Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells

McKenna, Declan; Gallus, Massimo; McKeown, Stephanie; Downes, C. Stephen; McKelvey‐Martin, Valerie J.

doi:10.1016/s1568-7864(03)00086-7

Cited by 59 publications

(56 citation statements)

References 21 publications

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“…The model depicted in figure 1, supported by the results of McKenna et al and Horvâthovâ et al [33,46], suggests that, aside from sequences attached to the nuclear matrix and always found in Comet heads, localizing any sequence of any size in the heads of Comets requires that the entire loop containing such sequences be free of damage. This presents us with a conundrum: if the two strands of a loop will continue to migrate to the tail until all the lesions and breaks are repaired, it might not be possible to detect TCR at the gene level by counting signal spots in Comet heads and tails.…”

Section: Discussionmentioning

confidence: 56%

“…Using this methodogy, it was shown that DNA interstrand crosslinks induced by mytomicin C were repaired faster in the p53 domain than in the genome overall [46].…”

Section: Comet and Comet-fish Assaysmentioning

confidence: 99%

“…An interesting question is whether the complementary strands in a loop migrate into the tail independently or together upon alkaline denaturation and electrophoresis. Comet-FISH with a probe for the p53 gene was applied to cells that had been damaged by ionizing radiation; using parameters such as percent DNA in the tails, number of breaks, approximate loop size and numbers of probe-specific "spots" in heads and tails, the investigators found that their results favored a model in which both strands in a loop migrate into the tail, but separately, even in cases in which one strand is broken and the complementary strand is intact [46]. In support of this interpretation, Collins and coworkers stained Comets with acridine orange and observed that neutralization after alkaline electrophoresis allowed intact supercoils in Comet heads to reanneal, while DNA in tails remained single-stranded [48].…”

Section: Comet and Comet-fish Assaysmentioning

confidence: 99%

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New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair

Spivak

Cox

Hanawalt

2009

Mutation Research/Reviews in Mutation Research

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Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30 kb. The single cell gel electrophoresis (Comet) assay allows detection of single-or double-strand breaks at a 10 to 100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

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Section: Discussionmentioning

confidence: 56%

“…Using this methodogy, it was shown that DNA interstrand crosslinks induced by mytomicin C were repaired faster in the p53 domain than in the genome overall [46].…”

Section: Comet and Comet-fish Assaysmentioning

confidence: 99%

Section: Comet and Comet-fish Assaysmentioning

confidence: 99%

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New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair

Spivak

Cox

Hanawalt

2009

Mutation Research/Reviews in Mutation Research

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“…DNA alkylation was assessed by modified comet assay as previously described (McKenna et al, 2003). Exponentially growing V79 cells transformed to over express either NQO1 (hdt), NQO2 (TH7) or transformed with empty plasmid (f179) were incubated with 10 mM MMC in a final volume of 1 ml for 1 h at 371C in separate eppendorfs.…”

Section: Nqo1 Activity Assaymentioning

confidence: 99%

Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

Jamieson

Tung

Knox³

et al. 2006

Br J Cancer

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NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species.

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“…In a special modification of the comet assay, a gene of interest can be marked by fluorescence in situ hybridization (FISH) . It is then possible to visualize whether a specific gene is located in the tail, thereby gaining information on damage to that specific region of the genome (McKenna et al 2003, Rapp et al 2005.…”

mentioning

confidence: 99%

Detection of Cyto- and Genotoxicity of Rod-Shaped Gold Nanoparticles in Human Blood Lymphocytes Using Comet-FISH

et al. 2015

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Summary Gold nanoparticles (GNPs) shall be applied in cancer therapy, conceivably by using a simple injection of GNPs into human veins. This will bring them in contact with red and white blood cells of the blood stream before they reach their main target, the cancer cells. However, possible cyto-and/or genotoxic effects of GNPs on lymphocytes are not known in detail and are thus studied here. Cytotoxicity was determined by Trypan blue exclusion assay. For genotoxicity, Comet and Comet-FISH (=fluorescence in situ hybridization) assays were done. In the latter test two gene markers for DNA damage, TP53 as tumor suppressor gene and TNF-α as tumor necrosis factor gene, were investigated. The cells were incubated in the presence of different concentrations of polyethylene glycol-coated rod-shaped GNPs of 50 nm or 30 nm in diameter. GNPs induced cytotoxic effects in human lymphocytes. The effects could be observed in concentration-and sizedependent manner; 30 nm sized GNPs were more toxic than 50 nm sized ones. Using the comet assay, it was demonstrated that GNPs induce high rates of DNA damage, which are represented e.g. as high ratios of tail moments, compared to non-treated lymphocytes. The target genes (TP53 and TNF-α) were observed preferentially in comet tails indicating high rates of induced DNA damage in this DNA area. Our results suggest that rod-shaped GNPs interact with human blood lymphocytes, reduce cell viability and cause relevant DNA damage in a concentration dependent manner. The small sized GNPs were more cyto-and genotoxic than big sized GNPs. The low concentration of big sized rod-shaped GNPs could be safe for cancer photothermal therapy rather than small rod GNPs. However, further investigations are recommended to be able to minimize potential risks of application.

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Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells

Cited by 59 publications

References 21 publications

New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair

New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair

Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

Detection of Cyto- and Genotoxicity of Rod-Shaped Gold Nanoparticles in Human Blood Lymphocytes Using Comet-FISH

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