An Msh2 Point Mutation Uncouples DNA Mismatch Repair and Apoptosis

[CANCER RESEARCH 64, 517–522, January 15, 2004] An Msh2 Point Mutation Uncouples DNA Mismatch Repair and Apoptosis

Diana P. Lin,1 Yuxun Wang,1 Stefan J. Scherer,1 Alan B. Clark,2 Kan Yang,3 Elena Avdievich,1 Bo Jin,1 Uwe Werling,1 Tchaiko Parris,1 Naoto Kurihara,3 Asad Umar,2 Raju Kucherlapati,4 Martin Lipkin,3 Thomas A. Kunkel,2 and Winfried Edelmann1 1Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York; 2Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Department of Health and Human Services, Research Triangle Park, North Carolina; 3Strang Cancer Research Laboratory at The Rockefeller University, New York, New York; and 4Harvard-Partners Center for Genetics and Genomics, Boston, Massachusetts

ABSTRACT These studies were performed with MMR-deficient cell lines that com- pletely lack particular MMR proteins and therefore lack all of the func- Mutations in the human DNA mismatch repair gene MSH2 are associated tions of those proteins. However, a significant proportion of HNPCC with hereditary nonpolyposis colorectal cancer as well as a significant pro- patients carry missense mutations in MMR genes (1), and it is unclear portion of sporadic colorectal cancer. The inactivation of MSH2 results in the accumulation of somatic mutations in the genome of tumor cells and resist- how these mutations affect individual MMR protein functions in DNA ance to the genotoxic effects of a variety of chemotherapeutic agents. Here we repair and damage responses. G674A show that the DNA repair and DNA damage-induced apoptosis functions of We therefore decided to generate a mouse line carrying the Msh2 Msh2 can be uncoupled using mice that carry the G674A missense mutation missense mutation to assess its impact on MMR and response to DNA in the conserved ATPase domain. As a consequence, although Msh2G674A damage and examine the consequences with respect to cancer suscepti- homozygous mutant mice are highly tumor prone, the onset of tumorigenesis bility. The mutation results in a glycine to alanine change at amino acid is delayed as compared with Msh2-null mice. In addition, tumors that carry residue 674 within the conserved ATPase domain at the COOH-terminal the mutant allele remain responsive to treatment with a chemotherapeutic region. This domain is characterized by the Walker “type A” motif agent. Our results indicate that Msh2-mediated apoptosis is an important GXXXXGKS/T (G denotes the modified G674 amino acid residue) component of tumor suppression and that certain MSH2 missense mutations known to coordinate the phosphate groups of ATP in many proteins that can cause mismatch repair deficiency while retaining the signaling functions that confer sensitivity to chemotherapeutic agents. hydrolyze ATP (18–20). Mutations in this MutS domain in bacteria and yeast result in MMR defects, and overexpression of these mutant proteins was shown to cause dominant mutator phenotypes (21–24). The impor- INTRODUCTION tance of ATP processing for MMR and tumorigenesis is underscored by the significant number of HNPCC missense mutations that are located in The DNA mismatch repair (MMR) system guards against genomic the ATP-binding domains of MSH2 (25). instability, and mutations in the human MMR genes MutS homolog 2 Here we show that the Msh2G674A mutation has differential effects (MSH2) and MutL homolog 1 (MLH1) are the cause of the majority of on the DNA repair and DNA damage response functions. Whereas it hereditary nonpolyposis colorectal cancer [HNPCC (1)]. Recent studies caused DNA repair deficiency that resulted in a strong cancer predis- indicate that MMR proteins not only protect mammalian genomes by position phenotype in the mice, it did not affect the DNA damage repairing mismatched bases that result from erroneous DNA replication, response function of Msh2. As a consequence, tumorigenesis in but also by mediating DNA damage-induced apoptosis as part of the Ϫ Ϫ Msh2G674A/G674A mice was delayed as compared with that in Msh2 / cellular response to endogenous and exogenous agents (2–4). These Ϫ Ϫ mice. In addition, unlike Msh2 / cells, Msh2G674A/G674A mouse studies showed that cell lines derived from HNPCC and MMR-defective