Contributions by Mutl Homologues Mlh3 and Pms2 to DNA Mismatch Repair and Tumor Suppression in the Mouse

Research Article

Contributions by MutL Homologues Mlh3 and Pms2 to DNA Mismatch Repair and Tumor Suppression in the Mouse

Peng-Chieh Chen,1 Sandra Dudley,2 Wayne Hagen,1 Diana Dizon,1 Leslie Paxton,1 Denise Reichow,3 Song-Ro Yoon,3 Kan Yang,4 Norman Arnheim,3 R. Michael Liskay,2 and Steven M. Lipkin1

1Department of Medicine and Biological Chemistry, University of California, Irvine, Irvine, California; 2Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon; 3Molecular and Computational Biology, University of Southern California, Los Angeles, California; and 4Strang Cancer Research Laboratory at the Rockefeller University, New York, New York

Abstract preponderance of MLH1 and MSH2 mutations is at first glance Germ line DNA mismatch repair mutations in MLH1 and MSH2 paradoxical because multiple MutS and MutL homologues exist in underlie the vast majority of hereditary non-polyposis colon humans. Studies in yeast and mammals have shown that MutS cancer. Four mammalian homologues of Escherichia coli MutL proteins form MSH2/MSH6 and MSH2/MSH3 heterodimers, which heterodimerize to form three distinct complexes: MLH1/PMS2, have partially overlapping specificities for binding to DNA MLH1/MLH3, and MLH1/PMS1. Although MLH1/PMS2 is mismatches (4, 8, 9). These same studies also show that deficiency generally thought to have the major MutL activity, the precise in MSH2 or double deficiencies in MSH3 and MSH6 results in a contributions of each MutL heterodimer to mismatch repair mismatch repair ‘‘null’’ phenotype, whereas a single deficiency in functions are poorly understood. Here, we show that Mlh3 either MSH6 or MSH3 produces attenuated mismatch repair defects. contributes to mechanisms of tumor suppression in the mouse. This partial functional overlap between MSH3 and MSH6 likely Mlh3 deficiency alone causes microsatellite instability, im- explains the preponderance of MSH2 mutations in HNPCC compared with either MSH6 or MSH3 mutations (10–12). paired DNA-damage response, and increased gastrointestinal tumor susceptibility. Furthermore, Mlh3;Pms2 double-deficient A similar situation exists for the heterodimeric MutL homo- mice have tumor susceptibility, shorter life span, microsatellite logues, which associate with the MutS heterodimers following instability, and DNA-damage response phenotypes that are initial mismatch binding. MLH1 can heterodimerize with PMS2 indistinguishable from Mlh1-deficient mice. Our data support (orthologue of yeast Pms1p; refs. 1, 4, 5), MLH3 (orthologue of yeast previous results from budding yeast that show partial Mlh3p), or PMS1 (8). However, our understanding of the relative functional redundancy between MLH3 and PMS2 orthologues contributions of each mammalian MutL heterodimers to mismatch for mutation avoidance and show a role for Mlh3 in gastro- repair and tumor suppression is incomplete. intestinal and extragastrointestinal tumor suppression. The Our previous studies have shown that Mlh1, but not Pms2 or data also suggest a mechanistic basis for the more severe mis- Pms1, deficiency predisposed to gastrointestinal malignancy in the mouse (13). We also showed that Mlh1-deficient mice have match repair–related phenotypes and cancer susceptibility in Mlh1- versus Mlh3-orPms2-deficient mice. Contributions by significantly higher levels of microsatellite instability, most both MLH1/MLH3 and MLH1/PMS2 complexes to mechanisms notably in mononucleotide repeat tracts, than either Pms2-or of mismatch repair–mediated tumor suppression, therefore, Pms1-deficient mice (14). Of particular relevance are studies in provide an explanation why, among MutL homologues, only budding yeast, which showed partial functional overlap between germ line mutations in MLH1 are common in hereditary non- pms1 (orthologue of mammalian PMS2) and mlh3 and suggested polyposis colon cancer. (Cancer Res 2005; 65(19): 8662-70) that a similar overlap may exist for the mammalian MutL orthologues (4, 8, 15). Based on the yeast and mouse studies, we and others (1, 4, 13–16) have proposed that the lower mutation Introduction levels and gastrointestinal tumor susceptibility in Pms2- versus DNA mismatch repair contributes to tumor suppression by Mlh1-deficient mice was due to functional overlap between Pms2, reducing mutations and promoting apoptosis in response to certain Pms1, and/or Mlh3. types of DNA damage (1–5). In humans, the majority of hereditary MLH3 is the most recently characterized eukaryotic MutL nonpolyposis colon cancer (HNPCC) families and sporadic colorec- homologue (15, 17). Studies of colorectal cancer patients have tal cancer with microsatellite instability have germ line MLH1 or indicated that MLH3 mutations are very rare in HNPCC families MSH2 mutations or somatic inactivation of MLH1 (6). Whereas a (18–21). However, the extent to which mammalian MLH3 recent study suggests that PMS2 mutations might be under- participates in tumor suppression, and the precise mechanisms estimated because of paralogous genes that complicate mutation involved, has not been addressed. Here, we use mouse models to detection (7), it is unlikely that this alone accounts for the >40:1 ratio show that Mlh3 deficiency alone causes increased microsatellite of identifiable MLH1 versus PMS2 mutations in HNPCC (6). The instability, defective DNA damage–induced response, increased cancer susceptibility in gastrointestinal and extragastrointestinal tissues, and early mortality. We also find that mice deficient for Note: Supplementary data for this article are available at Cancer Research Online both Mlh3 and Pms2 show phenotypes that are more severe than (http://cancerres.aacrjournals.org/). Requests for reprints: Steven M. Lipkin, Cancer Genetics Clinic, Chao Family those in either single-deficient mouse. In fact, all phenotypes National Cancer Institute Designated Comprehensive Cancer Center, University of studied in Mlh3;Pms2 double-deficient mice are indistinguishable California, Irvine, 204 Sprague Hall ZC 4038, Irvine, CA 92697-4038. Phone: 949-824- from Mlh1-deficient mice. In particular, the effect on mutation in 9219; Fax: 949-824-9224; E-mail: [email protected]. I2005 American Association for Cancer Research. mononucleotide repeats is more than additive in the double- doi:10.1158/0008-5472.CAN-05-0742 deficient mouse, suggesting partial redundancy between Mlh3 and

