Abasic sites in DNA are generated either spontaneously or after removal of altered bases during the base excision repair process. These as well as 3' damaged ends of DNA at single-strand breaks induced by reactive oxygen species are repaired by AP-endonucleases. The major human AP-endonuclease (named APE-1) has two unrelated activities. It may function as an activator of c-Fos and c-Jun transcription factors and as a repressor of the parathyroid hormone (PTH) gene by binding to the negative Ca(2+)-response elements (nCaRE) in its promoter. Preliminary studies indicate that the h-APE-1 gene is highly regulated. Analysis of its promoter activity by transient expression of the luciferase reporter gene in human, HeLa and TK6 cells suggested the presence of a negative regulatory element in the promoter. Two nCaRE-like sequences were identified in the promoter segment responsible for inhibiting reporter gene expression. Competitive electrophoretic mobility shift assay with HeLa nuclear extract indicated that the nCaRE sequences of the APE-1 and PTH genes are recognized by the APE-1 polypeptide. These results suggest that the APE-1 gene may be down-regulated by its own product.

The role of the bithiazole moiety of bleomycin in the interaction of the antibiotic with DNA has been studied by the use of synthetic bithiazole derivatives. The DNA affinity of individual C-terminal (bithiazole) analogues of bleomycin was measured in terms of the ability of these species to block the binding of bleomycin to DNA, as judged by diminution of the DNA degradation that attends bleomycin binding. DNA degradation was monitored both by release of [3H]thymine from radiolabeled PM-2 DNA and by alteration of bleomycin-treated DNA oligomers of defined sequence derived from Escherichia coli plasmid pLJ3. It was found that the affinity of the bithiazole derivatives for DNA depended on the presence of the bithiazole moiety itself but more importantly on the number and spacing of positively charged groups; 2'-(2-aminoethyl)-2,4'-bithiazole-4-[3-[(4-aminobutyl) amino]propyl]carboxamide (14), having three positively charged groups at neutral pH, was a reasonably effective inhibitor of DNA degradation by bleomycin. Consistent with the importance of the spacing of the positively charged groups, tetrapeptide S (12) was found to be significantly less inhibitory toward DNA degradation by bleomycin than tripeptide S, in spite of their equal number of positively charged groups and the greater structural similarity of the former to bleomycin A2. Bleomycin is known to cleave DNA perferentially at certain sequences. It was shown that the inhibitors employed in this study diminished DNA cleavage proportionately at each cleavage site; no alteration was observed in the specificity of cleavage. A number of the bithiazole analogues employed as inhibitors of bleomycin-mediated DNA degradation were also utilized in fluorescence quenching experiments with calf thymus DNA. Consistent with the belief that these species inhibit bleomycin degradation by competitive binding to the DNA substrate, the best inhibitors exhibited the greatest fluorescence quenching upon admixture of DNA.

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To investigate mechanisms of rat glutathione S-transferase P1 gene (rGSTP1) expression regulation during chemical carcinogenesis. we studied enhancer elements located in the region between -2.5 kb to -2.2 kb. The region was upstream from the start site of transcription and was divided into two major fragments, GPEI and GPEII. The GPEII fragment was further divided into two smaller fragments, GPEII- I and GPEII-2. Using a luciferase reporter system, we identified a strong enhancer of GPEI and a weak enhancer of GPEII in HeLa and a rat hepatoma cell line CBRH79 19 cell. The enhancer of GPEII was located within the GPEII-I region. Chemical stimulation by glycidyl methatylate (GMA) and phorbol 12-o-tetradecanoate 13-acetate (TPA) analysis revealed that induction of rGSTP1 expression was mainly through GPEI. Although H2O2 could enhance GPEII enhancer activity, the enhancement is not mediated by the NF-kappaB factor that bound the NF-kappaB site in GPEII. Using electrophoretic mobility shift assays (EMSA) and the UV cross-linking assays, we found that HeLa and CBRH7919 cells had proteins that specifically bound GPEI core sequence and a 64 kDa protein that interacted with GPEII-1. The cells from normal rat liver did not express the binding proteins. Therefore, the trans-acting factors seem to be closely related to GPEI, GPEII enhancer activities and may play an important role in high expression of rGSTPI gene.