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• W2000526989 abstract "Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression. hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly,hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was re-expressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5–5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell. Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression. hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly,hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was re-expressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5–5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell. mismatch repair competitive quantitative reverse transcription-polymerase chain reaction simian virus 40 hereditary nonpolyposis colorectal cancer The human mismatch repair (MMR)1 system ensures replication fidelity by correcting postreplication errors that have escaped the DNA proofreading function of DNA polymerase. Defects in the MMR system result in the development of a genetically unstable mutator phenotype and render the cell more susceptible to neoplastic transformation (reviewed in Refs. 1.Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (717) Google Scholar, 2.Marra G. Boland C.R. J. Natl. Cancer Inst. 1995; 87: 1114-1125Crossref PubMed Scopus (478) Google Scholar, 3.Modrich P. J. Biol. Chem. 1997; 272: 24727-24730Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The MMR system functions through the interactions between several proteins, such as hMSH2, hMSH3, hMSH6, hMLH1, hPMS2, and recently discovered hMLH3 (1.Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (717) Google Scholar, 2.Marra G. Boland C.R. J. Natl. Cancer Inst. 1995; 87: 1114-1125Crossref PubMed Scopus (478) Google Scholar, 3.Modrich P. J. Biol. Chem. 1997; 272: 24727-24730Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 4.Lipkin S.M. Wang V. Jacoby R. Banerjee-Basu S. Baxevanis A.D. Lynch H.T. Elliott R.M. Collins F.S. Nat. Genet. 2000; 24: 27-35Crossref PubMed Scopus (264) Google Scholar). hMutS-α is a heterodimer of hMSH2 and hMSH6 that binds to mismatched nucleotides or single insertion/deletion loops (5.Acharya 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 (462) Google Scholar, 6.Drummond J.T. Li G.M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (532) Google Scholar, 7.Guerrette S. Wilson T. Gradia S. Fishel R. Mol. Cell. Biol. 1998; 18: 6616-6623Crossref PubMed Scopus (120) Google Scholar). In addition, hMSH2 can dimerize with hMSH3 creating the hMutS-β complex, which binds larger DNA insertion/deletion mispaired loops (5.Acharya 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 (462) Google Scholar, 7.Guerrette S. Wilson T. Gradia S. Fishel R. Mol. Cell. Biol. 1998; 18: 6616-6623Crossref PubMed Scopus (120) Google Scholar, 8.Palombo F. Iaccarino I. Nakajima E. Ikejima M. Shimada T. Jiricny J. Curr. Biol. 1996; 6: 1181-1184Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Subsequently, activated hMutS-α or hMutS-β interacts with hMutL-α (a heterodimer of hMLH1 with hPMS2) to direct DNA repair (9.Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar, 10.Fishel R. Genes Dev. 1998; 12: 2096-2101Crossref PubMed Scopus (151) Google Scholar).Recent studies suggest that maintaining an appropriate balance of the components of the human MMR system is critical for its proper repair function. In hMSH6-defective HCT15 cells, hMSH3 protein is highly expressed, whereas in hMSH2-defective LoVo cells, hMSH3 and hMSH6 proteins are not detected (11.Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The suggested cause for the absence of the hMSH3 and hMSH6 proteins is that they are relatively unstable without forming a heterodimer complex with hMSH2 (11.Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 12.Drummond J.T. Genschel J. Wolf E. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10144-10149Crossref PubMed Scopus (140) Google Scholar). When hMSH3 was overexpressed by methotrexate-induced simultaneous amplification of the dihydrofolate reductase andhMSH3 genes in CHOR and HL60R cells, the relative amount of hMSH6 protein decreased (12.Drummond J.T. Genschel J. Wolf E. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10144-10149Crossref PubMed Scopus (140) Google Scholar, 13.Marra G. Iaccarino I. Lettieri T. Roscilli G. Delmastro P. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8568-8573Crossref PubMed Scopus (174) Google Scholar). In all likelihood, this decrease was caused by excess hMSH3 protein competing with hMSH6 for dimerization with the common partner protein, hMSH2 (12.Drummond J.T. Genschel J. Wolf E. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10144-10149Crossref PubMed Scopus (140) Google Scholar, 13.Marra G. Iaccarino I. Lettieri T. Roscilli G. Delmastro P. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8568-8573Crossref PubMed Scopus (174) Google Scholar).The human MMR system may be regulated in several different biological situations. Immunohistochemical studies show that the hMSH2 protein is readily detected in the proliferating portion of esophageal and intestinal epithelium (14.Leach F.S. Polyak K. Burrell M. Johnson K.A. Hill D. Dunlop M.G. Wyllie A.H. Peltomaki P. de la Chapelle A. Hamilton S.R. Kinzler K.W. Vogelstein B. Cancer Res. 1996; 56: 235-240PubMed Google Scholar, 15.Wilson T.M. Ewel A. Duguid J.R. Eble J.N. Lescoe M.K. Fishel R. Kelley M.R. Cancer Res. 1995; 55: 5146-5150PubMed Google Scholar, 16.Marra G. Chang C.L. Laghi L.A. Chauhan D.P. Young D. Boland C.R. Oncogene. 1996; 13: 2189-2196PubMed Google Scholar) and increases at least 12-fold in proliferating cells (16.Marra G. Chang C.L. Laghi L.A. Chauhan D.P. Young D. Boland C.R. Oncogene. 1996; 13: 2189-2196PubMed Google Scholar). Since the major role of the MMR system occurs during the immediate postreplicative stage, it seems plausible that the MMR system may be up-regulated in highly replicating cells.Although a stable relationship among the components of the MMR system is important in maintaining their function as a complex, the exact quantitative profile of each MMR component is not yet known. Limited information as to the genetic regulation of the MMR system is available. Recently, some quantitative approaches have been attempted to demonstrate that functional defects of the MMR system may result in microsatellite instability and carcinogenesis (17.Wei Q. Eicher S.A. Guan Y. Cheng L. Xu J. Young L.N. Saunders K.C. Jiang H. Hong W.K. Spitz M.R. Strom S.S. Cancer Epidemiol. Biomarkers Prev. 1998; 7: 309-314PubMed Google Scholar, 18.Wei Q. Bondy M.L. Mao L. Gaun Y. Cheng L. Cunningham J. Fan Y. Bruner J.M. Yung W.K. Levin V.A. Kyritsis A.P. Cancer Res. 1997; 57: 1673-1677PubMed Google Scholar).We have developed a new method, competitive quantitative reverse transcription-polymerase chain reaction (CQ-RT-PCR) for measuring the exact amount of each MMR mRNA. We then compared the amount of each mRNA to the expression of the corresponding protein. We successfully determined the exact amount of each MMR component in the balanced steady state for both mRNAs and proteins in MMR-proficient cell lines. Moreover, the quantitative changes in abnormal MMR-defective cell lines were studied at both the mRNA and protein level. We also found that all human MMR mRNA and proteins measured were up-regulated in highly proliferating SV40-transformed cell lines in comparison with their normal counterpart.DISCUSSIONThe DNA MMR system has been extensively studied in prokaryotes and yeast, but the human system is more complex and less well understood. The stoichiometry and quantitative interaction among the components of human DNA MMR are assumed to be similar to that in lower organisms (1.Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (717) Google Scholar). However, these issues have not been directly studied for all of the components of the system. Quantification of mRNA levels is necessary to evaluate the regulation of MMR gene expression. Northern blot hybridization, a conventional method for quantifying mRNA levels, is of limited value when sample sizes are small and the message is minimally expressed. We used multiplex RT-PCR to measure the relative amounts of mRNA for multiple individual genes between different samples. However, this technique is not sufficiently accurate to measure absolute transcript numbers. We developed a novel technique, CQ-RT-PCR using an internal RNA standard to measure the steady-state relationships among the components of the MMR system within individual cell lines. Furthermore, we used purified proteins as standards to measure absolute amounts of MMR proteins in individual cell lines.In six different DNA MMR-proficient human cell lines, we performed RT-PCR to determine relative mRNA levels for each cell. We were able to measure absolute amounts of DNA MMR mRNA by CQ-RT-PCR for five of these genes; we also determined the steady-state expression of the same five proteins. In DNA MMR-proficient cell lines, the relationship between mRNA and protein for each MMR gene was linear, suggesting that the level of each protein was primarily determined by transcription, with similar kinetic characteristics for each of the components of this system.In DNA MMR-proficient cell lines, the hMutS components were expressed substantially more abundantly than the hMutL components. The hMSH2 protein is an essential component of the human MMR system and is expressed 3–5 times more abundantly than hMLH1. hMSH2 must heterodimerize with either hMSH3 or hMSH6 for mismatched DNA to be recognized (5.Acharya 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 (462) Google Scholar), and presumably a complex between mismatched DNA and hMutS proteins transduces a signal through the hMutL system to initiate DNA repair by ADP-ATP exchange (10.Fishel R. Genes Dev. 1998; 12: 2096-2101Crossref PubMed Scopus (151) Google Scholar, 25.Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). The precise role of the hMutL complexes is not yet completely understood. In E. coli, MutL is thought to accelerate an ATP-dependent translocation of the MutS-MutL complex to a hemimethylated GATC Dam site bound by MutH to achieve strand-specific repair (3.Modrich P. J. Biol. Chem. 1997; 272: 24727-24730Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 9.Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). Recent data have suggested that the hMSH2-hMSH6 heteroduplex may function as a molecular switch. Fishel (10.Fishel R. Genes Dev. 1998; 12: 2096-2101Crossref PubMed Scopus (151) Google Scholar) proposed that hMutL complexes may act as an adenine nucleotide regulator for hMutS complexes in a signal transduction series. A partial crystal structure of the N-terminal fragment fromEscherichia coli suggests that MutL is homologous to the ATPase-containing DNA gyrase, which binds to DNA directly as does MutS (26.Ban C. Yang W. Cell. 1998; 95: 541-552Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 27.Ban C. Junop M. Yang W. Cell. 1999; 97: 85-97Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Coimmunoprecipitation experiments indicate that the hMLH1-hPMS2 heterodimer interacts with hMSH2, requiring mispaired DNA and ATP (28.Gu L. Hong Y. McCulloch S. Watanabe H. Li G.M. Nucleic Acids Res. 1998; 26: 1173-1178Crossref PubMed Scopus (183) Google Scholar). In every model of DNA MMR, the MutS components serve to recognize mispaired DNA, and MutL components function downstream to complete repair. The stoichiometric interaction of MutS and MutL is less clear. Since MutL proteins are unnecessary as long as mispaired nucleotides are not recognized, the initial complex may serve to initiate a repair cascade through a smaller number of MutL proteins.Understanding the steady-state relationships among the components of the DNA MMR system, in addition to informing their functional roles in repair, may provide insights into hereditary nonpolyposis colorectal cancer (HNPCC) as well. Among the most important causes of HNPCC are germline mutations in hMSH2 (29.Peltomaki P. Vasen H.F. Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 30.Aaltonen L.A. Salovaara R. Kristo P. Canzian F. Hemminki A. Peltomaki P. Chadwick R.B. Kaariainen H. Eskelinen M. Jarvinen H. Mecklin J.P. de la Chapelle A. N. Engl. J. Med. 1998; 338: 1481-1487Crossref PubMed Scopus (981) Google Scholar), an obligatory partner with either hMSH6 or hMSH3 in the mispair recognition complex (5.Acharya 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 (462) Google Scholar). In fact, hMSH2 and hMLH1 germline mutations account for most of the highly penetrant forms of HNPCC (29.Peltomaki P. Vasen H.F. Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 30.Aaltonen L.A. Salovaara R. Kristo P. Canzian F. Hemminki A. Peltomaki P. Chadwick R.B. Kaariainen H. Eskelinen M. Jarvinen H. Mecklin J.P. de la Chapelle A. N. Engl. J. Med. 1998; 338: 1481-1487Crossref PubMed Scopus (981) Google Scholar). hMSH6 is quantitatively the most important protein partner with hMSH2 and is expressed 4–12 times more abundantly than hMSH3. Germline mutations in hMSH6 account for relatively fewer cases of familial colorectal cancer than can be attributed to hMSH2 and hMLH1(29, 30), and the phenotype is relatively attenuated in the case ofhMSH6 germline mutations (31.Kolodner R.D. Tytell J.D. Schmeits J.L. Kane M.F. Gupta R.D. Weger J. Wahlberg S. Fox E.A. Peel D. Ziogas A. Garber J.E. Syngal S. Anton-Culver H. Li F.P. Cancer Res. 1999; 59: 5068-5074PubMed Google Scholar). To date, germline mutations in hMSH3 have never been linked to familial colorectal cancer (29.Peltomaki P. Vasen H.F. Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 30.Aaltonen L.A. Salovaara R. Kristo P. Canzian F. Hemminki A. Peltomaki P. Chadwick R.B. Kaariainen H. Eskelinen M. Jarvinen H. Mecklin J.P. de la Chapelle A. N. Engl. J. Med. 1998; 338: 1481-1487Crossref PubMed Scopus (981) Google Scholar).Recently, the hMLH1-hPMS1 heterodimer has been purified and termed hMutL-β (32.Raschle M. Marra G. Nystrom-Lahti M. Schar P. Jiricny J. J. Biol. Chem. 1999; 274: 32368-32375Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and a new human DNA MMR component hMLH3, which also forms a complex with hMLH1, has been reported (4.Lipkin S.M. Wang V. Jacoby R. Banerjee-Basu S. Baxevanis A.D. Lynch H.T. Elliott R.M. Collins F.S. Nat. Genet. 2000; 24: 27-35Crossref PubMed Scopus (264) Google Scholar). In our study, hMLH1 was consistently detected at higher levels than hPMS2, particularly at the protein level. Unfortunately, probes were not available to accurately quantitate hPMS1 and hMLH3 at the message or protein level. We speculate that the combined amounts of hPMS1, hPMS2, and hMLH3 proteins might be approximately equal to hMLH1.We investigated in detail the interactive regulation of each component of the MMR system in MMR-deficient cell lines missing different components of the system. In LoVo cells, which are defective inhMSH2 (23.Boyer J.C. Umar A. Risinger J.I. Lipford J.R. Kane M. Yin S. Barrett J.C. Kolodner R.D. Kunkel T.A. Cancer Res. 1995; 55: 6063-6070PubMed Google Scholar), hMSH3 and hMSH6 proteins were also nearly undetectable (our data, and Ref. 11.Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Of interest, compensatory transcriptional up-regulation of the hMSH3 gene can be seen in the absence of hMSH6, as is the case in HCT15 cells. Similarly, absence of the hMLH1 affects expression of the hPMS2 protein, as seen in HCT116 cells. Interactions among the components of the MMR system can become particularly complicated. Mutations of hMSH2 orhMLH1, which functionally inactivate the system, can also facilitate somatic mutations of hMSH3 and hMSH6, both of which bear repetitive mononucleotide tracts in their coding sequences (33.Perucho M. Nat. Med. 1996; 2: 630-631Crossref PubMed Scopus (162) Google Scholar).An inactivating mutation in hMSH6 in combination with intacthMSH2 is associated with an up-regulation ofhMSH3, which may compensate, to some degree, for the defective hMutS-α function. There is some redundancy in function between hMutS-α and hMutS-β as both can repair small insertion/deletion mispaired loops (34.Umar A. Risinger J.I. Glaab W.E. Tindall K.R. Barrett J.C. Kunkel T.A. Genetics. 1998; 148: 1637-1646Crossref PubMed Google Scholar). Moreover, the lack of hMSH6 protein permits a compensatory increase in hMutS-β by increasing the stability of hMSH3 following its dimerization with hMSH2 (11.Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Additionally, we confirmed the transcriptional up-regulation ofhMSH3 in the presence of inactivating mutation inhMSH6. This observation helps explain the relatively weaker mutator effect of a mutated hMSH6 gene, and the observation that germline mutations in hMSH6 are associated with an attenuated HNPCC phenotype (31.Kolodner R.D. Tytell J.D. Schmeits J.L. Kane M.F. Gupta R.D. Weger J. Wahlberg S. Fox E.A. Peel D. Ziogas A. Garber J.E. Syngal S. Anton-Culver H. Li F.P. Cancer Res. 1999; 59: 5068-5074PubMed Google Scholar).The colon cancer cell line HCT116 has undergone mutational inactivation of the hMLH1 gene and is defective in DNA MMR activity (23.Boyer J.C. Umar A. Risinger J.I. Lipford J.R. Kane M. Yin S. Barrett J.C. Kolodner R.D. Kunkel T.A. Cancer Res. 1995; 55: 6063-6070PubMed Google Scholar). The SW48 cell line expresses no hMLH1 due to hypermethylation of its promoter (24.Kane M.F. Loda M. Gaida G.M. Lipman J. Mishra R. Goldman H. Jessup J.M. Kolodner R. Cancer Res. 1997; 57: 808-811PubMed Google Scholar). The level of the hMutS-α mRNAs and proteins are variable in these cell lines, indicating that the hMutS and hMutL systems do not quantitatively regulate each other. The stability of hPMS2 appears to depend upon the presence of an intact hMLH1 (our data and Ref. 35.Yao X. Buermeyer A.B. Narayanan L. Tran D. Baker S.M. Prolla T.A. Glazer P.M. Liskay R.M. Arnheim N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6850-6855Crossref PubMed Scopus (116) Google Scholar). As seen in the HCT116+ch3 cell line, the reconstitution of hMLH1 also restores expression of the hPMS2 protein.We have previously reported that the human MMR system is regulated throughout the cell cycle in MMR-proficient cell lines (16.Marra G. Chang C.L. Laghi L.A. Chauhan D.P. Young D. Boland C.R. Oncogene. 1996; 13: 2189-2196PubMed Google Scholar). We noted that the MMR-proficient cancer cell lines showed higher levels of MMR mRNAs and proteins than we saw in the two normal lung fibroblast lines. The normal lung fibroblast cell line WI38 has been stably transformed and immortalized with the SV40 virus (19.Girardi A.J. Weinstein D. Moorhead P.S. Ann. Med. Exp. Biol. Fenn. 1966; 44: 242-254PubMed Google Scholar). The entire DNA MMR system underwent transcriptional up-regulation after SV40 transformation. The promoters of the MMR genes hMSH2,hMLH1, hPMS2, and hPMS1 have characteristic features of housekeeping genes, including abundant CG islands and no TATA box (36.Yanagisawa Y. Ito E. Iwahashi Y. Akiyama Y. Yuasa Y. Maruyama K. Biochem. Biophys. Res. Commun. 1998; 243: 738-743Crossref PubMed Scopus (10) Google Scholar, 37.Nicolaides N.C. Kinzler K.W. Vogelstein B. Genomics. 1995; 29: 329-334Crossref PubMed Scopus (41) Google Scholar, 38.Iwahashi Y. Ito E. Yanagisawa Y. Akiyama Y. Yuasa Y. Onodera T. Maruyama K. Gene (Amst .). 1998; 213: 141-147Crossref PubMed Scopus (40) Google Scholar, 39.Ito E. Yanagisawa Y. Iwahashi Y. Suzuki Y. Nagasaki H. Akiyama Y. Sugano S. Yuasa Y. Maruyama K. Biochem. Biophys. Res. Commun. 1999; 256: 488-494Crossref PubMed Scopus (84) Google Scholar). However, these sequences show potential binding sites for transcriptional activators including AP-1 (in the case of the hMSH2 and hPMS1 promoters) (36.Yanagisawa Y. Ito E. Iwahashi Y. Akiyama Y. Yuasa Y. Maruyama K. Biochem. Biophys. Res. Commun. 1998; 243: 738-743Crossref PubMed Scopus (10) Google Scholar, 38.Iwahashi Y. Ito E. Yanagisawa Y. Akiyama Y. Yuasa Y. Onodera T. Maruyama K. Gene (Amst .). 1998; 213: 141-147Crossref PubMed Scopus (40) Google Scholar) and T-antigen (in the hMSH2 promoter) (38.Iwahashi Y. Ito E. Yanagisawa Y. Akiyama Y. Yuasa Y. Onodera T. Maruyama K. Gene (Amst .). 1998; 213: 141-147Crossref PubMed Scopus (40) Google Scholar). The presence of these binding sites suggests that the MMR genes might be up-regulated in different physiological or biological settings. As cell cycle−dependent regulation of the MMR system has been previously reported (16.Marra G. Chang C.L. Laghi L.A. Chauhan D.P. Young D. Boland C.R. Oncogene. 