Abstract
The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials.
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References
Hartley JA, Spanswick VJ, Brooks N, Clingen PH, McHugh PJ, Hochhauser D, Pedley RB, Kelland LR, Alley MC, Schultz R, Hollingshead MG, Sausville EA, Gregson SJ, Howard PW, Thurston DE (2004) SJG-136 (NSC 694501) A novel rationally designed DNA minor groove interstrand cross-linking agent with potent and broad spectrum antitumour activity. Part 1: Cellular pharmacology, in vitro and initial in vivo antitumour activity. Cancer Res 64:6693-6699
Sarker D, Anderson D, Spanswick VJ, Davies S, Agarwal R, Aitken G, Kerr D, Hartley JA, Judson I, Middleton MR (2008) Preliminary results of a Cancer Research UK phase I trial combining the dinitrobenzamide prodrug CB1954 (tretazicar) and the NQO2 substrate EP-0152R (caricotamide) intraveneously (IV) every 3 weeks. J Clin Oncol 26(May 20 suppl):2505
Mayer A, Francis RJ, Sharma SK, Tolner B, Springer CJ, Martin J, Boxer GM, Bell J, Green AJ, Hartley JA, Cruickshank C, Wren J, Chester KA, Begent RH (2006) A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug. Clin Cancer Res 12:6509-6516
Hedley D, Ogilvie L, Springer C (2007) Carboxypeptidase-G2-based gene-directed enzyme-prodrug therapy: a new weapon in the GDEPT armoury. Nat Rev Cancer 7:870-879
Palmer DH, Mautner V, Mirza D, Oliff S, Gerritsen W, van der Sijp JR, Hubscher S, Reynolds G, Bonney S, Rajaratnam R, Hull D, Horne M, Ellis J, Mountain A, Hill S, Harris PA, Searle PF, Young LS, James ND, Kerr DJ (2004) Virus-directed enzyme prodrug therapy: intratumoural administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer. J Clin Oncol 22:1535-1537
McHugh PJ, Spanswick VJ, Hartley JA (2001) Repair of DNA interstrand cross-links: molecular mechanisms and clinical relevance. Lancet Oncol 2:483-490
Spanswick VJ, Craddock C, Sekhar M, Mahendra P, Shankaranarayana P, Hughes RG, Hochhauser D, Hartley JA (2002) Repair of DNA interstrand cross-links as a mechanism of clinical resistance to melphalan in multiple myeloma. Blood 100:224-229
Wynne P, Newton C, Ledermann JA, Olaitan A, Mould TA, Hartley JA (2007) Enhanced repair of DNA interstrand cross-linking in ovarian cancer cells from patients following treatment with platinum-based chemotherapy. Br J Cancer 97:927-933
Kohn KW, Ewig RAG, Erickson LC, Zwelling LA (1981) Measurement of strand breaks and cross-links by alkaline elution. In: Friedberg EC, Hanawalt PC (eds) DNA repair. Dekker, New York, pp 379-408
Hartley JM, Spanswick VJ, Gander M, Giacomini G, Whelan J, Souhami RL, Hartley JA (1999) Measurement of DNA cross-linking in patients on ifosfamide therapy using the single cell gel electrophoresis (Comet) assay. Clin Cancer Res 5:507-512
Ostling O, Johanson KL (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291-298
Webley SD, Francis RJ, Pedley RB, Sharma SK, Begent RH, Hartley JA, Hochhauser D (2001) Measurement of the critical DNA lesions produced by antibody-directed enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material. Br J Cancer 84:1671-1676
Corrie PG, Shaw J, Spanswick VJ, Sehmbi R, Jonson A, Mayer A, Bulusu R, Hartley JA, Cree I (2005) Phase I trial combining gemcitabine and treosulfan in advanced cutaneous and uveal melanoma patients. Br J Cancer 92:1997-2003
Lederman J, Gabra H, Jayson GC, Spanswick VJ, Rustin GJ, Jital M, James LE, Hartley JA (2007) Combination chemotherapy with carboplatin and gemcitabine in patients in platinum-resistant ovarian cancer chemotherapy - a phase II study demonstrating inhibition of DNA cross link repair by gemcitabine. Eur J Cancer Suppl 5:320
Puzanov I, Lee W, Berlin JD, Calcutt MW, Hachey DL, Vermeulen WL, Spanswick VJ, Hartley JA, Chen A, Rothenburg ML (2008) Final results of phase I and pharmacokinetic trial of SJG136 administered on a daily x3 schedule. J Clin Oncol 26, May 20 suppl., abstract 2504
Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumour and normal cells measured using the “comet” assay. Radiat Res 122:86-94
Klaude M, Erikkson S, Nygren J, Ahnstrom G (1996) The comet assay: mechanisms and technical considerations. Mutat Res 363:89-96
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Spanswick, V.J., Hartley, J.M., Hartley, J.A. (2010). Measurement of DNA Interstrand Crosslinking in Individual Cells Using the Single Cell Gel Electrophoresis (Comet) Assay. In: Fox, K. (eds) Drug-DNA Interaction Protocols. Methods in Molecular Biology, vol 613. Humana Press. https://doi.org/10.1007/978-1-60327-418-0_17
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DOI: https://doi.org/10.1007/978-1-60327-418-0_17
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