Ubiquitination-Dependent Proteolysis of O6-Methylguanine-DNA Methyltransferase in Human and Murine Tumor Cells following Inactivation with O6-Benzylguanine or 1,3-Bis(2-chloroethyl)-1-nitrosourea†

摘要: In this study, we investigated the role of ubiquitination in disposition inactivated O6-methylguanine-DNA methyltransferase (MGMT) protein human (HT-29 and CEM) murine (ts85) tumor cells. Using a combination immunoprecipitation immunoblotting techniques with antibodies against ubiquitin MGMT, anti-ubiquitin immunoaffinity chromatography, MGMT was found to coexist small amounts its ubiquitinated species both mouse cells, suggesting presence endogenous MGMT. Further, treatment HT-29 CEM cells MGMT-inactivating compounds, O6-benzylguanine (O6-BG, 20 μM) or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU, 100 μM), resulted increased levels within 1.5−3 h drug exposure. Kinetic studies treated O6-BG indicated slow gradual conversion polyubiquitinated forms over course 3−18 h, concomitant disappearance parent ...