Clinical Relevance ofMGMTin the Treatment of Cancer

The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC). On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref. 3). Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p21.3-23. We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family.

DNA mismatch repair is an important pathway of mutation avoidance. It also contributes to the cytotoxic effects of some kinds of DNA damage, and cells defective in mismatch repair are resistant, or tolerant, to the presence of some normally cytotoxic base analogues in their DNA. The absence of a particular mismatch binding function from some mammalian cells confers resistance to the base analogues O6-methylguanine and 6-thioguanine in DNA. Cells also acquire a spontaneous mutator phenotype as a consequence of this defect. Impaired mismatch binding can cause an instability in DNA microsatellite regions that comprise repeated dinucleotides. Microsatellite DNA instability is common in familial and sporadic colon carcinomas as well as in a number of other tumours. Several independent lines of investigation have identified defects in mismatch repair proteins that are causally related to these cancers.

In this study, we investigated the role of ubiquitination in the disposition of the inactivated O6-methylguanine-DNA methyltransferase (MGMT) protein in human (HT-29 and CEM) and murine (ts85) tumor cells. Using a combination of immunoprecipitation and immunoblotting techniques with antibodies against ubiquitin and MGMT, and anti-ubiquitin immunoaffinity chromatography, the MGMT protein was found to coexist with small amounts of its ubiquitinated species in both human and mouse tumor cells, suggesting the presence of endogenous inactivated MGMT. Further, treatment of HT-29 and CEM cells with MGMT-inactivating compounds, O6-benzylguanine (O6-BG, 20 microM) or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU, 100 microM), resulted in increased levels of ubiquitinated MGMT within 1.5-3 h of drug exposure. Kinetic studies in HT-29 cells treated with O6-BG indicated a slow and gradual conversion of the inactivated MGMT to its polyubiquitinated forms over a course of 3-18 h, with a concomitant disappearance of the parent MGMT protein. We also characterized the previously reported O6-BG-induced degradation of MGMT in HT-29 cell extracts [Pegg et al. (1991) Carcinogenesis 12, 1679-1683] and showed the extracts to be active in conjugation of the MGMT protein with ubiquitin. The proteolysis of O6-BG-inactivated MGMT in HT-29 cell extracts was energy-dependent and was markedly stimulated by ATP and Mg2+ ions. Using the ts85 temperature-sensitive mutant cell line, which expresses a thermolabile ubiquitin-activating enzyme, we observed a differential stability of the inactivated MGMT protein at permissive and nonpermissive temperatures. These results provide conclusive evidence that the MGMT protein, following its inactivation, is degraded via the ubiquitin proteolytic pathway.