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

Mutsumi Suzuki, Yoshikazu Sugimoto, Takashi Tsuruo

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23 Citations (Scopus)

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-nitrosourea (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.

Original languageEnglish
Pages (from-to)133-141
Number of pages9
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume401
Issue number1-2
DOIs
Publication statusPublished - 1998 Jun 5
Externally publishedYes

Keywords

  • Bicistronic retroviral vector
  • Gene therapy
  • Multidrug resistance
  • Nitrosourea resistance
  • Sister chromatid exchange

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

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