Kerstin Kenne

Altered DNA ligase III activity in the CHO EM9 mutant

Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.

Inhibition of cell-cell communication by methylsulfonyl metabolites of polychlorinated biphenyl congeners in rat liver epithelial IAR 20 cells

The effects of three polychlorinated biphenyl (PCB) congeners and their six methylsulfonyl (MeSO2)-metabolites on cell communication have been investigated in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells. The results demonstrated that at non-cytotoxic concentrations 2,2′,4′,5-tetrachlorobiphenyl, 2,2′,4′,5,5′-pentachlorobiphenyl (2,2′,4′,5,5′-pentaCB), 2,2′,4′,5,5′,6-hexachlorobiphenyl (2,2′,4′,5,5′, 6-hexaCB), and their 3- and 4-MeSO2 derivatives completely inhibited the cell communication within 1 h. 4-MeSO2-2,2′,4′,5,5′-pentaCB and 4-MeSO2-2,2′,4′,5, 5′,6-hexaCB appeared to inhibit the cell communication at slightly lower concentration than their parental PCB congeners and 3-MeSO2 derivatives. The results show that 3- and 4-MeSO2 derivatives of the PCB congeners tested inhibit gap junction intercellular communication at about the same potency as their parental compounds. Since inhibition of cell communication is often observed after treatment with many tumor promoters, our findings suggest that the metabolites may also act as tumor promoters.

The ability to alter the gap junction protein expression outside GST-P positive foci in liver of rats was associated to the tumour promotion potency of different polychlorinated biphenyls

The results demonstrate different modes of action by a dioxin-like polychlorinated biphenyl (PCB 126) and a non dioxin-like PCB (PCB 153) in the alteration of connexin (cx) 26 and cx 32 expression outside GST-P positive foci in liver of female Sprague-Dawley rats, treated according to an initiation-promotion protocol. A decreased relative amount of immunopositive cx 26 and cx 32 spots in the parenchymal cell plasma membranes was observed after treatment with the potent tumour promoters PCB 126 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). No reduction of cx 26 or cx 32 was noted after administration with the weaker tumour promoters PCB 153 or PCB 118 (PCB 118; both dioxin- and non dioxin-like). Additionally, we found that the down-regulation of connexins also occurred in rats treated with PCB 126 or TCDD without partial hepatectomy and initiation with nitrosodiethylamine. In summary, the results indicate that the ability to reduce the gap junction protein level in liver of rats can be associated to the tumour promotive potency of the different PCB-congeners and TCDD.

RecA-like activity in mammalian cell extracts of different origin

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Blooms syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Blooms syndrome fibroblast cell lines, Fanconis anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.

The human RAD51/RecA homologue gene is not a candidate gene for Bloom's syndrome

The human gene HSRAD51/RecA homologue has been investigated as a possible candidate gene involved in Blooms syndrome. No mutations were found in the cDNA isolated from three different Blooms syndrome cell lines, thus excluding the possibility that HSRAD51 is directly involved in the syndrome. Other possible candidates are discussed.