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Pms2 for mismatch repair-mediated mutation avoidance. The day, cells were exposed to different concentrations (0-5.0 Amol/L) of apparent requirement for both Mlh3 and Pms2 to achieve full 6-thioguanine (Sigma, St. Louis, MO) in DMEM complete media containing j mismatch repair activity in the mouse (presumably each function- 15% defined calf serum (Hyclone, Logan, UT) at 37 C for 24 hours. Cells ing as a heterodimer with Mlh1) has important implications for were rinsed twice with 5 mL PBS and were incubated in 15% complete medium. Seven to 10 days later, cells were fixed in 30% ethanol and stained MutL activity in mammals. In summary, our findings show that with 0.25% methylene blue. Only colonies containing >100 cells were budding yeast mismatch repair is a reliable paradigm for counted. For each dose, four to six 100 mm plates were analyzed. For each mammalian mismatch repair, provide a basis for the higher genotype, two to three independently derived mouse embryonic fibroblasts mutation frequencies observed in Mlh1 versus Pms2 deficiency, were used and four experiments were tested in each cell line. Our treatment and help to explain why Mlh1-deficient but not Pms2-deficient protocol is essentially identical to our previous studies of 6-thioguanine– mice develop gastrointestinal tumors. Most importantly, our results induced death in Mlh1/ mouse embryonic fibroblasts (31). showing contributions by both Mlh1/Pms2 and Mlh1/Mlh3 Flow cytometry analysis. Mouse embryonic fibroblasts were synchro- 4 complexes to DNA-damage response and tumor suppression nized by incubating with 0.5% complete medium. After 24 hours, 2 10 to 5 provide insight into why, among the MutL homologues, only germ 1 10 cells were seeded into 100 mm plates depending on the planned day of harvest and incubated with 15% complete medium. After 12 hours, cells were line MLH1 mutations are common in HNPCC. rinsed with PBS twice, treated with 5 Amol/L 6-thioguanine in 15% complete medium, and maintained in the 6-thioguanine–containing medium for 72 or Materials and Methods 96 hours. When harvesting cells, both floating cells and attached cells were +/ +/ collected and washed with PBS twice. Cells were pelleted and resuspended in Mouse strains and survival analyses. Wild-type (Wt), Mlh3 , Pms2 , sample buffer containing 0.1% glucose in PBS. Cells were repelleted and fixed +/ and Mlh1 mice were all generated and maintained on the 129 Sv/ by adding cold 70% ethanol dropwise and then stored in 4jC overnight for / / Ev genetic background and intercrossed to generate Mlh3 , Pms2 , and complete fixation. The next day, samples were stained with propidium iodine / / / Mlh1 mice, respectively. Mlh3 ;Pms2 mice were generated by (Sigma) for cell cycle analysis. The percentage of sub-G cells from each +/ +/ 1 intercrossing Mlh3 ;Pms2 mice. In addition to the mice used in the genotype was quantified from Flow Cytometry plots with FLOWJO software present study, for comparison, previously published cancer susceptibility (Tree Star, Inc., Ashland, OR). For each genotype, two independently derived / / studies done with Pms2 and Mlh1 mice on other strain back- mouse embryonic fibroblasts were used and three experiments were tested / grounds are also included in Table 1. Mlh1 mice previously described by for each cell line. Statistical significance of differences in the percentage of Edelmann et al. (22) were on a mixed 129/Ola and C57B/L6 background, sub-G1 cells was analyzed using Student’s paired t test. / / whereas the Pms2 and Mlh1 mice previously generated by Prolla et al. Immunoblotting. Mouse embryonic fibroblast cell extracts were (13) were on a mixed 129Sv/Ev and C57B/L6 background. Kaplan-Meier prepared as previously described (32) and 60 Ag protein from each lysate survival curves were generated as previously done (10, 22–27). The numbers were separated on 4% to 12% gradient SDS-PAGE gels followed by electro- +/ of mice included in survival analyses are Wt (n = 34), Mlh3 (n = 27), transfer onto nitrocellulose membranes. The membrane was first incubated / / / / Mlh3 (n = 34), Mlh3 ;Pms2 (n = 20), Pms2 (n = 16), and with rabbit anti-Mlh1 antibody (Ab-2; Calbiochem) and then with secondary / Mlh1 ,(n = 36). Statistical