Retroviral-mediated expression of the P140A, but not P140A/G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment

Rodney Maze, Chandrika Kurpad, Anthony E. Pegg, Leonard C. Erickson, David A. Williams

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

O6-Benzylguanine (6-BG) inactivates mammalian O6-methylguanine DNA methyltransferase (MGMT), an important DNA repair protein that protects cells against chloroethylnitrosourea (CENU) cytotoxicity. 6-BG is being tested as an approach to treat CENU-resistant tumors that overexpress endogenous MGMT. However, in addition to restoring CENU tumor cell sensitivity, 6-BG also increases the cytotoxic effects of CENUs on hematopoietic cells. Several 6- BG-resistant human MGMT mutants have been characterized in Escherichia coli and are predicted to protect mammalian cells against the combination of 6-BG and CENU treatment in vivo. Two mutants, P140A and P140A/G156A, demonstrated 20- and 1200-fold more resistance to 6-BG depletion of MGMT activity compared with wild-type MGMT (WTMGMT). Here, we analyzed retroviral vectors that express either WTMGMT, the P140A or P140A/G156A mutant forms of MGMT. Retroviral-infected L1210 hematopoietic cells demonstrated similar levels of RNA in all transduced clones. However, the amount of MGMT protein and DNA repair activity was reduced in clones expressing the P140A/G156A mutant compared with those expressing WTMGMT or P140A. Expression of P140A was associated with a 4- to 8-fold increase in resistance to 6-BG depletion of MGMT in transduced L1210 clones and a 1,3-bis(2-chloroethyl)-1-nitrosourea IC50 of 50 μM (compared with 27.5 μM for WTMGMT) in primary murine hematopoietic cells. These results demonstrate the utility of screening 6-BG- resistant MGMT proteins in hematopoietic cells and provide evidence that the P140A mutant form of MGMT generates 6-BG- and CENU-resistant hematopoietic cells. Retrovirus vectors expressing this mutant may be useful in future human gene therapy trials.

Original languageEnglish (US)
Pages (from-to)1467-1474
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume290
Issue number3
StatePublished - Sep 1 1999

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

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