DHFRyMSH3 amplification in methotrexate-resistant cells alters the hMutSayhMutSb ratio and reduces the efficiency of base–base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSa (hMSH2zhMSH6) and hMutSb (hMSH2zhMSH3). In HL-60 extracts the hMutSa to hMutSb ratio is roughly 6:1, whereas in methotrexateresistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base– base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSa and hMutSb display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion–deletion heterology, but only hMutSa restores base– base mismatch repair to extracts of HL-60R cells or hMSH2deficient LoVo colorectal tumor cells. Methotrexate (Mtx) is widely used for the treatment of human malignancies (see refs. 1 and 2 for recent perspectives). The primary target for this compound is dihydrofolate reductase (DHFR) (3), an enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate in a reaction essential to one-carbon metabolism (4). However, human tumor cells can acquire resistance to Mtx, a phenomenon that has been demonstrated both in vitro and in vivo (5–8). One well documented mechanism of resistance involves the amplification of a region of the human or rodent genome containing the DHFR gene (5–10), an event that leads to elevated expression of DHFR, which effectively circumvents the metabolic block produced by this agent. An unusual feature of the chromosomal organization of the human and murine DHFR gene is a shared promoter region 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 (mismatch repair protein 1) displays homology to the bacterial MutS polypeptide, and now is called hMSH3 (human MutS homolog 3). Although it has not been demonstrated that hMSH3 is produced in human cells, a role for the homologous protein in Saccharomyces cerevisiae has been described (13). Yeast MSH3 shares overlapping function with yMSH6, each of which forms a molecular complex with yMSH2 and contributes to the maintenance of microsatellite stability (14–16). More recently, yMSH3 has been coexpressed with yMSH2 to produce a heterodimer that recognizes insertion mismatches in a mobility shift assay (17). In vitro mismatch recognition activities of the corresponding human MSH2zMSH6 (hMutSa) and MSH2zMSH3 (hMutSb) heterodimers have been more extensively examined. hMutSa binds both base–base and nucleotide insertion mismatches and restores correction of both types of mispairs to extracts of repair-defective cell lines (18). Both heterodimers have been generated from cDNA constructs by baculovirus expression (19) and in vitro transcription and translation (20). In vitro analysis has indicated that whereas both hMutSa and hMutSb bind insertion–deletion mismatches, only hMutSa recognizes base–base mismatches (18–20). hMutSa and hMutSb may have complementary functions in recognition of insertion– deletion mismatches, differentially recognizing mispairs of this class depending on heterology size and sequence context (19, 21). The contribution of MSH3 to genetic stability in human cells is uncertain. Loss of MSH3 expression, but not associated hypermutability, has been reported in marrow cells from patients with hematological malignancies (22). More recently, the HHUA endometrial tumor cell line has been shown to contain mutations in both MSH3 and MSH6 (21). Introduction of chromosome 5 with a functional MSH3 gene into HHUA cells restored microsatellite stability at dinucleotide and tetranucleotide repeat sequences but not at mononucleotide or trinucleotide repeats. Extracts of chromosome 5-complemented HHUA cells were found to be proficient in repair of selected mononucleotide and tetranucleotide insertion– deletion mispairs, but to exhibit only limited activity on base–base mismatches. We show here that extensive overproduction of hMSH3 in Mtxr HL-60R promyelocytic leukemia cells sequesters virtually all of the nuclear hMSH2 into the hMutSb heterodimer. This phenomenon is associated with a defect in base–base mismatch repair and hypermutability at the HPRT locus. MATERIALS AND METHODS Cell Lines and Mismatch Repair Assays. Cell lines HL-60 and HL-60R were obtained from T. Shimada (Nippon Medical School, Tokyo) and cultured according to published procedures (11). HeLa S3 and LoVo cells were grown as described previously (18, 23). Mismatch repair assays contained 100 mg of nuclear extract protein and 24 fmol of heteroduplex DNA (23, 24). Extract complementation used 200 ng hMutSa or 100 ng hMutSb. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9410144-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: DHFR, dihydrofolate reductase; hMSH, human MutS homolog; HPRT, hypoxanthine phosphoribosyltransferase; Mtx, methotrexate. ‡To whom reprint requests should be addressed. e-mail: modrich@ biochem.duke.edu.