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DHFR/MSH3 Amplification in Methotrexate-Resistant Cells Alters

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DHFR/MSH3 Amplification in Methotrexate-Resistant Cells Alters Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10144–10149, September 1997 Biochemistry DHFRyMSH3 amplification in methotrexate-resistant cells alters the hMutSayhMutSb ratio and reduces the efficiency of base–base mismatch repair (drug resistanceygenetic instability) JAMES T. DRUMMOND*†,JOCHEN GENSCHEL†,ELISABETH WOLF*†, AND PAUL MODRICH*†‡ *Howard Hughes Medical Institute and †Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 Contributed by Paul Modrich, July 23, 1997 ABSTRACT The level and fate of hMSH3 (human MutS recently, yMSH3 has been coexpressed with yMSH2 to pro- homolog 3) were examined in the promyelocytic leukemia cell duce a heterodimer that recognizes insertion mismatches in a line HL-60 and its methotrexate-resistant derivative HL-60R, mobility shift assay (17). which is drug resistant by virtue of an amplification event that In vitro mismatch recognition activities of the corresponding spans the dihydrofolate reductase (DHFR) and MSH3 genes. human MSH2zMSH6 (hMutSa) and MSH2zMSH3 (hMutSb) Nuclear extracts from HL-60 and HL-60R cells were subjected heterodimers have been more extensively examined. hMutSa to an identical, rapid purification protocol that efficiently binds both base–base and nucleotide insertion mismatches and captures heterodimeric hMutSa (hMSH2zhMSH6) and restores correction of both types of mispairs to extracts of hMutSb (hMSH2zhMSH3). In HL-60 extracts the hMutSa to repair-defective cell lines (18). Both heterodimers have been hMutSb ratio is roughly 6:1, whereas in methotrexate- generated from cDNA constructs by baculovirus expression resistant HL-60R cells the ratio is less than 1:100, due to (19) and in vitro transcription and translation (20). In vitro overproduction of hMSH3 and heterodimer formation of this analysis has indicated that whereas both hMutSa and hMutSb protein with virtually all the nuclear hMSH2. This shift is bind insertion–deletion mismatches, only hMutSa recognizes associated with marked reduction in the efficiency of base– base–base mismatches (18–20). hMutSa and hMutSb may base mismatch and hypermutability at the hypoxanthine have complementary functions in recognition of insertion– phosphoribosyltransferase (HPRT) locus. Purified hMutSa deletion mismatches, differentially recognizing mispairs of this and hMutSb display partial overlap in mismatch repair class depending on heterology size and sequence context (19, specificity: both participate in repair of a dinucleotide inser- 21). tion–deletion heterology, but only hMutSa restores base– The contribution of MSH3 to genetic stability in human cells base mismatch repair to extracts of HL-60R cells or hMSH2- is uncertain. Loss of MSH3 expression, but not associated deficient LoVo colorectal tumor cells. hypermutability, has been reported in marrow cells from patients with hematological malignancies (22). More recently, Methotrexate (Mtx) is widely used for the treatment of human the HHUA endometrial tumor cell line has been shown to malignancies (see refs. 1 and 2 for recent perspectives). The contain mutations in both MSH3 and MSH6 (21). Introduction primary target for this compound is dihydrofolate reductase of chromosome 5 with a functional MSH3 gene into HHUA (DHFR) (3), an enzyme that catalyzes the reduction of cells restored microsatellite stability at dinucleotide and tet- dihydrofolate to tetrahydrofolate in a reaction essential to ranucleotide repeat sequences but not at mononucleotide or one-carbon metabolism (4). However, human tumor cells can trinucleotide repeats. Extracts of chromosome 5-comple- acquire resistance to Mtx, a phenomenon that has been mented HHUA cells were found to be proficient in repair of demonstrated both in vitro and in vivo (5–8). One well docu- selected mononucleotide and tetranucleotide insertion– mented mechanism of resistance involves the amplification of deletion mispairs, but to exhibit only limited activity on a region of the human or rodent genome containing the DHFR base–base mismatches. gene (5–10), an event that leads to elevated expression of We show here that extensive overproduction of hMSH3 in DHFR, which effectively circumvents the metabolic block Mtxr HL-60R promyelocytic leukemia cells sequesters virtually produced by this agent. all of the nuclear hMSH2 into the hMutSb heterodimer. This An unusual feature of the chromosomal organization of the phenomenon is associated with a defect in base–base mis- human and murine DHFR gene is a shared promoter region match repair and hypermutability at the HPRT locus. with a second ORF (11, 12) that is transcribed in the opposite direction (Fig. 1). The divergently transcribed human gene, originally called DUG (divergent upstream gene) or MRP-1 MATERIALS AND METHODS (mismatch repair protein 1) displays homology to the bacterial Cell Lines and Mismatch Repair Assays. Cell lines HL-60 MutS polypeptide, and now is called hMSH3 (human MutS and HL-60R were obtained from T. Shimada (Nippon Medical homolog 3). Although it has not been demonstrated that School, Tokyo) and cultured according to published proce- hMSH3 is produced in human cells, a role for the homologous dures (11). HeLa S and LoVo cells were grown as described protein in Saccharomyces cerevisiae has been described (13). 