Alkaline single-cell gel electrophoresis (comet assay)

Colleu-Durel, S.; Guitton, Nathalie; Nourgalieva, K.; Legué, Florence; Lévêque, J.; Danic, Bruno; Chenal, C

doi:10.1016/j.ejca.2003.09.033

Cited by 40 publications

(11 citation statements)

References 22 publications

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“…In contrast to the above findings, increased basal as well as elevated radiation-and doxorubicin-induced DNA damage levels have been observed in the blood lymphocytes from BC patients (Blasiak et al, 2004;Colleu-Durel et al, 2004;Hussien et al, 2005).…”

contrasting

confidence: 70%

“…Thus, in contrast to the present and several other studies (Alapetite et al, 1999;Rothfuß et al, 2000;Müller-Vogt et al, 2003), in which healthy individuals were used as references, the control group in Hussien et al (2005) includes patients with the benign breast disease. Besides this, even if the same version of the alkaline Comet assay (Singh et al, 1988) was used here and elsewhere (Colleu-Durel et al, 2004;Hussien et al, 2005), the quantitative TM data appear to differ greatly between laboratories. Thus, the TM values of 7.74 and 14.67 in nonirradiated cells from control and BC patients, respectively, obtained by Colleu-Durel et al (2004) are larger some 20 -30-fold than the TM values of 0.47 and 0.45 presented here in Figure 1A.…”

Section: Discussionmentioning

confidence: 97%

“…Besides this, even if the same version of the alkaline Comet assay (Singh et al, 1988) was used here and elsewhere (Colleu-Durel et al, 2004;Hussien et al, 2005), the quantitative TM data appear to differ greatly between laboratories. Thus, the TM values of 7.74 and 14.67 in nonirradiated cells from control and BC patients, respectively, obtained by Colleu-Durel et al (2004) are larger some 20 -30-fold than the TM values of 0.47 and 0.45 presented here in Figure 1A. For comparison, the TM values of 1.4 and 1.21 have been found, respectively, for bladder cancer patients and controls (Schabath et al, 2003), whereas TM values of about three have been obtained for both breast cancer patients and controls by Alapetite et al (1999).…”

Section: Discussionmentioning

confidence: 97%

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Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays

et al. 2006

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Spontaneous and radiation-induced genetic instability of peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n ¼ 50) was examined using the single-cell gel electrophoresis (Comet) assay and a modified G2 micronucleus (MN) test. Cells from apparently healthy donors (n ¼ 16) and from cancer patients (n ¼ 9) with an adverse early skin reaction to radiotherapy (RT) served as references. Nonirradiated cells from the three tested groups exhibited similar baseline levels of DNA fragmentation assessed by the Comet assay. Likewise, the Comet analysis of in vitro irradiated (5 Gy) cells did not reveal any significant differences among the three groups with respect to the initial and residual DNA fragmentation, as well as the DNA repair kinetics. The G2 MN test showed that cells from cancer patients with an adverse skin reaction to RT displayed increased frequencies of both spontaneous and radiation-induced MN compared to healthy control or the group of unselected BC patients. Two patients from the latter group developed an increased early skin reaction to RT, which was associated with an increased initial DNA fragmentation in vitro only in one of them. Cells from the other BC patient exhibited a striking slope in the dose -response curve detected by the G2 MN test. We also found that previous RT strongly increased both spontaneous and in vitro radiation-induced MN levels, and to a lesser extent, the radiation-induced DNA damage assessed by the Comet assay. These data suggest that clinical radiation may provoke genetic instability and/or induce persistent DNA damage in normal cells of cancer patients, thus leading to increased levels of MN induction and DNA fragmentation after irradiation in vitro. Therefore, care has to be taken when blood samples collected postradiotherapeutically are used to assess the radiosensitivity of cancer patients.

