No abstract

Generation of putative intrastrand cross-links and strand breaks was investigated in salmon sperm DNA exposed to Fenton-type oxygen radical-generating systems. 32P-Postlabeling analysis of DNA treated with hydrogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(II), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radical-mediated oxidative DNA lesions. The copper Fenton system generated the highest total yield of these DNA lesions (75.6 per 10(8) nucleotides), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (21.7), and vanadium (17.1). Two spots, common to all these Fenton systems, were the major oxidation products in each case. Similar Fenton-type treatment of the purine dinucleotides dApdG and dApdA resulted in products that were chromatographically identical on anion-exchange TLC and on reverse-phase HPLC to the two major products generated in DNA. These results extend our earlier studies suggesting that these products were the result of a free radical-mediated intrastrand cross-linking reaction. Incubations involving cadmium(II), chromium(III), or zinc(II) ions with hydrogen peroxide did not generate DNA oxidation products at levels greater than in incubations with hydrogen peroxide alone. Generation of the putative intrastrand cross-links increased in a concentration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) ions. However, in experiments with copper, iron, or vanadium ions, maximum levels were obtained at 250, 150, and 150 microM, respectively, and the yield declined with higher concentrations of these three metal ions. Agarose gel electrophoresis demonstrated extensive DNA strand breakage with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results demonstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independent mechanisms.

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone. Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions. Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

No abstract

Thymine glycols are formed in DNA by exposure to ionizing radiation or oxidative stress. Although these lesions are repaired by the base excision repair pathway, they have been shown also to be subject to transcription-coupled repair. A current model for transcription-coupled repair proposes that RNA polymerase II arrested at a DNA lesion provides a signal for recruitment of the repair enzymes to the lesion site. Here we report the effect of thymine glycol on transcription elongation by T7 RNA polymerase and RNA polymerase II from rat liver. DNA substrates containing a single thymine glycol located either in the transcribed or nontranscribed strand were used to carry out in vitro transcription. We found that thymine glycol in the transcribed strand blocked transcription elongation by T7 RNA polymerase ϳ50% of the time but did not block RNA polymerase II. Thymine glycol in the nontranscribed strand did not affect transcription by either polymerase. These results suggest that arrest of RNA polymerase elongation by thymine glycol is not necessary for transcription-coupled repair of this lesion. Additional factors that recognize and bind thymine glycol in DNA may be required to ensure RNA polymerase arrest and the initiation of transcription-coupled repair in vivo. Transcription-coupled repair (TCR)1 is a pathway of DNA excision repair that is targeted to removal of DNA lesions present in transcribed strands of expressed genes (1). TCR has been demonstrated in mammalian cells (2), Escherichia coli (3), and Saccharomyces cerevisiae (4 -6). TCR was originally observed for lesions repaired by nucleotide excision repair (2). An active RNA polymerase elongation complex is necessary for preferential repair of the transcribed strand (7). The arrest of transcription at DNA lesions has been proposed to serve as a specific signal to direct repair enzymes to the transcribed strand of an active gene to initiate a repair event (8). It is likely that this repair pathway evolved for the dedicated purpose of resolving the impasse of an RNA polymerase arrested at a lesion. The polymerase must be displaced both to permit verification that a lesion caused the arrest and to allow the repair enzymes to operate on the lesion (1).The role of RNA polymerases in TCR has been examined more directly by comparing the extent of RNA polymerase arrest in vitro by a lesion with TCR of that lesion in vivo. These studies have shown that various types of DNA damage in the template strand of DNA can act as blocks to transcription catalyzed by different RNA polymerases. In several cases, a correlation between the extent of polymerase arrest and the extent of TCR has been found (7). However, most of these studies have been carried out using viral and bacterial RNA polymerases. In an attempt to understand the role of mammalian RNA polymerase II (RNAP II) in TCR, we have developed an in vitro transcription system with templates containing a site-specific lesion positioned downstream of the adenovirus major late promoter (AdMLP) and purified proteins and i...

The results support a model in which hypoxia-enhanced uptake of radiotoxic (64)Cu induces sufficient DNA damage and toxicity to overcome the documented radioresistance in hypoxic MCF-7 cells. This suggests that (64)Cu-ATSM and related complexes have potential for targeted radionuclide therapy of hypoxic tumours.

The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. In addition to its regulatory role in cell cycle progression, p53 regulates apoptosis and can therefore influence cellular survival in response to DNA damage. More recent work has revealed that p53 is also involved in the nucleotide excision repair (NER) of structurally diverse types of DNA damage. The relative influence of p53 on NER and cellular sensitivity to DNA damage was investigated in this study using cells that differ in p53 status. Two cell models were selected: 041 TR fibroblasts in which the expression of p53 is regulated by a tetracycline-inducible promoter, and WI38 primary lung fibroblasts together with their isogenic derivative VA13, in which p53 is abrogated post-translationally by SV40 transformation. Cells were exposed to the clinically and environmentally relevant DNA-damaging agents cisplatin (0-5 microM, 2 h), (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (0-0.5 microM, 30 min) and UV-C (0-5 J/m2), each of which induce structurally distinct types of DNA damage known to be subject to p53-dependent NER. Sensitivity of the p53-proficient and p53-deficient cells to this DNA damage was then compared at each dose of DNA-damaging agent using the clonogenic survival assay and the colorimetric MTT assay. p53-proficient cells were more sensitive than p53-deficient cells to cisplatin, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and UV-C; these differences in cellular sensitivity were more apparent in the 041 TR cells (up to 3.6-, 5.8- and 1.9-fold, respectively) than the WI38/VA13 cells (up to 2.3-, 1.4- and 1.4-fold, respectively). Thus, despite the well-documented persistence of DNA damage in p53-deficient fibroblasts due to impaired NER, loss of p53 results in reduced DNA damage-mediated cytotoxicity.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.