Measurement of DNA Damage in Individual Cells Using the Single Cell Gel Electrophoresis (Comet) Assay

Hartley, Janet M.; Spanswick, Victoria J.; Hartley, John A.

doi:10.1007/978-1-61779-080-5_25

Cited by 36 publications

(45 citation statements)

References 15 publications

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“…Alkaline single-cell gel electrophoresis (comet) assay DNA strand break repair in cells irradiated with 15 Gy was measured using the comet assay as previously described (25). Data were presented as a percentage of tail moment, i.e., as a percentage of the amount of strand breaks resulting immediately after IR treatment.…”

Section: Irradiation Conditionsmentioning

confidence: 99%

Importance of EGFR/ERCC1 Interaction Following Radiation-Induced DNA Damage

Liccardi

Hartley

Hochhauser

2014

Clinical Cancer Research

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Purpose: The epidermal growth factor receptor (EGFR) plays an important role in cellular response to chemotherapy and radiotherapy through modulation of DNA repair. EGFR activates DNA-dependent protein kinase (DNA-PK) stimulating repair of DNA strand breaks (SB) and interstrand crosslinks (ICL). We investigated the role of EGFR in repair of ionizing radiation (IR)-induced SB independently of DNA-PK.Experimental Design: The EGFR interactome was investigated via mass spectrometry. IR-induced EGFR-ERCC1 binding was validated biochemically and via proximity ligation assay in different cell lines including the M059K and M059J glioma cell lines, proficient and deficient for the expression of DNAPKcs, respectively. EGFR-ERCC1 functional significance following IR-induced SB was investigated in knockdown experiments with the Comet and gH2AX foci assays. The effect of this interaction was tested with EGFR-ERCC1 knockdown in combination with gefitinib and NU7026 using the MTT and apoptosis assays.Results: This study demonstrates that EGFR inhibition further impairs IR-induced DNA repair in cells lacking expression of DNAPKcs or in combination with the DNAPK inhibitor NU7026. Our data suggest a role for EGFR in DNA repair independent of DNAPKcs but dependent on ERCC1. Alkaline comet and gH2AX foci assays in cells depleted of EGFR, ERCC1, or EGFR-ERCC1 expression demonstrated involvement of this interaction in DNA repair. Cellular survival and apoptosis data correlate with levels of residual DNA damage underlying the importance of this complex following SB.Conclusion: These data emphasize the importance of understanding the various mechanisms by which EGFR modulates DNA repair to optimize targeted therapy for patients with cancer.

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Section: Irradiation Conditionsmentioning

confidence: 99%

Importance of EGFR/ERCC1 Interaction Following Radiation-Induced DNA Damage

Liccardi

Hartley

Hochhauser

2014

Clinical Cancer Research

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“…To assess this, a modified version of the Comet assay was used (Hartley et al, 1999). For this, seedlings were grown in either the absence or presence of bleomycin to induce DNA fragmentation.…”

Section: Alt2 Localizes To the Nucleus In Response To Dna Damagementioning

confidence: 99%

“…To measure occurrence of DNA crosslinking, alkaline Comet assay conditions used were as previously described (Hartley et al, 1999). For this assay, DNA breakage was induced by treatment with 0.1 mg/mL bleomycin for 3 d, with a subset of samples also grown in the presence of either 20 mg/mL MMC or 100 mM AlCl 3 .…”

Section: Comet Assaymentioning

confidence: 99%

The Arabidopsis Cell Cycle Checkpoint Regulators TANMEI/ALT2 and ATR Mediate the Active Process of Aluminum-Dependent Root Growth Inhibition

et al. 2012

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Aluminum (Al) toxicity is a global issue that severely limits root growth in acidic soils. Isolation of suppressors of the Arabidopsis thaliana Al-hypersensitive mutant, als3-1, resulted in identification of a cell cycle checkpoint factor, ALUMINUM TOLERANT2 (ALT2), which monitors and responds to DNA damage. ALT2 is required for active stoppage of root growth after Al exposure, because alt2 loss-of-function mutants fail to halt root growth after Al exposure, do not accumulate CyclinB1;1 in the root tip, and fail to force differentiation of the quiescent center. Thus, alt2-1 mutants are highly tolerant of Al levels that are severely inhibitory to the wild type. The alt2-1 allele is a loss-of-function mutation in a protein containing a putative DDB1-binding WD40 motif, previously identified as TANMEI, which is required for assessment of DNA integrity, including monitoring of DNA crosslinks. alt2-1 and atr loss-of-function mutants, the latter of which affects the cell cycle checkpoint ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED, are severely sensitive to DNA crosslinking agents and have increased Al tolerance. These results suggest that Al likely acts as a DNA-damaging agent in vivo and that Al-dependent root growth inhibition, in part, arises from detection of and response to this damage by TANMEI/ALT2 and ATR, both of which actively halt cell cycle progression and force differentiation of the quiescent center.

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“…The plates were then incubated at 37°C and cells were harvested at 0, 4, 8, 16, 24, and 32 hours. The cells were stored at −80°C until assayed for cross-linking using the modified single-cell gel electrophoresis (comet) assay as previously described (19,20).…”

Section: Measurement Of Dna Interstrand Cross-linking In Cells Using mentioning

confidence: 99%

SG2285, a Novel C2-Aryl-Substituted Pyrrolobenzodiazepine Dimer Prodrug That Cross-links DNA and Exerts Highly Potent Antitumor Activity

et al. 2010

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The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand crosslink. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI 50 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD 50 was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development. Cancer Res; 70(17); 6849-58. ©2010 AACR.

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Measurement of DNA Damage in Individual Cells Using the Single Cell Gel Electrophoresis (Comet) Assay

Cited by 36 publications

References 15 publications

Importance of EGFR/ERCC1 Interaction Following Radiation-Induced DNA Damage

Importance of EGFR/ERCC1 Interaction Following Radiation-Induced DNA Damage

The Arabidopsis Cell Cycle Checkpoint Regulators TANMEI/ALT2 and ATR Mediate the Active Process of Aluminum-Dependent Root Growth Inhibition

SG2285, a Novel C2-Aryl-Substituted Pyrrolobenzodiazepine Dimer Prodrug That Cross-links DNA and Exerts Highly Potent Antitumor Activity

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