embryonic fibroblasts (MEFs) and teratocarcinomas remained sensi- sporadic tumors or MMR-deficient mice displayed increased mutation tive to treatment with genotoxic agents. rates in their genomes and also had increased resistance to the genotoxic effects of a variety of DNA damage-inducing agents, including cisplatin, temozolomide and N-methyl-NЈ-nitro-N-nitrosoguanidine [MNNG MATERIALS AND METHODS (5–10)]. In addition, as demonstrated initially in yeast and later in mam- Generation of Msh2G674A Mice. A 3.6-kb HincII fragment containing Msh2 malian cells, MMR has been implicated in the removal of endogenous exon 13 was isolated from a 129SvEv bacterial artificial chromosome genomic lesions such as mutagenic 8-oxoguanine that is incorporated from the library and subcloned. A mutation was introduced that changed codon 674 from oxidized deoxynucleotide triphosphate pool during DNA replication (11, glycine (GGT) to alanine (GCT) by site-directed mutagenesis (Stratagene Quick 12). It has been suggested that the failure to clear DNA damage-bearing Change Kit). A 5.0-kb NotI fragment containing two LoxP sites flanking a cells may be responsible in part for the increased mutation frequency in neomycin-PGKhygromycin resistance cassette was subcloned into the single SpeI MMR mutant cells and also may confer a selective advantage in tumor site. The modified HincII fragment was subsequently used to modify the Msh2 cells (13–16). This hypothesis is consistent with the observation that genomic locus in bacterial artificial chromosome clone mB183k13 of the RPCI-22 MMR deficiency in mouse tissues leads to an elevation in mutation 129 mouse genomic library by RecET-mediated recombination (26). A 24-kb frequency after the mice are exposed to DNA-damaging agents (10, 17). KpnI fragment containing the modified locus was excised from the bacterial artificial chromosome clone and used for gene targeting in WW6 embryonic stem (ES) cells (27). Three correctly targeted ES cell lines were injected into C57BL/6J Received 9/18/03; revised 10/20/03; accepted 11/5/03. blastocysts. Male chimeras from all three lines were mated to C57BL/6J females Grant support: NIH Grants CA76329 and CA93484 (to W. E.), CA84301 and ES11040 (to R. K.), CN05117 (to M. L.) and Center Grant CA13330 (to the Albert and transmitted the mutant allele through their germ line. Subsequently, F1 males Einstein College of Medicine); a Deutsche Krebshilfe fellowship (to S. J. S.); and an Irma carrying the mutant allele were mated to Zp3Cre transgenic females (C57BL/6J) T. Hirschl Career Scientist Award (to W. E.). to remove the resistance cassette by LoxP-mediated recombination. Male and The costs of publication of this article were defrayed in part by the payment of page female mice carrying the modified allele were intercrossed to generate Msh2ϩ/ϩ, charges. This article must therefore be hereby marked advertisement in accordance with G674A/ϩ G674A/G674A 18 U.S.C. Section 1734 solely to indicate this fact. Msh2 , and Msh2 mutant mice. Notes: D. P. Lin, Y. Wang, and S. J. Scherer contributed equally to this work. Reverse Transcription-PCR Analysis. Total RNA was isolated from Msh2 Supplementary data for this article are available at Cancer Research Online (http:// mutant ES cell lines using Trizol (GibcoBRL). Reverse transcription-PCR was cancerres.aacrjournals.org). performed with forward primer 5Ј-CGTAGAGCCAATGCAGACGCT-3Ј and Requests for reprints: Winfried Edelmann, Department of Cell Biology, Albert Ј Ј Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. reverse primer 5 -GGATGGAAGAAGTCTCCAGC-3 using the one Tube re- Phone: (718) 430-2030; Fax: (718) 430-8574; E-mail: [email protected]. verse transcription-PCR reaction kit (Roche) according to the manufacturer’s 517

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. DNA REPAIR AND DAMAGE-INDUCED APOPTOSIS IN MUTANT MICE instructions. The following cycling conditions were used: 30 min at 50°C(1 RESULTS cycle); 2 min at 94°C,30sat60°C, and 45 s at 68°C (35 cycles); and 7 min at 68°C G674A (1 cycle). The resulting 280-bp fragment was digested with either MnlI to detect Generation of Msh2 Mutant Mice. The mutant mouse line the wild-type RNA transcript or AluI to detect the mutant RNA transcript. was created by a knockin gene targeting strategy (Fig. 1A). Analysis of 22 G674A Western Blot Analysis. MEF cell extracts were prepared according to stand- litters of the F2 offspring showed that the Msh2 allele was trans- ϩ ϩ ϩ ard procedures, and 50 ␮g of protein of each cell lysate were separated on a 10% mitted in a normal Mendelian ratio with 40 Msh2 / ,89Msh2G674A/ , SDS-PAGE gel. Protein was transferred onto a PROTRAN membrane, and the and 37 Msh2G674A/G674A. None of the heterozygous or homozygous membranes were subsequently incubated with mouse monoclonal antibodies di- animals displayed any developmental abnormalities. Molecular analysis rected against Msh2 (Ab-2; Oncogene), Msh6 (clone 44; BD Biosciences), and showed that the Msh2G674A mutation allowed normal gene expression ␤-actin (C-2; Santa Cruz Biotechnology). and did not interfere with the stability of the mutant protein (Fig. 1, C and Gel Mobility Shift Assays. Nuclear extracts were prepared as described D). In eukaryotes, MSH2 forms complexes with either MSH6 or MSH3 Ј previously (28). The invariant sense oligonucleotide 5 -GGGAAGCTGCCAG- to initiate the repair of single base mutations (MSH2-MSH6) or larger Ј ␥ 32 GCCCCAGTGTCAGCCTCCTATGCTC-3 was end-labeled with [ - P]ATP insertion/deletion mutations (MSH2-MSH3) (31–37). The formation of ϫ and annealed in 1 DNA binding buffer [12% glycerol, 20 mM HEPES (pH 7.9), these complexes is important for the stability of the MutS proteins, and 100 mM KCl, 1 mM DTT, and 5 mM MgCl ] with 3ϫ molar ratios of antisense 2 immunohistochemical analysis in tumor cells showed that the loss of oligonucleotide 5Ј-GAGCATAGGAGGCTGACACTGGGGCCTGGCAGCTT- Ј ϫ MSH2 is frequently associated with the loss of MSH6 (38). Western blot CCC-3 to form a GC homoduplex probe or with 3 molar ratios of antisense G674A/G674A oligonucleotide 5Ј-GAGCATAGGAGGCTGACATTGGGGCCTGGCAGCTT- analysis of cell extracts derived from Msh2 mice showed that CCC-3Ј to form a GT mismatch-containing heteroduplex probe. Twenty ␮gof the mutation did not alter the stability of either mutant Msh2G674A or nuclear extract were preincubated in 1ϫ DNA binding buffer, 1 ␮g of poly(dI-dC), Msh6 protein in the cells (Fig. 1D). In addition, immunohistochemical and 20 ng of unlabeled homoduplex for 5 min on ice in a total volume of 19 ␮l. analysis indicated that the subcellular distribution of the mutant Ten ng of radiolabeled DNA probe were subsequently added, and the binding Msh2G674A or the Msh6 protein was not affected (data not shown). mixture was incubated on ice for 30 min. For reaction using cold probe compe- tition, cold competitor was included in the preincubation mixture. For adenine nucleotide exchange experiments, ATP or ATP-␥-S was added 15 min after the addition of the DNA probe. The reaction mixture was then subjected to electro- phoresis in a 6% polyacrylamide gel in 1ϫ Tris-borate EDTA buffer. The gels were dried, and the percentage of relative binding of Msh2-Msh6 and Msh2G674A-Msh6 complexes to G/T oligonucleotide probe in the presence of increasing amounts of cold competitor or increasing ATP concentration was quantified using a STORM PhosphorImager with ImageQuant software (Molec- ular Dynamics) and calculated as [(Msh2-Msh6-probe complex/Msh2-Msh6- probe complex ϩ free probe) ϫ 100]. Cell-Free Extracts and MMR Assay. The MMR proficiency of MES cell line cytosolic extracts was measured using M13mp2 DNA substrates and subsequent transfection of bacterial cells as described previously (29). Repair efficiency is expressed (in percentage) as 100 ϫ (1 Ϫ the ratio of the percentages of mixed bursts obtained from extract-treated and untreated samples). The substrates used are described in the Fig. 3 legend. Microsatellite Instability (MSI) Analysis. Mutations in microsatellite sequences were assayed by PCR of single target molecules. Equal amounts of tail DNA isolated from 10 mice each of Msh2ϩ/ϩ, Msh2Ϫ/Ϫ, and Msh2G674A/G674A mouse strain were pooled and diluted to 0.5–1.5 genome equivalents. Cycling reactions for the three markers analyzed, U12235, D7Mit91, and D17Mit123, were performed as described previously (30). MEF Survival Analysis. MEF cells (2 ϫ 104) of each Msh2 genotype were