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contrasting

confidence: 70%

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 97%

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Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays

et al. 2006

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“…Imaging DSBs in vivo is particularly informative, as their number and persistence would predict cell fate. Various techniques are being used to analyze DSBs, such as pulsed-field electrophoresis, comet assay, and neutral filter elution (27, 28). However, none has been widely adopted in epidemiologic studies, and assays with higher throughput are being sought.…”

Section: Discussionmentioning

confidence: 99%

Risk Assessment of Esophageal Adenocarcinoma Using γ-H2AX Assay

Gong

et al. 2013

Cancer Epidemiology, Biomarkers &Amp; Prevention

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Background Mutagen-induced DNA damage as measured in peripheral blood lymphocytes (PBLs) has been associated with increased risks of cancers. The formation of γ-H2AX is an early cellular response to DNA double-strand breaks (DSBs). We hypothesize that higher level of radiation-induced γ-H2AX in PBLs may be associated with an increased risk of esophageal adenocarcinoma (EAC). Methods Laser scanning cytometer–based immunocytochemical method was used to measure baseline and irradiation-induced γ-H2AX levels in PBLs from 211 EAC patients and 211 healthy controls. The ratio of induced γ-H2AX level to baseline level was used to evaluate individual susceptibility to DSBs. Relative risks for EAC associated with γ-H2AX were assessed by multivariable logistic regression analysis. Results Radiation-induced γ-H2AX level and the γ-H2AX ratio were significantly higher in cases than in controls. Dichotomized at the median in controls, a significantly increased risk for EAC was observed in association with high γ-H2AX ratio (odds ratio=2.94, 95% confidence interval=1.83–4.72). Quartile analyses showed significant dose-response associations between higher γ-H2AX ratio and increased risk of EAC (P for trend, 1.64E-06). In addition, joint effect between γ-H2AX ratio and smoking was observed: smokers who had high γ-H2AX ratio exhibited the highest risk of EAC (OR =5.53, 95% CI: 2.71–11.25) compared to never-smokers with low γ-H2AX ratio. Conclusion Radiation-induced DNA damage assessed by γ-H2AX ratio is associated with an increased risk of EAC. Impact γ-H2AX assay is a new and robust method to measure DSB damage in PBLs, which can be used to assess mutagen sensitivity and EAC risk.

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“…Comet assay was performed as in previous report with minor revisions [37]. Briefly, microscopic slides were coated with 1% normal agarose (GE Healthcare) followed by adding 1% low melting point (LMP) agarose onto each slide and then covering slides with coverslips.…”

Section: Methodsmentioning

confidence: 99%

PTEN Activation by DNA Damage Induces Protective Autophagy in Response to Cucurbitacin B in Hepatocellular Carcinoma Cells

Niu

Sun

et al. 2016

Oxidative Medicine and Cellular Longevity

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Cucurbitacin B (Cuc B), a natural product, induced both protective autophagy and DNA damage mediated by ROS while the detailed mechanisms remain unclear. This study explored the mechanism of Cuc B-induced DNA damage and autophagy. Cuc B decreased cell viability in concentration- and time-dependent manners. Cuc B caused long comet tails and increased expression of γ-H2AX, phosphorylation of ATM/ATR, and Chk1/Chk2. Cuc B induced autophagy as evidenced by monodansylcadaverine (MDC) staining, increased expression of LC3II, phosphorylated ULK1, and decreased expression of phosphorylated AKT, mTOR. Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation. Furthermore, Cuc B induced ROS formation, which was inhibited by N-acetyl-L-cysteine (NAC). NAC pretreatment dramatically reversed Cuc B-induced DNA damage, autophagy, and apoptosis. Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine (3-MA), chloroquine (CQ), and silencing phosphatase and tensin homolog (PTEN). 3-MA and CQ showed no effect on Cuc B-induced DNA damage. In addition, Cuc B increased PTEN phosphorylation and silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage. In summary, Cuc B induced DNA damage, apoptosis, and protective autophagy mediated by ROS. PTEN activation in response to DNA damage bridged DNA damage and prosurvival autophagy.

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Alkaline single-cell gel electrophoresis (comet assay)

Cited by 40 publications

References 22 publications

Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays

Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays

Risk Assessment of Esophageal Adenocarcinoma Using γ-H2AX Assay

PTEN Activation by DNA Damage Induces Protective Autophagy in Response to Cucurbitacin B in Hepatocellular Carcinoma Cells

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