Vol. 290, Issue 3, 1467-1474, September 1999

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 Treatment¹

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

Section of Pediatric Hematology/Oncology, Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indianapolis, Indiana (R.M., D.A.W.); Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana (R.M., D.A.W.); Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania (A.E.P); Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana (C.K., L.C.E.); and Howard Hughes Medical Institute, Indiana University School of Medicine, Indianapolis, Indiana (D.A.W.)

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.