RAN binding protein 1), WEE1, Cell Division Protein Kinase A, CDC28 protein kinase 1, CDC28 protein 369 232/116 kinase 2, M-Phase inducer phosphatase 2, M-phase phosphoprotein, mpp6, PPPlca(Protein phosphatase 1, catalytic subunit, alpha isoform), STM7-LSB, CENP-F kinetochore protein, Centromere autoantigen C, Centromere protein B (80kD), Centromere protein E (312kD), CHC1 (Chromosome condensation 1), Chromatin assembly factor-I pl50 subunit, Chromatin assembly factor-I p60 subunit, Chromosome segregation gene homolog CAS, HMGl (High-mobility group (nonhistone chromosomal) protein 1), Minichromosome Maintenance (MCM7), Mitotic centromere-associated kinesin, RMSAl (Regulator of mitotic spindle assembly 1), and SUPT5h(Chromatin structural protein homolog (SUPT5H)).

110. The method , inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 2, 14, 22, 30, 39, 55, 70, 80, and 102, wherein said gene is selected from the group consisting of PMCA1 (Calcium Pump), PMCA2 (Calcium Pump), PMCA3 (Calcium Pump), PMCA4 (Calcium Pump), ATP2bl (Calcium-Transporting ATPase Plasma Membrane), ATP2b2 (Calcium-Transporting ATPase Plasma

Membrane), ATP2b4 (Calcium-Transporting ATPase Plasma Membrane), ATP5b (ATP Synthase Beta Chain, Mitochondrial Precursor), Chloride Conductance Regulatory Protein ICLN, H-Erg (Potassium Channel Protein EAG), Nuclear Chloride Ion Channel Protein (NCC27), SCNlb(Sodium Channel, Voltage-Gated, Type I, Beta Polypeptide), Two P-Domain K+ Channel TWIK-1 , VDAC2 (Voltage-

Dependent Anion-Selective Channel Protein 2), ATPlbl (Sodium/Potassium- Transporting ATPase Beta-1 Chain), ATPlb2 (Sodium/Potassium-Transporting ATPase Beta-2 Chain), ATPase, Ca+ + transporting, plasma membrane 4, ATPase, Ca+ + transporting, plasma membrane 2, ATPase, Na+/K+ transporting, alpha 1 polypeptide, ATPase, Na+/K+ transporting, alpha 3 polypeptide, ATPase,

Na+/K+ transporting, beta 1 polypeptide, ATPase, Na+/K+ transporting, beta 2 polypeptide, Na+,K+ ATPase, 1 Subunit, Na+,K+ ATPase, 2 alpha, Na+ ,K+ ATPase, 3 beta, SLC9al (Solute carrier family 9 (sodium/hydrogen exchanger)), Solute earner family 4, anion exchanger, member 1, Solute canier family 4, anion 370 232/116 exchanger, member 2, Solute canier family 9 (sodium/hydrogen exchanger), Passive transporters, MaxiK Potassium Channel Beta Subunit, Chloride Channel 2, Chloride Channel Protein (CLCN7), TRPCl (Transient Receptor Potential Channel 1), Potassium Channel Kv2.1, ATP5d(ATP synthase, H+ transporting, mitochondrial Fl complex, delta subunit), ATP5fl(ATP synthase, H+ transporting, mitochondrial

F0 complex, subunit b), ATP5o(ATP synthase, H+ transporting, mitochondrial Fl complex, O subunit), ETFa(Electron-transfer-flavoprotein, alpha polypeptide (glutaric aciduria II)), ETFb(Electron-transfer-flavoprotein, beta polypeptide), Nadh- ubiquinone oxidoreductase 13 kd-B subunit, Nadh-ubiquinone oxidoreductase 39 kD subunit precursor, NADH-Ubiquinone oxidoreductase 75 kD subunit precursor,

NADH-Ubiquinone oxidoreductase MFWE subunit, NDUFV2(NADH dehydrogenase (ubiquinone) flavoprotein 2 (24kD)), Ubiquinol-cytochrome c reductase complex 11 kD, ATP Synthase Alpha Chain, NADH dehydrogenase- ubiquinone Fe-S protein 8, 23 kDa subunit, Ascorbic Acid (transporter), Folate Binding Protein, Folate receptor 1 (adult), Nicotinamide (transporter), Pantothenic

Acid transporter, Riboflavin (transporter), SCL19A1 (Solute Carrier Family 19, Member 1), Solute canier family 19 (folate transporter), member 1, Thiamine, B6, B12 (transporter), ATP7b (Copper-Transporting ATPase 2), Ceruloplasmin (ferroxidase), Ceruloplasmin receptor (Copper Transporter), Copper Transport Protein HAH1, Molybdenum, Selenium, Tranfenin Receptor (Iron Transporter),

Zinc Transporter, and mitochondrial import receptor subunit TOM20.

