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(CANCER RESEARCH56, 4881-4886, November 1. 19961 Advances in Brief The Role of DNA Mismatch Repair in Platinum Drug Resistance1 Daniel Fink,2 Sibylle Nebel, Stefan Aebi, Hua Zheng, Bruno Cenm, Alissar Nehmé,Randolph D. Christen, and Stephen B. Howell Department of Medicine and the Cancer Center, University of California at San Diego, La Jolla. California 92093-i'JWJS8 Abstract Loss of DNA mismatch repair occurs in many types of tumors. The effect ofthe loss ofDNA mismatch repair activity on sensitivity to cisplatin and a panel of analogues was tested using two pairs of cell lines proficient or deficient In this function. HCT116+ch2, a human colon cancer cell line deficient In hMLH1, was 2.1-fold resistant to cisplatin and 1.3-fold resist ant to carboplatin when compared to a subline complemented with chro mosome 3 expressing endometrial cancer a wild-type copy cell line HECS9, of hMLH1. which Likewise, Is deficient the human in hMSH2, was 1.8-foldresistantto cisplatinand 1.5-foldresistantto carboplatinwhen compared to a sublime complemented with chromosome 2 with a wild-type hMSH2. In contrast to cisplatin and carboplatin, which form the same types ofadducts in DNA, there was no difference In sensitivity between the DNA mismatch repair-proficient and -deficient cell lines for oxaliplatin, tetraplatin, transplatin, JM335, or JM216. The formation of protein-DNA complexes that contained hMSH2 and hMLH1 was documented by mo bility shift assay when nuclear extracts were incubated with DNA plati nated with cisplatin but not with oxaliplatin. These results demonstrate a correlation between failure of the DNA mismatch repair proteins to recognize the platinum adduct and low-level resistance, suggesting a role for the DNA mismatch repair system in generating signals that contribute to the generation of apoptotic activity. They also identify the use of drugs whose adducts are not recognized as a strategy for circumventing resist ance due to loss of DNAmismatchrepair. Introduction Cisplatin [cis-diamminedichloroplatinum] and carboplatin [cis-di ammine(1,1-cyclobutanedicarboxylato)platinum(II)] are chemothera peutic agents in widespread use for the treatment of a variety of human malignancies. However, many tumors are intrinsically resist ant to these drugs, and the development of acquired resistance during the course of treatment of even initially sensitive tumors is a common occurrence that constitutes a major obstacle to the curative use of these drugs. For example, the majority of human ovarian carcinomas respond initially to treatment with these agents, but most of these tumors recur, and when they do, most are resistant to treatment with both cisplatin and carboplatin (1). Changes of <2-fold in sensitivity to cisplatin are sufficient to account for treatment failure in human tumor xenografts (2). Resistance to cisplatin and carboplatin in cell lines selected with these agents in vitro is multifactorial (reviewed in Ref. 3), but partially because low levels of resistance are sufficient to cause Received 8/12/96; accepted 9/18/96. The costs of publication of this article were defrayed in part by the payment of page charges.Thisarticlemustthereforebe herebymarkedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. I Supported in part by Grant CTR4154 from the Council for Tobacco Research, grants lack of clinical responsiveness, it has been difficult to identify those mechanisms that are of the greatest clinical importance. We have recently reported the novel finding that loss of DNA mismatch repair produces low-level resistance to cisplatin (4). Loss of DNA mismatch repair is the predisposing factor in hereditary non polyposis colon cancer and occurs in a wide variety of sporadic human cancers as well (reviewed in Ref. 5). Loss of mismatch repair has been reported to cause moderate levels of resistance to the methylating agent MNNG3 (6) and high-level resistance to the anti metabolite 6-thioguanine (7). The fact that the mismatch repair system proteins can recognize the DNA distortion produced by the presence of 6-thioguanine and the adducts produced by both MNNG and cisplatin (8) suggests that the DNA mismatch repair proteins serve to detect the DNA damage caused by these agents and generate an