significance between genotypes was horseradish peroxidase–conjugated anti-rabbit IgG antibody (Jackson determined using the log-rank test as previously done (10, 22–27). Immunoresearch Laboratories, West Grove, PA). Immunoblotting of h-actin Analysis of tumors. All lines of mice were observed and not necropsied (Santa Cruz Biotechnology, Santa Cruz, CA) was done as a loading reference. until they became morbid or moribund. Sacrificed mice were surveyed for Microsatellite instability analysis. For microsatellite instability anal- tumors and suspicious masses were analyzed histologically as previously ysis, >1,000 individual microsatellite templates were analyzed for insertion/ done (10, 22–30). Tumors were pathologically confirmed by Dr. Kan Yang, deletion mutations by single-molecule PCR using the same protocols as an experienced mouse cancer pathologist who has analyzed many different previously done by our group and others (14, 26, 33). Briefly, for mismatch repair–defective strains for cancer susceptibility (10, 22–30). mononucleotide microsatellite marker JH117, equal amounts of tail DNA Statistical analyses of tumor onset and incidence among the different were isolated from five mice of each genotype, pooled, and diluted to 0.5 mouse lines were done by using the Mann-Whitney test as previously done to 1.5 genome equivalents (14). Four mice each were studied for (10, 22–30). Mlh3/ and Mlh3/;Pms2/ for U12235. Statistically significant Immunohistochemistry. Formalin-fixed tissues were deparaffinized and differences in the mutation frequencies between genomic DNA from rehydrated as previously described (22–30). Antigen retrieving was done by different genotypes were tested by the method of equality of two binomial autoclaving slides in 0.01 mol/L citric acid solution at 121jC for 20 minutes. proportions (14). We computed a confidence interval for each mutation The sections were stained overnight with antibody for mammary gland frequency and calculated a P value to test the statistical hypothesis that (anti-Neu/erbB2 antibody, 1:2,000 at 4jC; DAKO Corp., Carpinteria, CA) or the frequencies in the two genotypes were the same (two-sided test). antibody directly recognizing Mlh1 (clone Ab-2, 1:10 at room temperature; To test additivity of mutation frequencies in Mlh3/;Pms2/ DNA / / Calbiochem, San Diego, CA). The secondary antibodies were added and compared with Mlh3 or Pms2 DNA, we used the equation X12 = X1 signal-enhancing steps were done following the protocol described on the + X2 (X1X2), where X1, X2, and X12 are the mutation probabilities Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). Slides for Mlh3/,Pms2/, and Mlh3/;Pms2/ double-knockout mice. The were developed using VIP staining (Vector Laboratories) and counter- probabilities were corrected for the fact that the observed mutation stained with methyl green. frequencies include the background frequency observed in wild-type genomic DNA. Cell Lines and 6-thioguanine Treatment Intercrosses of Mlh3+/;Pms2+/ and Mlh3+/;Pms2+/ mice were used to generate Mlh3/, Pms2/, and Mlh3/;Pms2/ and Wt mouse Results embryonic fibroblasts. Generation of Mlh1/ mouse embryonic fibro- / Survival and cancer susceptibility in mice with MutL blasts has been previously described (31). Mlh1 mouse embryonic homologue defects. Because yeast studies predicted some degree fibroblasts were derived from embryos generated by intercrosses of of functional overlap between mammalian MLH3 and PMS2 (15), Mlh1/ mice on a mixed 129 Sv/Ev and C57B/L6 background. Mouse which could account for different phenotypes of Mlh1/ and embryonic fibroblasts were established from day 12.5 postcoitus embryos / isolated from the uteri of pregnant mice as previously described (32). Pms2 mice (14), we compared life span and cancer suscepti- / / / / To analyze the cytotoxicity in response to 6-thioguanine–induced DNA bility in wild-type (Wt), Mlh3 ,Pms2 , Mlh3 ;Pms2 , and / damage, spontaneously immortalized mouse embryonic fibroblasts were Mlh1 mice on the same 129 Sv/Ev strain background. For these sparsely plated (1,000 cells/plate) into 100 mm plates. The following studies, we used mice that were functionally null for the genes www.aacrjournals.org 8663 Cancer Res 2005; 65: (19). October 1, 2005

