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 m easured in mammalian cells. The plasmid pRSVCAT was UV irradiated or t reated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficien cy 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 expres sion of the CAT gene was found to decrease exponentially after transfe ction of pRSVCAT containing increasing numbers of DNA lesions per mole cule. The average number of BPDE-I adducts per plasmid molecule was me asured 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 endonucle ase. By expressing the inhibition of CAT activity in terms of the aver age number of lesions per gene, we were able to compare directly the e ffects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by B PDE-I adducts and pyrimidine dimers in DNA. D-0 values determined by l inear regression analysis of dose-response curves for inhibition of CA T activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene i n excision-proficient human fibroblasts; the corresponding values in m ouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar t hreshold densities of BPDE-I adducts and pyrimidine dimers were observ ed before inhibition of transcription from pRSVCAT was detected. No th reshold was observed in experiments with human fibroblasts deficient i n excision repair (xeroderma pigmentosum group A); calculated D-0 valu es were 1.2 pyrimidine dimers or 2.1 BPDE-I adducts. Our results permi t direct comparisons of the magnitude of inhibition of gene transcript ion by distinct DNA lesions, and suggest that BPDE-I adducts and UV-in duced cyclobutane pyrimidine dimers in template DNA block transcriptio n with similar efficacy.