Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action

M Suggitt, J Fearnley, D J Swaine, M Volpato, R M Phillips, M C Bibby, P M Loadman, D Anderson

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.

LanguageEnglish
Pages13-29
Number of pages17
JournalTeratogenesis Carcinogenesis and Mutagenesis
Volume23
Issue numberS2
DOIs
Publication statusPublished - 2003
Externally publishedYes

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Comet Assay
Flow cytometry
Mutagens
DNA Repair
Assays
Flow Cytometry
Repair
Cells
DNA
Mitomycin
Tumors
Neoplasms
Ethyl Methanesulfonate
Lymphocytes
DNA Damage
Single-Stranded DNA Breaks
Tumor Cell Line
S Phase
Cell Cycle
Adenocarcinoma

Cite this

Suggitt, M ; Fearnley, J ; Swaine, D J ; Volpato, M ; Phillips, R M ; Bibby, M C ; Loadman, P M ; Anderson, D. / Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action. In: Teratogenesis Carcinogenesis and Mutagenesis. 2003 ; Vol. 23, No. S2. pp. 13-29.
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abstract = "In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.",
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Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action. / Suggitt, M; Fearnley, J; Swaine, D J; Volpato, M; Phillips, R M; Bibby, M C; Loadman, P M; Anderson, D.

In: Teratogenesis Carcinogenesis and Mutagenesis, Vol. 23, No. S2, 2003, p. 13-29.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action

AU - Suggitt, M

AU - Fearnley, J

AU - Swaine, D J

AU - Volpato, M

AU - Phillips, R M

AU - Bibby, M C

AU - Loadman, P M

AU - Anderson, D

N1 - Copyright 2003 Wiley-Liss, Inc.

PY - 2003

Y1 - 2003

N2 - In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.

AB - In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.

KW - Cell Cycle/drug effects

KW - Cells, Cultured

KW - Comet Assay/methods

KW - Cross-Linking Reagents/toxicity

KW - DNA/drug effects

KW - DNA Damage

KW - DNA Repair

KW - DNA, Neoplasm/drug effects

KW - Ethyl Methanesulfonate/toxicity

KW - Flow Cytometry/methods

KW - Humans

KW - Lymphocytes/drug effects

KW - Micronuclei, Chromosome-Defective/drug effects

KW - Mitomycin/toxicity

KW - Mutagens/toxicity

KW - Neoplasms/genetics

U2 - 10.1002/tcm.10077

DO - 10.1002/tcm.10077

M3 - Article

VL - 23

SP - 13

EP - 29

JO - Teratogenesis Carcinogenesis and Mutagenesis

T2 - Teratogenesis Carcinogenesis and Mutagenesis

JF - Teratogenesis Carcinogenesis and Mutagenesis

SN - 1542-9733

IS - S2

ER -