111. The method .inhibitor, pharmaceutical composition, or nucleic acid probe of 3, 25, 23, 31, 41, 57, 71, 83, and 103, wherein said gene is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, Solute carrier family 5 (sodium/glucose cotransporter), Solute canier family 2 (facilitated glucose transporter), member 2, Solute canier family 2 (facilitated glucose transporter) member 5, Solute canier family 3 member 1, System b,(Na+ independent), System y,(Na+ independent), ATRCl(Catioinc), LEUT(Leucine Transporter), 371 232/116

SLClAl (Solute Carrier Family 1, Member 1), Solute carrier family 16 (monocarboxylic acid transporters), ACOl(Aconitase 1), ACO2(Aconitase 2, mitochondrial), Acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain, Acyl- Coenzyme A dehydrogenase, C-4 to C-12 straight chain, Acyl-Coenzyme A dehydrogenase, long chain, Acyl-Coenzyme A dehydrogenase, very long chain, aKGD (alpha ketoglutaratedehydrogenase), ALD-a (Aldolase), ALD-b (Aldolase), ALD-c (Aldolase), CS (Citrate Synthetase), Dihydrolipoamide S-succinyltransferase, DLAT(Dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex)), DLD(Dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex)), Elk (Oxoglutarate dehydrogenase), E2k (Dihydrolipoamide S-succinyltransferase), E3 (Dihydrolipoyl Dehydrogenase), ENOl(Enolase 1, alpha), ENO2(Enolase 2), ENO3(Enolase 3), Enolase 2, (gamma, neuronal), Enolase 3, (beta, muscle), FH(Fumarate hydratase), G3PDH (Glyceraldehyde-3-Phosphate Dehydrogenase), G6PD (Glucose-6-Phosphate

Dehydrogenase), Glucose-6-phosphate dehydrogenase, HK1 (Hexokinase 1), HK2 (Hexokinase 2), HK3 (Hexokinase 3), IDHl(Isocitrate dehydrogenase 1 (NADP+), soluble), IDH2(Isocitrate dehydrogenase 2 (NADP+), mitochondrial), MDHKMalate dehydrogenase 1, NAD (soluble)), MDH2(Malate dehydrogenase 1, NAD (mitochondrial)), NAD(H)-specific isocitrate dehydrogenase alpha subunit,

Oxoglutarate dehydrogenase (lipoamide), PDHB (Pyruvate Dehydrogenase), PDHB(Pyruvate dehydrogenase (lipoamide) beta), PDK4 (Pyruvate dehydrogenase kinase, isoenzyme 4), PFKL(Phosρhofructokinase), PGI (Phosphoglucoisomerase), PGKa (Phosphoglyceromutase), PGKb (Phosphoglyceromutase), PGM1 (Phosphoglyceromutase), PGM2 (Phosphoglyceromutase), PGM3

(Phosphoglyceromutase), PGM4 (Phosphoglyceromutase), Phosphofructokinase, muscle, Phosphoglucomutase 1, Phosphoglycerate kinase 1, PK1 (Pyruvate Kinase), PK2 (Pyruvate Kinase), PK3 (Pyruvate Kinase), Pyruvate dehydrogenase kinase isoenzyme 2 (PDK2), Pyruvate kinase, liver, Pyruvate kinase, muscle, 372 232/116

SDHl(Succinate dehydrogenase, iron sulphur (Ip) subunit), SDH2(Succinate dehydrogenase 2, flavoprotein (Fp) subunit), TKT(Transketolase (Wernicke- Korsakoff syndrome)), TPI (Trisephosphate Isomerase), Asparagine Synthetase, Aminoacylase-1, Aminoacylase-2, ACAC (Acetyl CoA Carboxylase Beta), ACAC (Acetyl CoA Carboxylase) , AC ADSB( Acy 1-co A dehydrogenase) , Mevalonate kinase ,

Phosphomevalonate kinase, Aspartoacylase, Ornithine decarboxylase 1, Short-acyl- CoA dehydrogenase, Medium acyl-CoA dehydrogenase, Long acyl-CoA dehydrogenase, Isovalveryl CoA dehydrogenase, 2-methyl branched chain, Adenosine Deaminase, Purine-nucleoside phosphorylase, Guanine Deaminase, Xanthine Oxidase, ITM1 (Integral Transmembrane Protein), GFPT (Glutamine-

Fructose-6-Phosphate Transaminase), Heparan, Polypeptide N- Acetyltransferase, ACAA(Acetyl-Coenzyme A acyltransferase), Lysophosphatidic acid acyltransferase- alpha, Lysophosphatidic acid acyltransferase-beta, FNTa (Farnesyltransferase Alpha Subunit), FNTb (Farnesyltransferase Beta Subunit), NMT1 (N-myristoyltransferase), Calcineurin A, Calcineurin B, Calreticulin Precursor, Phosphatase 2b,

PPP3ca(Protein phosphatase 3 , catalytic subunit), SNK Interacting 2-28(Calcineurin B Subunit), Protein Kinase C, PRKCA(Protein kinase C, alpha), PRKCBl (Protein kinase C, beta 1), PRKCD(Protein kinase C, delta), PRKCM(Protein kinase C, mu), PRKCQ(Protein kinase C-theta), PRKCSH(Protein kinase C substrate 80K-H), Geranylgeranyl, Geranylgeranyltransferase (Type I Beta), GGTB

(Geranylgeranyltransferase), Geranylgeranyltransferase (Type π Beta-Subunit), Gdp Dissociation Inhibitors, GDI Alpha (RAB GDP Dissociation Inhibitor Alpha), and Rab Gdp (RAB GDP Dissociation Inhibitor Alpha).

112. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 4, 16, 24, 32, 43, 59, 72, 86, and 104, wherein said gene is selected from the group consisting of GOT(Glutamic-oxaloacetic transaminase 2), GOTl(Glutamic-oxaloacetic transaminase 1), PYCS(Pynoline-5-carboxylate synthetase), Tyrosine aminotransferase, AARS, CARS, DARS, EPRS, FARS, 373 232/116

GARS, HARS, IARS, KARS, LARS, MARS, NARS, QARS , RARS, SARS, TARS, VARS, WRS, YARS, Ribosomal Protein Lll, Ribosomal Protein L12, Ribosomal Protein L17, Ribosomal Protein L18, Ribosomal Protein L18a, Ribosomal Protein L19, Ribosomal Protein L21, Ribosomal Protein L22, Ribosomal Protein L23, Ribosomal Protein L23a, Ribosomal Protein L25, Ribosomal Protein

L26, Ribosomal Protein L27, Ribosomal Protein L27a, Ribosomal Protein L28, Ribosomal Protein L29, Ribosomal Protein L30, Ribosomal Protein L31, Ribosomal Protein L32, Ribosomal Protein L35, Ribosomal Protein L35a, Ribosomal Protein L36a, Ribosomal Protein L39, Ribosomal Protein L4, Ribosomal Protein L41, Ribosomal Protein L44, Ribosomal Protein L6, Ribosomal Protein L7, Ribosomal

Protein L7a, Ribosomal Protein L8, Ribosomal Protein L9, Ribosomal Protein Pl, Ribosomal Protein S10, Ribosomal Protein Sll, Ribosomal Protein S13, Ribosomal Protein S14, Ribosomal Protein S15, Ribosomal Protein S15A, Ribosomal Protein S16, Ribosomal Protein S17, Ribosomal Protein S17A, Ribosomal Protein S17B, Ribosomal Protein S18, Ribosomal Protein S20, Ribosomal Protein S20A,

Ribosomal Protein S20B, Ribosomal Protein S21, Ribosomal Protein S23, Ribosomal Protein S25, Ribosomal Protein S26, Ribosomal Protein S28, Ribosomal Protein S29, Ribosomal Protein S3, Ribosomal Protein S3 A, Ribosomal Protein S4, Ribosomal Protein S4X, Ribosomal Protein S4Y, Ribosomal Protein S5, Ribosomal Protein S6, Ribosomal Protein S7, Ribosomal Protein S8, Ribosomal Protein S9,

Initiation of polypeptide polymerization, eIF-2 (Eukaryotic initiation factor), eIF-2- associated p67 (Eukaryotic initiation factor), eIF-2A(Eukaryotic initiation factor), eIF-2Alpha(Eukaryotic initiation factor), eIF-2B(Eukaryotic initiation factor), elF- 2B-Gamma(Eukaryotic initiation factor), eIF-2Beta(Eukaryotic initiation factor), elF- 3 pllO(Eukaryotic initiation factor), eIF-3 p36(Eukaryotic initiation factor), elF-

4A(Eukaryotic initiation factor), eIF-4C(Eukaryotic initiation factor), elF- 4E(Eukaryotic initiation factor), eIF-4Gamma(Eukaryotic initiation factor), elF- 5 (Eukaryotic initiation factor), eIF-5A, Eukaryotic peptide chain release factor subunit 1, P97(Eukaryotic initiation factor), eEFlA2(Eukaryotic elongation factor), 374 232/116 eEFlD(Eukaryotic elongation factor), eEF2(Eukaryotic elongation factor), eIF4A2 (Eukaryotic initiation factor), KIAA0031 (Elongation factor 2), KIAA0219(Putative translational activator C18G6.05C), Factor 1 -Alpha 2(Eukaryotic translation elongation factor 1 alpha 2), Cis-Trans Isomerase, DNAj Protein Homolog 1, DNAj Protein Homolog 2, DNAJ Protein homolog HSJ1, T-Complex,

Aspartylglucosaminidase, T-Complex 1, Alpha, T-Complex 1, Epsilon, T-Complex 1, Gamma, T-Complex 1, Theta, T-Complex 1, Zeta, 26S Protease regulatory subunit 4, Alpha-2-Macroglobulin, Calpain 1, Large, CLPP(ATP-Dependent CLP protease proteolytic subunit), KIAA0123 (Mitochondrial processing peptidase alpha subunit), MMP7, Proteasome Beta 6, Proteasome Beta 7, Proteasome C13,

Proteasome C2, Proteasome C7-1, Proteasome inhibitor hPI31 subunit, Proteasome P112, Proteasome P27, Proteasome P55, Enzyme E2-17 Kd(Cyclin-selective ubiquitin carrier protein), ISOT-3 (Ubiquitin carboxyl-terminal hydrolase T ), ORF (Ubiquitin carboxyl-terminal hydrolase 14), PGP(Ubiquitin carboxyl-terminal hydrolase isozyme Ll), UBA52(Ubiquitin A-52 residue ribosomal protein fusion product 1), Ubiquitin carboxyl-terminal hydrolase 3, Ubiquitin carboxyl-terminal hydrolase isozyme L3, Ubiquitin carboxyl-terminal hydrolase T, Ubiquitin canier protein (E2-EPF), Ubiquitin fusion-degradation protein (UFDIL), Ubiquitin Hydrolase, Ubiquitin-conjugating enzyme E2I, SEC23 (Protein transport protein SEC23), SEC23A(Protein transport protein SEC23), SEC7(Protein transport protein

SEC7), SEC61 (Beta Subunit), and LDLR (LDL receptor).

113. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 5, 17, 25, 33, 45, 73, 89, and 105, wherein said gene is selected from the group consisting of Adenylate Kinase-2, Adenylosuccinate synthetase,

Adenylosuccinate Lyase, DPRT (ADP-Ribosyltransferase), ADSL (Adenylosuccinate lyase/AMP synthetase), ADSS (Adenylosuccinate Synthetase), CAD PROTEIN, CTP Synthetase, CTPS(CTP synthetase), Cytidine Triphosphate Synthetase, GARS (Phosphoribosylglycinamide synthetase), GART (Phosphoribosylglycinamide 375 232/116 f ormy ltransferase) , G ART(Phosphor ibosy lglycinamide formyltransferase , phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase), GMP Synthetase, IMP Cyclohydrolase, IMP dehydrogenase, IMPDH1(IMP (inosine monophosphate) dehydrogenase 1), IMPDH2(IMP (inosine monophosphate) dehydrogenase 2), Phosphoribosyl diphosphotransferase,

Phosphor ibosy laminoimidazolecarboxamide formyltransferase ,

Phosphoribosy lformy lglycinamide synthetase , Phosphoribosylglycinamide carboxylase, Phosphoribosy lglycinamide-succinocarboxamide synthetase, PPAT (Amidophoribosy ltransferase), PPAT (Phosphoribosyl pyrophosphate amidotransferase), Ribonucleoside-diphosphate reductase Ml chain, Ribonucleoside- diphosphate reductase M2 chain, Thymidine Kinase, Thymidylate Synthase, UMK(Uridine kinase), UMPK (Uridine monophosphate kinase), UMPS(Uridine monophosphate synthetase (orotate phosphoribosyl transferase and orotidine-5'- decarboxylase)), Uridine Phosphorylase, DNA Origin Recognition Complex, ORC1, ORC2, ORC3, ORC4, ORC5, ORC6, ORC Regulators, CDC6, CDC7, CDC1,

DNA Polymerization, DNA Polymerases, Adprt (NAD(+) ADP- Ribosyltransferase), DNA Polymerase Alpha-Subunit, DNA Polymerase Delta, POLa(DNA Polymerase Alpha/Primase Associated Subunit), POLb(DNA Polymerase Beta Subunit), POLdl (Polymerase (DNA directed), Delta 1, Catalytic Subunit), POLd2(Polymerase (DNA directed), Delta 2), POLE(Polymerase (DNA directed)), POLg (DNA Polymerase Gamma Subunit), Terminal Transferase (DNA Nucleotidylexotransferase), Activator 1 36 Kd, CDC46 (DNA Replication Licensing Factor), CDC47 (DNA Replication Licensing Factor CDC47), DNA Topoisomerase III, DRAP1 (DNA Replication Licensing Factor MCM3), KIAA0030 Gene (Cell Division Control Protein 19), KIAA0083 Gene (DNA Replication Helicase DNA2

), MCM3 (DNA Replication Licensing Factor MCM3), PCNA (Proliferating Cell Nuclear Antigen), PRIM1 (DNA Primase 49 kD Subunit ), PRIM2 (DNA Primase), PRIM2a (DNA Primase 58 kD Subunit), PRIM2b (DNA Primase), RECa (Replication Protein A 14 kD Subunit), RFC1 (Replication Factor C (activator 1) 1), 376 232/116

RFC2 (Replication Factor C 2), RFC3 (Replication Factor C (activator 1) 3), RFC4 (Replication Factor C, 37-kD subunit), RFC5 (Replication Factor C), RPA1 (Replication protein Al (70kD)), RPA2 (Replication protein A2 (32kD)), RPA3 (Replication protein A3 (14kD)), TOPI (DNA Topoisomerase I), TOP2a (Topoisomerase (DNA) II Alpha (170kD)), TOP2b (Topoisomerase (DNA) II Beta

(180kD)), CHL1(CHL1-Related Helicase), DNA Helicase II, Mi-2(Chromodomain- Helicase- DNA-Binding Protein CHD-1 ), RECQL (ATP-Dependent DNA Helicase Ql), Smbp2 (DNA-Binding Protein SMUBP-2), H1(0) (Histone H5A), Histone Hid, Histone Hlx, Histone H2a.l, Histone H2a.2, Histone H2b.l, Histone H4, SLBP (Histone Haiφin-Binding Protein), TATA-binding Complex, Small Nuclear RNA-

Activating Complex, Polypeptide 1, 43KD (SNAPC1), Small Nuclear RNA- Activating Complex, Polypeptide 2, (SNAPC2), Small Nuclear RNA Activating Complex, Polypeptide 3, 50KD (SNAPC3), TAF2D(TBP-associated factor), TAFH100(TBP-associated factor), TAFII130(TBP-associated factor), TAFH20(TBP- associated factor), TAFII250(TBP-associated factor), TAFII28(TBP-associated factor), TAFII30(TBP-associated factor), TAFII32(TBP-associated factor), TAFII40(TBP-associated factor), TAFII55(TBP-associated factor), TAFII80(TBP- associated factor), TBP(TATA Binding Protein), TMF1 (TATA Element Modulatory Factor 1), RPB 7.0, RPB 7.6, RPB 17, RPB 14.4, RNA polymerase I subunit hRPA39, 13.6 Kd Polypeptide (DNA-Directed RNA Polymerase II 13.6 kD

