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 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 alpha-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 alpha-hOgg1 cDNA 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-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 O(6)-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.
Xu
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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, USA.
Hansen
W K
WK
Rosenquist
T A
TA
Williams
D A
DA
Limp-Foster
M
M
Kelley
M R
MR
D016475:3T3 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D015152:Blotting, Northern; D002330:Carmustine / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000633:toxicity; D019610:Cytoprotection; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004347:Drug Interactions; D029968:Escherichia coli Proteins; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000494:pharmacology*; D013852:Thiotepa / Q000494:pharmacology*; D014162:Transfection; D019076:Transgenes
D000970:Antineoplastic Agents; D029968:Escherichia coli Proteins; C048341:mafosfamide; D003520:Cyclophosphamide; D004247:DNA; D013852:Thiotepa; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D002330:Carmustine