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Enhanced cell killing and mutagenesis by ethylnitrosourea in xeroderma pigmentosum cells
Abstract:
It was shown previously that following treatment with ethylnitrosourea (ENU) SV40-transformed cells from xeroderma pigmentosum patient XP12RO, complementation group A remove O6-ethylguanine from their DNA 2 to 3 times more slowly than do such cells from a normal individual (GM637), but that each of the several other DNA adducts studied are removed at the same rate by both cell lines. If O6-ethylguanine contributes significantly to the cytotoxic effect of ENU, a population which can excise this lesion more rapidly than another during the posttreatment period should exhibit a higher survival. We compared these two cell lines for survival of colony-forming ability following exposure to ENU. XP12RO-SV40 cells proved approximately 3.5-fold more sensitive than GM637. To extend this study to non-transformed diploid human fibroblasts, we compared the survival of cells derived from normal individuals (NF) with that of fibroblasts from several XP patients: XP12BE (group A), XP7BE (group D), and XP4BE (an XP variant). The survival curve of NF, XP7BE, and XP4BE cells, but not XP12BE cells, exhibited a distinct shoulder. The slope of the survival curve for XP12BE cells was 3-fold steeper than that of the exponential part of the curve for NF; the slope of the curve for XP7BE cells, 2-fold steeper; and that of XP4BE cells, 1.7-fold steeper. This enhanced cytotoxicity was not observed in XP12BE cells treated with methylating agents such as methylnitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. We compared NF and XP12BE cells for sensitivity to the mutagenic action of ENU and found approximately 3-fold higher frequencies of 6-thioguanine resistant mutants in the XP cells. This result is expected if the XP cells are slower than normal in excising O6-ethylguanine and if this adduct is mutagenic. When we compared the frequency of mutations induced by ENU and u.v. radiation at doses which caused equal cell killing, ENU was 4-fold more efficient a mutagen than u.v.