Expression of human O6-methylguanine-DNA methyltransferase in a DNA excision repair-deficient Chinese hamster ovary cell line and its response to certain alkylating agents.
A plasmid has been constructed in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) complementary DNA is driven by the Rous sarcoma virus promoter sequence. We had previously shown that transfection of this plasmid into Chinese hamster ovary (CHO) cells results in the expression of MGMT and in increased cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine and 1-(2-chloroethyl)-1-nitrosourea (CNU) but not N-nitroso-N-ethylurea (ENU). In the present study, the Rous sarcoma virus promoter-MGMT was transfected into DNA excision repair-deficient CHO UV41 cells to investigate the phenotype associated with MGMT expression in the absence of DNA excision repair. Both the UV41/MGMT and CHO/MGMT cells expressed similar levels of MGMT and exhibited a similar increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine. The UV41 cells were 20-fold more sensitive to CNU than the wild-type CHO cells. Expression of MGMT increased the resistance to CNU about 6-fold in both cell lines, but the difference between the two cell lines attributable to the excision repair defect still persisted. The UV41 cells were 2- to 3-fold more sensitive than the wild-type CHO cells to the monofunctional alkylating agents 1-(2-hydroxyethyl)-1-nitrosourea and ENU, but the MGMT phenotype did not alter sensitivity. This suggests that alkylation at the O6 position of guanine has no role in cytotoxicity of ethylating agents and that monofunctional DNA damage has little role in the cytotoxicity of CNU. Since MGMT can prevent the formation of G-C interstrand cross-links formed by CNU, other excision repair-sensitive DNA adducts must play a major role in the sensitivity of UV41 cells to this bifunctional alkylating agent. These results suggest that DNA intrastrand cross-links may be major contributors to the cytotoxicity of CNU.
Wu
Z N
ZN
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03756.
Chan
C L
CL
Eastman
A
A
Bresnick
E
E
D000818:Animals; D016466:CHO Cells / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D008769:Methylnitronitrosoguanidine; D008780:Methyltransferases / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010641:Phenotype; D014162:Transfection
D008769:Methylnitronitrosoguanidine; C022146:1-(2-hydroxyethyl)-1-nitrosourea; C017139:1-(2-chloroethyl)-1-nitrosourea; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005038:Ethylnitrosourea