DNA Repair-deficient Cell Lines Research Articles

Sensitivity to Nitrogen Mustard Relates to the Ability of Processing DNA Damage in Chinese Hamster Ovary Cells

Abstract:The hallmark of the excision repair pathways is the removal of DNA adducts by excision of the damaged nucleotides. In the course of repair, transient DNA strand breaks occur, which can be measured by the Comet assay. We have investigated the processing of DNA damage, mediated by nitrogen mustard, in wild‐type AA8 Chinese hamster ovary cells, and in UV5, UV20 and UV41 DNA repair deficient cell lines. Whereas DNA repair could not be detected by unscheduled DNA synthesis at nitrogen mustard doses below 10 μM, processing of nitrogen mustard‐mediated DNA damage was observed by the Comet assay at a 100‐times lower concentration. Wild‐type Chinese hamster ovary AA8 cells were able to process nitrogen mustard‐mediated DNA damage within 4–24 hr depending on the dose of nitrogen mustard (0.1–10 μM). None of the repair‐deficient cell lines was able to completely process the DNA damage induced by 10 μM nitrogen mustard. At nitrogen mustard doses that conferred 10% colony forming ability, the repair‐deficient cells had an altered processing of nitrogen mustard‐mediated DNA damage: In the AA8, UV20, and UV41 cells, the amplitude of strand breaks peaked early (within 4 hr), the level of strand breaks in the nitrogen mustard exposed UV20 and UV41 cells did not return to the baseline of the unexposed reference culture, and the peak in strand breaks in the UV5 cell line occurred after 4 hr. Our results indicate that the single cell gel electrophoresis (Comet) assay is suitable for assessing repair capability of DNA alkylations.

Sensitivity to nitrogen mustard relates to the ability of processing DNA damage in Chinese hamster ovary cells.

The hallmark of the excision repair pathways is the removal of DNA adducts by excision of the damaged nucleotides. In the course of repair, transient DNA strand breaks occur, which can be measured by the Comet assay. We have investigated the processing of DNA damage, mediated by nitrogen mustard, in wild-type AA8 Chinese hamster ovary cells, and in UV5, UV20 and UV41 DNA repair deficient cell lines. Whereas DNA repair could not be detected by unscheduled DNA synthesis at nitrogen mustard doses below 10 microM, processing of nitrogen mustard-mediated DNA damage was observed by the Comet assay at a 100-times lower concentration. Wild-type Chinese hamster ovary AA8 cells were able to process nitrogen mustard-mediated DNA damage within 4-24 hr depending on the dose of nitrogen mustard (0.1-10 microM). None of the repair-deficient cell lines was able to completely process the DNA damage induced by 10 microM nitrogen mustard. At nitrogen mustard doses that conferred 10% colony forming ability, the repair-deficient cells had an altered processing of nitrogen mustard-mediated DNA damage: In the AA8, UV20, and UV41 cells, the amplitude of strand breaks peaked early (within 4 hr), the level of strand breaks in the nitrogen mustard exposed UV20 and UV41 cells did not return to the baseline of the unexposed reference culture, and the peak in strand breaks in the UV5 cell line occurred after 4 hr. Our results indicate that the single cell gel electrophoresis (Comet) assay is suitable for assessing repair capability of DNA alkylations.

UV-DNA Damage in Mouse and Human Cells Induces the Expression of Tumor Necrosis Factor α

Ultraviolet light induces the expression of tumor necrosis factor alpha (TNF alpha) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V increased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNF alpha expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNF alpha gene expression in mouse and human cells.

Mechanism of hyperoxia‐induced chromosomal breakage in chinese hamster cells

Exposure of cell cultures to hyperoxia, i.e., an atmosphere containing more than 20% O2, results in various genotoxic effects. The most prominent effect of hyperoxia is its clastogenicity. In this paper, earlier published data, obtained from research devoted to the mechanism of hyperoxia-induced clastogenesis, are reviewed. In addition, new data are presented concerning the hyperoxia-sensitivity of the DNA-repair deficient Chinese hamster cell lines xrs1, irs1, and EM9. None of these ionizing radiation-sensitive mutants showed hypersensitivity to hyperoxia, as measured by chromosomal aberration induction and loss of clonogenic cell survival. From the normal hyperoxia-sensitivity of xrs1, it may be concluded that DNA double strand breaks, of the type that are induced by ionizing radiation, do not play a role in chromosomal aberration formation by hyperoxia. In addition, since xrs1 is hypersensitive to drugs that inhibit topoisomerase II, it seems rather unlikely that exposure to hyperoxia affects topoisomerase II activity. Based on circumstantial evidence we hypothesize that perturbation of poly(ADP-ribose) metabolism may play a critical role in the mechanism of hyperoxia-induced clastogenesis.

Influence of dietary carrot on cytostatic drug activity of cyclophosphamide and its main directly acting metabolite: Induction of sister-chromatid exchanges in normal lymphocytes, Chinese hamster ovary cells, and their DNA repair-deficient cell lines

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5–3-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells ( P <0.01), and to a lesser extent in human lymphocytes ( p < 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.

Complementation of DNA-repair deficiencies in Chinese hamster ovary cells.

Genetic mapping studies in bacterial, lower eukaryotic, and mammalian systems have demonstrated that the enzyme or enzyme complex involved in the initial incision step of the DNA excision repair pathway is coded for by more than one genetic locus. This paper reports the results of complementation studies that were performed with a number of DNA repair deficient Chinese hamster ovary cell lines. Complementation abilities were measured by comparing the survival of selected mutant X mutant hybrids with that of the tetrapoloid wild type after exposure to a number of physical and chemical agents. In all cases studied, hybrids formed from two different mutant lines showed resistance similar to that of the wild-type line. These results not only demonstrate that the mutant lines were in different complementation groups, but that complementation of a DNA-repair defect associated with one particular agent can complement the defect for another DNA-damaging agent.

DNA repair deficiencies and cellular senescence are unrelated in xeroderma pigmentosum cell lines

Senescence of skin fibroblast cultures from normal individuals occurred after 23.9 ± 6.3 (S.D.) passages; senescence in DNA repair-deficient cell lines from xeroderma pigmentosum patients occurred at 22.9±5.5 passages. Cells from xeroderma pigmentosum variant and Cockayne syndrome patients reached senescence at similar passage numbers. Xeroderma pigmentosum patients contract skin cancer as a consequence of their repair deficiencies but show no symptoms of premature ageing; neither do their cells age prematurely in vitro. The clinical spectrum and the life-span of fibroblasts in culture therefore lend no support for a correlation between ageing and the DNA repair or DNA replication deficiencies found in xeroderma pigmentosum and Cockayne syndrome cells.