Figure 1

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The two-hit hypothesis arose of out Knudson's interest in the genetic mechanisms underlying retinoblastoma, a childhood form of retinal cancer. Under normal circumstances, apopulation of cells in the developing eye, called retinoblasts, stops growing and dividing during embryogenesis and differentiates into retinal photoreceptor (light-capturing) cells and nerve cells. Typically, these differentiated cells do not divide very often, if ever. In the case of retinoblastoma, however, the retinoblasts fail to differentiate; thus, these cells continue to divide, forming tumors in the retina. If left untreated, the retinal tumor cells will eventually metastasize (spread) to other parts of the body.

Between 1944 and 1969, Knudson studied 48 patients with retinoblastoma who had been admitted to the hospital (Knudson, 1971). For each patient, Knudson tabulated the age at diagnosis,sex,family history, whether the tumor occurred in one eye (unilaterally) or in both eyes (bilaterally), and an estimation of the number of tumors present in each eye. He also supplemented this information with similar data from two previous publications (Vogel, 1957; Schappert-Kimmijseret al., 1966).

At the time of Knudson's study, researchers believed that retinoblastoma could be caused by either somatic or germ-line mutations. Knudson's combined data itself showed that retinoblastoma was caused by a germ-line mutation in approximately 40% of U.S. cases. However, Knudson was puzzled by the observation that some children with an affected parent were disease-free, but these unaffected individuals later bore children with retinoblastoma; this finding suggested that an individual couldinherit a germ-line mutation but not have the disease. Knudson also noted that while the majority of children with an affected parent had bilateral tumors (25%–30%), some had only unilateral tumors (10%–15%). Furthermore, he determined that approximately 60% of retinoblastoma cases in the U.S. were unilateral and were not associated with a family history of the disease. These findings are summarized in Table 1; in the table, "hereditary" refers to those individuals with a family history of retinoblastoma.

Next, Knudson used mathematics to determine whether the data followed a trend associated with a one-hitmodel or a two-hit model ofgenemutation. In doing so, he used information from two groups; the first group consisted of 23 patients with bilateral hereditary retinoblastoma, and the second consisted of 25 patients with unilateral nonhereditary retinoblastoma. Because time is required for mutations to accumulate, Knudson examined the age at which the children in these two groups were diagnosed with retinoblastoma.

Figure 2: Two-hit tumor formation in both hereditary and nonhereditary retinoblastoma.

Whereas a \"one-hit\" clone is a precursor to the tumor in nonhereditary retinoblastomas, all retinoblasts (indeed, all cells) are one-hit clones in hereditary retinoblastoma.

© 2001Nature Publishing Group Knudson, A. Two genetic hits (more or less) to cancer.Nature Reviews Cancer1, 160 (2001). All rights reserved.

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With this information in mind, Knudson took a closer look at his two groups of patients. Not surprisingly, he found that diagnosis for bilateral hereditary cases of retinoblastoma occurred at a younger age and showed a curve consistent with a single-mutation process. Furthermore, he noted that the rate of diagnosis for unilateral nonhereditary retinoblastoma was delayed relative to that of the bilateral cases and showed a curve consistent with a two-mutation process (Figure 1).

Based on calculations using this clinical data, Knudson concluded that retinoblastoma was caused by two mutations: one in each copy of a singletumor suppressor gene (now calledRB1). He also estimated that each of the two mutations would occur at a rate of 2 x 10^-7 per year. Patients who inherited anRB1 mutation would develop tumors earlier, and they would often develop more than one tumor. In contrast, individuals who did not inherit a mutation would almost always be affected by a single tumor. This statement, which Knudson called the two-mutation hypothesis (Figure 2), is now known as the two-hit hypothesis (Knudson, 1971).

Based on the predicted mutation rate, Knudson expected that many individuals in the general population would acquire a singlesomatic mutation in theRB1 gene over their lifetime, and that the retinas of most people would therefore likely contain small groups of retinoblasts that had received one "hit" in theRB1 gene. In order to become cancerous, each retinoblast with onemutant copy of theRB1 gene would need to acquire a mutation in the remaining wild-type copy of the gene. Most individuals who had one hit did not develop retinoblastoma, however, because most of their mutated cells had already differentiated and quit dividing before they could receive a second hit (Knudson, 2001).

"Loss ofheterozygosity" is a phrase often used to describe the process that leads to the inactivation of the second copy of a tumor suppressor gene. During this process, aheterozygous cell receives a second hit in its remaining functional copy of the tumor suppressor gene, thereby becominghomozygous for the mutated gene. Mutations that inactivate tumor suppressor genes, called loss-of-function mutations, are often point mutations or small deletions that disrupt the function of theprotein that is encoded by the gene; chromosomal deletions or breaks that delete the tumor suppressor gene; or instances of somaticrecombination during which the normal gene copy is replaced with a mutant copy.

Figure 3

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With knowledge of the molecular identity of the retinoblastoma-associated gene in hand, researchers began to focus their efforts on the identification and characterization ofRB1 function. Over the years,RB1 has been shown to block cell proliferation andcell division and to regulate cell death (Chau & Wang, 2003).RB1 collaborates with a number of cellular proteins in order to carry out its functions. Furthermore,RB1 function has been shown to be inactivated by four distinct mechanisms: genetic inactivation, sequestration of theRB1-encoded protein by viral oncoproteins,phosphorylation of theRB1-encoded protein, and degradation of theRB1-encoded protein (Figure 3). Many valuable insights intoRB1 function have been gained through studies using mouse models in which the mouseRb1 gene is deleted.

RB1 is one gene among a growing list of tumor suppressor genes. According to the American Cancer Society (2005), at least 30 different tumor suppressor genes have been identified, including those listed in Table 2. Many of these genes function to inhibit cell division and cell proliferation, stimulate cell death, and repair damaged DNA.

As evidenced by the growing number of identified tumor suppressor genes, our cells use many means to overcome genetic insults and precisely regulate their proliferation, growth, and death. Our increasing knowledge of tumor suppressor gene function will continue to enhance our ability to diagnose and more effectively treat cancers at the molecular level in the years to come.