seeded onto a single well of a 24-well plate in 10% FCS/DMEM. On the following day, the cells were exposed to cisplatin, 6-thioguanine, or MNNG at different drug concentrations for 24 h or for different time periods. After drug exposure, the cells were washed once with PBS, washed once with PBS:methanol (1:1), fixed in 0.5 ml of 100% methanol, and air dried. The cells were subsequently stained with 0.1% crystal violet and washed extensively with PBS, and the dye was extracted in 10% acetic acid. The dye concentration was determined by measuring absorp- Fig. 1. Generation of Msh2G674A mutant mice. A, schematic representation of the pMsh2G674A targeting vector (top diagram), the Msh2 genomic locus (second diagram), and tion at A600 nm, and the percentage of cell survival was calculated as (treated cells/untreated cells ϫ 100). The experiments were performed for three different the modified Msh2 locus (bottom diagrams). The numbered boxes represent Msh2 exons. K, KpnI; B, BamHI; S, SpeI. Shaded triangles indicate LoxP sites. The G674A mutation in exon MEF strains for each Msh2 genotype and repeated at least three times for each 13 is indicated. Solid bar indicates the KpnI-BamHI hybridization probe for Southern blot analysis. B, Southern blot analysis of representative animals carrying the different Msh2 strain. Cisplatin (Bedford Laboratories), 6-thioguanine (Sigma), and MNNG ϩ ϩ ϩ ϩ alleles: wild-type (Msh2 / ); undeleted heterozygous Msh2G674Ahygro/ (Msh2GAhygro/ ); (Sigma) dilutions in culture medium were prepared fresh each time before use. For ϩ ϩ deleted heterozygous Msh2G674A/ (Msh2GA/ ); and deleted homozygous Msh2G674A/G674A ␮ 6 exposure to MNNG, 20 M O -benzylguanine (Sigma) was added to the medium. (Msh2GA/GA). The sizes of the modified undeleted (15.7 kb) and modified deleted (11.0 kb) ϩ ϩ Ϫ Ϫ Xenograft Experiments. Msh2 / , Msh2 / , and Msh2G674A/G674A ES cells alleles are indicated. C, reverse transcription-PCR analysis of total RNA in mouse embryonic (2 ϫ 106) were implanted into BALB/c nu/nu mice by s.c. injection into the flank fibroblast cells. Top diagram, PCR primers located in exon 12 and exon 13 were used to ␮ ⅐ Ϫ1 amplify a 280-bp cDNA fragment. Codon 674 can be digested with MnlI in wild-type cDNA regions. A single dose of cisplatin (10 g g body weight) was administered i.p. and with AluI in the mutant cDNA. The sizes of the expected restriction fragments are 48 h after tumor implantation in half of the mice. The remainder of the mice indicated for each restriction enzyme. Bottom panel, restriction digestion of cDNA generated ϩ ϩ received a single dose of saline as untreated control. Tumor growth at the from RNA isolated from wild-type (Msh2 / ) and homozygous mutant Msh2G674A/G674A (Msh2GA/GA) cells verifies expression of the mutant allele. D, Western blot analysis of cell inoculation sites was monitored daily by measuring the tumor size using a Vernier ϩ ϩ ϩ ϩ extracts isolated from wild-type (Msh2 / ), heterozygous Msh2G674A/ (Msh2GA/ ), and ϭ ϫ 2 caliper, and tumor volume was calculated [volume length (width) /2]. The homozygous mutant Msh2G674A/G674A (Msh2GA/GA) mouse embryonic fibroblast cells. The values for each time point were calculated as the mean of 10 replicates. analysis indicates normal expression of the Msh2G674A and Msh6 protein. 518

ϩ ϩ Fig. 2. Mismatch binding activities of Msh2 / and Msh2G674A/G674A nuclear extracts. Gel mobility shift assay in Msh2 mutant nuclear extracts was performed by incubating nuclear protein extracts isolated from Ϫ Ϫ ϩ ϩ Msh2 / , Msh2 / ,orMsh2G674A/G674A (referred to as Msh2GA/GA) ES cells with G/T-containing heteroduplex oligonucleotides. The positions of the Msh2/Msh6-DNA complex and the unbound free probe are indicated by the arrows. Unlabeled (cold) G/T-containing heteroduplex competitor oligonucleotide in the molar ratios indicated (A, left panel) or increasing amounts of ATP (B, left panel) was included in the reaction mixture. The percentage of ϩ ϩ relative binding of Msh2-Msh6 (Msh2 / ) and Msh2G674A-Msh6 (Msh2GA/GA) complexes to G/T oligonucleotide probe in the presence of increasing amounts of cold competitor or increasing ATP concentration is indicated (A and B, right panels, respectively).

