Differential formation and repair of the mutagenic DNA alkylation product O6-ethylguanine in transcribed and nontranscribed genes of the rat.
Intragenomic differences regarding the formation and repair of carcinogen-DNA adducts influence gene-specific mutational patterns and the cellular risk of malignant conversion. Using a newly developed, monoclonal antibody-based immunoaffinity method (Hochleitner, K., Thomale, J., Nikitin, A. Y., and Rajewsky, M. F. (1991) Nucleic Acids Res. 19, 4467-4472), it has become possible to quantitate the mutagenic DNA alkylation product O6-ethylguanine (O6-EtGua) at the level of single-copy genes. We have analyzed the formation and repair kinetics of O6-EtGua in DNA in relation to the transcriptional activity of selected genes in two isogenic rat hepatoma cell lines (Fao and H5) exposed to N-ethyl-N-nitrosourea. Whereas the frequency of O6-EtGua initially formed in the inactive immunoglobulin E gene was indistinguishable from the value for bulk DNA, the initial O6-EtGua/guanine molar ratio in the transcribed beta-actin gene was nearly three times higher. The overall elimination rates of O6-EtGua were the same for bulk DNA and the IgE gene sequence, i.e. rapid in Fao cells (68% removed within 20 min) and four times slower in H5 cells. Preferential repair was found in the transcribed gene: during the initial phase of elimination, the half-life of O6-EtGua in the beta-actin gene was three times shorter than in the IgE gene in Fao cells (5 versus 15 min) and 12 times shorter in H5 cells (20 min versus 4 h).
Thomale
J
J
Institute of Cell Biology (Cancer Research), University of Essen Medical School, Germany.
Hochleitner
K
K
Rajewsky
M F
MF
D000478:Alkylation; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000378:metabolism / Q000633:toxicity*; D015870:Gene Expression; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D008113:Liver Neoplasms; D016296:Mutagenesis; D051381:Rats; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; C007673:6-ethylguanine; D006147:Guanine; D005038:Ethylnitrosourea