3 previously (18, 23). Mismatch repair assays contained 100 mg Yeast MSH3 shares overlapping function with yMSH6, each of of nuclear extract protein and 24 fmol of heteroduplex DNA which forms a molecular complex with yMSH2 and contributes (23, 24). Extract complementation used 200 ng hMutSa or 100 to the maintenance of microsatellite stability (14–16). More ng hMutSb. The publication costs of this article were defrayed in part by page charge Abbreviations: DHFR, dihydrofolate reductase; hMSH, human MutS payment. This article must therefore be hereby marked ‘‘advertisement’’ in homolog; HPRT, hypoxanthine phosphoribosyltransferase; Mtx, accordance with 18 U.S.C. §1734 solely to indicate this fact. methotrexate. © 1997 by The National Academy of Sciences 0027-8424y97y9410144-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: modrich@ PNAS is available online at http:yywww.pnas.org. biochem.duke.edu. 10144 Downloaded by guest on October 1, 2021 Biochemistry: Drummond et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10145 Determination of HPRT Mutation Rates. Determination of HPRT mutation rates was performed by minor modifications of the procedure of Eshleman et al. (25). Mutants of HeLa, LoVo, HL-60, and HL-60R cells were identified after 1 month of growth in medium containing 5 mgyml 6-thioguanine, where clonal survival was visualized by staining viable cells with methylene blue dye (HeLa and LoVo) or nitro-blue tetrazo- lium dye (HL-60 and HL-60R). HeLa and LoVo were prop- agated in Eagle’s minimal essential medium with 10% fetal bovine serum, and HL-60 and HL-60R were grown in RPMI FIG. 1. Organization of human MutS homolog genes. The MSH3 1640 medium plus 15% fetal bovine serum. Cultures of gene is divergently transcribed from a promoter region shared with the HL-60R cells typically were supplemented with 1 mM Mtx to r DHFR gene (11, 41). The DHFR copy number (n) is about 200 in Mtx ensure maintenance of the high copy number of the DHFRy HL-60R cells (11). Genes encoding the two polypeptides that comprise MSH3 amplicon, but mutation rates were determined in both hMutSa, MSH2 and MSH6, reside in close proximity on chromosome the presence and the absence of the drug. In the latter case, 2 (20, 33). The relative orientation of transcription shown for MSH2 and MSH6 is arbitrary. cultures to be used for fluctuation analysis were established by inoculation of drug-free medium with about 100 cells from Gels and Western Analysis. Electrophoresis of protein culture samples that had been centrifuged and washed to samples was performed on 6% polyacrylamide in the presence remove Mtx. of sodium dodecylsulfate. Protein bands were visualized with Mutation rates were calculated using equation 8 (r 5 Coomassie stain or transferred to a poly(vinylidene difluoride) aNtln[NtCa]) of Luria and Delbru¨ck (26) and the numerical membrane (Immobilon P, Millipore), typically for 100 Vyhr at tables of Capizzi and Jameson (27), where r is the average 4°C in Trisyglycine buffer (25 mM Tris base plus 192 mM number of mutants per culture; a is the intrinsic mutation rate glycine). Blots were incubated in 100 mM Tris, pH 7.5y0.9% per locus per generation; Nt is the average number of mutants NaCly0.1% Tween 20 containing 5% nonfat dry milk and per culture; and C is the number of replicate cultures. probed with the specified antibody according to the enzyme chemiluminescence protocol supplied by Amersham. Antibod- RESULTS ies used in this study include a mouse monoclonal anti-hMSH2 (Calbiochem, Ab-1) and a goat polyclonal anti-hMSH6 (anti- MSH3 Amplification Is Associated with a Reduction in GTBP, Santa Cruz Biotechnology, N-20). A polyclonal anti- Levels of hMSH6. Using near-homogeneous hMutSa and body was raised in rabbits to the peptide sequence TEI- hMutSb as standards, the four cell lines used in this study were DRRKKRPLENDGPVKKK (residues 81–100), derived from subjected to Western analysis to obtain rough estimates of the the deduced peptide sequence for human hMSH3 (11). relative expression levels of hMSH2, hMSH3, and hMSH6. As shown in Fig. 2A, hMSH3 is present at much lower levels than Resolution of hMutSa and hMutSb. All steps were per- hMSH2 or hMSH6 in repair-proficient HeLa and HL-60 cell formed at 4°C. Samples of nuclear extract prepared
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