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.
Footnotes
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Send reprint requests to: David A. Williams, M.D., Howard Hughes Medical Institute, Cancer Research Institute, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202-5225. E-mail: dwilliam{at}iupui.edu
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↵1 This work was supported by National Cancer Institute Grants PO1CA75426 (to D.A.W.) and CA45628–10 (to L.C.E.).
- Abbreviations:
- MGMT
- O6-methylguanine DNA methyltransferase
- WTMGMT
- wild-type O6-methylguanine DNA methyltransferase
- 6-BG
- O6-benzylguanine
- CENU
- chloroethylnitrosourea
- BCNU
- 1,3-bis(2-chloroethyl)-1-nitrosourea
- IC50
- 50% inhibitory concentration
- FN
- fibronectin
- MSCV
- Moloney stem cell virus
- BM
- bone marrow
- CFU-GM
- colony-forming unit-granulocyte macrophage
- LTR
- long terminal repeat
- Received January 8, 1999.
- Accepted May 27, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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