UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.

XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Enhancement of excision repair of cisplatin-DNA adducts by cell-free extract from a cisplatin-resistant rat cell line.

To characterize the enhanced repair synthesis of defined DNA lesions, oligodeoxyribonucleotides were synthesized and inserted into plasmid DNA. The inserted plasmid DNA was treated with cis-diamminedichloroplatinum(II) (cisplatin) and subjected to in vitro DNA repair assay with soluble extract from the rat liver cell line Ac2F. All cisplatin adducts tested stimulated DNA repair synthesis. Moreover, two cisplatin-resistant cell lines, Ac2F-CR4 and Ac2F-CR10, were established by stepwise exposure of Ac2F cells to this drug. The DNA repair synthesis was enhanced 3- to 4-fold in the extract from cisplatin-resistant Ac2F cells relative to that from Ac2F cells. Such repair synthesis was suppressed by the specific DNA polymerase inhibitor aphidicolin. The results of the present study suggested that the enhanced repair activity induced by a cisplatin adduct can be detected by in vitro DNA repair assay with soluble cell extract.

Enhancement of DNA repair activity in rat-liver cells exposed to cisplatin.

Rat-liver cells (Ac2F cells) were transfected with cisplatin-damaged eucaryotic expression vectors carrying luciferase cDNA (cis-DDP-pVJ3luc) and incubated. The lysate of the incubated cells was subjected to a luciferase activity assay. Graded decrease in the activity was observed with increasing levels of platination of the plasmid DNA. In another experiment, Ac2F cells were pre-incubated in the presence of cisplatin ranging in concentration from 0.5 to 3 mu M. The viable cells were transfected with cis-DDP-pVJ3luc and incubated in the absence of this drug. The lysate of the incubated cells was subjected to the same assay. The level of the luciferase activity was raised with increasing cisplatin concentration. These results suggest that the repair activity for cis-DDP-pVJ3luc DNA is enhanced in Ac2F cells exposed to cisplatin.