P21Cip1 Is a Critical Mediator of the Cytotoxic Action of Thymidylate Synthase Inhibitors in Colorectal Carcinoma Cells

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Abstract
We have demonstrated previously that interferon (IFN)-γ sensitizes human colon carcinoma cell lines to the cytotoxic effects of 5-fluorouracil combined with leucovorin and to the thymidylate synthase inhibitor, ZD9331, dependent on thymineless stress-induced DNA damage, independent of p53. Here we demonstrate that the cyclin-dependent kinase (CDK) inhibitor p21Cip1 regulates thymineless stress-induced cytotoxicity in these cells. HCT116 wild-type (wt) and p53−/− cells underwent apoptosis and loss in clonogenic survival when exposed to ZD9331, whereas p21Cip1−/− cells were resistant. In contrast, IFN-γ induced marked cytotoxicity in p21Cip1−/− cells only. ZD9331 induced p21Cip1 up-regulation in all of the cell lines examined, as did thymidine deprivation in thymidylate synthase-deficient (thymidylate synthase−) cells. Furthermore, selective induction of p21Cip1 in RKO was sufficient to induce apoptosis. P21Cip1, cdk1, cdk2, and cyclin E mRNA expression increased coincident with S-phase accumulation in HT29 cells treated with ZD9331 or 5fluorouracil/leucovorin, as demonstrated by cDNA microarray analyses. Cell cycle analyses revealed that HCT116 wt and p21Cip1 −/− cells accumulated in S phase within 24 h of ZD9331 exposure; however, wt cells exited S-phase more rapidly, where apoptosis occurred before mitosis, either in late S or G2. Finally, the CDK inhibitor roscovitine potentiated the cytotoxic activity of ZD9331 in both wt and p21Cip1−/− cells, strongly suggesting a role for p21Cip1-dependent CDK inhibition in cytotoxicity induced by thymidylate synthase inhibition. In summary, p21Cip1 positively regulates the cytotoxic action of thymidylate synthase inhibitors, negatively regulates the cytotoxic action of IFN-γ, and enhances S-phase exit after thymineless stress, possibly via interaction with CDK-cyclin complexes.