1996; 13: 2189-2196PubMed Google Scholar), up-regulation of the MMR system in the synthetic and postsynthetic phases might reflect an increased need for active MMR function in actively proliferating cells. Increased recruitment of cells into the cell cycle from G0 phase or an increased proportion of S and G2/M phase cells by active proliferation may be responsible for up-regulation of the system.In summary, we have studied the steady-state expression of human DNA MMR transcripts and proteins, where a hierarchy of expression reflects the relative roles of these proteins in DNA repair. hMSH2 is the most abundantly expressed of these proteins, and the presence of hMSH2 protein serves to stabilize hMSH6 and hMSH3. The defect ofhMSH6 interactively up-regulates hMSH3transcription. The hMSH6 protein is substantially more highly expressed than hMSH3, an observation worthy of additional functional exploration. hMLH1 also behaves like a chaperone of stability for the other hMutL components. The components of the hMutS system are expressed severalfold more than components of the hMutL system, which suggests the presence of a signaling cascade in MMR function. Finally, SV40 transformation of a fibroblast cell line led to an up-regulation of the entire MMR system. The DNA MMR system is regulated primarily at a transcriptional level; however, mutational inactivation of the obligatory components of the system, hMSH2 andhMLH1, lead to post-translational down-regulation of its heterodimerizing partners. These findings have implications for the physiological regulation of the DNA MMR system and for the interpretation of germline mutations in hereditary colorectal cancer. The human mismatch repair (MMR)1 system ensures replication fidelity by correcting postreplication errors that have escaped the DNA proofreading function of DNA polymerase. Defects in the MMR system result in the development of a genetically unstable mutator phenotype and render the cell more susceptible to neoplastic transformation (reviewed in Refs. 1.Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (717) Google Scholar, 2.Marra G. Boland C.R. J. Natl. Cancer Inst. 1995; 87: 1114-1125Crossref PubMed Scopus (478) Google Scholar, 3.Modrich P. J. Biol. Chem. 1997; 272: 24727-24730Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The MMR system functions through the interactions between several proteins, such as hMSH2, hMSH3, hMSH6, hMLH1, hPMS2, and recently discovered hMLH3 (1.Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (717) Google Scholar, 2.Marra G. Boland C.R. J. Natl. Cancer Inst. 1995; 87: 1114-1125Crossref PubMed Scopus (478) Google Scholar, 3.Modrich P. J. Biol. Chem. 1997; 272: 24727-24730Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 4.Lipkin S.M. Wang V. Jacoby R. Banerjee-Basu S. Baxevanis A.D. Lynch H.T. Elliott R.M. Collins F.S. Nat. Genet. 2000; 24: 27-35Crossref PubMed Scopus (264) Google Scholar). hMutS-α is a heterodimer of hMSH2 and hMSH6 that binds to mismatched nucleotides or single insertion/deletion loops (5.Acharya 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 (462) Google Scholar, 6.Drummond J.T. Li G.M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (532) Google Scholar, 7.Guerrette S. Wilson T. Gradia S. Fishel R. Mol. Cell. Biol. 1998; 18: 6616-6623Crossref PubMed Scopus (120) Google Scholar). In addition, hMSH2 can dimerize with hMSH3 creating the hMutS-β complex, which binds larger DNA insertion/deletion mispaired loops (5.Acharya 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 (462) Google Scholar, 7.Guerrette S. Wilson T. Gradia S. Fishel R. Mol. Cell. Biol. 1998; 18: 6616-6623Crossref PubMed Scopus (120) Google Scholar, 8.Palombo F. Iaccarino I. Nakajima E. Ikejima M. Shimada T. Jiricny J. Curr. Biol. 1996; 6: 1181-1184Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Subsequently, activated hMutS-α or hMutS-β interacts with hMutL-α (a heterodimer of hMLH1 with hPMS2) to direct DNA repair (9.Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar, 10.Fishel R. Genes Dev. 1998; 12: 2096-2101Crossref PubMed Scopus (151) Google Scholar). Recent studies suggest that maintaining an appropriate balance of the components of the human MMR system is critical for its proper repair function. In hMSH6-defective HCT15 cells, hMSH3 protein is highly expressed, whereas in hMSH2-defective LoVo cells, hMSH3 and hMSH6 proteins are not detected (11.Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Fu" @default.
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