Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA

Collins, Andrew; Duthie, Susan J.; Dobson, V L

doi:10.1093/carcin/14.9.1733

Cited by 764 publications

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“…To test this idea directly, we performed Fpg (formamidopyrimidine DNA glycosylase) comet assays, which measure oxidative DNA lesions 25 . C57Bl/6 MEFs passaged in 20% oxygen for 10-14 days accumulated 3-4-fold more oxidative DNA damage than parallel cultures passaged in 3% oxygen (Fig.…”

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Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

et al. 2003

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Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres 1 . Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20% oxygen. MEFs did not senesce in physiological (3%) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs suggested that DNA damage limits MEF proliferation in 20% oxygen. Indeed, MEFs accumulated more DNA damage in 20% oxygen than 3% oxygen, and more damage than human fibroblasts in 20% oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing.Replicative senescence limits the proliferation of many cell types in culture and in vivo 2 .Human cells senesce replicatively largely because telomeres -DNA structures that cap chromosome ends -shorten and malfunction when replicated in the absence of telomerase, which most human cells do not express 1 . Senescent cells adopt a distinctive morphology and pattern of gene expression 2,3 , a phenotype also induced by telomere-independent events 2,4 such as DNA damage and activation of certain oncogenes. The senescence response is thought to suppress cancer 2 .MEFs are used widely in the study of replicative senescence, despite notable differences between human and rodent cells 1,2 . MEFs spontaneously overcome replicative senescence (immortalization) 5,6 , whereas human fibroblasts rarely do so. Moreover, MEF senescence relies predominantly on the p19 ARF /p53 tumour suppressor pathway, whereas human fibroblasts require loss of both p53 and retinoblastoma (Rb) tumour suppressor functions for Correspondence should be addressed to: J.C. (jcampisi@lbl.gov). Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Here, we compared the phenotypes of senescent mouse and human fibroblasts. We cultured MEFs from C57Bl/6 (or FVB; data not shown) mice under standard conditions, which include atmospheric (20%) oxygen (Fig. 1a). As expected 5 , the cells grew well for approximately 1 week (2-3 population doublings) before proliferation began to decline. After 4-5 weeks (8-10 population doublings), the cultures senesced (arrow), showing no change in cell number for more than 1 week and a senescent morphology. Proliferation eventually resumed, owing to outgrowth of immortal variants 5 . HHS Public AccessWe monitored the capacity for DNA synthesis at each passage by labelling cells with 3 Hthymidine for 3 days and counting radiolabelled nuclei. As reported 9 , the percentage of labelled nuclei d...

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Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

et al. 2003

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“…We used the modified alkaline comet assay described by Collins et al (1993) to increase the sensitivity of the assay by additionally measuring the oxidised pyrimidine or purine bases. The Endo III and the FPG enzymes recognize oxidatively damaged pyrimidines and purines, respectively, and nick the DNA at these sites creating single-strand breaks, which can be detected by the comet assay, thus increasing the sensitivity of the assay (Collins et al, 1993). In this study, the levels of DNA damage detected by basic and modified comet assay were significantly higher in breast cancer patients than controls.…”

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confidence: 99%

“…DNA damage biomarkers were measured in the MNC using the alkaline comet assay (Singh et al, 1988) and the modified comet assay (Collins et al, 1993) as described below.…”

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Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients

et al. 2005

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Folate is required for DNA synthesis, repair and methylation. Low folate status has been implicated in carcinogenesis, possibly as a result of higher rate of genetic damage. The aim of this study is to compare folate status and levels of DNA damage between breast cancer and benign breast disease control patients. Fasting blood samples from 64 histologically confirmed untreated breast cancer patients (mean age 57 years) and 30 benign breast disease control patients (mean age 51 years) were obtained. Red cell folate (RCF) and plasma homocysteine were measured. Mononuclear cells (MNC) were isolated for genetic damage analysis using the basic alkaline comet assay. Results are expressed as tail moment. Data were log transformed as appropriate before analysis for normalisation purposes. The geometric mean (95% confidence interval) of RCF (ng ml À1 ) in breast cancer patients was 339.07 (333.3 -404.6) vs 379.5 (335.8 -505.2) in control patients (P ¼ 0.24). Corresponding plasma homocysteine concentrations (mmol l À1 ) were 11.9 (10.6 -16.4) vs 10.1 (9.3 -11.9) (P ¼ 0.073), respectively. The mean tail moment (s.d.) of DNA damage in MNC of breast cancer patients detected by the basic comet assay was 1.4 (0.66) vs -0.17 (0.79) in controls (Po0.0001, t-test), the modified comet assay 'endonuclease III (Endo III)' was 1.7 (0.70) vs 0.86 (0.81) (Po0.0001, t-test), and the modified comet assay 'formamidopyrimidine glycosylase (FPG)' was 1.6 (0.62) vs 0.99 (0.94) (Po0.0001, t-test). There was a significant negative correlation between RCF levels and DNA damage detected by modified comet assay 'FPG' (Pearson Correlation Coefficient r 2 ¼ À0.26, P ¼ 0.02) and DNA damage was found to be significantly higher in MNC of breast cancer patients compared to benign breast disease control patients. Breast cancer patients tended to have lower RCF levels and higher levels of plasma homocysteine, but these differences were not significant. The study provides preliminary evidence that reduced folate status may be implicated in the aetiology of breast cancer perhaps by increasing the in vivo level of genetic instability.

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“…apurinic) sites in cells exposed to genotoxic carcinogens [15]. In this study, we did not use a modified comet assay with a purified DNA repair enzyme, formamidopyrimidine DNA-glycosylase (FPG), even though this method is more sensitive than the assay for detecting FPG-sensitive sites than our classic comet assay [31,32]. However, FPG is one of the multifunctional DNA repair enzymes participating in base excision repair [33].…”

Section: Discussionmentioning

confidence: 99%

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et al. 2002

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BackgroundSince lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs), the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level.ResultDNA damage and mitochondrial injury were measured after oxidative stress in the SV-40 transformed lung epithelial cell line challenged with hydrogen peroxide (H2O2). Single cell analysis of DNA damage was determined by assessing the number of 8-oxo-2-deoxyguanosine (8-oxo-dG) positive cells, a marker of DNA modification, and the length of a comet tail. Mitochondrial membrane potential, ΔΨm, was determined using JC-1. A 1 h pulse of H2O2 induced small amounts of apoptosis (3%). 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2. The number of cells with reduced ΔΨm increased after the addition of H2O2 in a concentration-dependent manner. In spite of a continual loss of ΔΨm, DNA fragmentation was reduced 2 h after exposure to H2O2.ConclusionThe data suggest that SV-40 transformed lung epithelial cells are resistant to oxidative stress, showing that DNA damage can be dissociated from mitochondrial injury.

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Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA

Cited by 764 publications

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Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients

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