Mismatch Binding Activity in Msh2G674A/G674A Cell Extracts. of 244) unstable alleles that were found previously in Msh2ϩ/ϩ animals We next studied the mismatch binding activities of nuclear extracts (39). This analysis indicated that the genomes of Msh2G674A/G674A mice isolated from Msh2ϩ/ϩ, Msh2Ϫ/Ϫ, and Msh2G674A/G674A ES cells. Using displayed a highly significant increase in mutation frequency at these two gel mobility shift assays, we did not detect any significant differences markers (P Ͻ 0.0001 for D7Mit91 and U12235, Msh2G674A/G674A com- between Msh2ϩ/ϩ and Msh2G674A/G674A extracts in their DNA binding pared with Msh2ϩ/ϩ). Furthermore, the MSI at these loci in the affinity using an oligonucleotide substrate containing a G/T mismatch. In Msh2G674A/G674A animals was comparable with the MSI observed in addition, proteins in both extracts bound with similar, albeit lower, Msh2Ϫ/Ϫ mice [D7Mit91, 27% (36 of 132); U12235, 21% (19 of 92)]. affinity to a homoduplex oligonucleotide substrate, whereas Msh2Ϫ/Ϫ These results indicate that the Msh2G674A mutation impairs the repair extracts did not show any binding activity (Fig. 2A; data not shown). In function of the protein and significantly increases the mutator phenotype contrast to the Msh2ϩ/ϩ extracts, the mutant Msh2G674A/G674A extracts in the genomes of the mutant mice. were partially resistant to ATP-dependent mismatch release, even at Survival and Cancer Susceptibility in Msh2G674A Mutant Mice. concentrations that exceed normal physiological conditions (Fig. 2B). When cohorts of Msh2G674A/G674A, Msh2G674A/ϩ, and Msh2ϩ/ϩ mice The addition of the poorly hydrolyzable ATP-␥-S analog also resulted in were followed for a period of 12 months, the overall survival and cancer mismatch release in Msh2ϩ/ϩ extracts but not in the Msh2G674A/G674A extracts (data not shown). These results are consistent with previous studies in yeast and suggest that the resistance of Msh2G674A/G674A cell extracts to ATP-dependent release from mismatched DNA is caused by defective or altered ATP binding resulting from the substitution of alanine for glycine in the P-loop (24). MMR Deficiency in Msh2G674A/G674A Cells. To test the impact of the Msh2G674A mutation on DNA MMR, we measured the repair activity in ES cell extracts using substrates containing G⅐G mismatches, single- base insertion/deletion mismatches, or 2-base insertion/deletion mismatches with a nick either 3Ј or 5Ј to the mismatched base (Fig. 3). Whereas both Msh2ϩ/ϩ and heterozygous Msh2G674A/ϩ extracts repaired all of these substrates, extracts prepared from homozygous G674A/G674A G674A/G674A Msh2 cells did not. The repair defect in the Msh2 Fig. 3. DNA mismatch repair (MMR) deficiency in Msh2G674A/G674A cell extracts. Ϫ Ϫ ϩ ϩ Ϫ Ϫ ϩ extracts was comparable with the defect that was observed in Msh2 / DNA MMR activity was assayed in Msh2 / , Msh2 / , Msh2GA/GA, and Msh2G674A/ cell extracts as described previously (52). Substrates designated with a ⍀ contain the extracts. Ј G674A/G674A number of extra nucleotides that accompany the symbol. Substrates are designated 3 or MSI in Msh2 Mice. We assessed the in vivo mutator 5Ј, indicating the position of the nick relative to the mismatch. The 3Ј nick is in phenotype in the Msh2G674A/G674A mice by analyzing MSI in tail the Ϫ strand at the AvaII site (position Ϫ264). The 5Ј nick is in the Ϫ strand at the Bsu36I ϩ genomic DNA. We found that at the dinucleotide marker D7Mit91, 28% site (position 276). The nucleotide position of the mismatch or unpaired bases in the lacZ complementation gene is indicated after the @, where position ϩ1 is the first G674A/G674A (38 of 134) of alleles tested were unstable in Msh2 mice; in transcribed base of the lacZ complementation gene. The minus sign designates the strand contrast, 9% (11 of 118) of alleles in Msh2ϩ/ϩ genomes were unstable. containing the extra nucleotide(s). The results are averages based on counting Ͼ500 plaques/variable in at least three independent experiments. Error bars represent the SDs. Similarly, 20% (24 of 123) of the alleles at the mononucleotide marker Blue/white ratios of plaque color (data not shown) demonstrate that, when observed, U12235 in Msh2G674A/G674A mice were unstable, compared with 3% (7 MMR was directed to the nicked strand. 519

