Abstract
In bladder cancer the observed microsatellite instability indicates that mismatch repair deficiency could be a frequently involved factor in bladder cancer progression. To investigate this hypothesis we analysed extracts of seven bladder cancer cell lines and, as a novel approach, five clinical cancer samples for mismatch repair activity. We found that one cell line (T24) and three of the clinical samples had a reduced repair capacity, measured to ~20% or less. The T24 cell extract was unable to repair a G-G mismatch and showed reduced repair of a 2-base loop, consistent with diminished function of the MSH2-MSH6 heterodimer. The functional assay was combined with measurement for mutation frequency, microsatellite analysis, sequencing, MTT assay, immunohistochemical analysis and RT-PCR analysis of the mismatch repair genes MSH2, MSH3, MSH6, PMS1, PMS2 and MLH1. A >7-fold relative increase in mutation frequency was observed for T24 compared to a bladder cancer cell line with a fully functional mismatch repair system. Neither microsatellite instability, loss of repair nor mismatch repair gene mutations were detected. However, RT-PCR analysis of mRNA levels did detect changes in the ratio of expression of the Mut S and Mut L homologues. The T24 cell line had the lowest MSH6 expression level of the cell lines tested. Identical RT-PCR analysis of seventeen clinical samples (normal urothelium, 7; pTa low stage, 5; and pT1-4 high stage, 5) indicated a significant change in the expression ratio between MSH3/MSH6 (P < 0.004), MSH2/MSH3 (P < 0.012) and PMS2/MLH1 P < 0.005, in high stage bladder tumours compared to normal urothelium and low stage tumours. Collectively, the data suggest that imbalanced expression of mismatch repair genes could lead to partial loss of mismatch repair activity that is associated with invasive bladder cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com
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References
Aebi S, Fink D, Gordon R, Kim HK, Zheng H, Fink JL and Howell SB (1997) Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells. Clinical Cancer Research 3: 1763–1767
Arzimanoglou II, Gilbert F and Barber HR (1998) Microsatellite instability in human solid tumors. Cancer 82: 1808–1820
Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, van Tuinen P, Ledbetter DH, Barker DF and Nakamura Y (1989) Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 244: 217–221
Bennett SE, Umar A, Oshima J, Monnat RJJ and Kunkel TA (1997) Mismatch repair in extracts of Werner syndrome cell lines. Cancer Res 57: 2956–2960
Børresen AL, Lothe RA, Meling GI, Lystad S, Morrison P, Lipford J, Kane MF, Rognum TO and Kolodner RD (1995) Somatic mutations in the hMSH2 gene in microsatellite unstable colorectal carcinomas. Hum Mol Genet 4: 2065–2072
Brewster SF, Gingell JC, Browne S and Brown KW (1994) Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder. Br J Cancer 70: 697–700
Buermeyer AB, Deschenes SM, Baker SM and Liskay RM (1999) Mammalian DNA mismatch repair. Annu Rev Genet 33: 533–564
Cairns P, Shaw ME and Knowles MA (1993) Preliminary mapping of the deleted region of chromosome 9 in bladder cancer. Cancer Res 53: 1230–1232
Christensen M, Jensen MA, Wolf H and Orntoft TF (1998) Pronounced microsatellite instability in transitional cell carcinomas from young patients with bladder cancer. Int J Cancer 79: 396–401
Drummond JT, Li GM, Longley MJ and Modrich P (1995) Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells [see comments]. Science 268: 1909–1912
Drummond JT, Genschel J, Wolf E and Modrich P (1997) DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair. Proc Natl Acad Sci USA 94: 10144–10149
