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• W2063973314 abstract "The role of specific mismatch repair (MMR) gene products was examined by observing several phenotypic end points in two MMR-deficient human endometrial carcinoma cell lines that were originally isolated from the same tumor. The first cell line, HEC-1-A, contains a nonsense mutation in the hPMS2 gene, which results in premature termination and a truncated hPMS2 protein. In addition, HEC-1-A cells carry a splice mutation in thehMSH6 gene and lack wild-type hMSH6 protein. The second cell line, HEC-1-B, possesses the same defective hMSH6locus. However, HEC-1-B cells are heterozygous at the hPMS2locus; that is, along with carrying the same nonsense mutation in hPMS2 as in HEC-1-A, HEC-1-B cells also contain a wild-type hPMS2 gene. Initial recognition of mismatches in DNA requires either the hMSH2/hMSH6 or hMSH2/hMSH3 heterodimer, with hPMS2 functioning downstream of damage recognition. Therefore, cells defective in hPMS2 should completely lack MMR (HEC-1-A), whereas cells mutant in hMSH6 only (HEC-1-B) can potentially repair damage via the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cells illustrate (i) the reduction of instability at microsatellite sequences, (ii) a significant decrease in frameshift mutation rate at HPRT, and (iii) the in vitro repair of looped substrates, relative to HEC-1-A cells, illustrating the repair of frameshift intermediates by hMSH2/hMSH3 heterodimer. Furthermore, the role of hMSH2/hMSH3 heterodimer in the repair of base:base mismatches is supported by observing the reduction in base substitution mutation rate at HPRT in HEC-1-B cells (hMSH6-defective but possessing wild-type hPMS2), as compared with HEC-1-A (hMSH6/hPMS2-defective) cells. These data support a critical role for hPMS2 in human MMR, while further defining the role of the hMSH2/hMSH3 heterodimer in maintaining genomic stability in the absence of a wild-type hMSH2/hMSH6 heterodimer. The role of specific mismatch repair (MMR) gene products was examined by observing several phenotypic end points in two MMR-deficient human endometrial carcinoma cell lines that were originally isolated from the same tumor. The first cell line, HEC-1-A, contains a nonsense mutation in the hPMS2 gene, which results in premature termination and a truncated hPMS2 protein. In addition, HEC-1-A cells carry a splice mutation in thehMSH6 gene and lack wild-type hMSH6 protein. The second cell line, HEC-1-B, possesses the same defective hMSH6locus. However, HEC-1-B cells are heterozygous at the hPMS2locus; that is, along with carrying the same nonsense mutation in hPMS2 as in HEC-1-A, HEC-1-B cells also contain a wild-type hPMS2 gene. Initial recognition of mismatches in DNA requires either the hMSH2/hMSH6 or hMSH2/hMSH3 heterodimer, with hPMS2 functioning downstream of damage recognition. Therefore, cells defective in hPMS2 should completely lack MMR (HEC-1-A), whereas cells mutant in hMSH6 only (HEC-1-B) can potentially repair damage via the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cells illustrate (i) the reduction of instability at microsatellite sequences, (ii) a significant decrease in frameshift mutation rate at HPRT, and (iii) the in vitro repair of looped substrates, relative to HEC-1-A cells, illustrating the repair of frameshift intermediates by hMSH2/hMSH3 heterodimer. Furthermore, the role of hMSH2/hMSH3 heterodimer in the repair of base:base mismatches is supported by observing the reduction in base substitution mutation rate at HPRT in HEC-1-B cells (hMSH6-defective but possessing wild-type hPMS2), as compared with HEC-1-A (hMSH6/hPMS2-defective) cells. These data support a critical role for hPMS2 in human MMR, while further defining the role of the hMSH2/hMSH3 heterodimer in maintaining genomic stability in the absence of a wild-type hMSH2/hMSH6 heterodimer. mismatch repair hypoxanthine-guanine phosphoribosyl transferase 6-thioguanine. Deficiencies in human mismatch repair (MMR)1 have been illustrated in a variety of sporadic and hereditary neoplasia in recent years (reviewed in Refs. 1Eshleman J.R. Markowitz S.D. Hum. Mol. Genet. 