Cytotoxic and mutagenic effects of methylnitrosourea in two human fetal fibroblast strains differing in O6-methylguanine-DNA methyltransferase activity.
A non-transformed human fibroblast strain, GM11, established from the skin of a therapeutically aborted fetus, has been reported to exhibit the Mer- phenotype, i.e. inability to support the growth of adenovirus 5 damaged with 1-methyl-3-nitro-1-nitrosoguanidine. In the present study we determined (i) loss of colony-forming ability and frequency of mutants resistant to 6-thioguanine (6TG) on exposure to the SN1 alkylating agent methylnitrosourea (MNU) and (ii) amount of O6-methylguanine-DNA methyltransferase (MGMT), the protein responsible for repairing O6-methylguanine (O6mG) produced by MNU, in GM11 cells compared to GM10, a Mer+ human fetal fibroblast strain. Irrespective of in vitro culture age, GM10 cells responded normally to the cytotoxic action of the alkylating agent, i.e. their clonogenic survival curves exhibited a shoulder at low MNU concentrations (less than or equal to 0.4 mM) and a D10 (dose reducing survival to 10%) of approximately 1.4 mM. By contrast, no shoulder was observed on the survival curves of GM11 cells and their D10 values decreased from approximately 0.6 mM at passage 4 to 0.1 mM at passage 27. In GM10 (Mer+) cells, unlike the biphasic dose response seen for cell killing, the frequency of 6TG-resistant mutants increased as a linear function of chemical concentration delivered (range 0.05-1.2 mM); the induced mutation frequency in these cells (passage 16-20) was equal to 220 x 10(-6)/mM MNU, a yield some 5-fold greater than that reported by others for non-fetal human fibroblasts. GM11 cells proved to be only approximately 1.5 times more mutable by MNU than GM10 cells at late passage, and the susceptibility of the former strain to MNU-induced mutations did not change significantly as a function of culture age (i.e. 316 x 10(-6) and 326 x 10(-6) mutants/mM MNU at passages 4 and 16-20 respectively). The GM10 strain contained approximately 75,000 MGMT molecules/cell at all passages (4-20) examined, whereas the GM11 strain harbored deficient amounts of the protein (approximately 22,500 molecules/cell) at the lowest passage available (4), and this residual activity decreased precipituously to undetectable amounts by passage 16. Together, these data demonstrate that in the two human fetal strains examined the constitutive level of cellular MGMT activity correlates much better with resistance to reproductive inactivation than with mutagenesis by MNU, implying that inefficient repair of O6meG lesions impacts more severely on cell lethality than on mutation induction in at least some biological systems.
Mirzayans
R
R
Molecular Genetics and Carcinogenesis Laboratory, Cross Cancer Institute, Edmonton, Alberta, Canada.
Middlestadt
M V
MV
Paterson
M C
MC
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008770:Methylnitrosourea / Q000494:pharmacology*; D008780:Methyltransferases / Q000378:metabolism*; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012867:Skin
D009153:Mutagens; D008770:Methylnitrosourea; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase