DNA Damage In Intact Cells Research Articles

UV‐induced DNA damage in human keratinocytes: Quantitation and correlation with long‐term survival

Ultraviolet (UV) radiation has a major role in the pathogenesis of skin cancer due to its capacity to induce immunosuppression and DNA damage in cells. In this study, we describe the use of a novel extra-long polymerase chain reaction (XL-PCR) assay for detection of UV-inducible DNA lesions in a human keratinocyte line (HaCaT cells). Ultraviolet B (UVB), in doses from 4 to 50 mJ/cm2 resulted in a linear increase in the number of DNA lesions in the genome [range 0.3 +/- 0.2 lesions-3.6 +/- 0.7 lesions (mean +/- SD)/10 kb]. At lower doses of UVB (<10 mJ/cm2), 89 +/- 13% lesions were repaired within 24 h of culture. At higher doses, more lesions remained unrepaired, but the repair efficacy expressed as a proportion of repaired lesions to the total amount of DNA lesions remained constant in the range 0-50 mJ/cm2. Moreover, we demonstrated a correlation between the dose of UV and cell survival. The D37 (dose that reduced clonogenic survival to 37%) of UVB equaled 19 mJ/cm2, corresponding to the introduction of 1.4 lesions/10 kb. In contrast to UVB, UVA1 irradiation neither induced measurable DNA damage nor induced cell death in the doses up to 15 J/cm2. In conclusion, the non-radioactive extra-long (XL)-based real-time (RT)-PCR assay system can be used to quantify the UV-induced DNA damage in intact cells. The DNA lesions detected by this assay are mainly induced by short-waved radiation in the UVB range, and unrepaired DNA lesions cause keratinocyte death or permanent cell-cycle block.

Potent Antitumor Activity of Novel Iron Chelators Derived from Di-2-Pyridylketone Isonicotinoyl Hydrazone Involves Fenton-Derived Free Radical Generation

The development of novel and potent iron chelators as clinically useful antitumor agents is an area of active interest. Antiproliferative activity of chelators often relates to iron deprivation or stimulation of iron-dependent free radical damage. Recently, we showed that novel iron chelators of the di-2-pyridylketone isonicotinoyl hydrazone (PKIH) class have potent and selective antineoplastic activity (E. Becker, et al., Br. J. Pharmacol., 138: 819-30, 2003). In this study, we assessed the effects of the PKIH analogues on the redox activity of iron in terms of understanding their antitumor activity. We tested the PKIH analogues for their ability to promote iron-mediated ascorbate oxidation, benzoate hydroxylation, and plasmid degradation. Subsequent experiments assessed their ability to bind DNA, inhibit topoisomerase I, and cause DNA damage. To measure intracellular reactive oxygen species, we used the redox-sensitive probe, 2',7'-dichloro-fluorescein-diacetate, to measure intracellular PKIH-dependent redox activity. The PKIH analogues had relatively little effect on ascorbate oxidation in the presence of Fe(III) but stimulated benzoate hydroxylation and plasmid DNA degradation in the presence of Fe(II) and H2O2. These ligands could not inhibit DNA topoisomerase I or cause DNA damage in intact cells. PKIH markedly increased the intracellular generation of reactive oxygen species, and this was inhibited by catalase. This enzyme also decreased the antiproliferative effect of PKIH, indicating H2O2 played a role in its cytotoxic activity. Our results suggest that the antiproliferative effects of these chelators relates to intracellular iron chelation, followed by the stimulation of iron-mediated free radical generation via the so-formed iron complex.

Inhibitory effects of isoflavones on nitric oxide- or peroxynitrite-mediated DNA damage in RAW 264.7 cells and φX174 DNA

The inhibitory effects of isoflavones (genistin, daidzin and their aglycones genistein, daidzein) on sodium nitroprusside (SNP; nitric oxide donor)- or peroxynitrite-mediated DNA damage in intact cells and in plasmid DNA was investigated. RAW 264.7 cells, a murine macrophage cell line, are capable of producing nitric oxide and superoxide anion. However, macrophages themselves are also shown to be more sensitive to nitric oxide or peroxynitrite, and were therefore used in these studies. Results from single-cell gel electrophoresis (the comet assay) showed that these isoflavones, at the concerning of 25–200 μ m, inhibited the induction of nitric oxide- or peroxynitrite-mediated macrophage genotoxicity, with genistein showing the greatest inhibition. Genistein and daidzein, at a concentration of 1–25 μm, dose-dependently inhibited peroxynitrite-induced φX174 DNA degradation based on the results of agarose gel electrophoretic analysis. Although SNP could increase the cellular GSH level, no significant differences in the glutathione content or the GSH:GSSG ratio were observed for genistein and daidzein in the presence or absence of SNP as compared with SNP-only treated RAW 264.7 cells. Exposure of RAW 264.7 cells to SNP caused the enzyme activities of GSH peroxidase, GSH reductase and catalase decrease to 44, 20 and 34% of that of untreated cells, respectively. On the contrary, exposure of RAW 264.7 cells to SNP in the presence of 100 μm of genistein or daidzein caused the enzyme activities of GSH peroxidase, GSH reductase and catalase decrease to 18, 9 and 12% (genistein) or 13, 9 and 19% (daidzein) of that of untreated cells, respectively. These results suggest that the inhibition by isoflavones of SNP- or peroxynitrite- mediated DNA damage could be attributed to their nitric oxide or peroxynitrite scavenging activities and their prevention of antioxidant enzyme inactivation.

Involvement of DT-diaphorase (EC 1.6.99.2) in the DNA cross-linking and sequence selectivity of the bioreductive anti-tumour agent EO9.

The chemistry of the mitomycin C-related drug indoloquinone EO9 would suggest that its mechanism of action is likely to involve DNA damage after reductive activation. The ability of this agent to induce DNA damage in intact cells has been examined using alkaline filter elution. After treatment with pharmacologically relevant concentrations of EO9, both DNA strand breaks and interstrand cross-links were detected in rat Walker tumour cells and human HT29 colon carcinoma cells. These cell lines express relatively high levels of DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase), which is believed to be involved in EO9 activation. The extent of DNA damage was increased by approximately 30-fold under hypoxia in BE colon carcinoma cells that express non-functional DT-diaphorase, but this dramatic hypoxia enhancement was not seen in HT-29 cells. These data are consistent with cytotoxicity studies that indicate that DT-diaphorase appears to be important in EO9 activation under aerobic conditions, but other enzymes may be more relevant under hypoxia. The involvement of DT-diaphorase in DNA damage induction was further investigated using cell-free assays. DNA cross-links were detectable in plasmid DNA co-incubated with EO9, cofactor and DT-diaphorase but not in the absence of this enzyme. In contrast, using a Taq polymerase stop assay, monofunctional alkylation was detected in plasmid DNA without metabolic activation, although the sequence selectivity was altered after reduction catalysed by DT-diaphorase.

Analysis of metal-induced oxidative DNA damage in cultured mammalian cells.

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.

Augmentation of bleomycin-induced DNA damage in intact cells

Bleomycin, an important cause of pulmonary fibrosis, is known to produce DNA damage. The mechanism for this damage in vitro is related to free radical production by a bleomycin and iron complex. To determine whether bleomycin causes damage to DNA in vivo by a similar mechanism, we used a viral minichromosome that is replicated in cultured cells. Bleomycin causes dose-dependent damage to intracellular DNA, and this damage is augmented by Fe2+ but not Fe3+. The augmentation of the bleomycin-induced DNA damage caused by Fe2+ is also dose dependent in that increasing DNA damage occurs with increasing amounts of Fe2+. These studies demonstrate that bleomycin causes damage to DNA in vivo and suggest that bleomycin must rely on Fe2+ to donate an electron for oxygen radical-induced DNA strand scission.

DNA damage in intact cells induced by bacterial metabolites of chloramphenicol.

Four chloramphenicol (CAP) metabolites known to be produced by intestinal bacteria were examined with respect to their capacity to induce DNA damage in intact cells. The induction of DNA single-strand breaks in Raji cells, activated human lymphocytes, and human marrow cells was assayed by the alkaline elution technique. One of the four compounds tested, dehydro-CAP, was capable of inducing DNA single-strand breaks in all three cell systems at concentrations of 10(-4) M. This effect is comparable to that observed previously with nitroso-CAP, the nitroreduction intermediate of CAP. The nitroreduction of dehydro-CAP by human bone marrow cell homogenate was detected by the production of the corresponding amino derivative amounting to 5.6 X 10(-5) M from 2 X 10(-3) M substrate under aerobic conditions. In sharp contrast, nitroreduction of CAP by bone marrow could not be demonstrated. The genotoxicity of dehydro-CAP, its relative stability compared to the nitroso-CAP, and its nitroreducibility by bone marrow suggest that this bacterial metabolite of CAP may play a key role as a mediator of aplastic anemia in the predisposed host.

DNA damage and repair following treatment of V-79 cells with sulfonated phthalocyanines.

Abstract. Two gallium‐phthalocyanines were tested for their effects on cell survival, trypsinization, and DNA strand breaks in intact and permeable cells, measured by alkaline elution. Gallium‐tetrasulfophthalocyanine was not phototoxic to cells and caused no measurable DNA damage in intact cells, while a mixture of less sulfonated gallium‐sulfophthalocyanines, containing an average of 2.7 sulfonates per molecule, was very phototoxic and induced DNA strand breaks and/or alkali‐sensitive sites; however, both drugs were equally effective at inducing DNA strand breaks in permeable cells. The DNA damage was rapidly repaired in intact cells; this process was not inhibited by aphidicolin, an inhibitor of DNA polymerases alpha and delta, under conditions in which DNA replicative synthesis was inhibited by more than 90%.

The sequence specificity of bleomycin-induced DNA damage in intact cells.

Bleomycin causes lesions to DNA in intact cells and in purified DNA under appropriate conditions. Using a middle repetitive DNA sequence called alpha-DNA as a target sequence, we have compared the sequence specificity of bleomycin-induced DNA cleavage in intact human cells and in purified human DNA. Bleomycin induces numerous cleavage sites in alpha-DNA which vary widely in intensity and give rise to a complex pattern of bands on a DNA sequencing gel. Unexpectedly, the intensity and position of bleomycin-induced DNA cleavage sites are very similar in intact human cells and in purified human DNA.

Endogenous ADP ribosylation of high mobility group proteins 1 and 2 and histone H1 following DNA damage in intact cells

Endogenous ADP ribosylation of nonhistone high-mobility group (HMG) proteins and histone H1 was studied in cultured mouse mammary tumor cells following treatment with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). MNNG treatment of cells caused a rapid and transient increase in ADP ribosylation of histone H1 and HMG 1 and 2, whereas (ADP-ribose) n on HMG 14 and 17 was not affected. 3-Aminobenzamide, an inhibitor of (ADP-ribose) n synthetase, prevented the increase in ADP ribosylation of histone H1 and HMG 1 and 2. This inhibitor enhanced the cell-killing effect of MNNG, but had no significant effect on the removal of methylated purines. The preferential increase in ADP ribosylation of HMG 1 and 2 and histone H1 may be necessary for cell recovery from DNA damage.