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.

Abstract

O(6)-Benzylguanine (6-BG) inactivates mammalian O(6)-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 IC(50) of 50 microM (compared with 27.5 microM 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.

• Authors: R Maze, C Kurpad, A E Pegg, L C Erickson, D A Williams
• Journal: The Journal of pharmacology and experimental therapeutics (J Pharmacol Exp Ther) Vol. 290 Issue 3 Pg. 1467-74 (Sep 1999) ISSN: 0022-3565 [Print] United States
• PMID: 10454526 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antineoplastic Agents
• O(6)-benzylguanine
• 1-(2-chloroethyl)-1-nitrosourea
• Guanine
• O(6)-Methylguanine-DNA Methyltransferase
• Ethylnitrosourea

Topics

• Animals
• Antineoplastic Agents (toxicity)
• Carcinoma
• Ethylnitrosourea (analogs & derivatives, toxicity)
• Female
• Genetic Vectors
• Guanine (analogs & derivatives, pharmacology)
• Hematopoietic Stem Cells (drug effects, enzymology)
• Humans
• Leukemia L1210
• Melanoma, Experimental
• Mice
• Mice, Inbred C57BL
• Mutation
• O(6)-Methylguanine-DNA Methyltransferase (biosynthesis, genetics, metabolism, physiology)
• Retroviridae (genetics, metabolism)
• Tumor Cells, Cultured