survival between the Msh2Ϫ/Ϫ and Msh2G674A/G674A mice was highly significant (Fig. 4; P ϭ 0.001, log-rank test). DNA Damage Response in Msh2G674A/G674A MEF Cells. The difference in survival between the Msh2Ϫ/Ϫ and Msh2G674A/G674A mice suggested that the mutant protein retained some function important for tumor suppression. In recent years, several studies demonstrated that MSH2-deficient human colorectal cancer cell lines as well as mouse embryonic fibroblast lines have an increased resistance to treatment with a variety of DNA-damaging agents including cisplatin, MNNG, and 6-thioguanine. It was proposed that the resistance to DNA damage- induced apoptosis in MMR-deficient cancer cells might provide a selective advantage in the initial stages of tumorigenesis (16). We therefore analyzed the genotoxic response to treatment with DNA-damaging agents in Msh2ϩ/ϩ, Msh2Ϫ/Ϫ, and Msh2G674A/G674A MEF lines. Consist- ent with previous results, Msh2Ϫ/Ϫ MEF cells were largely resistant to

Fig. 4. Survival of Msh2G674A mutant mice. The time of death or the time when the mice treatment with cisplatin at the drug levels tested (Fig. 5, A and B). In ϩ/ϩ G674A/G674A became moribund was recorded. The survival curves were generated by using GraphPad Prism contrast, both Msh2 and Msh2 MEF lines were sensitive ϩ ϩ ϩ 3.0 software. Solid black line, Msh2 / mice (n ϭ 21); solid green line, Msh2G674A/ mice ϩ/ϩ ϭ G674A/G674A ϭ Ϫ/Ϫ to cisplatin exposure. The differences in sensitivity between the Msh2 (n 27); solid red line, Msh2 mice (n 34); broken blue line, Msh2 (B6) G674A/G674A Ϫ/Ϫ mice (n ϭ 18), C57BL/6J (N9) backcross (42); broken red line, Msh2G674A/G674A (B6) mice or Msh2 cells and Msh2 cells were highly significant Ϫ Ϫ Ϫ Ϫ (n ϭ 15), C57BL/6J (N7) backcross. The difference between the Msh2 / (B6) and (P Յ 0.0003). The Msh2 / cells also displayed increased resistance to G674A/G674A ϭ Msh2 (B6) curves is significant (P 0.001, log-rank test). treatment with 6-thioguanine and, to a lesser extent, to treatment with MNNG, whereas Msh2ϩ/ϩ and Msh2G674A/G674A cells showed higher susceptibility of the Msh2G674A/G674A mice were clearly affected. None of sensitivity at the same drug concentrations (Supplementary Fig. 1). The ϩ ϩ the Msh2G674A/G674A mice died in the first 3 months of life, and by 6 cisplatin sensitivity in Msh2 / and Msh2G674A/G674A cells was associ- months, Ͼ90% of Msh2G674A/G674A mice were still alive (Fig. 4). By 9 ated with significant increases in apoptosis as assessed by terminal months of age, Ͼ60% of Msh2G674A/G674A mice were alive; however, the deoxynucleotidyl transferase-mediated nick end labeling assay (Fig. 5C; ϩ ϩ number of surviving animals declined rapidly in the next 3 months, and P Ͻ 0.0001 for both Msh2 / and Msh2G674A/G674A compared with all of the remaining mice died by 12 months of age. Only one untreated cells). In contrast, no significant increase in the number of Ϫ Ϫ Msh2G674A/ϩ and none of the Msh2ϩ/ϩ mice died during the same period apoptotic cells was seen in Msh2 / cells when compared with untreated of time. The reduced survival in the Msh2G674A/G674A mutant mice was cells. Interestingly, there was also a significant increase in the number of caused by an increase in cancer predisposition. Most of the animals that terminal deoxynucleotidyl transferase-mediated nick end labeling-posi- died and were available for analysis had developed non-Hodgkin’s lym- tive cells in the untreated Msh2G674A/G674A MEF cultures compared with ϩ ϩ Ϫ Ϫ phomas (10 of 16 animals, 63%) between the ages of 9 and 12 months the untreated Msh2 / or Msh2 / cell cultures (Ps Ͻ 0.001). (Supplementary Table 1). A smaller number of mice between the ages of Cisplatin Sensitivity in Msh2G674A/G674A Teratocarcinomas. In 7 and 10 months (3 of 16 animals, 19%) developed gastrointestinal the next set of experiments, we studied the in vivo cisplatin sensitivity of adenocarcinomas. One animal at 9 months of age developed a squamous Msh2 mutant teratocarcinomas in athymic nude mice. ES cells have the basal cell carcinoma of the skin (1 of 16 animals, 6%). In 2 animals that capacity to develop into teratocarcinomas when injected into immunod- were 10 months of age, no obvious tumors could be detected (2 of 16 eficient athymic nude mice and were shown to provide a suitable model animals, 12%). Although the tumor spectrum in the Msh2G674A/G674A system to study drug sensitivity. In addition, the use of Msh2Ϫ/Ϫ ES cells mice resembled that seen in previously studied Msh2Ϫ/Ϫ mouse strains as xenografts in athymic nude mice demonstrated a major impairment in (40–42), we noted a striking difference in survival. Whereas the 50% the cisplatin