Mechanisms of inactivation of mismatch repair genes in human colorectal cancer cell lines: The predominant role of hMLH1

Fifteen to twenty-five percent of sporadic colorectal carcinomas are replic ation error (RER) positive. Because the frequency of mutations in the misma tch repair genes (hMLH1 and hMSH2) is low in these tumors, we have investig ated the role of mutational inactivation, methylation of the promoter regio n, and loss of heterozygosity (LOH) as a possible explanation for the mutat or phenotype of RER+ colorectal cancer cell Lines. Genomic DNA was extracte d from a panel of 49 human colorectal cancer cell lines. The RER status was determined by amplification of BAT-26, All exons of hMLH1 and hMSH2 mere a mplified with the PCR and screened by using single-strand conformational po lymorphism and direct sequencing. The methylation status was ascertained by methylation-specific PCR after bisulfite modification of DNA, Western blot ting for hMLH1 was performed on methylated cell lines before and after the addition of the demethylating agent 5-azacytidine, LOH was sought by GENESC AN analysis of amplified CA repeat markers and indirectly by determining th e number of homozygotes in the cell lines and human random controls, Twelve cell lines from ten tumors (24%) were RER+. Hypermethylation of the hMLH1 promoter occurred in five of ten (50%) RER+ tumors, whereas three of thirty -two (6%) RER tumors showed partial methylation. None of the fully methylat ed cell lines expressed hMLH1, although all reexpressed hMLH1 after treatme nt with 5-azacytidine, There was no LOH in the RER+ tumors in either hMLH1 or hMSH2. Our results suggest that mutations of hMLH1 together with hyperme thylation of the promoter region, but not LOH, are the cause of the mutator phenotype in the majority (70%) of RER+ tumors.