UBIQUITINATION-DEPENDENT PROTEOLYSIS OF O-6-METHYLGUANINE-DNA METHYLTRANSFERASE IN HUMAN AND MURINE TUMOR-CELLS FOLLOWING INACTIVATION WITHO-6-BENZYLGUANINE OR 1,3-BIS(2-CHLOROETHYL)-1-NITROSOUREA

In this study, we investigated the role of ubiquitination in the dispo sition of the inactivated O-6-methylguanine-DNA methyltransferase (MGM T) protein in human (HT-29 and GEM) and murine (ts85) tumor cells. Usi ng a combination of immunoprecipitation and immunoblotting techniques with antibodies against ubiquitin and MGMT, and anti-ubiquitin immunoa ffinity chromatography, the MGMT protein was found to coexist with sma ll 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, O -6-benzylguanine (O-6-BG, 20 mu M) or 1,3-bis(chloroethyl)-1-nitrosour ea (BCNU, 100 mu M), resulted in increased levels of ubiquitinated MGM T within 1.5-3 h of drug exposure, Kinetic studies in HT-29 cells trea ted with O-6-BG indicated a slow and gradual conversion of the inactiv ated MGMT to its polyubiquitinated forms over a course of 3-18 h, with a concomitant disappearance of the parent MGMT protein, We also chara cterized the previously reported O-6-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 prote in with ubiquitin, The proteolysis of O-6-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 observ ed a differential stability of the inactivated MGMT protein at permiss ive and nonpermissive temperatures, These results provide conclusive e vidence that the MGMT protein, following its inactivation, is degraded via the ubiquitin proteolytic pathway.