injury signal that eventually contributes to the triggering of the apoptotic reaction that destroys the cell. No information is currently available on whether loss of DNA mismatch repair causes resistance to other platinum-containing drugs in addition to cisplatin. We have investigated the effect of the loss of mismatch repair on sensitivity to a variety of cisplatin analogues that produce either identical, similar, or quite different adducts in DNA and report here that resistance due to the loss of mismatch repair is specific to cisplatin and carboplatin. Failure of the loss of mismatch repair to cause resistance to platinum-containing analogues containing a diaminocyclohexane group is linked to failure of the DNA mismatch repair proteins to recognize this type of adduct and form complexes on platinated DNA probes. Of particular importance is the observation that loss of mismatch repair does not cause resistance to oxaliplatin or JM216, two new platinum analogues now in clinical evaluation (9, 10). Materials and Methods Cell Lines. The humanovarianadenocarcinomacell line 2008 (11) was maintained as were nologies, hMLH1 akademischen Nachwuchses zur Forderung des 1640 supplemented with the hMSH2-deficient human endometrial adenocarcinoma cell line HEC59 (13) and a subline complemented with chromosome 2 (clone HEC59/ 2-4). Both cell lines were maintained in Iscove's modified Dulbecco's medium (Irvine Scientific, Irvine, CA) supplemented with 2 m@vt L-glutamineand 10% heat-inactivated fetal bovine serum. All the chromosome-complemented lines were maintained in medium supplemented with geneticine (400 @xg/ml for HCT1l6+ch2 and HCI'l l6+ch3 and 600 @g/ml for HECS9+ch2; Life Tech of Cancer ofthe Award from the Kommission at 37°Cin RPMI deficient human colorectal adenocarcinoma cell line HCT116 was obtained from the American Type Culture Collection (ATCC CCL 247); sublines complemented with chromosome 3 (clone HCT116/3-6) and chromosome 2 (clone HCT116/2-1) were obtained from Drs. C. R. Boland and M. Koi (12), from the American Society of Clinical Oncology and from the Association for the Cure Prostate, by a Fellowship in a 5% CO2 atmosphere 2 m@iL-glutamine and 10% heat-inactivated fetal bovine serum. The hMLH1- Inc., Gaithersburg, in HCT1I6+ch2 MD). The absence and HCTI16+ch3 and presence cells of expression of as well as expression of of the University of Zurich to D. F., and by a Fellowship Award from the Ernst ScheringResearchFoundation,Berlin, and the EMDO Stiftung, Zurich, to S. N. This work was conducted in part by the Clayton Foundation for Research-California Division.R.D.C. andS. B.H.areClaytonFoundationInvestigators. D. F. and S. N. contributed equally to this work. 2 To whom requests for reprints should be addressed, at the Department of 3 The abbreviations used are: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; JM2I6, bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV); JM335, trans-ammine (cyclohexylaminedichlorodihydroxo)platinum(IV); oxaliplatin, [trans-(L)-l,2-diaminocy clohexane]oxalatoplatinum(II); tetraplatin,(d,l)trans-l,2-diaminocyclohexanetetrachlo Medicine 0058, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093. Phone:(619)822-1110;Fax:(619)822-1111. roplatinum(IV); poly(dI@dc),poly(deoxyinosinic-deoxycytidylic acid); GTBP, GIF bind ing protein. 4881 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE hMSH2 in HEC59 and HEC59+2 cells were verified by immunoblot analysis (data not shown). All cell lines tested negative for contamination with Myco plasma HN spp. @ transplatin Centre were purchased (Reading, from Sigma United Chemical Kingdom). Tetraplatin Co. (St. Louis, ,oc Pt HN sanne, Switzerland). JM216 and JM335 were kindly provided by the Johnson Technology HN\ 0 3\/ Materials. Cisplatin and carboplatin were obtained from Bristol-Myers Squibb Co. (Princeton, NJ). Oxaliplatin was a gift from Debiopharm (Lau Matthey ci / Pt /\ ci HN o—c 3 and Cisplafin MO). Carboplatin Cytotoxicity Assays. Cisplatin, carboplatin, oxaliplatin, tetraplatin, and JM335weredissolvedimmediatelybeforeusein 0.9%NaCIsolutionat 1mz@i, @ whereas stock solutions of JM216 and transplatin were prepared in DMSO. The final concentration of DMSO in the cultures was <0.1% at all drug concentrations and in controls. Clonogenic assays were