1996; 5: 1489-1494Crossref PubMed Google Scholar, 2Marra G. Boland C.R. Gastroenterol. Clin. N. Am. 1996; 25: 755-772Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 3Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1329) Google Scholar). This observation has led to a wealth of studies focusing on the intricacies of human MMR. However, the proteins and specific biochemistry within the MMR pathway are still currently being defined. The list of functional human MMR genes now includeshMSH2, hMSH3, hMSH6 (GTBP),hMLH1, and hPMS2, which are believed to function in MMR as depicted in Fig. 1. Several additional homologues of the bacterial MMR proteins MutS and MutL have been identified in human cells (4Risinger J.I. Umar A. Boyd J. Berchuck A. Kunkel T.A. Barrett J.C. Nat. Genet. 1996; 14: 102-105Crossref PubMed Scopus (160) Google Scholar, 5Horii A. Han H.J. Sasaki S. Shimada M. Nakamura Y. Biochem. Biophys. Res. Commun. 1994; 204: 1257-1264Crossref PubMed Scopus (63) Google Scholar, 6Nicolaides N.C. Carter K.C. Shell B.K. Papadopoulos N. Vogelstein B. Kinzler K. Genomics. 1995; 30: 195-206Crossref PubMed Scopus (104) Google Scholar, 7Fishel R. Wilson T. Curr. Opin. Gen. Dev. 1997; 7: 105-113Crossref PubMed Scopus (150) Google Scholar); however, their function in the human pathway is yet undefined. Nonetheless, their presence suggests that the aforementioned list of functional human MMR genes may be incomplete. Mismatch recognition in human MMR involves homologues of the bacterial protein MutS. These proteins appear to function in at least two heterodimers, designated hMutSα (hMSH2/hMSH6) and hMutSβ (hMSH2/hMSH3) (8Kolodner R.D. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (543) Google Scholar, 9Drummond J.T. Li G.M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (535) Google Scholar, 10Palombo F. Iaccarino I. Nakajima E. Ikejima M. Shimada T. Jiricny J. Curr. Biol. 1996; 6: 1181-1184Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 11Acharya S. Wilson T. Gradia S. Kane M.F. Guerrette S. Marsischky G.T. Kolodner R. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13629-13634Crossref PubMed Scopus (466) Google Scholar, 12Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar), which appear to have overlapping and redundant repair capabilities with respect to the type of DNA mismatch or loop substrate (12Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar). A redundancy in mismatch recognition by the human heterodimers hMSH2/hMSH6 and hMSH2/hMSH3 was first predicted by overlapping in vitro binding affinities (10Palombo F. Iaccarino I. Nakajima E. Ikejima M. Shimada T. Jiricny J. Curr. Biol. 1996; 6: 1181-1184Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 11Acharya S. Wilson T. Gradia S. Kane M.F. Guerrette S. Marsischky G.T. Kolodner R. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13629-13634Crossref PubMed Scopus (466) Google Scholar). Recently, a human cell line, HHUA, defective in both hMSH6 and hMSH3, has been defined (4Risinger J.I. Umar A. Boyd J. Berchuck A. Kunkel T.A. Barrett J.C. Nat. Genet. 1996; 14: 102-105Crossref PubMed Scopus (160) Google Scholar), and the predicted redundancy in human mismatch recognition was supported by complementing either defect by transfer of a specific human chromosome (12Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar). ThesehMSH6/hMSH3-defective cells have an elevated mutation rate at the HPRT locus and a high rate of instability at microsatellite sequences, both of which were significantly reduced by complementing either the hMSH6 orhMSH3 defect by chromosome transfer. This overlapping phenotypic restoration illustrates the redundancy of function by either hMSH2/hMSH6 or hMSH2/hMSH3 for base:base mismatch repair or frameshift intermediate repair (12Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar). In yeast, genetic studies also suggest that the MSH2 gene product forms a heterodimer with either MSH6 or MSH3 (13Habraken Y. Sung P. Prakash L. Prakash S. Curr. Biol. 1996; 6: 1185-1187Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (499) Google Scholar, 15Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), with each heterodimer complementing the function of the other. This redundancy in yeast mismatch recognition was first indicated by the observation that mutation rates are only slightly elevated at several loci for either MSH6- orMSH3-deficient yeast strains. However, a strain with mutations in both MSH6 and MSH3 has significantly elevated mutation rates, comparable with the mutation rates observed in MSH2-deficient strains (14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (499) Google Scholar, 15Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 16Greene C.N. Jinks-Robertson S. Mol. Cell. Biol. 1997; 17: 2844-2850Crossref PubMed Scopus (100) Google Scholar, 17Sia E.A. Kokoska R.J. Dominska M. Greenwell P. Petes T.D. Mol. Cell. Biol. 1997; 17: 2851-2858Crossref PubMed Scopus (330) Google Scholar). Although the complexities observed with initial mismatch recognition in the human MMR pathway appear analogous to that observed in yeast, there has not been any evidence that yeast MSH2/MSH3 is capable of base:base mismatch repair such as that illustrated by hMSH2/hMSH3 in humans (12Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar). Following recognition of DNA mismatches in human cells, an additional heterodimer of hMLH1/hPMS2, designated hMutLα (18Li G.M. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1950-1954Crossref PubMed Scopus (352) Google Scholar), functions in repair complex formation (8Kolodner R.D. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (543) Google Scholar, 18Li G.M. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1950-1954Crossref PubMed Scopus (352) Google Scholar, 19Modrich P. J. Biol. Chem. 1997; 272: 24727-24730Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The proteins that comprise this heterodimer are homologues of the bacterial protein MutL. Although only two MutL homologues have been implicated in the process of repair complex formation, at least eleven additional homologues belonging to the PMS gene family have been characterized in human cells (5Horii A. Han H.J. Sasaki S. Shimada M. Nakamura Y. Biochem. Biophys. Res. Commun. 1994; 204: 1257-1264Crossref PubMed Scopus (63) Google Scholar, 6Nicolaides N.C. Carter K.C. Shell B.K. Papadopoulos N. Vogelstein B. Kinzler K. Genomics. 1995; 30: 195-206Crossref PubMed Scopus (104) Google Scholar). It would seem possible that hMLH1/hPMS2 function may be complemented to some extent by any of these PMS homologues, perhaps by a second hMutL heterodimer consisting of two presently undefined proteins, or an additional protein complexed with either of the subunits in hMLH1/hPMS2. One such PMS gene homologue,hPMS1, has been implicated in human mismatch repair by the observation of a mutant hPMS1 allele isolated from a family predisposed to colon cancer (21Papadopoulos N. Nicolaides N.C. Wei Y.F. Ruben S.M. Carter K.C. Rosen C.A. Haseltine W.A. Fleischmann R.D. Fraser C.M. Adams M.D. Ventor J.C. Hamilton S.R. Petersen G.M. Watson P. Lynch H.T. Peltomaki P. Mecklin J. de la Chapelle A. Kinzler K.W. Vogelstein B. Nature. 1994; 371: 75-80Crossref PubMed Scopus (1445) Google Scholar), although a function for hPMS1 has yet to be determined. A transgenic mouse mutant in PMS1 does exhibit elevated instability at mononucleotide sequences, suggesting that perhaps PMS1 plays a role in mononucleotide stability (22Prolla T.A. Baker S.M. Harris A.C. Tsao X. Yao C.E. Bronner C.E. Bronner Zheng B. Gorodon M. Reneker N. Arnheim N. Shibata D. Bradley A Liskay M. Nat. Genet. 1998; 18: 276-279Crossref PubMed Scopus (301) Google Scholar). Additionally, knockout mice mutant in either hMLH1 orhPMS2 have differing phenotypes (22Prolla T.A. Baker S.M. Harris A.C. Tsao X. Yao C.E. Bronner C.E. Bronner Zheng B. Gorodon M. Reneker N. Arnheim N. Shibata D. Bradley A Liskay M. Nat. Genet. 