title: P21Cip1 is a critical mediator of the cytotoxic action of thymidylate synthase inhibitors in colorectal carcinoma cells.
abstract: We have demonstrated previously that interferon (IFN)-gamma 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(Cip1) 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-gamma 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-gamma, and enhances S-phase exit after thymineless stress, possibly via interaction with CDK-cyclin complexes.
authors.affiliation: Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
authors.forename: James I
authors.initials: JI
authors.lastname: Geller
authors.forename: Kinga
authors.initials: K
authors.lastname: Szekely-Szucs
authors.forename: Istvan
authors.initials: I
authors.lastname: Petak
authors.forename: Belinda
authors.initials: B
authors.lastname: Doyle
authors.forename: Janet A
authors.initials: JA
authors.lastname: Houghton
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D000077546:Roscovitine; D016196:S Phase / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D019014:fas Receptor / Q000096:biosynthesis / Q000502:physiology
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011687:Purines; D011799:Quinazolines; C094299:ZD 9331; D019014:fas Receptor; D000077546:Roscovitine; D007371:Interferon-gamma; D013940:Thymidylate Synthase