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 MSIpositive 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.Germ-line defects in DNA mismatch repair (MMR) genes account for the inherited familial cancer syndrome of hereditary nonpolyposis colon cancers in which affected individuals show accelerated development of cancers of the proximal colon, endometrium, and stomach (1-5). These cancers typically demonstrate inactivation of the residual wild-type MMR allele inherited opposite the germ-line mutant (1-6), absence of DNA MMR activity in in vitro assays (7,8), and acquisition of an in vivo mutator phenotype showing up to 1,000-fold increased gene mutation rates (9, 10). Additionally, these cancers display an associated instability of genomic microsatellite sequences (MSI) (1-5). MSI is similarly found in approximately 15-20% of sporadic colon cancers that arise in individuals without any family history of colon cancer (1-5). Similarly to hereditary nonpolyposis colon cancer-associated colon cancers, sporadic MSI colon cancers arise predominantly in the proximal colon and show a high rate of frameshift mutations at a mutational hotspot in the transforming growth factor-␤ type II receptor tumor suppressor gene (1, 11). Familial and sporadic MSI colon cancers thus appear to share a common carcinogenic pathway. In this regard, previous studies from our group established that MMR gene inactivation via somatic mutation was the cause of some cases of sporadic MSI colon cancers (12). However, unexpectedly, in many spo...

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