Abstract

A Down's syndrome associated gene, Single Minded 2 gene short form (SIM2-s), is specifically expressed in colon tumors but not in the normal colon. Antisense inhibition ofSIM2-s in a RKO-derived colon carcinoma cell line causes growth inhibition, apoptosis, and inhibition of tumor growth in a nude mouse tumoriginicity model. The mechanism of cell death in tumor cells is unclear. In the present study, we investigated the pathways underlying apoptosis. Apoptosis was seen in a tumor cell-specific manner in RKO cells but not in normal renal epithelial cells, despite inhibition of SIM2-s expression in both of these cells by the antisense. Apoptosis was depended on WT p53 status and was caspase-dependent; it was inhibited by a pharmacological inhibitor of mitogen-activated protein kinase activity. Expression of a key stress response gene, growth arrest and DNA damage gene (GADD)45α, was up-regulated in antisense-treated tumor cells but not in normal cells. In an isogenic RKO cell line expressing stable antisense RNA toGADD45α, a significant protection of the antisense-induced apoptosis was seen. Whereas antisense-treated RKO cells did not undergo cell cycle arrest, several markers of differentiation were deregulated, including alkaline phosphatase activity, a marker of terminal differentiation. Protection of apoptosis and block of differentiation showed a correlation in the RKO model. Our results support the tumor cell-selective nature ofSIM2-s gene function, provide a direct link between SIM2-s and differentiation, and may provide a model to identify SIM2-s targets.

Methods

Cell Culture. Isogenic colon cancer-derived cell lines (RKO, RKO-E6-p53-knockout, and RKO-AS45.1-stable GADD45α antisense clone) and OM-1 and SW 480 colon carcinoma-derived cells were obtained from American Type Culture Collection and were maintained as recommended by American Type Culture Collection. Early-passage primary human renal epithelial (HRE) cells were obtained from Cambrex (Walkersville, MD) and were cultured following the manufacturer's instructions.

Inhibitors and Antibodies. AntisenseSIM2-s was synthesized as described (22). Methylmethane sulfonate was obtained from Sigma Aldrich. Caspase-family inhibitor set IV was obtained from Biovision (Mountain View, CA). p38 kinase inhibitor (SB202190) was obtained from A. G. Scientific (San Diego). General tyrosine kinase inhibitor (Genistein) was from Sigma Aldrich. Antibodies were obtained from Santa Cruz Biotechnology.

RT-PCR. End-point RT-PCR analysis and primers used were described previously (22,23). Quantitative real-time PCR was done by using β2 microglobulin standards (Roche Applied Sciences, Indianapolis). Exon-specific RT-PCR primers were used (see Table 1, which is published assupporting information on the PNAS web site).

Cell Cycle and FACS Analysis of GADD45αProtein. Cells were harvested after 100 nM antisense or control oligo treatment and fixed at 4°C overnight in 70% ethanol. The fixed cells were resuspended in PBS and treated with 20 μg/ml RNaseA for 30 min at 37°C, stained with 60 μg/ml propidium iodide at room temperature for 30 min, and scanned by an Epics XL-MCL Flow Cytometer (Beckman Coulter). Cell cycle distribution was calculated by usingmulticycle v.3.1.1 software (Phoenix Flow Systems, San Diego). GADD45α protein expression was analyzed in antisense-treated RKO or HRE cells by using either GADD45α-phycoerythrin (PE)-conjugated antibody or its IgG1 isotype control PE-conjugated antibody by using an Epics XL-MCL Flow Cytometer (Beckman Coulter).

Caspase and p38 Kinase Inhibitor Experiments. For inhibitor studies, the cells were pretreated with 20 μM caspase inhibitors, 20 μM SB202190 (p38 MAPK inhibitor), or 10 μM Genistein (general tyrosine kinase inhibitor) for 1 h before transfection with the oligonucleotides and then continuously incubated with the inhibitors. At select time points, apoptosis was measured by using the Cell Death Detection ELISA^Plus (Roche Applied Science), as described (23). Each experiment was repeated two to three times.

