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.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Srivenugopal, KS; Yuan, XH; Friedman, HS; Ali-Osman, F

Published Date

  • January 1, 1996

Published In

Volume / Issue

  • 35 / 4

Start / End Page

  • 1328 - 1334

PubMed ID

  • 8573590

Pubmed Central ID

  • 8573590

Electronic International Standard Serial Number (EISSN)

  • 1520-4995

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi9518205

Language

  • eng