BIALLELIC INACTIVATION OF HMLH1 BY EPIGENETIC GENE SILENCING, A NOVELMECHANISM CAUSING HUMAN MSI CANCERS

Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA r epair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated wi th inherited MSI colon cancer, and somatic mutations are causally asso ciated with sporadic MSI colon cancer. Previously however, we demonstr ated that in many sporadic MSI colon cancers hMLH1 and all other DNA m ismatch repair genes are wild type. To investigate this class of tumor s 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 abs ent, even though hMLH1-coding sequences were wild type. In each such c ase, absence of hMLH1 protein was associated with the methylation of t he hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hML H1 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 account ed for the silencing of both maternal and paternal tumor hMLH1 alleles , both of which could be reactivated by 5-azacytidine. In summary, sub stantial numbers of human MSI cancers appear to arise by hMLH1 silenci ng via an epigenetic mechanism that can inactivate both of the hMLH1 a lleles. Promoter methylation is intimately associated with this epigen etic silencing mechanism.