performed by seeding 250 cells from a single-cell suspension into 60-mm plastic dishes in 5 ml of media. After 24 h, appropriate amounts of the drugs were added to the dishes, and the cells were exposed for 1 h. Thereafter, the cells were washed, and new drug-free medium was added. Colonies of at least 50 cells were scored visually after 8—10 days for HCT116 and 12—14 days for HEC59. Each experiment was performed a minimum of three times using triplicate cultures for each drug concentration. IC,0 values were estimated using logarithmic interpolation at a relative plating efficiency of 0.5. DNA Fragment Preparation. A l23-bp DNA ladder(Life Technologies, Inc.) consisting of various numbers of a single l23-bp fragment ligated in various copy numbers head-to-tail was platinated overnight by incubation NH / end-labeled using [y-32P]ATP (3000 Ci/mmol; DuPont New England /F@\ NH@1 ci :@ Oxaliplatin Tetraplatin KII:@— Pt \/ 3 ci OH 3 /I\ NH I 2 NH \/ Nuclear, molar ratio of 0.05 served as a competitor to identify specific binding. A Perkin Elmer Cetus model 373 atomic absorption spectrophotom eter was used to quantitate the extent of platination after exposure to cisplatin or oxaliplatin. JM335 HN@a 0 /f____\\,/ ci NH Pt ci OH HN 3 at Boston, MA) and T4 polynucleotide kinase (Promega, Madison, WI). Unin corporated label was removed by chromatography on Sephadex G50 (Phar macia Biotech Inc., Piscataway, NJ). Salmon sperm DNA platinated at a drug:nucleotide N I \Io—c NH 37°C with aquated cisplatin or aquated oxaliplatin diluted in 10 mMNaCIO4at a drug:nucleotide molar ratio of 0.05. The platinated DNA ladder was precip itated in ethanol and digested to completion with AvaI (Life Technologies, Inc.) to reduce all fragments to 123 bp. This fragment contains 13 0(1 pairs that are potential sites for intrastrand d(GpG) cross-links and 11 potential sites for d(ApG) cross-links. The digest was analyzed by electrophoresis in 2% agarose. The 123-bp DNA fragment was recovered from the gel, cleaned, and ethanol-precipitated. DNA quantitated by spectrophotometry at 260 nm was @ 0—c 0 ci Transpiatin cH 3 NH2 \@__/ @0 JM21Ô Fig. I. Structures of the platinum-containing drugs studied. Nudear Extract Preparation. Cells were harvestedduring exponential growth, washed in buffer [10 msi HEPES (pH 7.4), 10 mMKC1,and 0.5 mM DTT], and lysed at 4°Cin the same buffer containing 1% NP4O.The nuclei were collected by centrifugation and lysed in high-salt buffer [20 mMHEPES (pH 7.9), 500 mMNaC1,0.2 mMEDTA, 0.5 mi@iphenylmethylsulfonyl fluo ride, 0.5 mMDTF, 1.5 mr@i MgCI2,and 20% glycerol] at 4°C.The supernatant was designated the nuclear extract and stored at —70°C (14). Protein concen trationsweredeterminedby the methodof Bradford. Pt(II), and the chlorides and moieties attached to the platinum atom via oxygen atom linkages are displaced by water to produce aquated forms of the drug. The moieties attached via the nitrogen atoms remain associated with the platinum atom even after it has formed an adduct with DNA. Thus, after aquation, cisplatin and carboplatin produce the same types of adducts in DNA as do oxaliplatin and MObIlity Shift Assays. The platinated and radiolabeled l23-bp DNA frag tetraplatin, whereas the structure of the adducts produced by trans ment (1.5 ng) was incubated on ice for 30 mm with nuclear extract (5 @.&g) in platin, JM335, and JM216 are somewhat different. The effect of the the presence of 2.5 ,.&gpoly(dI-dc)poly(dI@dc)(Sigma) in a final volume of 10 loss of DNA mismatch repair activity on sensitivity to cisplatin and @d.hMLH1 was detected by supershift using the rabbit polyclonal antibody these analogues was tested using two pairs of cell lines. One member PC56 (Calbiochem, San Diego, CA), and hMSH2 was detected using the rabbit of each pair was proficient with respect to DNA mismatch repair, polyclonal antibody PC57 (Calbiochem). The rabbit polyclonal anti-Bcl-xL PC whereas the other was deficient; lack of DNA mismatch repair in one 67 (Calbiochem) and rabbit anti-actin (A-2066; Sigma) were chosen as nega tive controls. Antibody (0.05 @xg) was added to the incubation mixtures. All of the deficient lines was due to lack of hMSH2 function, whereas in incubation mixtures were diluted in shift buffer [12 mMHEPES (pH 7.9), 12% the other deficient line, it