Alkaline Phosphatase (ALP) Activity. ALP activity was measured as described (24). The assays were repeated in three to five independent experiments.

Immunohistochemistry (IHC). Antisense-treated cells in culture were analyzed by IHC by using affinity-purified rabbit antiserum to SIM2-s-specific hydrophobic peptides SHGGGWQMETEPSRF (Sigma Genosys, The Woodlands, TX), as described (22,23).

Results

Inhibition of SIM2-s Expression Results in Tumor Cell-Selective Apoptosis. In an earlier study (22), we have shown that RKO colon carcinoma cells undergo rapid apoptosis (14–24 h) in response to inhibition ofSIM2-s gene expression by antisense. To clarify whether the inhibition of SIM2-s mRNA precedes apoptosis, we have performed an early time-course analysis. RKO cells were treated with either the control or theSIM2-s antisense at 4, 6, 8, 10, and 14 h. The treated cells were analyzed forSIM2-s mRNA expression by real-time PCR and for apoptosis by ELISA (Fig. 1A). Inhibition of SIM2-s mRNA was seen as early as 8 h after transfection, whereas evidence of apoptosis was seen only at 10 h onwards. The half-life of SIM2-s has been shown to be 2 h (25), which can account for the rapidity of apoptosis induction. Whereas in the normal colon tissue, SIM2-s expression was not detected, normal human kidney is one of the few organs in whichSIM2-s expression was detected (21,22). Hence we tested the effects of theSIM2-s antisense in early-passage primary HRE cells and compared them with RKO cells (Fig. 1B). Similar levels of inhibition ofSIM2-s mRNA were seen upon antisense treatment in both the tumor and normal cells (50%), but pronounced apoptosis was seen only in the RKO-derived tumor cells and not in the normal HRE cells. The antisense inhibited SIM2-s protein expression in both these cells (Fig. 1C). Over 80% of the antisense-treated RKO cells underwent apoptosis. The antisense-treated HRE cells did not show inhibition of cell proliferation, evidence of DNA laddering, morphological changes, nuclear condensation, or cytoplasmic blebbing (data not shown). These results suggest that apoptosis may be tumor cell-selective.

To further clarify the apoptotic mechanism, we next used specific pathway inhibitors. The RKO cells were significantly protected from apoptosis induced bySIM2-s antisense by both general (Z-VAD-FMK) and specific caspase inhibitors (caspase 9 and 10). This effect was not seen when caspase 2 or 8 inhibitors were used (Fig. 1D). Inhibitors of other caspases (caspases 3, 4, 5, 6, and 13) showed only marginal protection (10–15%) against apoptosis (data not shown). To further map the signal transduction involved in apoptosis, we next used key kinase pathway pharmacological inhibitors. Significant protection from apoptosis was seen when the RKO cells were pretreated with a p38 MAPK pathway inhibitor (SB 202190). As a control, we used Genistein, a nonspecific tyrosine kinase inhibitor, which did not cause such a protection (Fig. 1E). Treatment of RKO cells with these inhibitors alone did not cause apoptosis (data not shown).

We next addressed whether apoptosis mediated by the inhibition ofSIM2-s expression depends on p53 tumor suppressor gene function. Isogenic RKO (WT p53) or RKO-E6 (p53 functional knockout by HPV-E6 oncogene) cells were treated either with the control oligo orSIM2-s antisense and at the indicated time analyzed by apoptosis ELISA (Fig. 1F). In the isogenic RKO E6 cells, SIM2-s antisense-induced apoptosis was 4- to 5-fold reduced.

Identification of GADD45αas a Critical Mediator of SIM2-s Antisense Mechanism. Up-regulation of GADD genes is often seen in tumor cells undergoing apoptosis. Hence we investigated the regulation of GADD family members in theSIM2-s antisense-treated RKO cells (Fig. 2A). The inhibition ofSIM2-s by antisense in RKO cells resulted in up-regulation of the expression of three GADD family members (GADD45α, -45β, and -34). GADD45γ and -153 gene expression was not detected in the RKO cells. GADD45α protein expression was up-regulated in the RKO (Fig. 2B) but not in normal HRE cells (Fig. 2C) upon treatment with the antisense.

We next tested whether GADD45α is functionally relevant to apoptosis by using the RKO-GADD45α knockout isogenic cell line. The RKO/RKO GADD45α antisense cells (RKO-AS45.1) were treated with 100 nM either control orSIM2-s antisense oligonucleotides and at 10, 14, 18, and 24 h, the cells were analyzed for apoptosis by ELISA (Fig. 2D). Apoptosis was significantly inhibited (>66%) in the RKO-AS45.1 cells. Because p38 MAPK is upstream of GADD45α, these results are consistent with the protection of apoptosis seen by using a p38 MAPK inhibitor, SB 202190 (Fig. 1E).

Inhibition of SIM2-s Expression Does Not Cause Cell Cycle Arrest. Enforced expression of GADD45α in diverse cell types causes growth suppression and activation of a G₂ checkpoint, which depends on WT p53 status (for a review, see ref.18). GADD45α overexpression results in growth suppression through activation of the G₂ checkpoint of the cell cycle (15). Although the growth suppression is seen in a p53-independent manner, the G₂ arrest is seen only in tumor and normal cells with WT p53 function (26,27). The up-regulation of GADD45α mRNA and protein expression seen in our study in a tumor cell-selective manner raised a possibility that the observed apoptosis induced by theSIM2-s antisense may be a consequence of cell cycle arrest. Hence we performed cell cycle analysis of the antisense-treated RKO or HRE cells (Fig. 3A). Nonsynchronous cells were treated either with control or SIM2-s antisense oligonucleotides and, at different time points, the cells were analyzed by FACS. Methylmethane sulfonate, which has been shown to cause G₂ arrest in RKO cells (27), was used as a positive control. For simplicity, RKO at 14 h and HRE at 24 h are shown. No discernible differences of cells at G₁, S, and G₂ phases were seen between the control and the antisense-treated RKO or HRE cells. The sub-G₀ apoptotic peak was seen in the antisense-treated RKO but not in the HRE cells. Consistent with the lack of cell cycle block, expression of p21 mRNA and protein was not affected by the antisense treatment of RKO cells (Fig. 3B). These results suggested that apoptosis induction by inhibition of SIM2-s is not a consequence of the cell cycle block.

Induction of Differentiation in the Antisense-Treated Cancer Cells. Colon cancer cells undergo terminal differentiation and apoptosis in response to diverse differentiation stimuli (28,29). Key pathways including caspase and p38 MAPK/ERK1/2 are critical to the initiation of differentiation and the subsequent apoptosis signals (30–32). In colon cancer-derived cell lines, ALP activity is used to measure differentiation status (33). In addition, depending on the cell type, other markers, such as mucins (MUC2/MUC3), and carcinoembryonic antigen (CEACAM) family members are shown to be up-regulated upon terminal differentiation (34). Another marker, immature colon carcinoma transcript (ICT1), a colon-specific gene, has been shown in colon carcinoma-derived cell lines to be down-regulated in response to the induction of terminal differentiation (35). The appearance of these cell markers is often associated with indications of cell death, such as detachment from the culture dish (36). Hence, we investigated the differentiated status of theSIM2-s antisense-treated RKO cells for marker gene expression and ALP activity (Fig. 3B–D). Expression of ICT1 was down-regulated in a time-dependent manner after antisense treatment (Fig. 3B). mRNA expression of ALP1, MUC2, and CEACAM7 was also up-regulated upon antisense treatment in the RKO cells but not in normal HRE cells (Fig. 3C). The up-regulation of these genes was seen 8 h after antisense treatment, a time point where no apoptosis is seen (seeFig. 1A). ALP activity in the antisense-treated RKO cells was stimulated to levels similar to those attained with a known inducer of differentiation, sodium butyrate (Fig. 3D). Consistent with the protection of antisense-mediated apoptosis seen in the RKO-E6 and -AS45.1 isogenic cell lines, the induction of ALP activity was also inhibited in these cell lines. Basal and antisense-induced ALP was significantly inhibited by the p38 MAPK inhibitor (SB202190). Antisense-induced ALP activity was also abolished by the caspase 9 inhibitor (Z-LEHD-FMK).

Discussion

Whereas the RKO cell expresses only the short isoform of SIM2 (SIM2-s), normal kidney and HRE cells express both the long and the short isoforms (SIM2-s/SIM2-l), and thus the tumor cell-selective apoptosis may be due to a redundancy mechanism. Alternatively, in normal HRE cells, theSIM2-s gene may transcriptionally regulate distinct sets of genes. A possibility remains that this could be due to differences in SIM2-s RNA/protein stability in normal and tumor cells.

We further map the pathways involved inSIM2-s antisense-mediated apoptosis as dependent on caspase activity (caspases 9 and 10). Caspases are key proteases involved in both initiating apoptotic mechanisms in response to proapoptotic signals (initiator caspases) and cell disassembly (effector caspases). Caspase 9 is a key mitochondrial-dependent initiator caspase that activates caspase 3, an effector molecule, as well as other caspases. Caspase 10 is an initiator caspase that plays a key role in receptor-mediated apoptotic signals (for reviews, see refs.37 and38). The significant protection ofSIM2-s antisense-mediated apoptosis by caspase 9 and 10 inhibitors in the RKO cells identifies both extrinsic and intrinsic caspase-dependent pathway in tumor-selective apoptosis. Results with isogenic p53 null RKO cells (RKO-E6) further provide a hint into the mechanism of apoptosis induced by the inhibition of SIM2-s expression. Inactivation of p53 either by overexpression of E6 oncoprotein (RKO-E6) or mutation (as in SW 480 and OM-1 colon carcinoma cells) inhibits apoptosis induced by the antisense. It is tempting to speculate that targets of SIM2-s require WT p53 tumor suppressor gene function.

Our results establish GADD45α as a critical mediator ofSIM2-s gene function in regulating growth and differentiation. A recent study involving overexpression of melanoma differentiation-associated gene (MDA-7/IL-24) showed induction of melanoma-selective apoptosis and up-regulation of GADD45α, and apoptosis was inhibited by a p38 MAPK inhibitor (39). MAPKs are serine–threonine kinases that are activated by phosphorylation in response to a wide array of extracellular signals (40,41). Three distinct MAPKs (ERK, JNK, and p38) play a critical role in apoptosis and differentiation; the ERKs (ERK1/ERK2) are implicated in growth and differentiation, and JNK and p38 are stress-activated MAPKs that play a key role in apoptosis and stress response (for a review, see ref.42).

The tumor-selective apoptosis seen in the RKO cells by inhibition ofSIM2-s gene expression did not result in cell cycle arrest or alteration of the levels of p21 mRNA or protein. The GADD45α protein has been shown to associate with nuclear proteins, including the cdc2–cyclin B1 kinase complex (43), p21 (44), and proliferating cell nuclear antigen (45); however, up-regulation of GADD45α is not always associated with cell cycle arrest. For example, Jinet al. (26) demonstrated that overexpression of GADD45α in HCT116, but not in HeLa cells, resulted in G₂ arrest despite growth suppression in both of these cell lines. Thus, activation of the GADD45-mediated stress response and the subsequent apoptosis we observe may be exerted at the level of p38 MAPK/JNK independent of cell cycle blockage. Inhibition of apoptosis induced by theSIM2-s antisense with the p38 MAPK inhibitor strongly supports this.

The induction of several markers of differentiation and the up-regulation of ALP activity in antisense-treated RKO cells implicate differentiation as a key mechanism. However, the SIM2-s independent mechanism must exist in leukemia-derived cell lines, because these cells do not express SIM2-s (22). Deregulation of diverse differentiation markers before apoptosis suggests apoptosis is a consequence of differentiation. The lack of induction of ALP-1, MUC2, and carcinoembroyonic antigen 7 mRNAs in the normal HRE cells by the antisense implies that, in these normal cells, the SIM2-s gene regulates a distinct set of genes. Frequently, when cells differentiate, they are withdrawn from the cell cycle, but there are several exceptions to this phenomenon. In murine keratinocytes, activation of the Raf/MAPK pathway leads to expression of differentiation markers, but the cells continue to proliferate in a p53-dependent manner (46). In the U-937 leukemia cell line, overexpression of WT 53 induces differentiation, but the cells are not arrested in the G₁ phase of the cell cycle (47).

In recent years, a new model has been emerging, a continuum model whereby cells receive signals to differentiate at all stages of the cell cycle (48). Our results of induction of differentiation without cell cycle arrest are consistent with this model. Our results further link the GADD-mediated stress signal, p38 MAPK, and p53 function in the induction of differentiation and apoptosis by inhibition ofSIM2-s gene expression. A model underlying this link is shown inFig. 4. The putative role of SIM2 protein is as a transcriptional repressor (49). SIM2-s protein, either as a homodimer or a heterodimer, causes transcriptional repression of key differentiation-inducing genes, leading to proliferation. Down-regulation of SIM2-s expression causes a release from this repression, and SIM2-s-regulated proteins such as X or X/Y can cause transcriptional activation of gene(s) in a p53-dependent manner. Such a transcription activates GADD-mediated signals in a p38 MAPK-dependent manner, leading to terminal differentiation and caspase-dependent apoptosis.

Trisomy 21 is linked to human cancer, as evidenced by a 20-fold elevated risk of leukemia in people with Down's syndrome (50). Higher risks for acute lymphoblastic leukemia, myelodysplastic syndrome, acute myeloid leukemia, and early-onset Alzheimer's degeneration are seen in Down's patients, whereas solid tumor formation is relatively rare. However, in an earlier study (22), we demonstrated thatSIM2-s expression was not seen in diverse leukemia-derived cell lines, suggesting the epidemiological link between Down's patients and leukemia may not involve the function of theSIM2-s gene. It is unclear whether overexpression of SIM2-s is a cause or an effect of transformation. However, reduced exposure to environmental factors that contribute to cancer risk, tumor suppressor genes on chromosome 21, and a slower rate of replication or higher likelihood of apoptosis in Down's syndrome cells offer a few reasons for the low frequency of solid tumors seen in Down's patients (51).

We have also recently embarked on a genome-wide global gene expression study with a view toward identification of SIM2-s-regulated targets usingSIM2-s antisense-treated RKO cells as a model. By using gene ontology of GeneChip (Affymetrix, Santa Clara, CA) output, various apoptosis and differentiation-related genes were found to be up-regulated inSIM2-s antisense-treated RKO cells, consistent with the data presented here (see Table 2, which is published assupporting information on the PNAS web site).

Conclusion

Our results suggest the tumor cell-selective nature of the SIM2-s gene. Key pathways, including GADD, caspase, and p53 function, are identified as critical to the function of the SIM2-s gene. Identification of differentiation as a key mechanism for the apoptosis provides a first link between SIM2-s, and differentiation and may allow for future experiments aimed at SIM2-s target discovery.

Supplementary Material

Acknowledgments

We thank J. Narayanan for editorial assistance. This paper is contribution no. P200504 from the Center of Excellence in Biomedical and Marine Biotechnology.

References

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