Genetic consequences of tolerance to methylation DNA damage in mammalian cells.
We previously characterized a clone of CHO cells, clone B, that displayed tolerance to the cytotoxic effects of N-methylnitrosourea (MNU) and 6-thioguanine (6-TG). To determine whether this phenotype affected the mutagenic response of the cells, MNU-induced mutation to 8-azaadenine resistance (8-AAr) was measured in the parental and clone B cells. Comparable mutation frequencies were found in the two cell lines up to 0.5 mM MNU, while at higher MNU concentrations mutations could be reproducibly measured only in clone B cells. Similar amounts of DNA methylated bases were found in the two cell lines after a 30 min treatment with different concentrations of [3H]MNU and the same linear relationship was observed when mutation induction by MNU was plotted as a function of the amount of O6-methylguanine (O6-MeGua) in DNA, indicating that mutation induction in both cell lines was related to the presence of this methylated base. Fifteen MNU-induced 8-AAr mutants were isolated from each cell line and the sequences of the adenine phosphoribosyltransferase (aprt) mutations determined. The type (in 90% of the cases, GC to AT transitions), the sequence context and the strand localization of the mutations indicated that all mutations were targeted at O6-MeGua in DNA and no difference was found between the two lines. These results are consistent with a mechanism of tolerance of O6-MeGua that does not alter the processing of this methylated base into a mutation. Growth in 6-TG induced point mutations in clone B but not in the parental cells. A model is proposed in which the alkylation tolerant variant is altered in a mismatch correction pathway responsible for the cytotoxicity of the methylated base.
Aquilina
G
G
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Rome, Italy.
Biondo
R
R
Dogliotti
E
E
Bignami
M
M
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D001372:Aza Compounds / Q000633:toxicity; D001483:Base Sequence; D016466:CHO Cells; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004351:Drug Resistance; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D008745:Methylation; D008770:Methylnitrosourea / Q000633:toxicity; D008969:Molecular Sequence Data; D009154:Mutation; D010042:Ouabain / Q000633:toxicity; D013866:Thioguanine / Q000378:metabolism*
D001372:Aza Compounds; C025946:3-methyladenine; D010042:Ouabain; D006147:Guanine; C008450:7-methylguanine; D008770:Methylnitrosourea; D004247:DNA; C008449:O-(6)-methylguanine; D000228:Adenine Phosphoribosyltransferase; D013866:Thioguanine; D000225:Adenine