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 alpha-hOgg1: Implications for protective gene therapy applications

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 age nt, is used in the treatment of breast, ovarian, and bladder carcinomas and is also being tested for efficacy in the treatment of central nervous syst em tumors. Thiotepa produces ring-opened bases such as formamidopyrimidine and 7-methyl-formamidopyrimidine, which can be recognized and repaired by t he formamidopyrimidine glycosylase/AP lyase (Fpg) enzyme of Escherichia col i. Using this background information, we have created constructs using the E. coli fpg gene along with the functional equivalent human ortholog alpha -hOgg1. Although protection with the Fpg protein has been previously observ ed in Chinese hamster ovary cells, we demonstrate significant (100-fold) pr otection against thiotepa using the E. coli Fpg or the human alpha -hOgg1 c DNA in NIH3T3 cells. We have also observed a 10-fold protection by both the Fpg and alpha -hOgg1 transgenes against 1,3-N,N'-bis(2-chloroethyl)- N-nit rosourea (BCNU) and, to a lesser extent, mafosfamide (2-fold), an active fo rm of the clinical agent cyclophosphamide. These latter two findings are no vel and are particularly significant since the added protection was in an O (6-)methylguanine-DNA methyltransferase-positive background. These results support our general approach of using DNA base excision repair genes in gen e therapy for cellular protection of normal cells during chemotherapy, part icularly against the severe myelosuppressive effect of agents such as thiot epa, BCNU, and cyclophosphamide.