Alkaline Comet Assay for Assessing DNA Damage in Individual Cells

Pu, Xinzhu; Wang, Zemin; Klaunig, James E.

doi:10.1002/0471140856.tx0312s65

Cited by 87 publications

(42 citation statements)

References 22 publications

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“…Under alkaline conditions this method detects a variety of genotoxic changes, including double and single DNA strand breaks and incomplete excision repair sites [22]. Figure 2A illustrates the increased number of comet tails when D2/CA follow AraC exposure of AML blasts, and the intensity of the comet tails was quantitated and is shown in Figure 2B.…”

Section: Resultsmentioning

confidence: 99%

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts

2016

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Acute Myeloid Leukemia (AML) has grave prognosis due to aggressive nature of the disease, the toxicity of standard treatment, and overall low cure rates. We recently showed that AML cells in established culture treated with cytarabine (AraC) and a differentiation agent combination show enhancement of AraC cytotoxicity. Here we elucidate molecular changes which underlie this observation with focus on AML blasts in primary culture. The cells were treated with AraC at concentrations achievable in clinical settings, and followed by the addition of Doxercalciferol, a vitamin D2 derivative (D2), together with Carnosic acid (CA), a plant-derived antioxidant. Importantly, although AraC is also toxic to normal bone marrow cell population, the enhanced cell kill by D2/CA was limited to malignant blasts. This enhancement of cell death was associated with activation of the monocytic differentiation program as shown by molecular markers, and the increased expression of vitamin D receptor (VDR). Apoptosis elicited by this treatment is caspase-dependent, and the optimal blast killing required the increased expression of the apoptosis regulator Bim. These data suggest that testing of this regimen in the clinic is warranted.

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Section: Resultsmentioning

confidence: 99%

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts

2016

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“…Since the single cell gel electrophoresis assay (comet assay) has been used as a useful reliable genotoxicity test that detects DNA migration caused by strand breaks [22], we applied the cells to be exposed to irradiation, allowed to recover for 2 hours, and then for detection of DNA damage by alkaline comet assay (Figure 4). Cells did not show significant different tail movement before irradiation.…”

Section: Resultsmentioning

confidence: 99%

Increased expression of SKP2 is an independent predictor of locoregional recurrence in cervical cancer via promoting DNA-damage response after irradiation

Yang

Chen

et al. 2016

Oncotarget

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Although radiation therapy was known to be effective to cervical cancer, loco-regional recurrences are frequently found in patients. We aimed to identify a molecular marker predicting the response of cervical cancer to radiotherapy. We included the patients (n = 149) with cervical cancer who had undergone radiotherapy from 2004 to 2006. Tumor samples were collected to examine the association between the expression of S-phase kinase-associated protein 2 (SKP2) and prognosis in cervical cancer. We found higher expression of SKP2 associated with recurrence (HRs: 2.52, p < 0.001), death (HRs: 2.01, p < 0.001) and higher locoregional recurrence rate (HRs: 3.76, p < 0.001). Cervical cancer cell lines with higher expression of SKP2 showed higher colony formation, cell survival rate and fewer DNA damages after irradiation. SKP2-C25, an inhibitor for SKP2 activity, dose-dependently decreased cell viability after irradiation and knockdown of SKP2 impaired DNA-damage response and sensitized the cervical cancer cells to irradiation. Our data showed the SKP2 represents a promising tool to identify patients with cervical cancer who have a higher risk of locoregional recurrence after radiotherapy. Targeting SKP2 may serve as a potential radiosensitizer for developing effective therapeutic strategies against cervical cancer.

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“…110 111 DNA damage induced by bacterial genotoxins in vivo has been commonly evaluated using sur-112 rogate markers of the DDR, such as the phosphorylated form of the Histone H2AX (gH2AX) 113 (Rogakou et al, 1999). We have now assessed direct induction of DNA fragmentation in cells 114 isolated from the intestinal mucosa, using an alkaline comet-based assay, considered a gold-115 standard method to detect DNA strand breaks (Pu et al, 2015). Our data showed that a func-116 tional typhoid toxin promotes DNA fragmentation to a greater extent than that observed in mice 117 infected with the control Salmonella (Figure 2A).…”

Section: Infection With Genotoxin-producing Bacteria Induces Senescenmentioning

confidence: 99%

Influence of the microenvironment on the modulation of the host response by the typhoid toxin

Martin

Butter

Paparouna

et al. 2020

Preprint

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30Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA 31 damage response (DDR) in vitro. These toxins are produced by Gram negative bacteria, en-32 riched in the microbiota of Inflammatory Bowel Disease (IBD) and colorectal cancer (CRC) 33 patients. However, their role in infection remains poorly characterized. We have addressed the 34 role of the typhoid toxin in the modulation of the host-microbial interaction in health and dis-35 ease. 36Infection with a genotoxigenic Salmonella protected mice from intestinal inflammation. The 37 toxin-induced DNA damage caused senescence in vivo, which was uncoupled from the inflam-38 matory response, and associated with the maintenance of an anti-inflammatory environment. 39This effect was lost when infection occurred in mice suffering from inflammatory conditions 40 that mimic Ulcerative Colitis, a form of IBD. 41These data highlight a complex context-dependent crosstalk between bacterial genotoxins-in-42 duced DDR and the host immune response, underlining an unexpected role for bacterial geno-43 toxins. 44 45 cumulates in the cytoplasm of senescent cells (Evangelou et al., 2017). As shown in Figures 2C 130 and S3A, we detected a significant increase in GL13 epithelial positive cells in the colon and 131 small intestine of mice infected with the MC1 TT and MC71 TT strains compared to the levels 132 detected in mice infected with the control Salmonella, with the most prominent effect observed 133 in the C57BL/6 mouse strain (cp Figure 2C and Figure S3A). In our model, induction of senes-134 cence was uncoupled from the activation of a pro-inflammatory response (Figures 1, 3 and S2). 135 136The typhoid toxin maintains an anti-inflammatory microenvironment 137 Supplementary Information

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Alkaline Comet Assay for Assessing DNA Damage in Individual Cells

Cited by 87 publications

References 22 publications

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts

Increased expression of SKP2 is an independent predictor of locoregional recurrence in cervical cancer via promoting DNA-damage response after irradiation

Influence of the microenvironment on the modulation of the host response by the typhoid toxin

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