1998; 18: 276-279Crossref PubMed Scopus (301) Google Scholar, 23Prolla T.A. Abuin A. Bradley A. Semin. Cancer Biol. 1996; 7: 241-247Crossref PubMed Scopus (28) Google Scholar), with respect to cancer predisposition and meiotic phenotypes, suggesting that the roles of each specific gene are not exclusive to the hMLH1/hPMS2 heterodimer. Taken together, the proteins involved in the formation of a repair complex are not as well defined as those involved in mismatch detection. Therefore, additional MutL homologues or perhaps additional biochemical roles for either hMLH1 or hPMS2 other than in the hMLH1/hPMS2 heterodimer may be involved in human MMR. To further investigate the biochemical complexities involved in human MMR, several phenotypes were determined in two cells lines originating from the same endometrial carcinoma. These cell lines, HEC-1-A and HEC-1-B, both contain a mutation in the hMSH6 gene and ahPMS2 allele that contains a premature nonsense codon; however, they differ by either the lack or presence of a wild-type copy of hPMS2, respectively. Fig. 1 illustrates these differences within the MMR pathway between HEC-1-A and HEC-1-B as compared with the current model of MMR. The roles of individual MMR heterodimers can then be examined by comparing differences in mismatch repair in vitro, microsatellite instability, and the mutation rate and mutational specificity at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. The data presented here support the notion that hPMS2 plays a critical role in maintaining genomic stability in human cells, as illustrated by the significant instability observed in HEC-1-A cells. Furthermore, the data suggest the biochemical role of the hMutSβ (hMSH2/hMSH3) heterodimer, as illustrated by observing the differences in phenotypes between the HEC-1-B cells (hMSH6-defective) and the HEC-1-A cells (hMSH6/hPMS2-defective). Human endometrial carcinoma cell lines HEC-1-A and HEC-1-B were from the American Type Culture Collection. Both cell lines were maintained in DF medium (Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) + 10% dialyzed fetal bovine serum; HyClone). The mutation of the mismatch repair genehPMS2 in HEC-1-A has been previously described (24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The status of hPMS2 in HEC-1-B was determined by a protein truncation test as described (24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, total RNA was prepared from cell lines using a single-step procedure. cDNA was prepared and used as a template for coupled transcription and translation using the TNTTM system (Promega). The resulting protein products were then analyzed. Upon noting a wild-type copy of hPMS2 protein in HEC-1-B, the cDNA was then reamplified and sequenced (as described below for HPRT cDNA). Other MMR genes in both cell lines were analyzed in a similar manner. Extracts from cell lines were prepared as described previously (25Roberts J.D. Kunkel T.A. Methods Mol. Genet. 1993; 2: 295-313Google Scholar). Procedures for substrate preparation and for measuring mismatch repair activity were as described (26Thomas, D. C., Umar, A., and Kunkel, T. A. (1995)Methods: A Companion to Methods in Enzymology, Vol. 7, pp. 187–197, Academic Press, New YorkGoogle Scholar). Briefly, repair reactions (25 μl) contained 30 mm Hepes (pH 7.8), 7 mm MgCl2, 4 mm ATP, 200 μm each CTP, GTP, and UTP, 100 μm each dATP, dGTP, dTTP, and dCTP, 40 mmcreatine phosphate, 100 mg/ml creatine phosphokinase, 15 mmsodium phosphate, 1 fmol of the indicated heteroduplex DNA, and 50 μg of the cell extract protein. After incubating at 37 °C for 15 min, samples were processed and introduced into Escherichia coliNR9162 (mutS) via electroporation. Cells were plated, M13mp2 plaque colors were scored, and the repair efficiency was calculated (26Thomas, D. C., Umar, A., and Kunkel, T. A. (1995)Methods: A Companion to Methods in Enzymology, Vol. 7, pp. 187–197, Academic Press, New YorkGoogle Scholar). Single cell clones of the HEC-1-A and HEC-1-B cell lines were isolated by limiting dilution. DNA was isolated from independent single cell clones and amplified by PCR as described (24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Several microsatellite sequences were then examined for variations in allele length. Mutant frequency and mutation rate determinations at the HPRT locus were determined as described previously (28Glaab W.E. Tindall K.R. Carcinogenesis. 