responsiveness of the tumor in vivo (43, 44). Because the survival of Msh2Ϫ/Ϫ mice on various mixed genetic backgrounds was results in the Msh2G674A/G674A MEF lines indicated that the Msh2G674A reported at approximately 6 months of age, it took between 9 and 10 mutation would behave differently in this model system, we studied months for 50% of the Msh2G674A/G674A mice to die. To directly compare tumor growth in nude mice injected with Msh2ϩ/ϩ, Msh2Ϫ/Ϫ,or the survival between the Msh2G674A/G674A and Msh2Ϫ/Ϫ lines on a Msh2G674A/G674A ES cells and measured their responsiveness to cisplatin similar genetic background, we generated a cohort of Msh2Ϫ/Ϫ and treatment. We found that without cisplatin treatment, ES cells of all three Msh2G674A/G674A mice that were backcrossed several times onto the Msh2 genotypes rapidly developed into teratocarcinomas after implanta- C57BL/6 background. This comparison confirmed that the difference in tion (Fig. 6, AϪC). However, treatment with cisplatin had different

Fig. 5. Cisplatin sensitivity and increased apoptosis in Msh2G674A/G674A mouse embryonic fibroblast (MEF) cells. MEF strains of the different Msh2 genotypes were exposed to cisplatin at varying concentrations and for different time periods. Three different MEF lines per genotype were analyzed for each concentration or time point, and each data point was calculated from three different experiments for each MEF line. A, survival of cells after a 48-h exposure to different cisplatin concentrations. B, survival of cells after exposure to 80 ␮M cisplatin at different time intervals. C, apoptotic response to cisplatin treatment (20 ␮M cisplatin for 24 h) as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling assay; the assay was performed using two MEF lines for each genotype, and at least 1000 cells in each of two independent experiments were counted per MEF line. 520

Fig. 6. Msh2G674A/G674A-derived teratocarcinomas remain sensitive to cisplatin treatment. Tumor growth and cisplatin treatment in athymic nude ϩ ϩ Ϫ Ϫ mice injected with Msh2 / (A), Msh2 / (B), P Ͻ 0.01 ,ء .and Msh2G674A/G674A (C) ES cells (Student’s t test).

effects on tumor growth in the three cell lines. Whereas Msh2Ϫ/Ϫ cells directed to the newly synthesized strand (3, 5). Because DNA adducts in did not respond to cisplatin treatment, and tumor growth occurred at the template strand cannot be removed, the MMR reaction is continu- similar rates in cisplatin-treated and untreated mice (Fig. 6B), the growth ously initiated upon repair synthesis, leading ultimately to the formation ϩ ϩ of both Msh2 / and Msh2G674A/G674A tumors was significantly sup- of double strand breaks that provide a signal for apoptosis. Alternatively, pressed after cisplatin treatment (Fig. 6, A and C). These results are it was proposed that the binding of MSH2-MSH6 and also MLH1-PMS2 consistent with the observations made in the MEF cells and demonstrate complexes to damaged bases at the replication fork could block DNA that although the Msh2G674A mutation has a severe impact on DNA replication or other processes such as transcription and repair, leading to mismatch repair, it still confers a wild-type-specific response to cell cycle arrest and cell death (47). The molecular analysis of cisplatin-induced DNA damage. In this regard, Msh2G674A is a separation Msh2G674A/G674A cells shows that although the mutant Msh2G674A pro- of function mutation. tein is capable of mismatch binding, it does not allow normal MMR to proceed and therefore supports the latter notion. Our results are consistent DISCUSSION with the idea that MMR components can function as sensors for genetic damage (16, 48) and are also in agreement with a recent model by Brown G674A We generated a mouse line that carries the Msh2 missense et al. (49), which proposes that MSH2-MLH1 complexes act as molec- mutation and studied the consequences on individual MMR functions and ular scaffolds that physically link downstream effectors involved in DNA G674A cancer susceptibility. The analysis of Msh2 mice showed that this damage response pathways such as the ATM (ataxia telangietasia mu- mutation results in MMR deficiency and increased cancer susceptibility tated) gene product and checkpoint kinase 2 (CHK2). In this model, in homozygous mutant mice. In contrast, DNA mismatch repair was not MSH2-bound CHK2 and MLH1-bound ATM complexes interact at the significantly impaired in heterozygous mutant mice, indicating that the sites