Role of p21Cip1 in thymidylate synthase (TS) inhibitor induced apoptosis in human colon carcinoma cell lines (cc)

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p21Cip1 (p21) was initially identified as a potent cyclin dependent kinase inhibitor and a direct downstream target of tumor suppressor p53 after DNA damage, but may also be regulated in a p53-independent manner. Further, both a pro-apoptotic role and an anti-apoptotic role has been described for p21. The functional role of p21 in G1-phase cell cycle arrest is well characterized, however the function of p21 in the induction of apoptosis is less well defined. p21 has demonstrated functional roles both in the nucleus and in the cytoplasm, in the latter case where it has been found to have anti-apoptotic function. We have previously demonstrated that p21 is critical in thymidylate synthase (TS) inhibitor-induced apoptosis. Thus, HCT116 wt cells were sensitive to the TS inhibitor ZD9331, whereas isogenic HCT116 p21-/- cells were resistant. The aim of this study was to elucidate the mechanism by which p21 regulates thymineless stress-induced cytotoxicity in CRC. HCT116 wt and p21-/- both accumulated in S phase within 24 hr following ZD9331 exposure, however wt cells exited S-phase, progressed to G2 more rapidly and died by apoptosis at 48 hr, which occurred prior to M-phase. In contrast p21 -/- cells progressed more slowly through S-phase and did not die by apoptosis. By 48 hr following exposure to ZD9331, HCT116 wt cells demonstrated enhanced nuclear localization of p21 determined by microscopy and also following cellular fractionation. At the onset of apoptosis and as cells moved from S-phase to G2-phase, p21 was also upregulated in the cytoplasm, and a lower Mr product of ∼15 kD was detected, dependent on caspase activation. Cells demonstrated a loss in mitochondrial membrane permeabilization, release of cytochrome c, and significant upregulation in Bak expression in mitochondrial fractions compared to Bax. In contrast, no change in these parameters was determined in p21-/- cells. Further, HCT116 Bax -/- cells demonstrated sensitivity to ZD9331. In conclusion data suggest that p21 regulates ZD9331-induced apoptosis. The hypothesis is that following ZD9331-induced DNA damage, nuclear accumulation of p21 and S-phase arrest in HCT116 wt cells, cleavage of p21 allows exit of p21 from the nucleus to the cytoplasm at the onset of apoptosis, thereby disinhibiting cyclin-cdk complexes and allowing cells to progress from S-phase to G2-phase, where they undergo apoptosis. Studies to test this hypothesis are currently underway. Supported by NCI awards CA 32613, CA 21765 and by ALSAC.