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

摘要

Chemotherapeutic agents used in the treatment of cancer often lead to dose-limiting bone marrow suppression and may initiate secondary leukemia. N,N1,NI'-triethylenethiophosphoramide (thiotepa), a polyfunctional alkylating agent, is used in the treat- ment 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 formamidopyri- midine 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 a-hOgg1. Although protection with the Fpg Drotein has been previously observed in Chinese hamster ovary cells, we demonstrate significant (1OO-fold) protection against thiotepa using the E. coli Fpg or the human a-hOgg1 cDNA in NIH3T3 cells. We have also observed a 1O-fold protection by both the Fpg and a-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 cyclo- phosphamide. These latter two findings are novel and are par- ticularly significant sin~ the added protection was in an 06- 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 se- vere myelosuppressive effect of agents such as thiotepa, BCNU, and cyclophosphamide.