was due to lack of hMLH1 function. glycerol, 4 mMTris (pH 7.9), 5 ms@MgCI2, 100 nmi KCI, 0.6 inst EDTA, and Parental HCT1 16 human colon carcinoma cells are DNA mismatch 0.6 msi DTF], electrophoresed with 2 pi of 0.06% bromphenol blue in a 5% repair-deficient due to a deletion of one hMLHJ allele and mutation of polyacrylamide gel (29:1, acrylamidthisacrylamide), dried onto Whatman the other (13). In the HCTI 16+ch3 subline, the hMLH1 deficiency 3-MM paper, and autoradiographed at —70°C. was complemented by transfer into the cell of a complete chromo some 3 containing a wild-type copy of hMLHJ; the HCT1 l6+ch2 Results subline into which chromosome 2 had been transferred served as a control (12). Parental HEC59 human endometrial carcinoma cells are Cytotoxicity Studies. Fig. 1 shows the structures of cisplatin and the various analogues included in this study. They differ with respect DNA mismatch repair-deficient due to two different mutations in each allele of hMSH2 (13). In the HEC59+ch2 subline, the hMSH2 defi to the leaving groups that are displaced as the drug becomes aquated ciency was complemented by transfer of a full-length chromosome 2 and to the structure of the adducts produced in DNA. In the intracel lular compartment, the axial ligands are lost as Pt(IV) is reduced to containing a wild-type copy of the gene. 4882 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE Cisplatin 100 100 0 0 > > 2: ... \@ @10 C 10 ci, 0 ci@ 0@ 0 5 10 15 20 1 25 0 5 10 1@5 20 25 Oxaliplatin 100 Fig. 2. Clonogenic survival curves for cisplatin, oxaliplatin, and JM216for the mismatch-deficient (& HCT116+ch2)and -proficient (0 , HCT1I6+ch3)coloncarcinomacell linesandthe mismatch-defi cient (A, HEC59) and -proficient (•, HEC59+ch2) endometrial carci norm cell lines. Each point represents the mean of three to five exper imentsperformedwithtriplicatecultures.Bars,SD. 0 > 2: :, 2: :@ C,) U) C C a) 0 a) ci) 0 ci) 0@ 0@ 10 10 jiM JM21Ô 100 100 N 0 0 > > 2: :@ 2: :@ U) C/) C ci) C ci) 0 0 ci) ‘I) 0@ 0 20 Fig. 2 shows the survival of clonogenic cells as a function of drug concentration for three representative drugs (cisplatin, oxaliplatin, and JM216) for both pairs ofcell lines. The IC50 values for all ofthe drugs tested are presented in Table 1. The hMLH1-deficient HCT116+ch2 cells were 2.1-fold more resistant to cisplatin than the DNA mismatch repair-proficient HCT1 16+ch3 cells (IC50, 23.2 ± 3.7 versus Table 1 IC50 values for the platinum-containing drugs used in DNA mismatch repair deficient (HCTII6+ch2 and HECS9) and -proficient cells (HCTII6+ch3 and HEC59+ch2)a HCTI16+ch2 (@&M)Cisplatin Carboplazin Oxaliplatin Tetraplatin JM335 Transplatin 1.9a JM2I623.2 Values arethe (MM)HCTI16+ch3 (g.CM)HEC59 ±3.7 125.2 ±12.0 ±3.5 97.9 ±6.1 14.1 ±3.6 30.1 ±8.8 23.3 ±2.2 16.3 ±4.3 33.7 ±10.7 23.3 ±2.0 (@&M)HEC59+ch2 ±1.0 51.6 ±3.3 14.9 ±2.2 ±1.2 35.0 ±4.3 21.5 ±4.4 15.8 ±1.6 26.0 ±1.7 17.5 ±2.5 17.9 ±2.6 263.4 ±74.0 287.4 ±68.2 221.6 ±38.8 298.3 ±55.1 53.2 ±5.511.2 50.2 ±3.114.0 38.8 ±3.27.9 39.9 ± mean ±SD of at least three independent experiments in triplicate. 40 60 80 100 10 20 40 60 jiM 80 100 11.2 ±3.5 @LM SD; n 5; P < 0.05 in a two-sided t test). Likewise, the DNA mismatch repair-deficient HEC59 cells were 1.8-fold more resistant to cisplatin than the proficient HEC59+ch2 cells (IC50, 14.0 ±1.0 versus 7.9 ±1.2 @tM SD; n = 3; P < 0.05 in a two-sided t test). Carboplatin contains a 1,1-cyclobutanedicarboxylato-leaving group and undergoes aquation more slowly, but the structures of the aquated forms of cisplatin and carboplatin are the same as are the types of adducts produced in DNA. Consistent with this, carboplatin was 5.4-fold less potent than cisplatin against the HCT116+ch2 cells and 3.7-fold less potent against the HEC59 cells. However, as for cisplatin, both DNA mismatch repair-deficient cells were resistant to carboplatin. The HCT116+ch2 cells were 1.3-fold resistant relative to the HCT1 16+ch3 cells (IC50, 125.2 ±12.0 versus 97.9 ±6.1 @M SD; n = 3; P < 0.05 in a two-sided t test), and the HEC59 cells were 1.5-fold resistant relative to the HEC59+ch2 cells (IC50, 5 1.6 ±3.3 versus 35.0 ±4.3 @.LM SD; n 3; P < 0.05 in a two-sided t test). Although the potency of the other drugs tested differed as a func lion of chemical structure, the data presented in Table 