1997; 18: 1-8Crossref PubMed Scopus (127) Google Scholar). Mutant frequencies were obtained by plating 106 cells in 40 μm6-thioguanine (6-TG; Sigma) at a density of 5 × 104per 10-cm dish. Cells were incubated 12–14 days and 6-TG-resistant (6-TGr) colonies were visualized by staining with 0.5% crystal violet (in 50% methanol, v/v; Sigma). Mutation rate determinations were performed using cell populations cleansed of pre-existing HPRT mutants by culturing in HAT medium (DF medium + 100 μm hypoxanthine, 0.4 μmaminopterin, and 16 μm thymidine; Sigma). Following HAT removal, the initial HPRT mutant frequency was determined and 2–3 × 106 cells were subcultured. Several additional mutant frequencies were then obtained while maintaining the cells in logarithmic growth. Population doublings were determined between mutant frequency determinations. After obtaining 5–6 subsequent mutant frequencies, mutation rate was then obtained by plotting the observed mutant frequency as a function of population doubling and calculating the slope by linear regression. The slope of the curve yields the mutation rate (mutations/cell/generation). Independent mutants resistant to 6-TG were obtained by first eliminating pre-existing HPRT mutants from cell cultures by subculturing in HAT medium. Following removal of HAT medium, 100-cell inocula were plated to further ensure that no pre-existing HPRT mutants were present in subcultures. Each 100-cell inoculum was grown to approximately 2.5 × 106 cells and then was selected in 40 μm 6-TG at a density of 5 × 105 cells per 10-cm dish. Colonies were allowed to grow for 14–18 days, and individual 6-TGr clones were isolated. These 6-TGr clones were transferred to 24-well dishes and grown to confluency. Independent mutants were defined as those spontaneous mutants arising in different initial 100-cell inocula. Amplification of HPRT mRNA from mutant clones was performed by a procedure modified from Yang et al. (29Yang J.L. Maher V.M. McCormick J.J. Gene (Amst.). 1989; 83: 347-354Crossref PubMed Scopus (131) Google Scholar). The pairs of primers used for HPRT cDNA amplification and sequencing and their annealing sites relative to the A of the ATG initiation codon (30Jolly D. Okayama H. Berg P. Esty A. Filpula D. Bohlen P. Johnson G. Shively J.E. Hunkapillar T. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 477-481Crossref PubMed Scopus (292) Google Scholar) are as follows: HP1, 5′-CTCCTCTGCTCCGCCACCGGCTTCC-3′ (−65 to −41); HP2, 5′-AACATTGATAATTTTACTGGCGATGTC-3′ (701 to 727); HP3, 5′-CTCCTCCTGAGCAGTCAGCCCGCGCG-3′ (−41 to −16); and HP4, 5′-CGATGTCAATAGGACTCCAGATGTT-3′ (683 to 707). Mutant clones were washed with ice-cold phosphate-buffered saline, resuspended at a density of approximately 500 cells/μl and stored as 1-μl aliquots at −80 °C. 1-μl aliquots were quickly thawed, and 9 μl of cDNA synthesis mixture was added. This mixture contained 500 μm dNTPs (Amersham Pharmacia Biotech), 1 μg of bovine serum albumin, 10 mm dithiothreitol, 2.5% (v/v) Nonidet P-40, 20 mm Tris-HCl (pH 8.4), 50 mmKCl, 2.5 mm MgCl2, 25 units of SuperScript II RNase H− reverse transcriptase (Life Technologies), 10 units RNasin RNase inhibitor (Promega), and 100 ng of HP2 primer. cDNA synthesis was performed at 37 °C for 1 h. PCR1 mixture was then added directly to the cDNA synthesis mixture, which contained 300 μm dNTPs, 15 mm Tris-HCl (pH 8.55), 60 mm KCL, 3.5 mm MgCl2, 2.5 units Taq polymerase (Perkin-Elmer), 100 ng of HP1 primer, and 50 ng of HP2 primer. PCR1 was performed as follows: 1 cycle of 94 °C for 4 min, 65 °C for 1 min, 72 °C for 2.5 min, followed by 29 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 2 min. Nested primer PCR was then performed by adding 5 μl