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

Abstract

Fifteen to twenty-five percent of sporadic colorectal carcinomas are replication error (RER) positive. Because the frequency of mutations in the mismatch repair genes (hMLH1 and hMSH2) is low in these tumors, we have investigated the role of mutational inactivation, methylation of the promoter region, and loss of heterozygosity (LOH) as a possible explanation for the mutator phenotype of RER+ colorectal cancer cell lines. Genomic DNA was extracted 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 were amplified with the PCR and screened by using single-strand conformational polymorphism and direct sequencing. The methylation status was ascertained by methylation-specific PCR after bisulfite modification of DNA. Western blotting for hMLH1 was performed on methylated cell lines before and after the addition of the demethylating agent 5-azacytidine. LOH was sought by GENESCAN analysis of amplified CA repeat markers and indirectly by determining the 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 methylated cell lines expressed hMLH1, although all reexpressed hMLH1 after treatment 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 hypermethylation of the promoter region, but not LOH, are the cause of the mutator phenotype in the majority (70%) of RER+ tumors.

Figure 1

A silver-stained SSCP polyacrylamide gel of exon 16 in hMLH1. The heterozygous band shift at GP2d is arrowed. Sequencing confirmed this to be a 2-bp substitution at codon 618 (AAG → GCG; Lys → Ala), previously described in HNPCC kindred. This sequence change was also found in one of the 89 United Kingdom human random controls, giving further weight to the association between subpolymorphic missense variants and CRC.

Figure 2

Sequence chromatogram of exon 8 in hMLH1. The heterozygous single-bp substitution at codon 226 (CGG → TGG; Arg → Trp) in LS411 is demonstrated (arrow). LS411 was also shown to be methylated in the hMLH1 promoter region and did not express hMLH1 protein on Western blotting.

Figure 3

PCR-based HpaII restriction enzyme assay demonstrating (a) a methylated hMLH1 promoter region in three RER+ cell lines (SW48, LS411, and HCA7), and (b) an unmethylated hMLH1 promoter region in two RER+ (HCT116 and GP2d) and one RER− cell lines (C99). (U, undigested; H, incubated with HpaII; M, incubated with MspI).

Figure 4

Western blot of cell lines: 1 wk after treatment with 5-azacytidine (VACO5, HCA7, and HCT116) and 2 wk after treatment with 5-azacytidine (HCA7, LS411 and SW48) and positive controls (C70, GP2d). Reexpression of hMLH1 can been seen 1 wk after treatment with 5-azacytidine in the methylated VACO5 and HCA7, but not in the unmethylated RER+ control, HCT116. Two weeks after treatment, expression of hMLH1 is virtually absent (faint band in HCA7, LS411, and SW48), suggesting that this epigenetic mechanism of gene silencing is driven by a dynamic process.