of DNA damage, resulting in the phosphorylation of CHK2 by ATM mutation does not act in a dominant manner. Our analysis of this mouse and the subsequent activation of the S-phase checkpoint and apoptotic line demonstrates that the roles of MMR in the prevention of DNA pathways. Furthermore, MMR-mediated apoptosis appears to be acti- replication errors and DNA damage-induced apoptosis can be separated vated through p53-dependent and p53-independent pathways and also by Msh2 missense mutations, and both functions are important for tumor involves the activation of p73 (9, 50, 51). In contrast to MMR-deficient suppression. Although, the Msh2G674A/G674A mutant mice show a strong cell lines, which display variable defects in the induction of p53 and p73, cancer predisposition phenotype with a tumor spectrum similar to that of G674A/G674A Ϫ Ϫ Msh2 MEF cells have normal induction of both proteins, Msh2 / mice, the extended survival in these animals in the first 9 indicating that normal Msh2 ATPase activity is not required for this months of life is consistent with the idea that the loss of the DNA response (data not shown). The presence of mutant Msh2G674A protein damage-induced apoptotic response could provide a selective advantage in Msh2G674A/G674A ES cells might allow the formation of mutant Msh- for tumor cells in the initial stages of tumorigenesis (45, 46). We also Mlh complexes that are capable of signaling cell cycle arrest and apo- observed a rapid decline in survival of the Msh2G674A/G674A animals between 9 and 12 months of age, which might be explained by the ptosis and provide an explanation for the increased number of apoptotic cells that we observed in the untreated Msh2G674A/G674A MEF cultures. In eventual accumulation of genomic mutations in cells that are not cleared G674A by apoptosis. These cells could then accelerate tumorigenesis in the older these cells, the DNA repair defect caused by the Msh2 mutation Msh2G674A/G674A mice, once the initial barrier to tumorigenesis is over- prevents the removal of misincorporated bases or oxidized bases such as come. Our results indicate that the increased mutation rates caused by 8-oxoguanine; however, it does not interfere with the recognition and MMR deficiency are sufficient to drive tumorigenesis and that it is the binding of such lesions. The persistence of these DNA lesions in the combination of increase in mutation rates and defective apoptosis that genome and their continuous recognition by the mutant MMR complexes cooperates to result in tumorigenesis. Our results also demonstrate that may in turn result in checkpoint activation and increased apoptosis. This the DNA damage-induced apoptosis function of Msh2 can delay but not hypothesis is supported by preliminary studies that revealed decreased G674A/G674A prevent tumorigenesis. cell proliferation and alterations in the cell cycle in Msh2 5 G674A The DNA repair defect in the Msh2G674A/G674A mutant mice is con- MEF cells. The availability of Msh2 mutant mice will aid in future sistent with previous studies in bacteria and yeast and indicates that the studies to investigate the role of Msh2 in these processes. ATPase domain is essential for the activation of the repair processes that Our results also suggest that a subset of tumors that carry MSH2 facilitate the removal of mismatched bases (21–24). However, in contrast missense mutations will remain responsive to treatment with chemother- to the Msh2-null allele, the Msh2G674A mutation did not significantly apeutic agents, a finding that may have important implications for the affect the cellular response to DNA damage-inducing agents, indicating treatment of HNPCC patients. Different MSH2 missense mutations will that normal ATP processing with subsequent repair is not essential for the likely have varied effects on DNA repair and apoptosis. Therefore, apoptosis signaling function of Msh2. Different models have been de- determining the genotype/phenotype correlations of MMR point muta- veloped in the past to explain the resistance of MMR-deficient cell lines tions in HNPCC patients may provide valuable information for treatment to DNA-damaging agents. One model suggested that DNA repair-com- and prognosis. petent cells engage in futile repair cycles after treatment with alkylating agents because MMR is a strand-specific mechanism and is always 5 S. J. Scherer and W. Edelmann, unpublished observations. 521

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