DBD-FISH on Neutral Comets: Simultaneous Analysis of DNA Single- and Double-Strand Breaks in Individual Cells

Fernández, José Luís; Vázquez-Gundı́n, Fernando; Rivero, María Teresa; Genescà, Anna; Gosálvez, Jaime; Goyanes, Vicente

doi:10.1006/excr.2001.5328

Cited by 41 publications

(28 citation statements)

References 20 publications

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“…It is important to note that ionizing radiation causes the generation of DSBs, but single-strand breaks are generated far more frequently than DSBs (Fernandez et al, 2001). Single-strand breaks are converted into DSBs in replicating templates (Larner et al, 1999).…”

Section: Indications For An Antagonistic Role Of P53 During Replicatimentioning

confidence: 99%

Wild-type p53 inhibits replication-associated homologous recombination

Janz

Wiesmüller

2002

Oncogene

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In mammalian cells homologous recombination is stimulated, when the replication fork stalls at DNA breaks or unrepaired lesions. The tumor suppressor p53 downregulates homologous recombination independently of its transcriptional transactivation function and has been linked to enzymes of DNA recombination and replication. To study recombination with respect to replication, we utilized a SV40 virus based assay, to follow the synchronous events after primate cell infection. g-ray treatment at different times after viral entry unveiled an increase of interchromosomal exchange frequencies, when the damage was introduced during DNA synthesis. Elevated recombination frequencies were fully suppressed by p53. With respect to the downregulation of spontaneous recombination, we noticed a requirement for active p53 molecules, when replication started. After a transient treatment with replication inhibitors, we observed inhibition of the drug induced recombination by p53, particularly for the elongation inhibitor aphidicolin. Consequently, we propose that p53 is a surveillance factor of homologous recombination at replication forks, when they stall as a consequence of endogenous or of exogenously introduced damage.

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Section: Indications For An Antagonistic Role Of P53 During Replicatimentioning

confidence: 99%

Wild-type p53 inhibits replication-associated homologous recombination

Janz

Wiesmüller

2002

Oncogene

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“…The assessment of DNA damage in fish sperm for fertility assessment is so far achieved using the comet assay (Cabrita et al 2005, Dietrich et al 2005. The comet assay is consistent and able to determine singleor double-stranded breakage, and may also enable DNA sequence identification associated with DNA breaks (Fernández et al 2001). However, the comet assay is technically challenging in requiring DNA electrophoresis and sophisticated image analysis software for achieving systematically consistent results.…”

Section: Introductionmentioning

confidence: 99%

Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test

López‐Fernández¹,

Gage²,

Arroyo³

et al. 2009

Reproduction

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Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employing in situ nick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0-60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.

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“…MDA-MB468 and CRL2336) cell lines [12,16,17]. Following alkaline lysis, the breast cancer cell lines showed evidence of increased endogenous and IR-induced alkali-labile sites in comparison to normal cells (see Table 1).…”

Section: Resultsmentioning

confidence: 97%

Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells sing Comet–FISH

Kumaravel

Bristow

2005

Breast Cancer Res Treat

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A proportion of breast cancers acquire genetic alterations at 17q11.2-q12 (HER-2/neu), 20q13.2 (ZNF217 gene) and 17p13.1 (p53). We describe a unique technique (Comet-FISH) in which we documented relative genetic instability at p53 and HER-2/neu gene loci within a panel of malignant breast cancer cell lines (MCF-7; MDA-MB-468 and CRL-2336). Furthermore, Comet-FISH data were consistent with preferential repair of the p53 locus following gentoxic insult and suggest that this assay may be quite useful for the study of genetic instability.

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DBD-FISH on Neutral Comets: Simultaneous Analysis of DNA Single- and Double-Strand Breaks in Individual Cells

Cited by 41 publications

References 20 publications

Wild-type p53 inhibits replication-associated homologous recombination

Wild-type p53 inhibits replication-associated homologous recombination

Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test

Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells sing Comet–FISH

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