title: Efficient protection of cells from the genotoxicity of nitrosoureas by the retrovirus-mediated transfer of human O6-methylguanine-DNA methyltransferase using bicistronic vectors with human multidrug resistance gene 1.
abstract: Retrovirus-mediated transfer of O6-methylguanine-DNA methyltransferase (MGMT; E.C. 2.1.1.63) and a human multidrug-resistance gene (MDR1) confers resistance to nitrosoureas and natural product antitumor agents, respectively. In a previous study, we constructed two bicistronic retroviral vectors, Ha-MDR-IRES-MGMT and Ha-MGMT-IRES-MDR, that allow co-expression of the MGMT gene and the MDR1 gene to protect cells from the toxicity of combination chemotherapy. Each cell transduced with Ha-MDR-IRES-MGMT or Ha-MGMT-IRES-MDR showed high-level resistance to vincristine and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), indicating that the two drug-resistance genes can be functionally co-expressed from these vectors. In the present study, we examined whether the expression of MGMT from these MDR1-MGMT bicistronic retroviral vectors could protect cells from the genotoxicity of nitrosoureas. Three independent Ha-MDR-IRES-MGMT-transduced clones and three independent Ha-MGMT-IRES-MDR-transduced clones of HeLa MR cells showed 12-23-fold and 27-30-fold higher MGMT activity than the parental cells. These clones are more resistant to ACNU mutagenicity measured by the frequency of the emergence of 6-thioguanine-resistant colonies after ACNU treatment over the frequency seen in the parental cells. The ACNU-induced sister chromatid exchange (SCE) was markedly suppressed in these clones. Murine bone marrow cells were transduced with either Ha-MDR-IRES-MGMT or Ha-MGMT-IRES-MDR. Non-selected populations of the transduced cells showed only marginal increases in drug resistance and MGMT activity. Remarkable increase in drug resistance and MGMT activity were observed after a short exposure of the transduced cells to vincristine. The Ha-MDR-IRES-MGMT-transduced, vincristine-selected bone marrow cells showed 27-fold resistance to vincristine, 7-fold resistance to ACNU, and 10-fold higher MGMT activity than the non-transduced, non-selected cells. The Ha-MGMT-IRES-MDR-transduced, vincristine-selected cells showed 8-fold resistance to vincristine, 16-fold resistance to ACNU and 19-fold higher MGMT activity than the non-transduced, non-selected cells. The rates of ACNU-induced SCE in the vincristine-selected cells were as follows: non-transduced cells (non-selected) and HaMDR-transduced cells>Ha-MDR-IRES-MGMT-transduced cells>Ha-MGMT-IRES-MDR-transduced cells. Again, the only marginal levels of increases in the rates of ACNU-induced SCE were observed in non-selected population of the transduced cells. These results indicate that the MDR1-MGMT bicistronic retrovirus vectors would be useful to protect normal hematopoietic cells from nitrosourea-induced mutagenesis, and drug-selectable bicistronic constructs would have great advantage over non-selectable vectors.
authors.affiliation: Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Suzuki
authors.forename: Y
authors.initials: Y
authors.lastname: Sugimoto
authors.forename: T
authors.initials: T
authors.lastname: Tsuruo
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009152:Mutagenicity Tests; D015376:Nimustine / Q000633:toxicity*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000091402:Prohibitins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012190:Retroviridae; D012854:Sister Chromatid Exchange / Q000187:drug effects*; D014162:Transfection; D014750:Vincristine / Q000633:toxicity
chemical_list: C000717331:PHB2 protein, human; D000091402:Prohibitins; D011994:Recombinant Proteins; D015376:Nimustine; D014750:Vincristine; D019853:O(6)-Methylguanine-DNA Methyltransferase