Table 1. Comparison of tumor incidence in mouse lines

Genotype Total no. Mean life span (mo) Number of mice with tumors

Overall Gastrointestinal Lymphomas

Wild type 33 21.8 1 (3%) 0 1 (3%) Mlh3/ 27 15.1 22 (84%) 14 (52%) 11 (40%) Pms2/ 14 9.7 11 (77%) 0 11 (77%) Pms2/* 17 8.0 16 (94%) 0 16 (94%) Mlh3/;Pms2/ 19 6.9 16 (84%) 8 (42%) 11 (57%) Mlh1/ 34 6.7 32 (85%) 14 (41%) 18 (52%) c Mlh1/ 18 7.2 14 (78%) 6 (33%) 8 (44%) Mlh1/* 24 9.5 23 (95%) 20 (83%) 15 (62%)

NOTE: Wild-type, Mlh3/, Pms2/, Mlh3/;Pms2/, and Mlh1/ mice are on a 129 Sv/Ev genetic background. Previously published cancer susceptibility studies done with Pms2/ and Mlh1/ mice are also included for comparison with the Pms2/ and Mlh1/ lines used in the current study. Mlh1/ mice previously described by Edelmann et al. (22) were on a mixed 129/Ola and C57B/L6 background, whereas the Pms2/ and Mlh1/ mice previously generated by Prolla et al. (13) were on a mixed 129Sv/Ev and C57B/L6 background. The total number of mice analyzed comprehensively for cancer is indicated. All lines of mice were not sacrificed for tumors until they became morbid or moribund. All tumors were pathologically confirmed as previously done (10, 22–30). Gastrointestinal tumors are stomach, small intestine, and colorectal tumors. ‘‘Overall,’’ the sum of both gastrointestinal tumors and lymphomas. *Previous data from Prolla et al. (13). cPrevious data from Edelmann et al. (22).

under study based on lack of detectable protein by Western analysis Mlh3/ mice (P < 0.003 and P < 0.001, both Mann-Whitney test; (14, 17). Consistent with previous studies (13, 22), Mlh1-deficient Table 2), and lymphomas significantly earlier than in Pms2/mice mice have early mortality (mean 6.7 months) due to gastrointestinal (P < 0.01, Mann-Whitney test; Table 2). In fact, susceptibility to and extragastrointestinal cancers, and Pms2-deficient mice have less gastrointestinal cancer and lymphoma in Mlh3/;Pms2/ mice pronounced early mortality mean (9.7 months) associated with only was indistinguishable from Mlh1/ mice on the same 129 Sv/Ev extragastrointestinal cancers (Fig. 1; Table 1). Interestingly, Mlh3/ strain background (P = 0.57 for gastrointestinal cancer, P = 0.4 for mice have a significantly shorter life span compared with either lymphoma, Mann-Whitney test; Table 2; refs. 13, 22). In contrast, Wt or Mlh3+/ mice (P < 0.0001 for both comparisons, log-rank test; significant differences were seen in mean gastrointestinal tumor (Fig. 1) but live significantly longer than Pms2/ or Mlh1/ occurrence for comparisons of Mlh3/ versus Mlh3/;Pms2/ animals (P < 0.0001 for both; Fig. 1; Table 1). (P < 0.003) or Mlh1/ (P < 0.003), respectively (Table 2). For Next, we analyzed the tumor spectra in a cohort of 27 morbid lymphomas, significant differences were seen for comparisons of or moribund Mlh3/ mice. More than 50% of Mlh3/ mice developed primary malignancies in the lower gastrointestinal tract, including stomach, small intestine, colon, and rectum. Both adenomas (n = 9) and adenocarcinomas (n = 4) were observed (Fig. 2A-B; Supplementary Fig. S1A; Supplementary Table S1). In parallel, no gastrointestinal cancers were observed in >30 age/ gender-matched Wt littermate controls (Fig. 1; Table 1). Therefore, the gastrointestinal cancer susceptibility of Mlh3/ mice is highly significant (P < 0.0001, log-rank test; Fig. 1; Table 1). Tissue surveys showed that Mlh3/ mice also have significantly higher risk of extragastrointestinal cancer, including lymphomas, basal cell carcinoma of the skin, mammary gland carcinomas, osteosarco- mas, testicular cancer, and hepatic adenomas compared with wild- type mice (P < 0.0001, log-rank test; Fig. 3; Table 1; Supplementary Table S1). These data suggest that Mlh3 contributes to suppression of both gastrointestinal and extragastrointestinal tumors. Consis- tent with previous reports, gastrointestinal tumors were not seen in / the Pms2 animals (Table 1; ref. 13). Figure 1. Kaplan-Meier survival plot of Mlh1/, Mlh3+/, Mlh3/, Pms2/, Of particular interest, survival of Mlh3/;Pms2/ mice was Mlh3/;Pms2/, and Wt Mice. Using the log-rank test, significant survival / differences were observed between Mlh3/ (n = 34) versus Wt (n = 34; indistinguishable from Mlh1 mice (P = 0.21, Mann-Whitney test; / / / / / / P < 0.0001), Mlh3 ;Pms2 (n = 20) versus Mlh3 (P < 0.0001), Mlh1 Fig. 1) and 84% of Mlh3 ;Pms2 mice examined developed (n = 36) versus Mlh3/ (P < 0.001), Mlh1/ versus Pms2/ (n = 16; P < 0.001), Mlh3/;Pms2/ versus Pms2/ (P < 0.01), and Pms2/ versus Mlh3/ gastrointestinal or extragastrointestinal cancers (Table 1; Supple- / / / / / (P < 0.01) mice. Mlh1 versus Mlh3 ;Pms2 survival was not significantly mentary Fig. S1B-C). In Mlh3 ;Pms2 mice, gastrointestinal different (P = 0.77). All mice lines used in this study are on the 129 Sv/Ev cancers and lymphomas were detected significantly earlier than in genetic background.

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Figure 2. Gastrointestinal cancer types in Mlh3/ and related mismatch repair mutant mouse lines. A and B, gastrointestinal tumors in Mlh3/ mice. A, representative stomach adenoma (4); B, duodenal adenoma (4). Sections are H&E-stained section from Mlh3/ mice ranging from 12 to 14 months of age. Ca, adenocarcinoma; Mu, normal mucosa; SM, submucosal tissue; Ad, adenoma tissue. C and D, immunohistochemistry of Mlh1 protein in intestinal tumor of Mlh3/ (C;60) and intestinal tumor of Mlh3/;Pms2/ mice (D;60). Positive staining is indicated by dark red nuclear immunostaining. Sections are counterstained with methyl green.