1 show that 4883 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE there was no difference in sensitivity between the DNA mismatch repair-proficient and -deficient cells for oxaliplatin, tetraplatin, trans platin, JM335, or JM2I6 over the first two logs of cell kill. This indicates that, among the drugs tested, platinum drug resistance asso ciated with loss of DNA mismatch repair activity is specific for cisplatin and carboplatin and suggests that this is due to differences in the ability of the DNA mismatch repair proteins to recognize the different types of adducts produced. The difference in sensitivity of the mismatch repair-deficient HCT116 and HEC59 cells was not due to a decrease in the uptake of cisplatin into these cells or the extent of DNA platination (data not shown). The l.h cellular accumulation was 303 ±58 fmol4tg protein (SD) for the HCTI l6+ch2 cells and 289 ±82 fmol/p@gprotein (SD) for the HCT116+ch3 cells (n = 4; P = 0.80 in a two-sided t test). Mobility Shift Assays. The ability of mismatch repair proteins to recognize different types of platinum adducts was examined using mobility shift assays. Nuclei from the human ovarian adenocarcinoma cell line 2008 were used as a source of nuclear extract because these cells express hMLH1 and hMSH2 in amounts that are readily detect able by immunoblot and had been previously characterized with respect to their ability to form complexes with platinated DNA (15). The formation of protein complexes on platinated DNA was detected by gel mobility shift assay using a l23-bp double-stranded oligonu cleotide platinated with either cisplatin or oxaliplatin at a drug: nucleotide molar ratio of 0.05. Fig. 3A shows that, in the absence of nuclear extract, the l23-bp probe migrated to the bottom of the gel. When incubated with nuclear extract in the presence of carrier DNA in the form of poly(dldc)poly(dldc), three complexes were detected by virtue of their retarded gel mobility (Fig. 3A, Lane 4). Addition of a 1000-fold excess of salmon sperm DNA platinated with cisplatin at a drug:nucleotide molar ratio of 0.05 eliminated the formation of all three complexes (Lane 3), and incubation of nonplatinated probe with nuclear extract did not result in the formation of any complexes (data not shown), both of which establish that complex formation was specific for platinated DNA. The presence of hMSH2 and hMLH1 proteins in all three of the complexes formed on DNA treated with cisplatin was documented by the fact that addition of either anti hMLH1 antibody (Lane 5) or anti-hMSH2 antibody (Lane 6) caused a supershift of each of the complexes that was not observed with a control antibody directed against Bcl-xL (Lane 8). Fig. 3B shows the results of the same type of experiment performed under identical conditions and run on the same gel with the 123-bp probe platinated with oxaliplatin. In the presence of carrier poly(dFdc)poly(dldc) DNA, the probe formed two complexes (Fig. 3B, Lane 4), and complex formation was eliminated by addition of a 1000-fold excess of salmon sperm DNA platinated with oxaliplatin (Lane 3). However, neither the anti-hMSH2 (Lane 6) nor the anti hMLH1 antibody (Lane 5) detected the presence of hMSH2 or hMLHI in these complexes. This result indicates that different types of complexes are formed depending on the type of N-containing adduct in the DNA, and that the DNA mismatch repair complex that forms on cisplatin adducts does not form on oxaliplatin adducts. This specificity parallels the fact that loss of DNA mismatch repair func tion resulted in resistance for cisplatin but not for oxaliplatin, sug gesting that resistance is related to a failure of the repair system to recognize the presence of the adduct or generate a signal that can trigger apoptosis. DNA, but is not associated with resistance to analogues that produce several other types of adducts. This observation is important for several reasons: (a) it identifies a novel molecular mechanism that can cause cisplatin and carboplatin resistance; (b) it indicates a role for at least the hMSH2 and hMLH1 components of the DNA mismatch repair system in the recognition of DNA damage or the generation of signals that leads to apoptosis; (c) it documents that the components of the DNA mismatch repair system responsible for the