Estimating DNA Repair by Sequential Evaluation of Head and Neck Tumor Radiation Sensitivity Using the Comet Assay

Terris, David J.; Ho, Edith Y.; Ibrahim, Hani; Dorie, Mary J.; Kovacs, Mary S.; Le, Quynh Thu; Koong, Albert C.; Pinto, Harlan A.; Brown, J. Martin

doi:10.1001/archotol.128.6.698

Cited by 19 publications

(13 citation statements)

References 21 publications

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“…This discrepancy, at a first glance, can be explained by the fact that the Comet assay does not only detect radiation-induced DNA damages, but also the nicks and gaps that are generated during base excision repair (BER) and nucleotide excision repair (NER) (Asaeda et al, 1998;Collins and Squires, 1986;de With and Greulich, 1995;Kleinau et al, 1997;Olive and Banath, 1993). The repair time measured in our experiments is slow compared with other reports in the literature (Hamilton et al, 2001;Terris et al, 2002), where half-life times of a few minutes to 30 minutes (for SSBs and base damages) have been reported. A possible reason for this is the high UV-A fluences of 960 kJ/m 2 used for the repair study.…”

Section: Resultscontrasting

confidence: 63%

After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Rapp¹,

Greulich²

2004

Journal of Cell Science

105

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After induction of DNA double-strand breaks (DSB) two repair systems, the error-prone `nonhomologous end joining' (NHEJ) and the more accurate `homologous recombination repair' (HRR) can compete for the same individual DSB site. In the human keratinocyte cell line, HaCaT, we have tested the spatial co-localisation and the temporal sequence of events. We used UV-A (365 nm) as a damaging agent, which can be applied in clearly defined doses and can lead to rare DSBs via propagation of clustered single-strand breaks (SSBs). DNA fragmentation and repair was measured by the Comet assay and persisting DSBs were quantified by the micronucleus assay. Direct DSB detection was performed by immunohistochemical labelling of γ-H2AX, a phosphorylated histone that is assumed to form one foci per DSB. Intra- and inter-pathway interactions were quantified by co-localisation, FRET imaging and by co-immunoprecipitation (Co-IP) of XRCC4, DNA-PK and Ku70 as representatives of NHEJ, Rad51 and Rad52 for HRR and γ-H2AX, Mre11 and Rad50 as representatives of both pathways. In G2 cells, where both systems are available, the temporal sequence after irradiation is: (1) γ-H2AX (2) Mre11 (3) DNA-PK Rad51 (4) XRCC4. That is, the first two proteins involved in both pathways `label' the damaged site and initiate repair, followed by the NHEJ, which is temporally overlapping with HRR activity. Taking all these observations together we suggest that a cell tries to repair DSBs with a combination of both HRR and NHEJ, if available.

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Section: Resultscontrasting

confidence: 63%

After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Rapp¹,

Greulich²

2004

Journal of Cell Science

105

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“…When cells have been extracted from excised human tumours, the alkaline comet assay has successfully identified variations in radiosensitivity in studies of bladder (42), head and neck (14), breast (6) and a range of metastatic tumours (52). These studies further demonstrate how the comet assay could support clinical decision making.…”

Section: Prediction Of Tumour Radiosensitivitymentioning

confidence: 87%

Potential use of the comet assay in the clinical management of cancer

2008

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“…With this modification, cell lines of different SF2 values could be separated using doses above 1.0 Gy. Recently, it has been shown that the halflife of repair of human head and neck tumours is about 4 min, showing that the rate of DNA repair following irradiation of human tumours in situ is of a similar order of magnitude to that in vitro (Terris et al, 2002).…”

Section: Discussionmentioning

confidence: 97%

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation

et al. 2003

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Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0 -6 Gy), a significant correlation was found (r 2 ¼ 0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0 -2 Gy) and radiosensitivity at higher doses (4 -6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.

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Estimating DNA Repair by Sequential Evaluation of Head and Neck Tumor Radiation Sensitivity Using the Comet Assay

Abstract: The comet assay is a promising tool for evaluating radiation sensitivity in individual cells. The rate of DNA repair early after irradiation is consistent with data in the literature.

Cited by 19 publications

References 21 publications

After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Potential use of the comet assay in the clinical management of cancer

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation

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