Measurement of Drug-Induced DNA Interstrand Crosslinking Using the Single-Cell Gel Electrophoresis (Comet) Assay

Spanswick, Victoria J.; Hartley, Janet M.; Ward, Timothy H; Hartley, John A.

doi:10.1385/1-59259-687-8:143

Cited by 37 publications

(48 citation statements)

References 16 publications

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“…The modification of the single-cell gel electrophoresis (comet) assay has been fully validated for the detection of ICLs, including those produced by platinum-based agents (29,39). It is now increasingly used to monitor ICL formation and unhooking in clinical samples (30,40) as a pharmacodynamic end point in clinical trials (41,42), and following ex vivo treatment (39,43).…”

Section: Discussionmentioning

confidence: 99%

“…Alkaline Single-Cell Gel Electrophoresis (Comet) Measurement of DNA ICLs was achieved using a modification of the comet assay as previously described (29,30). Immediately before analysis, cells were irradiated (12.5 Gy) to deliver a fixed number of random DNA strand breaks.…”

Section: Methodsmentioning

confidence: 99%

“…The comet assay, with modifications to allow the quantitation of ICL formation and repair (29), was used. Results are presented in Fig.…”

Section: Effect Of Her2 Inhibition On Dna Repairmentioning

confidence: 99%

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Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents

Boone

Bhosle

Tilby

et al. 2009

Molecular Cancer Therapeutics

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HER2 (ErbB2) is overexpressed in up to 30% of human breast cancers. Preclinical and clinical studies suggest synergy between some chemotherapeutic agents and the humanized anti-HER2 antibody trastuzumab (Herceptin). This study investigated the effects of etoposide and cisplatin on the repair of DNA damage in breast cancer cell lines. We examined the potential significance of HER2 nuclear expression in DNA repair. MCF-7, SK-BR-3, and MDA-MB-453 cells were treated with cisplatin and etoposide. Repair of DNA interstrand crosslinks (ICL) and strand breaks, following incubation with cisplatin and etoposide, respectively, were quantitated by the single-cell gel electrophoresis (comet) assay. Intrastrand crosslinks produced by cisplatin were assessed by ELISA. The effects of trastuzumab were measured in combination with these drugs. Similar experiments were done using HER2-negative MDA-MB-468 cells transfected with HER2 and a construct lacking the nuclear localization sequence. Incubation of breast cancer cell lines with trastuzumab delayed the repair of ICL produced by cisplatin. There were no effects on the repair of intrastrand crosslinks produced by cisplatin, or repair of DNA strand breaks following etoposide treatment. Transfection of HER2 into MDA-MB-468 cells inhibited the repair of cisplatin-induced ICL, whereas transfection of a HER2 construct lacking the nuclear localization sequence did not affect DNA repair. These results indicate that HER2 expression modulates the repair of specific DNA lesions produced by chemotherapy. The effect on ICL repair requires nuclear expression of HER2. Understanding the mechanisms of interaction between DNA-interacting agents and HER2 inhibitors will inform the design of clinical trials and optimize the therapeutic effects of these combinations.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents

Boone

Bhosle

Tilby

et al. 2009

Molecular Cancer Therapeutics

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“…The comet assay was performed under alkaline conditions as described with modifications (Spanswick et al, 1998). Briefly, 2.4 × 10 4 cells were embedded in 1% agarose on a microscope slide then lysed in high salt buffer containing 2.5 M NaCl, 100 mm disodium EDTA, 10 mM Tris-HCl (pH 10.5), 1% triton X-100.…”

Section: Measurement Of Interstrand Crosslinks Using the Comet Assaymentioning

confidence: 99%

Measurement of the critical DNA lesions produced by antibody-directed enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material

et al. 2001

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Summary An antibody-directed enzyme prodrug therapy (ADEPT) system against CEA-positive tumours is currently in phase I clinical trials. It consists of a prodrug, 4-[N,N-bis(2-iodoethyl) amino] phenoxycarbonyl L-glutamic acid (ZD2767P) and a conjugate of the F(ab') 2 anti-CEA antibody A5B7 and the bacterial enzyme carboxypeptidase G2 (CPG2). ZD2767P is converted by antibody-targeted CPG2 into an active bifunctional alkylating drug (ZD2767) at the tumour site. The IC 50 value of the prodrug against the human colorectal tumour LS174T cell line was 55 ± 9 µM following a 1 h exposure. In contrast, co-incubation of ZD2767P with CPG2 resulted in 229-fold increase in activity. Using a modified comet assay, DNA interstrand cross links (ISC) were detected within 1 h of ZD2767P + CPG2 treatment and were repaired by 24 h. A clear dose-response was seen between the level of ISC, growth inhibition and ZD2767 concentration. Administration of a therapeutic dose of ZD2767P 72 h after the F(ab′) 2 A5B7 conjugate to mice bearing LS147T xenografts resulted in extensive ISC in the tumour after 1 h; repair was seen at 24 h. Tumour biopsies and peripheral lymphocytes were studied in 5 patients on the ADEPT phase I clinical trial. In 4 patients no ISC were detected. These patients also demonstrated poor localization of conjugate and no tumour response was seen. However a significant level of ISC was detected in one tumour biopsy, which also showed evidence of conjugate localization and clinical response. These studies demonstrate the application of the comet assay in the measurement of ISC in vitro and in clinical material and confirm that activation of ZD2767P results in the formation of DNA crosslinks.

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“…Details of the Comet assay are described elsewhere (Spanswick et al, 1999). All procedures were carried out on ice and in subdued lighting.…”

Section: Drug-induced Dna Damagementioning

confidence: 99%

Phase I trial combining gemcitabine and treosulfan in advanced cutaneous and uveal melanoma patients

et al. 2005

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Gemcitabine and treosulfan are DNA-damaging agents. Preclinical studies suggest that synergism exists when melanoma cells are exposed to both drugs concurrently. We conducted a phase I trial in advanced melanoma patients to determine the optimal dose of gemcitabine to be combined with treosulfan. Cohorts of three patients received increasing doses of gemcitabine, commencing at 0.5 g m À2 , followed by a fixed dose of 5.0 g m À2 treosulfan on day one of a 21-day cycle. Patients alternately received a first cycle of single-agent gemcitabine or treosulfan before subsequent cycles of both drugs. Peripheral blood lymphocytes were collected in cycles 1 and 2 at various time points until 48 h post-treatment. The single-cell gel electrophoresis (Comet) assay was used to measure chemotherapy-induced DNA damage. A total of 27 patients were enrolled, no objective responses were observed, but two uveal melanoma patients had minor responses. Dose-limiting myelosuppression was reached at 3.0 g m À2 gemcitabine. DNA single-strand breaks were detected 4 h post-gemcitabine, repaired by 24 h. DNA interstrand crosslinks were detected 4 h post-treosulfan, fully removed by 48 h. Following combination chemotherapy, treosulfan-induced DNA crosslinks persisted, still being detectable 48 h post-treatment, supporting the hypothesis that gemcitabine potentiates treosulfan-induced cytotoxicity. The recommended regimen for further study is 2.5 g m À2 gemcitabine combined with 5.0 g m À2 treosulfan.

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Measurement of Drug-Induced DNA Interstrand Crosslinking Using the Single-Cell Gel Electrophoresis (Comet) Assay

Cited by 37 publications

References 16 publications

Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents

Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents

Measurement of the critical DNA lesions produced by antibody-directed enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material

Phase I trial combining gemcitabine and treosulfan in advanced cutaneous and uveal melanoma patients

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