Retroviral-mediated expression of the P140A, but not P140A/G156A, mutant form of O-6-methylguanine DNA methyltransferase protects hematopoietic cellsagainst O-6-benzylguanine sensitization to chloroethylnitrosourea treatment

O-6-Benzylguanine (6-BG) inactivates mammalian O-6-methylguanine DNA methyl transferase (MGMT), an important DNA repair protein that protects cells aga inst 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 in creases the cytotoxic effects of CENUs on hematopoietic cells. Several 6-BG -resistant human MGMT mutants have been characterized in Escherichia coil a nd are predicted to protect mammalian cells against the combination of 6-BG and CENU treatment in vivo. Two mutants, P140A and P140A/G156A, demonstrat ed 20- and 1200-fold more resistance to 6-BG depletion of MGMT activity com pared with wild type MGMT (WTMGMT). Here, we analyzed retroviral vectors th at express either WTMGMT, the P140A or P140A/G156A mutant forms of MGMT. Re troviral-infected L1210 hematopoietic cells demonstrated similar levels of RNA in all transduced clones. However, the amount of MGMT protein and DNA r epair activity was reduced in clones expressing the P140A/G156A mutant comp ared with those expressing WTMGMT or P140A. Expression of P140A was associa ted with a 4- to 8-fold increase in resistance to 6-BG depletion of MGMT in transduced L1210 clones and al,3-bis(2-chloroethyl)-1-nitrosourea IC50 of 50 mu M (compared with 27.5 mu M for WTMGMT) in primary murine hematopoieti c cells. These results demonstrate the utility of screening 6-BG-resistant MGMT proteins in hematopoietic cells and provide evidence that the P140A mu tant form of MGMT generates 6-BG- and CENU-resistant hematopoietic cells. R etrovirus vectors expressing this mutant may be useful in future human gene therapy trials.