of PCR1 to PCR2 mixture (PCR1 mixture with 100 ng each of HP3 and HP4 primers substituted for HP1 and HP2 primers). PCR2 was performed under the same PCR conditions as PCR1. PCR2 products were checked on an agarose gel, and full-length cDNA was visualized. A gel plug containing the full-length product was removed and PCR2 was repeated for 15 cycles. The purified HPRT cDNA PCR product was used for sequencing. The entire HPRTcoding region from each mutant was sequenced utilizing automated sequencing with both the HP3 and HP4 primers. A dideoxy terminator cycle sequencing reaction was performed using a dye terminator ready mix with Taq polymerase (Perkin-Elmer) as follows: 94 °C for 30 s, 60 °C for 4 min for 25 cycles. DNA sequences were obtained on Applied Biosystems automated DNA sequencer model 377. Each sequencing reaction allowed for analysis of the entire 657 base coding region of HPRT, and therefore, each mutation determined was confirmed by sequence analysis in the opposite direction. The endometrial carcinoma HEC-1-A cell line and the substrain HEC-1-B cell line were isolated from the same adenocarcinoma (31Kuramoto H. Tamura S. Notake Y. Am. J. Obstet. Gynecol. 1972; 114: 1012-1019Abstract Full Text PDF PubMed Scopus (219) Google Scholar). Although originating from the same tumor, the individual cell lines differ in their karyotype: the HEC-1-A cell line is near diploid (modal chromosome number of 49), whereas the HEC-1-B cell line is near triploid to tetraploid (modal chromosome number of 82). There are a few cell line-specific markers present, such as a deletion on chromosome 9 (p13p22) in HEC-1-B. However, most markers are shared between the HEC-1-A and HEC-1-B cell lines, such as a deletion on chromosome 7 (q32) and a deletion on chromosome 6 (p21.3–p23). There are two X chromosomes present in both the HEC-1-A and HEC-1-B cell lines. The defect in the hPMS2 gene in HEC-1-A cells has been previously defined (24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). This cell line contains a C to T mutation at codon 802 in the hPMS2 gene, which results in a TGA termination codon. The same truncated protein product present in HEC-1-A was observed in HEC-1-B, as determined by protein truncation test, which upon sequencing revealed the same C to T mutation at codon 802. But in contrast to HEC-1-A, the hPMS2 protein truncation test in HEC-1-B cells yielded two gene products (Fig. 2). The additional polypeptide was a full-length product, and DNA sequencing of the cDNA used in the analysis determined it to be wild-type hPMS2. Thus, HEC-1-B cells contain both wild-type hPMS2 protein, as well as truncated protein. These results were confirmed by Western analysis of hPMS2 protein (data not shown). Recent work involving the HEC-1-A cell line suggested the presence of a second MMR gene mutation (32Risinger J.I. Umar A. Glaab W.E. Tindall K.R. Kunkel T.A. Barrett J.C. Cancer Res. 1998; 58: 2978-2981PubMed Google Scholar). Analyses of other MMR genes in HEC-1-A were conducted by Western analysis, protein truncation tests, and by direct DNA sequencing. Both the hMLH1 and hPMS1genes were investigated and appear to be wild type at the molecular level by DNA sequencing of the entire coding region. However, analysis of the hMSH6 gene did not reveal a wild-type sequence. Sequencing of the cDNA illustrated a 199-base deletion in the 3′ region of the cDNA, corresponding to nucleotides 3802–4001 (32Risinger J.I. Umar A. Glaab W.E. Tindall K.R. Kunkel T.A. Barrett J.C. Cancer Res. 1998; 58: 2978-2981PubMed Google Scholar). The presence of a wild-type copy of the cDNA was not observed. Therefore, HEC-1-A cells are mutant in both hPMS2 and hMSH6. Similar analysis of the HEC-1-B cell line indicated the same mutation in thehMSH6 gene, that of a deletion of base 3802–4001. No wild-type copy of hMSH6 cDNA was observed in HEC-1-B. By observing these MMR gene mutations, the cell lines were then defined as mutant in both hPMS2 and hMSH6 (HEC-1-A) or mutant in only hMSH6 (HEC-1-B). Fig. 1 demonstrates the defects in both