Mlh3/ versus Mlh3/;Pms2/ (P < 0.001), Mlh1/ (P < 0.001), microsatellite instability is poorly defined (18, 34, 35). Our previous and Pms2/ mice (P < 0.01; Table 2). Significant differences were analyses on pooled Mlh3/ mouse embryonic fibroblasts using also seen for comparisons of Mlh1/ versus Pms2/ (P < 0.005) ‘‘short’’ (V14 bp) mononucleotide microsatellite repeat sequences and Mlh3/;Pms2/ versus Pms2/ mice (P < 0.01, all Mann- did not detect significant microsatellite instability (35). However, Whitney test; Table 2). In summary, our data indicate a tumor short microsatellite repeat sequences in mammalian cells are less suppressor role for mouse Mlh3, suggest that Mlh3 and Pms2 both susceptible to microsatellite instability mutations than longer contribute to mismatch repair–mediated tumor suppression, and repeat sequences (14, 36). To assess whether Mlh3 deficiency show that the combination of Mlh3 and Pms2 deficiencies is causes an attenuated mutator phenotype in mononucleotide essentially indistinguishable from Mlh1 deficiency for both tumor repeats similar to yeast Mlh3 deletion (15), we did more sensitive spectra and onset. microsatellite instability assays by making two changes in our Mlh3 and Pms2 roles in mismatch repair–mediated muta- experimental procedures: First, we used the more quantitative tion avoidance. The association between MLH3 deficiency and technique of single-genome microsatellite instability analysis (14)

Figure 3. Extragastrointestinal tumors in Mlh3/ mice. A, non-Hodgkin’s lymphoma metastasized to liver (20); B, basal cell carcinoma of the skin (4); C, mammary gland carcinoma (10); D, mammary gland carcinoma immunostained for Neu/ erbB2 (40). Neu/erbB2 immunopositivity is indicated by dark red cell membrane immunostaining, as highlighted by arrows. (A)to (C) are H&E stained, whereas (D) is counterstained with methyl green. All tumors are from Mlh3/ mice. Bcc, basal cell carcinoma; K, islands of keratinization; Li, normal liver.

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Table 2. Comparisons of gastrointestinal and lymphomatous tumor occurrence

Genotype Mean gastrointestinal FSE P Mean lymphoma age (mo) FSE P tumor age (mo)

Mlh3/ 12.46 11.62-13.31 16.90 16.2-17.6 <0.01* <0.01* <0.003* Pms2/ NA NA 9.6 9.08-10.12 <0.001* Mlh3/;Pms2/ 8.00 7.42-8.61 <0.003* 5.63 5.09-6.16 <0.01* <0.005* 0.57 0.4 Mlh1/ 8.71 8.07-9.36 5.72 4.95-6.50

NOTE: The ages of autopsied mice when tumors were verified are shown as mean with SE. All mouse strains are on the 129 Sv/Ev background. Abbreviation: NA, not applicable. *Statistically significant differences.

and, second, we analyzed longer mononucleotide repeat tracts, A24 damaging agents (37–43), including the DNA alkylation mimetic / and A33, respectively. For Mlh3 tail DNA, the microsatellite drug 6-thioguanine (44). To determine the relative importance of instability rate was significantly elevated compared with DNA from mouse Mlh3 and Pms2 in response to alkylation damage, we Wt mice at the A24 (U12235A) and A33 (JH117) mononucleotide compared survival of spontaneously immortalized mouse embry- repeat tracts (P = 0.015 and P = 0.008, respectively, test of equality onic fibroblasts after 6-thioguanine treatment using a colony of two binomial proportions; Table 3). formation assay. We found that Mlh3/ mouse embryonic To test the functional relationship of Mlh3 and Pms2 for mismatch fibroblasts displayed increased resistance to 6-thioguanine com- repair–mediated mutation avoidance, we compared microsatellite pared with Wt cells (P = 0.007, Student’s paired t test; Fig. 4A). The instability in the double- and single-knockout animals. We found level of resistance for Mlh3/ mouse embryonic fibroblasts was not / / / that mutation levels at both An markers in Mlh3 ;Pms2 mice significantly different from Pms2 mouse embryonic fibroblasts were significantly higher than either Mlh3/ (P < 0.001) or Pms2/ (P = 0.2) but was less than that observed for Mlh1/ cells (P < 0.001, (P V 0.003) animals, and were indistinguishable from Mlh1/ Student’s paired t test; Fig. 4A). Under the same conditions, Mlh3/; mice (P z 0.19; Table 3). In the Mlh3/;Pms2/ mice, the Pms2/ mouse embryonic fibroblasts were more resistant than mutation levels seemed to be greater than additive based on a either Mlh3/ or Pms2/ mouse embryonic fibroblasts (P = 0.003 binomial proportions test (P < 0.02). In all three genotypes, most in both comparisons) and indistinguishable from Mlh1/ mouse mutations in the mononucleotide repeat tracts were 1 to 2 bp embryonic fibroblasts (P = 0.62, Student’s paired t test; Fig. 4A). deletions. In summary, Mlh3 deficiency significantly increases To characterize the DNA-damage responses of the different mononucleotide repeat instability but to an extent less than that genotypes further, we used flow cytometry to analyze cell cycle in Pms2/ mice. Furthermore, results with the Mlh3/;Pms2/ profile of these mouse embryonic fibroblasts with treatment of mice suggest partial overlap of function for mutation avoidance, 5 Amol/L 6-thioguanine for 72 or 96 hours. Consistent with previous similar to the results seen in budding yeast (15). studies, treatment of this high concentration of 6-thioguanine Mlh3 and Pms2 roles in mismatch repair–mediated caused a significantly increased sub-G1 cell population (indicative of response to DNA damage. Human and/or mouse cell lines cellular apoptosis or necrosis) in Wt mouse embryonic fibroblasts deficient in MLH1, PMS2, MSH2,orMSH6 show increased resistance by 72 hours (Fig. 4C; data not shown). After 96 hours of treatment, and decreased levels of apoptosis in response to certain DNA- all mouse embryonic fibroblast lines have significantly increased