Fishel R, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J, Kane M and Kolodner R (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer [published erratum appears in Cell 1994 Apr 8; 77(1):167]. Cell 75: 1027–1038
Fogh J (1978) Cultivation, characterization, and identification of human tumor cells with emphasis on kidney, testis, and bladder tumors. Natl Cancer Inst Monogr 5–9
Genschel J, Littman SJ, Drummond JT and Modrich P (1998) Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha. J Biol Chem 273: 19895–19901
Genuardi M, Anti M, Capozzi E, Leonardi F, Fornasarig M, Novella E, Bellacosa A, Valenti A, Gasbarrini GB, Roncucci L, Benatti P, Percesepe A, Ponz dL, Coco C, de Paoli A, Valentini M, Boiocchi M, Neri G and Viel A (1998) MLH1 and MSH2 constitutional mutations in colorectal cancer families not meeting the standard criteria for hereditary nonpolyposis colorectal cancer. Int J Cancer 75: 835–839
Ghimenti C, Tannergard P, Wahlberg S, Liu T, Giulianotti PG, Mosca F, Fornaciari G, Bevilacqua G, Lindblom A and Caligo MA (1999) Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours. Br J Cancer 80: 11–16
Gonzalez-Zulueta M, Ruppert JM, Tokino K, Tsai YC, Spruck CH, Miyao N, Nichols PW, Hermann GG, Horn T and Steven K (1993) Microsatellite instability in bladder cancer. Cancer Res 53: 5620–5623
Gu L, Hong Y, McCulloch S, Watanabe H and Li GM (1998) ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair. Nucleic Acids Res 26: 1173–1178
Habraken Y, Sung P, Prakash L and Prakash S (1998) ATP-dependent assembly of a ternary complex consisting of a DNA mismatch and the yeast MSH2-MSH6 and MLH1-PMS1 protein complexes. J Biol Chem 273: 9837–9841
Kallioniemi A, Kallioniemi OP, Citro G, Sauter G, DeVries S, Kerschmann R, Caroll P and Waldman F (1995) Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization. Genes Chromosomes Cancer 12: 213–219
Keen AJ and Knowles MA (1994) Definition of two regions of deletion on chromosome 9 in carcinoma of the bladder. Oncogene 9: 2083–2088
Knowles MA, Shaw ME and Proctor AJ (1993) Deletion mapping of chromosome 8 in cancers of the urinary bladder using restriction fragment length polymorphisms and microsatellite polymorphisms. Oncogene 8: 1357–1364
Kolodner RD and Marsischky GT (1999) Eukaryotic DNA mismatch repair. Curr Opin Genet Dev 9: 89–96
Kyriazis AA, Kyriazis AP, McCombs WB and Peterson WDJ (1984) Morphological, biological, and biochemical characteristics of human bladder transitional cell carcinomas grown in tissue culture and in nude mice. Cancer Res 44: 3997–4005
Lettieri T, Marra G, Aquilina G, Bignami M, Crompton NE, Palombo F and Jiricny J (1999) Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15. Carcinogenesis 20: 373–382
Li M, Zhang ZF, Reuter VE and Cordon-Cardo C (1996) Chromosome 3 allelic losses and microsatellite alterations in transitional cell carcinoma of the urinary bladder. Am J Pathol 149: 229–235
Lothe RA (1997) Microsatellite instability in human solid tumors. Mol Med Today 3: 61–68
Mahdy E, Yoshihiro S, Zech L, Wester K, Pan Y, Busch C, Dohner H, Kallioniemi O, Bergerheim U and Malmstrom PU (1999) Comparison of comparative genomic hybridization, fluorescence in situ hybridization and flow cytometry in urinary bladder cancer. Anticancer Res 19: 7–12
Marra G, Iaccarino I, Lettieri T, Roscilli G, Delmastro P and Jiricny J (1998) Mismatch repair deficiency associated with overexpression of the MSH3 gene. Proc Natl Acad Sci USA 95: 8568–8573
Miyamoto H, Shuin T, Torigoe S, Iwasaki Y and Kubota Y (1995) Retinoblastoma gene mutations in primary human bladder cancer. Br J Cancer 71: 831–835
O'Toole C, Price ZH, Ohnuki Y and Unsgaard B (1978) Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma. Br J Cancer 38: 64–76
