Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers

Abstract

Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.

Figure 1

hMLH1 protein expression in MSI cancers. Shown is a Western blot of hMLH1 protein expression. The presence (+) or absence (−) of MSI and the status of the coding region of the hMLH1 gene (wt, wild type; mut, mutant) are denoted below each lane. The Western analysis was performed as described in Materials and Methods.

Figure 2

Analysis of methylation status of the hMLH1 promoter. Shown is the product resulting from PCR amplification of the hMLH1 promoter region before or after digestion. U, Undigested; H, digested with HpaII; M, digested with MspI. The methylation status (+, methylated; −, unmethylated) of the hMLH1 promoter is designated below each sample.

Figure 3

Induction of hMLH1 protein expression after 5-azacytidine (AzaC) treatment. The Western analysis was performed as described in Materials and Methods. U, Cells untreated; T, cells treated with 5-azacytidine.

Figure 4

Time course of hMLH1 protein expression after 5-azacytidine treatment. AN3CA cells were treated with 5-azacytidine as detailed in Materials and Methods. An untreated sample (day 0), a sample immediately after 5-azacytidine treatment (day 8), and samples 1 week (day 15) and 2 weeks (day 21) after 5-azacytidine treatment were collected for Western analysis to evaluate production of hMLH1 protein.

Figure 5

Reversible expression of hMLH1 protein in single-cell clones treated with 5-azacytidine. Western analysis of hMLH1 protein expression was performed as described in Materials and Methods and is shown for AN3CA cells untreated (U) or on day 8 of treatment with 5-azacytidine (T). As described in Materials and Methods, subclones 1, 2, and 3 were established from the day 8 treated AN3CA cell population immediately after washout of the drug and were analyzed for continued hMLH1 expression when they had reached confluence. The same three subclones were then retreated with 5-azacytidine and again analyzed (treated subclones). Subclones 1 and 3 are representative normal-growth-rate subclones; whereas subclone 2 is representative of the group of slow-growth-rate subclones.

Figure 6

Allele-specific analysis of hMLH1 expression after 5-azacytidine treatment in Vaco 5 and Vaco 6. (A) Reverse transcription–PCR analysis of hMLH1 expression in Vaco5 and Vaco432 before (+) and after (−) treatment with 5-azacytidine (AzaC). (B) DNA sequences of hMLH1 cDNAs from Vaco5 and Vaco432 amplified by reverse transcription–PCR after treatment with 5-azacytidine. The arrow points to a coding region polymorphism at codon 219 (ATC to GTC).