[CANCER RESEARCH 64, 6296 – 6303, September 1, 2004] P21 Cip1 Is a Critical Mediator of the Cytotoxic Action of Thymidylate Synthase Inhibitors in Colorectal Carcinoma Cells James I. Geller, 1,4 Kinga Szekely-Szucs, 1 Istvan Petak, 2 Belinda Doyle, 1 and Janet A. Houghton 1,3 1 Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee; 2 Semmelweis University and 1 st Institute of Pathology and Experimental Cancer Research, Budapest, Hungary ABSTRACT 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, inde- pendent 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 apo- ptosis and loss in clonogenic survival when exposed to ZD9331, whereas p21 Cip1 / cells were resistant. In contrast, IFN- induced marked cy- totoxicity in p21 Cip1 / cells only. ZD9331 induced p21 Cip1 up-regulation in all of the cell lines examined, as did thymidine deprivation in thymi- dylate synthase-deficient (thymidylate synthase  ) cells. Furthermore, se- lective induction of p21 Cip1 in RKO was sufficient to induce apoptosis. P21 Cip1 , cdk1, cdk2, and cyclin E mRNA expression increased coincident with S-phase accumulation in HT29 cells treated with ZD9331 or 5- fluorouracil/leucovorin, as demonstrated by cDNA microarray analyses. Cell cycle analyses revealed that HCT116 wt and p21 Cip1 / 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 G 2 . Finally, the CDK inhibitor roscovitine potentiated the cytotoxic activity of ZD9331 in both wt and p21 Cip1 / cells, strongly suggesting a role for p21 Cip1 -dependent CDK inhibition in cytotoxicity induced by thymidylate synthase inhibition. In summary, p21 Cip1 posi- tively 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. INTRODUCTION Colorectal cancer is a leading cause of cancer-related morbidity and mortality each year in the United States. Thymidylate synthase inhib- itors, such as 5-fluorouracil, continue to maintain their role as first- line chemotherapy for the treatment of colorectal cancer. Efforts to maximize efficacy of thymidylate synthase inhibitor-based regimens have addressed ways to selectively modulate the cellular response to thymidylate synthase inhibition (1), as well as to develop more spe- cific thymidylate synthase inhibitors including ZD9331 and ZD1694 (tomudex and raltitrexed). These folate-based inhibitors of thymidy- late synthase hold promise for greater therapeutic selectivity com- pared with 5-fluorouracil/leucovorin (2). At this time, however, our understanding of key effectors downstream of DNA damage required for thymidylate synthase inhibitor-induced cytotoxicity remains limited. Previous work in our laboratory has demonstrated synergism be- tween 5-fluorouracil/leucovorin combined with interferon (IFN)- in tumor cell killing in human colorectal cancer, with promising preclin- ical (3) and clinical (4) activity. The interaction mechanism requires 5-fluorouracil/leucovorin-induced DNA damage and does not affect cells that demonstrate 5-fluorouracil-induced RNA-mediated cytotox- icity that occurs in normal gastrointestinal tissues (1, 5, 6) or certain human colorectal cancer cell lines (e.g., HCT8 and HCT116; ref. 3). Thymineless stress-induced apoptosis in colorectal cancer cells de- void of thymidylate synthase activity (thymidylate synthase - cells; refs. 1, 7), 5-fluorouracil/leucovorin-induced cytotoxicity (3), and the synergistic interaction between 5-fluorouracil/leucovorin and IFN- (3, 4) all involve the Fas death receptor and its signaling pathway. More recently, we have demonstrated that IFN- is also synergistic in potentiating the cytotoxic effect of ZD9331 (2). In contrast to 5-flu- orouracil/leucovorin + IFN-, ZD9331 +/- IFN- displays a broader range of cytotoxicity that occurs via DNA damage-induced cell death. In expanded studies with ZD9331, we demonstrated addi- tional “apoptosis-related” sites of interaction between thymidylate synthase inhibitors and IFN-, independent of Fas, and downstream of the mitochondria, involving up-regulation and activation of specific caspases (2). All of the studies confirm that thymidylate synthase inhibition, either via conventional agents (5-fluorouracil/leucovorin) or via the newer thymidylate synthase inhibitors (ZD9331), induce apoptosis and cytotoxicity in human colorectal cancer cells independ- ent of the cell-cycle/DNA-damage response protein p53. However, the role of the multifunctional cell cycle-related protein p21 Cip1 in these models is less well characterized. P21 Cip1 functions at multiple levels (8), and although knowledge regarding the multifunctionality of this protein continues to increase, many of its functions remain to be determined. P21 Cip1 is well known as a p53 response gene (9) capable of inhibiting multiple cyclin- dependent kinases (CDKs), resulting in the induction of G 1 or G 2 cell cycle arrest, assuring the normal progression of cells through the cell cycle (10). CDKs are protein kinases that, when complexed to the appropriate cyclin, enhance cell cycle progression (reviewed in ref. 10). P21 Cip1 null cells, paradoxically, appear more susceptible to accumulation in an arrested phase of the cell cycle, including S phase (11). Additionally, deletion of p21 Cip1 has been shown to induce uncoupling of S phase and mitosis in cells treated with anticancer agents (12), and p21 Cip1 -induced G 1 arrest itself can lead to subse- quent depletion of mitosis-control proteins and abnormal mitosis with endoreduplication in recovering cells (13). These cell cycle phenom- ena are presumed to be important in determining whether or not a cell will recover from a given form of DNA damage. As an important mediator of such cell cycle phenomena, which are now known to play a critical role in apoptotic responses (14), it is not surprising that p21 Cip1 has been reported to have both pro- and antiapoptotic roles. Overexpression of p21 Cip1 in human cancer cell lines has been reported to induce apoptosis (15), implying a proapop- totic role, whereas overexpression of p21 Cip1 , leading to G 1 arrest, can protect against p53-mediated apoptosis in other human cancer models (16). P21 Cip1 may also be involved in the regulation of apoptosis induced by tumor necrosis factor  (17), tumor necrosis factor-related apoptosis-inducing ligand (18, 19), and Fas (20), as well as cytotoxic agents (11, 19, 21). Received 3/10/04; revised 6/21/04; accepted 7/8/04. Grant support: NIH Awards RO1 CA 32613, 4 T32-CA70089, Cancer Center Support (CORE) Grant CA 21765, and by the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Janet A. Houghton, Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: 901-495–3456; Fax: 901-495–3966; E-mail: janet.houghton@stjude.org. ©2004 American Association for Cancer Research. 6296 Research. on February 21, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from