[CANCER RESEARCH 58. 767-778. February 15, 1998] Defective Expression of the DNA Mismatch Repair Protein, MLH1, Alters G2-M Cell Cycle Checkpoint Arrest following Ionizing Radiation1 Thomas W. Davis, Carmell Wilson-Van Patten, Mark Meyers, Keith A. Kunugi, Scott Cuthill,2 Catherine Reznikoff, Christopher Garces, C. Richard Roland, Timothy J. Kinsella, Richard Fishel, and David A. Boothman3 Department of Human Oncology. University of Wisconsin-Madison, Madison, Wisconsin 53792 ¡T.W. D., C. W-V. P., M. M.. K. A. K.. S. C. C. K., C. G.. T. J. K.. D. A. B.¡; Department of Medicine and the Cancer Center, University of California-San Diego. La Jolla, California 92093 ¡C.R. B./; and DNA Repair and Molecular Carcinogenesis Program, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsvlvania 19107 ¡R.F.] ABSTRACT A role for the Mut L homologue-1 (MLH1) protein, a necessary com ponent of DNA mismatch repair (MMR), in G2-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G, arrest and/or G,-S transition after damage was, however, not discounted. We report that MLHl-deficient human colon carcinoma (IK'II16) cells showed decreased survival and a con comitant deficiency in G2-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected hu man colon carcinoma (IK"11 Id 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embry onic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from Ml,111 knockout mice also demonstrated classic DNA damage tol erance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLHI ' ) com pared with HCT116 (MI.Hl ) cells after IR or 6-TG. Retroviral vector- mediated expression of the E6 protein did not, however, affect the en hanced G2-M cell cycle arrest observed in IK" 1 116 3—6compared with MLHl-deficient IK II16 cells. A role for MLH1 in G2-M cell cycle checkpoint control, without alteration in G,, after IR was also suggested by similar S-phase progression between irradiated MLHl-deficient and MLHl-proficient human or murine cells. Introduction of a nocodazole- induced G2-M block, which corrected the MLHl-mediated G2-M arrest deficiency in IK I 116 cells, clearly demonstrated that IK Til ft and IK II16 3-6 cells did not differ in G, arrest or G,-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G, cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLHl-mediated G2-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells. INTRODUCTION Hereditary nonpolyposis colorectal cancer is a familial colon cancer syndrome that accounts for 1-5% of all colorectal tumors (1). This syndrome is caused by inherited mutations in several MMR4 genes, Received 7/23/97; accepted 12/16/97. The coses 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. 1This work was supported by Grant DE-FO02-93ER61707-06 from the U. S. Depart ment of Energy, Health Effects Research Division (to D. A. B.), and by NIH Grant 90-DKHD-10 (to T. W. D.). A preliminary version of this work was presented, in part, at the 1996 American Association for Cancer Research Annual Meeting, April 20-24, Washington, DC. 2 Present address: Roche Discovery Welwyn. Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AY, United Kingdom. 3 To whom requests for reprints should be addressed, at Department of Human Oncology, University of Wisconsin-Madison, K4/626 Clinical Sciences Center, 600 Highland Avenue. Madison, WI 53792. Phone: (608)262-4970; Fax: (608)263-8613; E-mail: boothman@humonc.wisc.edu. 4 The abbreviations used were: MMR, mismatch repair; 6-TG, 6-lhioguanine; MNNG, /V-methyl-/V"-nitrosoguanidine; DSB. DNA double-strand break; MLHI, mutL homo logue-1; MSH2, human mutS homologue; PMS2, postmeiotic segregation homologue-2; IR, ionizing radiation; SER, survival enhancement ratio (i.e., the dose of IR required to kill including MSH2, MLHI, and PMS25 (2-6). Mutations within these genes have also been observed in sporadic cancers of various origins (7-10). The MMR system is important for maintaining genomic fidelity through the recognition and repair of incorrectly paired nu- cleotides, which can arise in DNA by physical damage to existing nucleotides, DNA polymerase incorporation errors, or heteroduplex formation during genetic recombinational events (11-14). Expression of the MLHI gene, mapped to chromosome 3p21, is essential for competent MMR and maintenance of microsatellite stability (15, 16). Because MLH1 has no known enzymatic activity or functional motifs, it has been hypothesized to play a role as a "molecular matchmaker" for other DNA repair proteins (17). Transgenic mice lacking MLHI expression develop normally but present with lymphomas at an early age and are sterile, presumably due to faulty meiotic chromosome segregation (18, 19). To date, the only data on functional activity in mammalian cells suggest that the MLH 1 protein may regulate G2-M cell cycle checkpoint arrest following 6-TG or MNNG exposure, which results in DNA base damage and mispaired lesions (20). Previously, Hawn et al. (20) and Hawn et al. (21) generated and characterized a genetically matched human colon carcinoma model system, HCT116 (MLH1~) and HCT116 3-6 (MLHT). HCT116 cells were deficient in DNA MMR (22) due to the lack of MLH 1 transcript expression (4, 5). HCT116 3-6 (MLH1+) cells were gen erated by introducing (via microcell fusion) a single copy of normal human chromosome 3 into HCT116 cells (21). These corrected cells demonstrated expression of the MLHI transcript (by single-strand conformation polymorphism-PCR), reconstituted MMR, increased stability in the maintenance of lengths of microsatellite sequences, and an increased sensitivity to MNNG or 6-TG (20, 21). HCT116 cells were apparently defective in the G2-M cell cycle arrest checkpoint when compared with the MMR-competent, HCT116 3-6 cells after 6-TG exposure (20). These previous data did not, however, eliminate a role for the MLHI protein in G, arrest or transition from G, into S phase. In general, the mechanisms of damage-induced G2-M cell cycle checkpoint arrest in mammalian cells are poorly understood. A num ber of regulatory proteins have been implicated in the G2-M cell cycle checkpoint, including cyclin Bl, lyn kinase, cdc2, cdc25, and topoi- somerase II-a (23-30). We, therefore, sought to determine whether the MLHI protein, and MMR in general, played a role in G2-M cell cycle checkpoint responses following other forms of DNA damage, such as IR. The role of MMR in processing damage caused by IR, compared with agents that directly cause base mispairing (e.g., 6-TG), is not known. IR treatment generates a myriad of DNA lesions, including the formation of modified nucleotides, abasic sites, DNA- protein cross-links, SSBs, and DSBs (31, 32). The most lethal of these lesions are thought to be DSBs (33). DNA mismatches may arise directly from IR treatment or may result from later DNA repair 50% of one cell line divided by the dose required to kill 50% of another); SSB, DNA single-strand break. ' Throughout this report we use MLHI, MSH2, and PMS2 designations for both hunan (HMLHI. HMSH2, and hPMS2) and murine (Mlhl. Msh2. and Pms2) genes. 767 Research. on February 19, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from