Table 3. Mononucleotide repeat mutation levels

Mouse model JH117(33A) Frequency (%) P U12235(24A) Frequency (%) P

Mlh1/ 43/100 43 74/360 20.5 >0.19 >0.19 Mlh3/;Pms2/ 34/100 34 42/193 21.7 <0.003* <0.003* Pms2/ 16/100 16 <0.001* 44/449 9.8 <0.001* 0.13 0.027* Mlh3/ 9/100 9 18/322 5.6 0.008* 0.015* Wt 1/100 1 4/223 1.8

NOTE: Mutation frequency data in tail DNA from 4-week-old Mlh1/, Mlh3/;Pms2/, Pms2/, Mlh3/, and Wt mice are presented on two unique mouse mononucleotide microsatellite markers JH117 [(A)33] and U12235 [(A)24]. The total number of mutant size variant alleles divided by the total number of alleles is shown for each microsatellite marker. We compared the mutation frequency among pairs of genotypes by testing the equality of two binomial proportions. We computed a 95% confidence interval for each mutation frequency and calculated a P value to test the statistical hypothesis that the frequency in the two genotypes is the same (two-tailed test). The U12235 mutation data for Mlh1/ and Pms2/ was reported previously using the same methodology (14). *Statistically significant differences.

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Figure 4. Response to 6-thioguanine–induced alkylated DNA damage. A, cytotoxicity. Independently derived Mlh3/ (n = 3), Pms2/ (n = 2), Mlh3/;Pms2/ (n = 2), and Wt (n = 3) mouse embryonic fibroblast cell lines were treated with 6-thioguanine as described in Materials and Methods. The number of survivor cell colonies for each 6-thioguanine treatment is scored, and the average survivals of each different exposure concentration for cultures were scored relative to that of untreated cultures (set to 100% for comparison; ref. 31). Data were then pooled from all independent experiments. Using the two-sided Mann-Whitney test, significant differences in survival rates are seen for Mlh3/ versus Wt (P < 0.01), Pms2/ versus Wt (P <0.01),Mlh3/ versus Mlh1/ (P <0.01),Pms2/ versus Mlh1/ (P <0.01),and Mlh3/;Pms2/ versus Mlh3/ or Pms2/ cells (P <0.01).6-TG, 6-thioguanine. B, Mlh1 and Pms2 protein expression in different mismatch repair mutant cells. Western blotting analysis of Mlh1 and Pms2 protein in mouse embryonic fibroblast lines are shown with h-actin protein levels for comparison. C, cell cycle analysis by flow cytometry. Mouse embryonic fibroblasts were treated with 5 Amol/L 6-thioguanine for 72 or 96 hours and stained with propidium iodide for analysis. Representative histograms with peaks of cells in G1, S, and G2 phases are shown for each genotype. Representative untreated mouse embryonic fibroblast cells were harvested at 96 hours; similar results were seen at 72 hours. D, quantification of sub-G1 cells 96 hours after 6-thioguanine treatment. Mouse embryonic fibroblasts were treated with 5 Amol/L 6-thioguanine for 96 hours and stained with propidium iodide for analysis. The percentages of sub-G1 cells from each genotype were quantified from histograms with FLOWJO software. Bars, SE.

numbers of sub-G1 cells in response to 6-thioguanine–induced DNA induced G2 cell cycle arrest (31). We observed significantly increased damage. However, mismatch repair–deficient mouse embryonic G2 arrest in wild-type mouse embryonic fibroblasts treated with fibroblasts showed a reduced fraction of sub-G1 cells compared 5 Amol/L 6-thioguanine for 72 hours, but this arrest is impaired in with Wt mouse embryonic fibroblasts indicative of an impaired mismatch repair–deficient cells (Fig. 4C and D). Consistent with our 6-thioguanine DNA-damage response in mismatch repair–deficient other DNA-damage studies, the G2 arrest is less pronounced in cells (Fig. 4C and D). Consistent with the colony formation studies, Mlh3/,Pms2/, Mlh3/;Pms2/, and Mlh1/ mouse embry- / / the fraction of sub-G1 cells in Mlh3 and Pms2 mouse onic fibroblasts. Therefore, both Pms2 and Mlh3 contribute to / / embryonic fibroblasts were similar (P = 0.87), and Mlh3 ;Pms2 6-thioguanine–induced G2 cell cycle arrest in mouse embryonic mouse embryonic fibroblasts have a sub-G1 population that is fibroblasts (Fig. 4C and D). Similar results were seen with treatment indistinguishable from Mlh1/ mouse embryonic fibroblasts (P = of methylating agent N-methyl-NV-nitro-N-nitrosoguanidine (data 0.16, t test; Fig. 4C and D). Previous studies in mouse embryonic not shown). In summary, these results show impaired DNA-damage fibroblast cells have shown that Mlh1 is required for 6-thioguanine– response in the context of either Mlh3 or Pms2 mutations. www.aacrjournals.org 8667 Cancer Res 2005; 65: (19). October 1, 2005

