Carcinogenesis, Vol 18, 889-896, Copyright © 1997 by Oxford University Press
Y Tominaga, T Tsuzuki, A Shiraishi, H Kawate and M Sekiguchi
An enzyme O6-methylguanine-DNA methyltransferase (MGMT) catalyzes transfer
of a methyl group from O6-methylguanine and O4-methylthymine of alkylated
DNA to its own molecule, thereby repairing the pre- mutagenic lesions in a
single step reaction. Making use of gene targeting, we developed mouse
embryonic stem (ES) cell lines deficient in the methyltransferase.
Quantitative immunoblot analysis and enzyme assay revealed that MGMT-/-
cells, in which both alleles were disrupted, contained no methyltransferase
protein while cells with one intact allele (MGMT+/-) contained about half
the amount of protein carried by the parental MGMT+/+ cells. MGMT-/- cells
have an extremely high degree of sensitivity to simple alkylating agents,
N-methyl-N'- nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea
(MNU), whereas MGMT+/- cells are slightly more sensitive to these agents,
as compared with findings from normal cells. A high frequency of mutation
was induced in MGMT-/- cells on exposure to a relatively low dose of MNNG.
Electrophoretic analyses of the DNAs as well as fluorochrome staining of
the cells revealed that MGMT-/- cells treated with MNNG undergo apoptotic
death, which occurs after G2-M arrest in the second cycle of cell
proliferation.
ARTICLES
Alkylation-induced apoptosis of embryonic stem cells in which the gene for DNA-repair, methyltransferase, had been disrupted by gene targeting
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
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