Inhibition of reporter gene expression in mammalian cells. Effects of distinct carcinogen lesions in DNA.
The effect of UV photoproducts or benzo[a]pyrene-diol-epoxide-I (BPDE-I) adducts in DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of cell extracts used in the chloramphenicol acetyl transferase (CAT) assays to contain equal beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid DNA treated with Micrococcus luteus UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by BPDE-I adducts and pyrimidine dimers in DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar threshold densities of BPDE-I adducts and pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (xeroderma pigmentosum group A); calculated D0 values were 1.2 pyrimidine dimers of 2.1 BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that BPDE-I adducts and UV-induced cyclobutane pyrimidine dimers in template DNA block transcription with similar efficacy.
Sorscher
D H
DH
Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill.
Cordeiro-Stone
M
M
D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism / Q000633:toxicity; D002273:Carcinogens / Q000633:toxicity*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D011740:Pyrimidine Dimers / Q000633:toxicity; D014162:Transfection
D001580:Benzopyrenes; D002273:Carcinogens; D011740:Pyrimidine Dimers; C072019:benzo(a)pyrene diolepoxide I; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase