Modulation of the toxic and mutagenic effects induced by methyl methanesulfonate in Chinese hamster ovary cells by overexpression of the rat N-alkylpurine-DNA glycosylase.
Exposure of mammalian cells to alkylating agents causes transfer of alkyl groups to N- as well as O-atoms of DNA bases. Especially the O-alkylated G and T bases have strong mutagenic properties, since they are capable of mispairing during replication. The mutagenic potential of N-alkylbases is less clear although specific base excision repair (BER) pathways exist which remove those lesions from the DNA. We investigated the relative contribution of N-alkylations to mutation induction at the Hprt gene in cultured Chinese hamster ovary cells (CHO). To this end BER activity in CHO cells was modulated by introduction of an expression vector carrying the rat N-alkylpurine-DNA glycosylase (APDG) gene, which codes for a glycosylase that is able to remove 3-methyladenine and 7-methylguanine from DNA thereby generating apurinic sites. Upon selection of a CHO clone which 10 times overproduced APDG compared to control CHO cells, mutation induction, the mutational spectrum, and cell survival were determined in both cell lines following treatment with methyl methanesulfonate (MMS). The results show that over-expression of APDG renders CHO cells more sensitive for mutation induction as well as cytotoxicity induced by MMS. The involvement of apurinic sites in induction of base pair changes at positions where 3-methyladenine was induced is inferred from the observation that the mutational spectrum of MMS-induced mutations in APDG-CHO cells showed twice as much base pair changes at AT base pairs (33.3%) compared to the spectrum of MMS-induced mutations in CHO-control cells (15.8%).
Calléja
F
F
MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, Netherlands.
Jansen
J G
JG
Vrieling
H
H
Laval
F
F
van Zeeland
A A
AA
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D020137:Base Pair Mismatch; D016466:CHO Cells / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D045647:DNA Glycosylases; D011088:DNA Ligases / Q000378:metabolism; D019175:DNA Methylation; D004260:DNA Repair; D004790:Enzyme Induction; D016368:Frameshift Mutation; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity*; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection
D000477:Alkylating Agents; D009153:Mutagens; D011993:Recombinant Fusion Proteins; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D008741:Methyl Methanesulfonate; D007041:Hypoxanthine Phosphoribosyltransferase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II; D011088:DNA Ligases; D000225:Adenine