difference in sensitivity are quite specific in their ability to discriminate between different types of closely related adducts; and (d) it identifies a series ofplatinum-containing drugs that might be of therapeutic value for the treatment of tumors resistant to cisplatin and carboplatin by virtue of the loss of DNA mismatch repair. MSH2, either by itself (16) or in conjunction with GTBP/pl6O (17, 18), recognizes small DNA mismatches, and Duckett et a!. demon strated that hMutSa activity, consisting of a heterodimer of hMSH2 and GTBP/pl6O, can bind to DNA platinated by cisplatin (8), sug gesting that at least some types of cisplatin adducts are recognized as the functional equivalent of a nucleotide mismatch by the DNA mismatch repair system. The results presented in this study extend these observations by documenting that whether or not a cell can recognize the adduct via the mismatch repair system has a functional consequence, in this case, manifest as a change in drug sensitivity. A priori one might have expected that loss of a DNA repair system would result in hypersensitivity to cisplatin. There is good evidence that cisplatin adducts can be removed from DNA by nucleotide excision repair (19, 20), and cells defective in nucleotide excision repair are in fact markedly hypersensitive to this drug (21, 22). However, in the case of the DNA mismatch repair system, loss of function results in cisplatin resistance. This is true also for two other drugs that produce distortions in DNA that are recognized by the DNA mismatch repair complex of proteins 6-thioguanine and MNNG (8). The current paradigm is that the DNA mismatch repair complex recognizes the adduct in the template strand and attempts repair of the newly synthesized nonadducted strand. As long as the adduct persists, insertion of new bases in the nonadducted strand fails to resolve the apparent mismatch, and the futile cycling of the attempted repair results in the generation of a signal that normally causes apoptosis (8). When the DNA mismatch repair system is defective, presumably this signal is not generated, and the cell seems to be resistant to the drug. The structural basis for the ability of hMSH2 to recognize mis matches is not well defined beyond the fact that the double helix is distorted. The adducts produced by cisplatin and carboplatin also distort DNA, and the data are consistent with the concept that it is this distortion that is being recognized. Information is not currently avail able on whether hMSH2 or the hMSH2/GTBP complex can distin guish between the different types of cisplatin adducts, which include G-G, A-G, G-X-G intrastrand cross-links, G-G interstrand cross-links, and G monoadducts, although it has been demonstrated that the hMutSa activity can distinguish between the cisplatin l,2-intrastrand and the transplatin 1,3-intrastrand adducts (8). The failure to recog nize the adducts produced by oxaliplatin may be a result of a differ ence in the distortion produced by its adducts in DNA, or it could be due to steric hindrance of the binding of hMSH2/GTBP by the diaminocyclohexane ring present in the oxaliplatin adduct. Resolution of this question will require detailed structural information about the differences in cisplatin and oxaliplatin adducts complexed with hMSH2. The current information provides a reasonable basis for predicting that loss of DNA mismatch repair will cause resistance to Discussion other platinum-containing analogues that do not have substitutions on the amine groups. The present study indicates that loss of DNA mismatch repair due The sequence of events in DNA mismatch repair is believed to to lack of either hMSH2 or hMLHI activity results in resistance to both cisplatin and carboplatin, drugs that form the same adducts in begin with the binding of hMSH2/GTBP to the mismatch, followed 4884 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE 12345678 9 A p4 Ifri@ + + + @1 + + + + + + + + + + + + + + + + + + + + + + Carrier Competftor + + antl-hMLH1 + + an11@hMSH2 + + Fig. 3. Recognition of cisplatin (A) and oxaliplatin (B) adducts by nuclear protein extracts documented by mobility shift assays. The 123-bp DNA fragment + was platinated to the same drug:nucleotide molar . ratio of 0.05 with cisplatin or oxaliplatin. The carrier Cisplatintreated