cell lines and how these defects relate to the current model of MMR. The ability of HEC-1-A and HEC-1-B cells to repair various DNA mismatches or loops in vitro was examined in cellular extracts derived from either cell line. This repair activity was determined using a substrate located within a lacZ gene sequence. The repair of the substrate can be assessed colorimetrically dependent upon α-complementation. The in vitro repair efficiencies for HEC-1-A and HEC-1-B are presented in Fig. 3. These repair activities are compared with the repair activities observed in MMR-proficient HeLa cell extracts. This figure illustrates repair at a representative base:base mismatch, and at a 1-, 2-, and 4-base DNA loop. For the repair of single base:base mismatches, both cell lines were defective relative to HeLa cells. However, there was differential repair of DNA looped intermediates. HEC-1-B cells were proficient in the repair of 1, 2, and 4 bases, whereas HEC-1-A cells were deficient. This in vitro repair assay suggests that the hMSH2/hMSH3 heterodimer is functional in HEC-1-B. The instability at microsatellite sequences was examined in both HEC-1-A and HEC-1-B cells at several loci by determining microsatellite tract length in single-cell clones. These repetitive tracts of DNA are prone to slippage during DNA synthesis, and the looped intermediates are subsequently repaired by MMR proteins. Lack of mismatch repair predisposes these repetitive tracts of DNA to instability, which is observed as expansion or contraction of the tract length. The instability at several microsatellite loci has been previously illustrated in HEC-1-A (24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and is presented in Table I for comparison. The microsatellite loci examined in HEC-1-A were highly unstable, indicative of cells deficient in MMR. Table I also presents the instability at microsatellite sequences for HEC-1-B cells. These sequences are relatively stable in comparison with HEC-1-A. For instance, 8 of 24 clones were unstable at the D17S791 marker in HEC-1-A, whereas only 2 of 24 were unstable in HEC-1-B. In total, 22% of the clones examined in HEC-1-A were unstable compared with only 4% in HEC-1-B. The instability associated with HEC-1-B cells is only slightly elevated from MMR-proficient cells, similar to that reported previously for cells defective in hMSH6 (12Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar, 33Papadopoulos N. Nicolaides N.C. Liu B. Parsons R. Lengauer C. Palombo F. D'Arrigo A. Markowitz S. Willson J.K.V. Kinzler K.W. Jiricny J. Vogelstein B. Science. 1995; 268: 1915-1917Crossref PubMed Scopus (475) Google Scholar, 34Bhattacharyya N.P. Skandalis A. Ganesh A. Groden J. Meuth M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6319-6323Crossref PubMed Scopus (404) Google Scholar). These findings suggest that repair of 2–4-base loops in DNA is only slightly compromised in HEC-1-B, whereas HEC-1-A cells appear deficient in 2–4-base-loop repair.Table IMicrosatellite instability in single-cell clones of HEC-1-A and HEC-1-B cell linesMicrosatellite markerRepeatHEC-1-AHEC-1-BD14S76(CA)n3 /400 /17D2S147(CA)n3 /400 /17D14S78(CA)n6 /242 /24D17S791(CA)n8 /242 /24D7S1794(CTT)n17 /400 /17 Total37 /168 (22%)4 /99 (4%)Listed are the number of clones having alterations over the total number of clones examined.Previously reported (in Ref. 24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Open table in a new tab Listed are the number of clones having alterations over the total number of clones examined. Previously reported (in Ref. 24Risinger J.I. Umar A. Barrett J.C. Kunkel T.A. J. Biol. Chem. 1995; 270: 18183-18186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). A substantial increase in the rate of spontaneous mutations has been observed in cells defective in MMR. To observe potential differences in the rate of mutations arising spontaneously between HEC-1-A and HEC-1-B cells, the mutation rate at HPRT was determined for both cell l" @default.
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