Polypeptide), POLR2C(RNA polymerase H, polypeptide C (33kD)), Polypeptide A (220kd), RNA Polymerase II 23k, RNA polymerase II holoenzyme component (SRB7), RNA polymerase II subunit (hsRPBlO), RNA polymerase II subunit (hsRPBδ), RNA polymerase II subunit hsRPB4, RNA polymerase II subunit hsRPB7, RNA Polymerase II Subunit(DNA- Directed RNA Polymerases I, II, and

III 7.3 kD polypeptide), TCEBlL(Transcriρtion elongation factor B (SIII), polypeptide 1-like), RNA polymerase III subunit (RPC39), RNA polymerase III subunit (RPC62), Elongation Factor 1-Beta, Elongation Factor S-H, TCEA (HOkD), TCEB1, TCEB (18kD), TCEB1L, TCEB3, TCEC (15kDa), TFIIS (Transcription 377 232/116

Elongation Factor IIS), E2F1 (E2F Transcription Factor), TFAP2A (Transcription Factor A2 Alpha), TFCP2 (Transcription Factor CP2), TFC12 (Transcription Factor 12), PRKDC (Protein Kinase, DNA activated catalytic subunit), SUPT6H, TFIIA gamma subunit, TFIIA delta, TFIIB related factor hBRF (HBRF), TFIIE Alpha Subunit, TFIIE Beta Subunit, TFIIF, Beta Subunit, GTF2F1 (TFIIF), GTF2F2

(TFIIF), General Transcription Factor IIIA, TFIIH(52 kD subunit of transcription factor), TFIIH(p89), TFIIH(p80), TFIIH(p62), TFIIH(p44), TFIIH(p34), Transcription Factor Ilf (General transcription factor IIF, polypeptide 1 (74kD subunit))Transcription Factor Ilf (General transcription factor IIF, polypeptide 1 (74kD subunit)), BTF 62 kDSubunit (Basic transcription factor 62 kD subunit),

CAMP-dependent transcription factor ATF-4, CCAAT box-binding transcription factor 1, CRM1 (Negative regulator CRM1), Cyclic- AMP-dependent transcription factor ATF-1, GABPA(GA-binding protein transcription factor, alpha subunit (60kD)), ISGF-3 (Signal transducer and activator of transcription 1 -alpha/beta), NFIX(Nuclear factor I/X (CCAAT-binding transcription factor)), NFYA(Nuclear transcription factor Y, alpha), NTF97(Nuclear factor p97), Nuclear factor I-B2 (NFD32), Nuclear factor NF45, Nuclear factor NF90, POU2Fl(POU domain, class 2, transcription factor 1), Sp2 transcription factor, TCF12(Transcription factor 12 (HTF4, helix-loop-helix transcription factors 4)), TCF3(Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47)), TCF6L1 (Transcription factor

6-like 1), TF P65(Transcriρtion factor p65), TFCOUP2(Transcription factor COUP 2 (a.k.a. ARP1)), Transcription factor IL-4 Stat, Transcription Factor S-II (Transcription factor S-H-related protein), Transcription factor Stat5b, Transcription Factor, Transcription factor (CBFB), 9G8 Splicing Factor (Pre-mRNA Splicing factor SRP20), CC 1.3 (Splicing factor (CO.3)), HnRNP F protein,

HNRPA2B1 (Heterogeneous nuclear ribonucleoproteins A2/B1),

HNRPG(Heterogeneous nuclear ribonucleoprotein G), HNRPK(Heterogeneous nuclear ribonucleoprotein K), Pre-mRNA splicing factor helicase, Pre-mRNA splicing factor SF2, P33 subunit, Pre-mRNA splicing factor SRP20, Pre-mRNA 378 232/116 splicing factor SRP75, PRP4(Serine/threonine-protein kinase PRP4), PTB-Associated Splicing Factor, Ribonucleoprotein A' , Ribonucleoprotein Al , Ribonucleoprotein C1/C2, RNP Protein, L (Heterogeneous nuclear ribonucleoprotein L), RNP-Specific C(U1 small nuclear ribonucleoprotein C ), SAP 145(Spliceosome associated protein ), SAP 61(Splicesomal protein), SC35(Splicing factor), SF3al20, SFRS2(Splicing factor, arginine/serine-rich 2), SFRS5 (Splicing factor, arginine/serine-rich 5), SFRS7(Splicing factor, arginine/serine-rich 7), Small nuclear ribonucleoprotein SM Dl, SnRNP core protein Sm D2, SnRNP core protein Sm D3, SNRP70(U1 snRNP 70K protein), SNRPB(Small nuclear ribonucleoprotein polypeptides B and Bl), SNRPE(Small nuclear ribonucleoprotein polypeptide E), SNRPN(Small nuclear ribonucleoprotein polypeptide N), Splicing factor SF3al20, Splicing factor U2AF 35 kD subunit, Splicing factor U2AF 65 kD subunit, SRP30C(Pre-mRNA splicing factor SF2, p33 subunit), SRP55-2(Pre-mRNA splicing factor SRP75), Transcription factor BTEB, Transcription initiation factor TFIID 250 kD subunit, Cleavage and polyadenylation specificity factor, Cleavage stimulation factor, 3' pre-RNA, subunit

1, 50kD, Cleavage stimulation factor, 3' pre-RNA, subunit 3, 77kD, HNRNP Methyltransferase, PABPLl(Poly(A)-binding protein-like 1), Pap mRNA(Poly(A) Polymerase), RNA unwinding, RNA Helicase, GU Protein (ATP-Dependent RNA helicase dead), KIAA0224 Gene(Putative ATP-dependent RNA helicase), RNA Helicase A, RNA Helicase P110, and Stel3(Nuclear RNA Helicase).

114. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 6, 18, 26, 34, 47, 63, 92, and 106, wherein said gene is selected from the group consisting of AP47(Clathrin Coat Assembly AP47), AP50(Clathrin Coat Assembly Protein AP50), Cell Surface Protein (Clathrin Heavy Polypeptide-Like

Protein ), Cltb(Clathrin Light Chain B), Cite (Clathrin Heavy Chain), Adenylate Cyclase, Adenylate Cyclase, Adenylate Cyclase, π, Adenylate Cyclase,IV, Complex I, MTND1 (Subunit ND1), MTND2 (Subunit ND2), MTND3 (Subunit ND3), MTND4 (Subunit ND4), MTND4L (Subunit ND4L), MTND5 (Subunit ND5), 379 232/116

MTND6 (Subunit ND6), Complex H, Complex HI, Cytochrome b subunit, Complex IV, COl (Cytochrome c Oxidase Subunit I), CO2 (Cytochrome c Oxidase Subumt 2), CO3 (Cytochrome c Oxidase Subunit 3), Complex V, ATP Synthase Subunit ATPase 6, Kinesin Heavy Chain, Kinesin Light Chain, Syntaxin la, Syntaxin lb, Syntaxin 3, Syntaxin 5a, Syntaxin 7, CANX (Calnexin), ER Lumen Protein 1, ER

Lumen Protein 2, Ribophorin I, Ribophorin H, Signal recognition particle receptor, SRP Protein, TIM17 preprotein translocase, Golgin-245, TGN46 (Trans-Golgi Network Integral Membrane Protein TGN38 Precursor), Beta-Cop, Coatomer Beta' Subunit, Coatomer Delta Subunit, Gp36b Glycoprotein (Vesicular integral-membrane protein VIP36 precursor), Homologue of yeast sec7, Protein transport protein SEC13

(Chromosome 3p25), SEC14 (S. Cerevisiae), Synaptic vesicle membrane protein VAT-1, Synaptobrevin-3, Synaptotagmin I, Transmembrane(COP-coated vesicle membrane protein p24 precursor), Vacuolar-Type (Clathrin-coated vesicle/synaptic vesicle proton pump 116 kd subunit ), 140 kD Nucleolar phosphoprotein, Autoantigen p542, Export protein Rael (RAEl), Heterogeneous nuclear ribonucleoprotein Al , Nuclear pore complex protein hnupl53, Nuclear pore complex protein NUP214, Nuclear pore glycoprotein p62, Nuclear Transport Factor 2, Nucleoporin 98 (NUP98), NUP88, Ribonucleoprotein A, Ribonucleoprotein B", Karyopherin, Importin Alpha Subunit, TRN (Transportin), Actin, Beta-Centractin, Capping Protein Alpha, CFL1 (Cofilin, Non-Muscle Isoform), Desmin, Dystrophin,

Gelsolin, hOGGl (Myosin Light Chain Kinase), IC Heavy Chain, Itga2 (Integrin, Alpha 2 (CD49B, alpha 2 Subunit of VLA-2 receptor)), Itga3 (Integrin Alpha-3 Precursor), Keratin 19, Keratin, Type H, Lamin A, LBR(Lamin B Receptor), Light Chain Alkali, MacMarcks mRNA, MAPla (Microtubule-Associated Protein 1A), MAP2(Microtubule-Associated Protein 2), MEGl(Protein-Tyrosine Phosphatase

MEG1), Microtubule-Associated Protein TAU, Suppressor Of Tubulin STU2, TUBg (Tubulin Gamma Chain), Tubulin Alpha-4 Chain, USHlb (Myosin H Heavy Chain), Villin, Villin 2 (Ezrin), Actin Depolymerizing, Capping (Actin Filament), MYH9(Myosin, Heavy Polypeptide 9, Non-Muscle), MYL5(Myosin Regulatory 380 232/116

Light Chain 2), Myosin Heavy Chain 95F, Myosin Heavy Chain IB, Myosin IB, Sh3pl7(Myosin IC Heavy Chain), Sh3pl8(Myosin IC Heavy Chain), KIAA0059(Dematin:Actin-Bundling Protein), TTN (Titi Myosin Light Chain Kinase), ATP6c(Vacuolar H+ ATPase proton channel subunit), ATPόal (ATPase, H+ Transporting, Lysosomal (Vacuolar Proton Pump), Alpha Polypeptide, 70kD),

ATPόbl (ATPase, H+ transporting, lysosomal (vacuolar proton pump), beta polypeptide, 56/58kD), ATP6d(ATPase, H+ transporting, lysosomal (vacuolar proton pump) 42kD), ATP6e( ATPase, H+ transporting, lysosomal (vacuolar proton pump) 31kD), ATPase, H+ transporting, lysosomal (vacuolar proton pump) 31kD, and Superoxide Dismutase.

115. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a conditionally essential gene, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of: (a) determining at least two alleles of a said gene;

116. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a conditionally essential gene, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

117. A pharmaceutical composition, comprising 381 232/116 at least one allele specific inhibitor targeting at least one but less than all allelic forms of a conditionally essential gene in a population; and a pharmaceutically acceptable canier or excipient.

118. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a conditionally essential gene having at least two alternative alleles, comprising the steps of:

(a) identifying a conditionally essential gene that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell;

(c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in whom cancerous cells have only an allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene and contain an allelic form not inhibited by said inhibitor.

119. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of: a. subjecting cells of said precancerous condition to an altered condition such that a first conditionally essential becomes essential; b. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of said first conditionally essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells; and 382 232/116 wherein cells of said precancerous condition have undergone LOH of said first gene.

120. The method of claim 119, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of: c. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of a conditionally essential gene or an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different gene, and wherein said patient is heterozygous for each targeted gene and each targeted gene has undergone LOH in cells of said precancerous condition.

121. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a conditionally essential gene, comprising the steps of: a) subjecting cells of said cancer to altered conditions such that said gene is essential; and administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.

122. The method of claim 121, further comprising the steps of: (a) determining whether non-cancerous cells of said patient are heterozygous for a particular conditionally essential gene; or

(c) both (a) and (b). 383 232/116

123. A method of inhibiting growth of a cell comprising the steps of: a) subjecting said cell to conditions such that said gene is essential; and b) administering at least one inhibitor active on an allele of said conditionally essential gene, wherein said inhibitor is less active on at least one other allele of said gene.

124. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a conditionally essential gene, wherein said patient is suffering from a cancer, said method comprising the step of: identifying a patient heterozygous for a said gene, wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

125. The method of claim 124, further comprising the step of deteπnining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

126. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a conditionally essential gene, wherein said patient is suffering from a cancer, said method comprising the step of: determining whether cancer cells in said patient have undergone LOH of a said gene, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

126. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a conditionally essential gene, wherein said portion comprises a sequence variance site, and wherein 384 232/116 said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

127. A method for selecting a patient for treatment with an antiproliferative treatment, comprising the steps of: a) determining whether normal somatic cells in a potential patient are heterozygous for an essential or conditionally essential gene, wherein a first allelic form of said gene is more active than a second allelic form, and wherein a reduction in the activity of said gene in a cell increases the sensitivity of said cell to a said antiproliferative treatment; and b) determining whether cancer cells of said patient have only said second allelic form of said gene, wherein if said somatic cells are heterozygous and said cancer cells have only said second allelic form, it is indicative that said patient is suitable for treatment with said antiproliferative treatment.

128. A method for selecting an antiproliferative treatment for a patient suffering from a cancer, comprising the steps of: a) determimng whether normal somatic cells in a potential patient are heterozygous for an essential or conditionally essential gene which reduces the sensitivity of cells to an antiproliferative treatment, wherein a first allelic form of said gene is more active than a second allelic form, and wherein a reduction in the activity of said gene in a cell increases the sensitivity of said cell to a said antiproliferative treatment; and b) determining whether cancer cells of said patient have only said second allelic form of said gene, wherein if said somatic cells are heterozygous for said gene and said cancer cells have only said second allelic form, it is indicative that said antiproliferative 385 232/116 treatment is suitable for said patient.

129. The method of any of claims 115-129, wherein said gene is selected from the group consisting of: galactose- 1 -phosphate uridy ltransferase, galactose kinase, UDP galactose-4- epimerase, methionine synthase, asparagine synthase, glutamine synthetase, multidrug resistance gne/Pglycoprotein, multidrug resistance associated proteins 1-5, bleomycin hydrolase, dihydropyrimidine dehydrogenase, β-ureidopropoinase, β-alanine synthetase, cytidine deaminase, thiopurine methyltransferase, CYP1A1, CYP1A2, CYP2A6, CYP2A7, CYP2B6, CYP2B7, CYP2C8, CYP2C9, CYP2C17, CYP2C18,

CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP3A3, CYP3A4, CYP3A5, CYP3A7, CYP4B1, CYP7, CYP11, CYP 17, CYP 19, CYP21, CYP27, glutathione transferase alpha, glutathione transferase theta, glutathione transferae mu, glutathione transferase pi, methylguanine methyltransferase, 3-alkylguanine alkyltransferase, 3-methyladenine DNA glucosylase, DNA dependent protein kinase, catalytic subunit of DNA-PK, DNA binding subunit of DNA-PK Ku-70 or Ku-80 subunit, KARP-1, Poly(ADP-ribose) polymerase, Fanconi Anemia genes A, B, C, D, E, F, G, and H, ERCC-1, ERCC2/XPD, ERCC3/XPB, ERCC4, ERCC5, ERCC6, XPA, XPC, XPE, HHR23A, HHR23B, uracil glycosylase, 3-methyl adenine DNA glycosylase, NF-kappa B, XRCC4, XRCC5/Ku80, XRCC6, XRCC7, glutathione-X-transferase, I-kappa B alpha,

HSP70, HSP27, and 9-oxoguanine DNA glycosylase.

131. A method for identifying a potential patient undergoing transplantation for treatment with an inhibitor active on at least one but less than all alleles of an essential gene, comprising the step of: identifying a patient undergoing an allogenic bone manow transplantation in which the donor tissue contains at least one alternative allele of an essential gene different from the alleles in somatic cells of said patient. 386 232/116

132. The method of claim 131, wherein said donor or said recipient is homozygous for an alternative allelic form of an essential gene that is not present in the other of said donor or said recipient.

133. A method for treating graft versus host disease in a patient receiving allogenic bone manow transplantation, said method comprising the step of administering to said patient at least one allele specific inhibitor specific for at least one but less than all of the allelic forms of an essential gene in a population, wherein said inhibitor inhibits stimulation of the donor immune system, and cells of the said patient comprise an allelic form of said gene not present in the donor bone manow.

134. The method of claim 133, wherein said allele specific inhibitor is selected by identifying at least one alternative alleles of an essential gene present in the donor tissues but absent in the normal somatic cells of said patient; and selecting a said inhibitor active on a said alternative allele of an essential gene present in said donor tissues but absent in the normal somatic cells of said patient.

135. The method of claim 134, wherein said at least one inhibitor recognizes both alleles of said essential gene that are present in said donor, but not both alleles of said gene that are present in said patient.

136. A method for enhancing engraftment of an allogenic bone marrow transplant, comprising the step of administering to a patient receiving said transplant an allele specific inhibitor which kills or suppresses the patient's bone manow but not the donor bone manow, thereby providing space for engraftment of the donor cells within the marrow cavity.

137. The method of claim 136, wherein the allele specific inhibitor is selected by 387 232/116 identifying alternative alleles of an essential gene that are present in the recipient but not the donor marrow.

138. The method of claim 137, wherein said allele specific inhibitor recognizes both allelic forms of the essential gene that are present in the recipient, but not both allelic forms of the same gene that are present in the recipient.

139. A method for treating or preventing chimerism in allogenic bone manow transplantation, comprising selectively killing or suppressing proliferation of the patient's own cells without toxicity to the donor cells by administering to a patient receiving said transplantation at least one allele specific inhibitor active on at least one but less than all alternative alleles of a gene vital for cell growth or viability, wherein said inhibitor targets the allelic form or forms of a gene in bone manow of said patient but does not target at least one allelic form of said gene in the donor bone manow.

140. A method for treating cancer in a patient receiving allogenic or autologous transplantation, comprising the step of administering to said patient at least one allele specific inhibitor which kills or inhibits the growth of cancer cells without toxicity to the transplanted manow.

141. The method of claim 141, wherein said transplantation is autologous transplantation and said at least one allele specific inhibitor is selected to be active on the allele of an essential gene remaining in the cancer cells due to LOH in patients whose normal somatic cells are heterozygous for said essential gene, but not on the alternative allele of said gene present in said normal somatic cells, whereby said administration enables continuing therapy of cancer without suppression of the transplanted manow. 388 232/116

142. The method of claim 140, wherein said transplantation is allogenic transplantation and said allele specific inhibitor recognizes both alleles of said essential gene that are present in the recipient, but not both forms of the said gene that are present in said patient.

143. A method for eliminating malignant cells from transplanted manow during autologous transplantation of a patient heterozygous for an essential gene, comprising contacting cells from harvested autologous bone manow ex vivo with at least one allele specific inhibitor active on at least one but less than all alternative alleles of said essential gene, wherein said inhibitor targets an allelic form of said gene present in cancer cells of said patient but does not target an alternative allele of said gene present in normal cells from said autologous bone manow, wherein said gene has undergone LOH in cancer cells of said patient.

144. The method of claim 143, wherein said autologous bone manow is harvested from said patient prior to high dose radiation or chemotherapy.

145. The method of claim 143, further comprising the steps of: a. identifying one alternative allele of an essential gene remaining in the cancer cell due to LOH in patients who are heterologous with two different alternative forms of the essential gene in normal cells of the autologous bone manow; b. cultivating said autologous bone marrow ex vivo in the presence of an allele specific inhibitor that inhibits the allele that is present in the cancer cells, but not the heterologous allele that is present in the normal bone manow. .

146. The method of claim 143, wherein said autologous bone marrow is contacted with a plurality of said allele specific inhibitors. 389 232/116

147. A method for separating a first cell from a mixture of cells, comprising the steps of: a) providing an allele specific binding compound which binds to at least one but less than all alleles of a gene, wherein a said allele of said gene expressed in said first cell is not expressed in other cells of said mixure of cells or is expressed in other cells in said mixture of cells and not in said first cell; b) contacting said mixture of cells with said binding compound under conditions such that said binding compound binds to said allele and not to non-target alleles; and c) separating bound cells from unbound cells.

148. The method of claim 147, wherein said mixture of cells comprises normal somatic cells and cancer cells from a patient, said first cells are said normal somatic cells, and said first cells express a said allele deleted in said cancer cells due to LOH of said gene, comprising separating said normal somatic cells from said cancer cells.

149. The method of claim 147, wherein said allele specific binding compound is an antibody or antibody fragment.

150. The method of claim 147, wherein said binding compound is attached to a solid support.