P21Cip1 is a critical mediator of the cytotoxic action of thymidylate synthase inhibitors in colorectal carcinoma cells

• Published in: Cancer research (Chicago, Ill.) Vol. 64; no. 17; pp. 6296 - 6303
• Main Authors: GELLER, James I, SZEKELY-SZUCS, Kinga, PETAK, Istvan, DOYLE, Belinda, HOUGHTON, Janet A
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 01.09.2004
• Subjects: Antineoplastic agents; Apoptosis - drug effects; Apoptosis - physiology; Biological and medical sciences; Colorectal Neoplasms - drug therapy; Colorectal Neoplasms - enzymology; Cyclin-Dependent Kinase Inhibitor p21; Cyclins - biosynthesis; Cyclins - deficiency; Cyclins - metabolism; Cyclins - physiology; Drug Synergism; Enzyme Inhibitors - pharmacology; fas Receptor - biosynthesis; fas Receptor - physiology; G1 Phase - drug effects; G1 Phase - genetics; G1 Phase - physiology; HCT116 Cells; HT29 Cells; Humans; Interferon-gamma - pharmacology; Medical sciences; Pharmacology. Drug treatments; Purines - pharmacology; Quinazolines - pharmacology; S Phase - drug effects; S Phase - genetics; S Phase - physiology; Thymidylate Synthase - antagonists & inhibitors; Tumors; Rectal disease; Enzyme; Transferases; Colorectal cancer; Cytotoxicity; Malignant tumor; Thymidylate synthase; Colonic disease; Methyltransferases; Digestive diseases; Intestinal disease; Inhibitor; Mediator
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-04-0863

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.