Comparisons with Mlh3;Pms2 double-deficient or Mlh1-deficient because the MLH1-PMS2 heterodimer is generally accepted as the animals indicate that both Mlh3 and Pms2 contribute to mismatch only significant MutL activity functioning in mismatch repair– repair-mediated response to DNA alkylation damage, presumably by related mutation avoidance and DNA-damage responses (for each functioning as heterodimers with Mlh1 protein. reviews see, refs. 4, 5, 47). A potential explanation for the Mlh1 protein expression in Mlh3/ and Mlh3/;Pms2/ differences between MLH1 and PMS2 phenotypes is some degree tumors and cell lines. Previous studies have shown that Pms2 of functional redundancy between the MutL homologues PMS2, levels are reduced in Mlh1-deficient cells (16). To evaluate whether MLH3, and PMS1, each of which partner with MLH1 (1, 4, 9, 13–16). the increased mismatch repair–related phenotypes observed in the Studies in yeast show that pms1 and mlh3 (orthologues of human Mlh3/ and Mlh3/;Pms2/ mice and cells might be due to PMS2 and MLH3, respectively) have partially overlapping functions decreased Mlh1 protein abundance, we determined Mlh1 levels in in mutation avoidance (15), which led to the suggestion that MLH3 mouse embryonic fibroblast lines by Western blot (Fig. 4B), and in might perform similar roles in mammalian mismatch repair gastrointestinal tumors and normal bowel by immunohistochem- mediated tumor suppression. To test this possibility and to istry (Fig. 2C-D; Supplementary Fig. S1D). As expected, Mlh1 determine more precisely the consequence of Mlh3 deficiency in protein expression was not detectably reduced in Pms2/ mouse the mouse, we compared the phenotypes of animals deficient in embryonic fibroblasts (Fig. 4B). Mlh1 protein expression was also Mlh3, Pms2, or both (Mlh3/;Pms2/) with Mlh1/ mice (which not detectably reduced in Mlh3/ or Mlh3/;Pms2/ mouse most likely represent the ‘‘mismatch repair-null’’ phenotype). In embryonic fibroblasts (Fig. 4B). Similarly, intestinal tumors from brief, we found that Mlh3 deficiency alone has significant Mlh3/ and Mlh3/;Pms2/ mice showed strong nuclear Mlh1 consequences for mutation avoidance, DNA-damage response, immunostaining in both tumor and surrounding normal gastroin- gastrointestinal and extragastrointestinal tumor susceptibility, and testinal tissue (Fig. 2C-D; data not shown), whereas only life span. Our findings suggest that mouse Mlh3 and Pms2 proteins background immunostaining was present in Mlh1/ normal both contribute to mismatch repair mechanisms relevant to tumor gastrointestinal tissue (Supplementary Fig. S1D). These results suppression, presumably as Mlh1/Mlh3 and Mlh1/Pms2 complexes, suggest that the phenotypes determined here for Mlh3 or and that both proteins must be present for complete mismatch Mlh3;Pms2 deficiency in the mouse were not secondary effects of repair function in somatic cells. decreased Mlh1 protein levels. Additionally, we looked at Pms2 Mlh3/ mice showed increased incidence of cancer at later mean protein levels in the same cell lines. As expected, Pms2 protein age than either Pms2-orMlh1-deficient mice. Mlh3/ mice had levels were much lower in Mlh1/ cells. However, Pms2 protein significantly higher occurrence of both gastrointestinal and extra- levels seem not to be notably different in Mlh3/ cells (Fig. 4B). gastrointestinal (largely lymphomas and basal cell carcinoma of skin) cancers compared with Wt mice. The extragastrointestinal Discussion tumors were of later onset and with reduced multiplicity in Mlh3- deficient mice compared with either Mlh1-orPms2-deficient mice. Among MutL homologues, MLH1 mutations are by far the most In gastrointestinal epithelium, tumors occurred later in Mlh3/ common cause of HNPCC, whereas PMS2, PMS1, and MLH3 compared with Mlh1/ mice, and as previously reported (14) mutations are rare (1, 8, 18, 34, 45, 46). Analyses of the knockout gastrointestinal tumors were not seen in Pms2/ mice. We found mouse models for all the MutL homologues show that Mlh1- it somewhat surprising that gastrointestinal cancers were seen in deficient mice develop gastrointestinal tumors, whereas Pms2-or Mlh3- but not in Pms2-deficient mice, especially given their Pms1-deficient animals do not (13). These findings in HNPCC respective mutator phenotypes. However, because Pms2-deficient patients and the MutL mouse models are somewhat surprising mice died (mainly from lymphomas) earlier than Mlh3-deficient mice, gastrointestinal tumors might not have had adequate time to develop. Consistent with increased tumor susceptibility, Mlh3/ mice showed two additional hallmarks of a mismatch repair defect. First, DNA from Mlh3/ mice had increased levels of mutations in mononucleotide repeat sequences. By using an assay with increased sensitivity, we found that mutation of mononucleotide repeats in Mlh3/ mice was on average f50% of the level seen in Pms2/ mice, which, in turn, was on average f50% of that seen in Mlh1/ mice. An f2-fold difference in mutation avoidance between Mlh1/ and Pms2/ mice was well documented in our previous reports (13, 14, 31, 48, 49). Second, using colony formation and flow cytometry analyses to assess the DNA-damage response, mouse embryonic fibroblast cells from Mlh3/ mice displayed increased survival relative to wild-type cells in response to the DNA-damaging agent 6-thioguanine. Therefore, Mlh3 knockout mice show multiple phenotypes, including increased tumor susceptibility, increased microsatellite instability, Figure 5. Model of Mlh1/Mlh3 and Mlh1/Pms2 complexes in mismatch repair mechanisms relevant to tumor suppression. Mlh1/Mlh3 and Mlh1/Pms2 and decreased responses to induced DNA damage, each of which heterodimers both function in repair of 1 base loop mispairs in mononucleotide are characteristic of mismatch repair defects relevant to carcino- repeats and in the response to DNA alkylation damage. Available evidence genesis. Overall, our results clearly show that Mlh3 deficiency in the suggests that both the Msh2/Mhs6 and Msh3/Msh6 heterodimers function in ‘‘1 base loop’’ repair. *DNA damage by the alkylation mimetic drug 6-thioguanine, mouse causes gastrointestinal and extragastrointestinal cancer 6-methylthioguanine. susceptibility and defects in mechanisms of mismatch repair tumor

