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    SubjectsHaematopoietic Stem Cells (1)View MoreJournal
    Carcinogenesis (2)
    Authors
    Margison, Geoffrey P (2)
    Dexter, T Michael (1)Jelinek, J (1)Kleibl, K (1)Morten, John E N (1)Year (Issue Date)
    1988-01 (2)
    TypesArticle (2)

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    Increased O6-alkylguanine alkyltransferase activity in Chinese hamster V79 cells following selection with chloroethylating agents.

    Morten, John E N; Margison, Geoffrey P (1988-01)
    Chinese hamster V79 lung fibroblasts express low levels (specific activity 2-4 fmol/mg protein) of O6-alkylguanine (O6-AG) alkyltransferase (ATase). In cells surviving selection with low doses (10 micrograms/ml) of the chloroethylating agent, mitozolomide (Mz), ATase activity was increased 5- to 8-fold. Repeated selection of such cells produced a maximal specific activity of 36-40 fmol/mg protein, whilst selection at 20 or 40 micrograms/ml result in specific activities of approximately 50 and 70 fmol/mg respectively. Only slight decreases in ATase activity were seen by 51 days after an initial selection with 10 micrograms/ml Mz. A similar effect was observed using chlorozotocin. Selected cells had a higher D37 for Mz (2.5-6.0 micrograms/ml) in comparison with control cell (D37, 0.8 micrograms/ml) but the D37s for nitrogen mustard and vincristine were closely similar in selected and control cells. Possible explanations for the increase in ATase activity are discussed.
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    Transfection of murine multi-potent haemopoietic stem cells with an E. coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents.

    Jelinek, J; Kleibl, K; Dexter, T Michael; Margison, Geoffrey P (1988-01)
    O6-alkylguanine-DNA-alkyltransferase (ATase)-deficient murine haemopoietic stem cells were transfected, following electroporation, with a G418-selectable expression vector containing the protein coding region of the Escherichia coli ATase gene ada. Clones of cells that were resistant to G418 or the chloroethylating agent mitozolomide (Mz) were selected and most were shown to express very high levels of bacterial gene-encoded ATase. In comparison with control cells that were transfected with the parent vector, the ATase-expressing clones were considerably more resistant to the toxic effects of the methylating agents N-methyl-N-nitrosourea and methylmethanesulphonate or the chloroethylating agents Mz or taurine chloroethylnitrosourea, but unchanged in their susceptibility to the bis-chloroethylating agent nitrogen mustard. Thus alkylation damage in DNA that can be repaired by the E. coli ATase constitutes the principal lethal lesion produced by alkylating agents in murine haemopoietic stem cells and the ATase deficiency in these cells can be complemented by electroporation-mediated gene transfection.
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