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.
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.
Maze
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Section of Pediatric Hematology/Oncology, Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indianapolis, Indiana, USA.
Kurpad
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Pegg
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Erickson
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Williams
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D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D002277:Carcinoma; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000633:toxicity; D005260:Female; D005822:Genetic Vectors; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007939:Leukemia L1210; D008546:Melanoma, Experimental; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; C064976:O(6)-benzylguanine; C017139:1-(2-chloroethyl)-1-nitrosourea; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005038:Ethylnitrosourea