Vol. 296, Issue 3, 825-831, March 2001

Protection of Mammalian Cells against Chemotherapeutic Agents Thiotepa, 1,3-N,N'-Bis(2-chloroethyl)-N-nitrosourea, and Mafosfamide Using the DNA Base Excision Repair Genes Fpg and -hOgg1: Implications for Protective Gene Therapy Applications

Yi Xu, W. Kent Hansen, Thomas A. Rosenquist, David A. Williams, Melissa Limp-Foster and Mark R. Kelley

Department of Pediatrics, Section of Hematology/Oncology, Herman B. Wells Center for Pediatric Research and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana (Y.X., W.K.H., M.L.-F., M.R.K.); Department of Pediatrics, Herman B. Wells Center for Pediatric Research and Molecular Genetics, Howard Hughes Institute, Indiana University School of Medicine, Indianapolis, Indiana (D.A.W.); and Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York (T.A.R.)

Chemotherapeutic agents used in the treatment of cancer often lead to dose-limiting bone marrow suppression and may initiate secondary leukemia. N,N',N"-triethylenethiophosphoramide (thiotepa), a polyfunctional alkylating agent, is used in the treatment of breast, ovarian, and bladder carcinomas and is also being tested for efficacy in the treatment of central nervous system tumors. Thiotepa produces ring-opened bases such as formamidopyrimidine and 7-methyl-formamidopyrimidine, which can be recognized and repaired by the formamidopyrimidine glycosylase/AP lyase (Fpg) enzyme of Escherichia coli. Using this background information, we have created constructs using the E. coli fpg gene along with the functional equivalent human ortholog -hOgg1. Although protection with the Fpg protein has been previously observed in Chinese hamster ovary cells, we demonstrate significant (100-fold) protection against thiotepa using the E. coli Fpg or the human -hOgg1 cDNA in NIH3T3 cells. We have also observed a 10-fold protection by both the Fpg and -hOgg1 transgenes against 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) and, to a lesser extent, mafosfamide (2-fold), an active form of the clinical agent cyclophosphamide. These latter two findings are novel and are particularly significant since the added protection was in an O⁶-methylguanine-DNA methyltransferase-positive background. These results support our general approach of using DNA base excision repair genes in gene therapy for cellular protection of normal cells during chemotherapy, particularly against the severe myelosuppressive effect of agents such as thiotepa, BCNU, and cyclophosphamide.