Skip to main content

Advertisement

SpringerLink
Log in

Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

RH1 is a novel anticancer agent with potent DNA-cross linking activity. RH1 has the potential to be activated within tumors over expressing NQO1, giving maximal antitumour activity with reduced toxicity in normal tissues. RH1 has recently completed a Cancer Research UK sponsored phase I clinical trial at two different centers in the United Kingdom. The comet-X assay was a secondary endpoint in this trial and assay validation was necessary. We describe here this validation process. Whilst it is impossible to cover all variations/conditions of a pharmacodynamic assay, we have strived to evaluate and demonstrate that this assay conforms to the three R’s of validation, that is robustness, reliability and reproducibility.

Methods

K562 and peripheral blood mononuclear cells were treated with either radiation alone, or with a combination of radiation and drug. These samples were then embedded in low melting point agarose and subjected to a modified version of the alkaline single cell gel electrophoresis (Comet) assay, described here as the comet-X assay. Variations in the preparation, electrophoresis, storage and scoring of these samples was investigated. In addition radiation and drug dose response curves were constructed. Finally stability of QC standards was investigated over a 30-month period.

Results

We have demonstrated a linear radiation-dose response in cells up to 20 Gy and drug induced DNA cross-linking up to 50 nM. From the radiation dose response curves we were able to show that the relative inaccuracy measured against a global mean value was less than 25% and the relative (within day) imprecision was less than 30% over all doses. Between day runs produced an intra assay imprecision of 21.2%. Variables involved in the electrophoresis process showed the voltage across all slides in the tank ranged from 3.1 to −2.0 (mV) whilst the current ranged from 0.8–5.5 mA. QC standards were prepared from PBMCs of healthy donors and frozen at −80°C. The stability of these frozen QC standards was measured over a 30-month period. No significant deterioration in any of the control, irradiated or drug treated samples was observed.

Conclusions

The comet-X assay has been shown to be a robust, reliable and reproducible assay. It is ideally suited for the evaluation of the pharmacodynamic effects of DNA cross-linking agents undergoing early clinical trials. Furthermore, this assay may provide valuable data, in conjunction with pharmacokinetics, when measuring toxicity and efficacy as part of the RH1 phase I clinical trial.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298

    Article  PubMed  CAS  Google Scholar 

  2. Fairbairn DW, Olive PL, O’Neill KL (1995) The comet assay: a comprehensive review. Mutat Res 339:37–59

    PubMed  CAS  Google Scholar 

  3. