p21Cip1-dependent regulation of response to thymidylate synthase (TS) inhibitors

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The cyclin-dependent kinase inhibitor, p21Cip1, is a direct transcriptional target of p53 during DNA damage and may also play an active role in the induction of apoptosis. p21Cip1 function is regulated by phosphorylation, localization and protein-protein interaction. We have previously demonstrated that IFN-γ sensitizes human colon carcinoma cell lines to the cytotoxic effects of TS inhibitors and that p21Cip1 regulates thymineless stress-induced cytotoxity. In HCT116 isogenic cell lines, IFN-γ sensitized wt cells to ZD9331-induced cytotoxicity, while p21Cip1 −/− cells were resistant. In contrast, IFN-γ induced marked cytotoxicity in p21Cip1 −/− cells only. The aim of this study was to determine the mechanisms of ZD9331-, IFN-γ and ZD9331 + IFN-γ induced apoptosis, and the role of p21Cip1 in these pathways. Cell cycle analyses revealed that HCT116 wt and p21Cip1 −/− cells accumulated in S phase within 24 hr of ZD9331 +/–IFN-γ exposure, however wt cells exited S-phase more rapidly and died by apoptosis, which occurred prior to mitosis, either in late S or G2. In wt cells, caspases -7, -2 and -3 exclusively, play a role in ZD9331 +/–IFN-γ induced apoptosis, however caspase-3 activation was reduced compared to caspase-7 or caspase-2. Active caspase-7 was isolated in the microsomal fraction of cell lysates, and both cytochrome c and Smac/DIABLO were released from mitochondria, independent of caspase-8 activation and Bax expression, with no detectable expression of active caspase-9 downstream of the mitochondria. The IAP protein, XIAP, was downregulated following ZD9331 +/–IFN-γ treatment. P21Cip1 was extensively upregulated in wt cells 48 hr after treatment with ZD9331 +/–IFN-γ and confocal microscopy demonstrated predominant increased expression in the nucleus, with expression in the cytoplasm. While treatment with IFN-γ alone did not induce apoptosis in wt cells, there was increased expression of the pro form and cleaved product of caspase-4. In contrast, in p21Cip1 −/− cells, the total level of procaspase-4 expression was reduced, and no cleavage product was detected. In conclusion, data suggest that the mechanism of ZD9331-induced apoptosis in HCT116 wt cells may involve crosstalk between the endoplasmic reticulum (microsomal fraction) and mitochondria, dependent on p21Cip1, and enhanced by IFN-γ. While caspase-3 is a main executioner caspase, caspase-7 or caspase-2 may actually decide the fate of these cells. In contrast, IFN-γ induced caspase-4 expression and cleavage is not pro-apoptotic in wt cells. Supported by NCI awards CA 32613, CA 21765 and by ALSAC.