DNA Nuclear Extract anfl-Bcl-x1 + DNA was poly(dklc)-poly(dI-dc), and the competitor DNA was salmon sperm DNA platinated to a drug: nucleotidemolarratioof0.05witheithercisplatin(A) 123456789 B or oxaliplatin (B) and added in 1000-fold excess relative to the DNA. @ •@@ipd ‘@‘ •@11@ @. + + + 4 + + + + 4 + Oxaliplatin + + + + + + + Nuclear + + + + + + + Carrier + Competitor + + antl-hMLH1 + 1@ anli-hMSH2 + + + 4. treated DNA Extract antl-Actin by the recruitment of a hMLH1IPMS2 heterodimer (23). The fact complex fully functional for nucleotide mismatch repair is required that deficiency in DNA mismatch repair due to loss of hMLH1 for the generation of this signal. hMLH1 has already been identi function results in resistance to cisplatin and carboplatin suggests fled as having a function in meiotic recombination that does not that the hMSH2/GTBP complex by itself is not able to generate the seem to involve hMSH2 (24), and it is possible that the signaling signal that contributes to the triggering of apoptosis and whose loss function of the mismatch repair complex can be mediated by only results in resistance. However, it is not clear that a complete a subset of all of the proteins required for full DNA repair activity. 4885 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research. DNA MISMATCHREPAIRAND PLATINUMDRUG RESISTANCE The spectrum of mutations that result in loss of DNA mismatch repair activity may differ from the spectrum of mutations that produce drug resistance. A variety of other proteins also bind to cisplatin adducts, the best-studied of which are members of the high mobility group family of proteins (25, 26). In the case of one of these, the Saccharomyces cerevisiae gene Ixrl, mutational loss is also associated with low-level resistance (27). Proteins containing the high mobility group domain can antagonize the excision of cisplatin adducts by the nucleotide excision repair mechanism (28). How any of these interact with the DNA mismatch repair proteins is unkown, but it is likely that they also play a role in the recognition of cisplatin damage and generation of an injury signal. How likely is it that the relatively small degree of resistance to cisplatin and carboplatin associated with loss of mismatch repair is clinically significant? Unlike many other drugs, only quite small degrees of cisplatin resistance (<2-fold) are required to account for clinical failure of treatment (2). The 2.1-fold resistance observed in the HCT116 system and 1.8-fold resistance observed in the HEC59 system would be expected to be sufficient to result in the gradual enrichment of cells in the tumor population deficient in mismatch repair during repeated courses of treatment, and this may contribute to the phenomenon of acquired cisplatin resistance that is so common among patients with tumors such as ovarian carcinoma and squamous cell carcinomas of the head and neck that are initially quite responsive to this drug. The results of this study showing that loss of mismatch repair does not confer resistance to oxaliplatin or JM216 suggest that the use of these drugs may avoid this problem and recommend these drugs for use in tumors deficient in mismatch repair. 8. Duckett, D. R., Drummond, J. T., Murchie, A. I. H., Reardon, J. T., Sancar, A., Lilley, D. M. J.. and Modrich, P. Human MutSa recognizes damaged DNA base pairs containing 0@-methylguanine, O@°-methylthymine,or the cisplatin-d(GpG) adduct. Proc. NatL Acad. Sci. USA, 93: 6443-6447, 1996. 9. Levi,F.,Perpoint,B.,Garufi,C..Focan,C.,Chollet,P.,Depres-Brummer, P.,Zidani, R., Brienza, S., ItZhaki, M., Iacobelli. S., Kunstlinger, F., Gastiaburu, J., and Misset, J-L. Oxaliplatin activity against metastatic colorectal cancer: a Phase II study of 5-day continuous venous infusion at circadian rhythm-modulated rate. Eur. J. Cancer, 29: 1280—1284, 1993. 10. Raynaud, F. I., Mistry, P., Donaghue, A., Poon, 0. K., Kelland, L. R., Barnard, C. F., Murrer, B. A., and Harrap, K. R. Biotransformation of the platinum drug JM216 following oral administration to cancer patients. Cancer Chemother. Pharmacol., 38: 155—162, 1996. 11. DiSaia, P. J., Sinkovics, J. 0., Rutledge, F. N., and Smith, J. P. Cell-mediated immunity to human malignant cells. Am. J. Obstet. 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