Orlow I, Lianes P, Lacombe L, Dalbagni G, Reuter VE and Cordon-Cardo C (1994) Chromosome 9 allelic losses and microsatellite alterations in human bladder tumors. Cancer Res 54: 2848–2851
Ørntoft TF, Meldgaard P, Pedersen B and Wolf H (1996) The blood group ABO gene transcript is down-regulated in human bladder tumors and growth-stimulated urothelial cell lines. Cancer Res 56: 1031–1036
Polascik TJ, Cairns P, Chang WY, Schoenberg MP and Sidransky D (1995) Distinct regions of allelic loss on chromosome 4 in human primary bladder carcinoma. Cancer Res 55: 5396–5399
Presti JCJ, Reuter VE, Galan T, Fair WR and Cordon-Cardo C (1991) Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Res 51: 5405–5409
Rasheed S, Gardner MB, Rongey RW, Nelson-Rees WA and Arnstein P (1977) Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses. J Natl Cancer Inst 58: 881–890
Rigby CC and Franks LM (1970) A human tissue culture cell lines from a transitional cell tumour of the urinary bladder: growth, chromosome pattern and ultrastructure. Br J Cancer 24: 746–754
Roberts JD and Kunkel TA (1993) Fidelity of DNA Replication in Human Cells. Methods Mol Genet 2: 295–313
Ruppert JM, Tokino K and Sidransky D (1993) Evidence for two bladder cancer suppressor loci on human chromosome 9. Cancer Res 53: 5093–5095
Shcherbakova PV and Kunkel TA (1999) Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations. Mol Cell Biol 19: 3177–3183
Simon R, Burger H, Brinkschmidt C, Bocker W, Hertle L and Terpe HJ (1998) Chromosomal aberrations associated with invasion in papillary superficial bladder cancer. J Pathol 185: 345–351
Taylor JA, Li Y, He M, Mason T, Mettlin C, Vogler WJ, Maygarden S and Liu E (1996) p53 mutations in bladder tumors from arylamine-exposed workers. Cancer Res 56: 294–298
Thomas DC, Umar A and Kunkel TA (1995) Measurement of heteroduplex repair in human cell extracts. Methods: A Companion to Methods in Enzymology 7: 187–197
Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB and Issa JP (1999) CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 96: 8681–8686
Umar A, Boyer JC and Kunkel TA (1994) DNA loop repair by human cell extracts [see comments]. Science 266: 814–816
Umar A, Koi M, Risinger JI, Glaab WE, Tindall KR, Kolodner RD, Boland CR, Barrett JC and Kunkel TA (1998a) Correction of hypermutability, N -Methyl- N′-nitro- N -nitro- N -nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and. MSH6 Cancer Research 57: 3949–3955
Umar A, Risinger JI, Glaab WE, Tindall KR, Barrett JC and Kunkel TA (1998b) Functional overlap in mismatch repair by human MSH3 and MSH6. Genetics 148: 1637–1646
Voorter C, Joos S, Bringuier PP, Vallinga M, Poddighe P, Schalken J, du MS, Ramaekers F, Lichter P and Hopman A (1995) Detection of chromosomal imbalances in transitional cell carcinoma of the bladder by comparative genomic hybridization. Am J Pathol 146: 1341–1354
Wang TF, Kleckner N and Hunter N (1999) Functional specificity of MutL homologs in yeast: evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction. Proc Natl Acad Sci USA 96: 13914–13919
Wingo PA, Ries LA, Giovino GA, Miller DS, Rosenberg HM, Shopland DR, Thun MJ and Edwards BK (1999) Annual report to the nation on the status of cancer, 1973–1996, with a special section on lung cancer and tobacco smoking. J Natl Cancer Inst 91: 675–690
Yu Z, Chen J, Ford BN, Brackley ME and Glickman BW (1999) Human DNA repair systems: an overview. Environ Mol Mutagen 33: 3–20
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Thykjaer, T., Christensen, M., Clark, A. et al. Functional analysis of the mismatch repair system in bladder cancer. Br J Cancer 85, 568–575 (2001). https://doi.org/10.1054/bjoc.2001.1949
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DOI: https://doi.org/10.1054/bjoc.2001.1949
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