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Mismatch Repair and Tumor Suppression by Mlh3 and Pms2 suppression. However, the phenotypes of Mlh3-deficient mice were ated with Mlh3 or Mlh3;Pms2 deficiency was not simply due to less severe than those of either Pms2-orMlh1-deficient mice. decreased protein levels of Mlh1. Studies in budding yeast suggested that partial functional Overall, we find that the phenotypes of the Mlh3/;Pms2/ redundancy between mouse Pms2 and Mlh3 could explain the mice are more severe than either Mlh3/ or Pms2/ mice and difference between Mlh1 and Pms2 knockout mice (15). indistinguishable from Mlh1/ mice. Extending the paradigm Therefore, we compared Mlh3;Pms2 double-deficient mice with developed for the yeast MutL homologues MLH3 and PMS1 (15), Mlh3, Pms2, and Mlh1-deficient mice. Deficiency in both Mlh3 and the stronger phenotype of the Mlh3/;Pms2/ mice suggests Pms2 caused a reduced life span compared with the Mlh3 or Pms2 partial functional redundancy between Mlh3 and Pms2 for single-deficient mice. Furthermore, the spectrum and timing of mismatch repair function. Whereas further studies are clearly gastrointestinal and extragastrointestinal cancer occurrences in the required, our initial findings suggest that DNA mismatch repair double-deficient mice was similar to that of Mlh1/ animals functions that help prevent tumorgenesis require both Pms2 and (13, 22). Consistent with increased tumor susceptibility, the absence Mlh3, presumably with each acting as a heterodimer with Mlh1 of both Mlh3 and Pms2 resulted in mutation levels that were higher (Fig. 5). Contributions by both MLH1/MLH3 and MLH1/PMS2 than either single-deficient animals and, importantly, similar to complexes to mismatch repair functions underlying tumor Mlh1/ animals (14). When the double-deficient mice were suppression provide insight and an explanation as to why among compared with the single-deficient mice, the effect on mutation HNPCC kindreds, only MLH1 mutations are vastly more common frequency within mononucleotide repeats is more than additive. than mutations in the other MutL homologues. Finally, our These results are consistent with Mlh3 and Pms2 performing results show that deficiency for Mlh3 alone in the mouse can partially overlapping roles, presumably when complexed with Mlh1, impair mismatch repair–mediated processes and result in the during repair of single base loop mispairs in mononucleotide acceleration of both gastrointestinal and extragastrointestinal repeats. Previous studies have shown that defects in MLH1 or PMS2 cancers. Perhaps, further and more targeted genetic testing will can compromise normal DNA-damage responses (37–43). Our data reveal mutation of human MLH3 in certain familial or sporadic here show that mouse embryonic fibroblast cells from Mlh3/ and cancers. Pms2/ animals are both more resistant to 6-thioguanine– induced cell death and G2 cell cycle arrest than Wt cells, but less resistant than Mlh1/ mouse embryonic fibroblasts. Importantly, Acknowledgments the Mlh3;Pms2 double-deficient cells are more resistant to killing Received 3/7/2005; revised 6/15/2005; accepted 7/28/2005. than either single-mutant, showing a level of resistance indistin- Grant support: USPHS grants CA87588 and R01CA98626, and American Cancer Society (S.M. Lipkin), R37GM032741 (R.M. Liskay), R01GM045413 (R.M. Liskay), and guishable from Mlh1-deficient cells, and suggesting that both Mlh3 RO1GM36745 (N. Arnheim). and Pms2 are important for the 6-thioguanine–induced DNA- The costs of publication of this article were defrayed in part by the payment of page damage response. Western and immunohistochemical analyses charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. provided evidence that the observed increased mutator phenotype, We thank Shaheen Sikander and Jaquelyn Nguyen for technical assistance and impaired DNA-damage response, and cancer susceptibility associ- Peter Calabrese for testing the microsatellite instability data for additivity.

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