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

We have demonstrated previously that interferon (IFN)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 p21 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 p21 / cells were resistant. In contrast, IFNinduced marked cytotoxicity in p21 / cells only. ZD9331 induced p21 up-regulation in all of the cell lines examined, as did thymidine deprivation in thymidylate synthase-deficient (thymidylate synthase ) cells. Furthermore, selective induction of p21 in RKO was sufficient to induce apoptosis. P21, 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 p21 / 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 p21 / cells, strongly suggesting a role for p21-dependent CDK inhibition in cytotoxicity induced by thymidylate synthase inhibition. In summary, p21 positively regulates the cytotoxic action of thymidylate synthase inhibitors, negatively regulates the cytotoxic action of IFN, and enhances S-phase exit after thymineless stress, possibly via interaction with CDK-cyclin complexes.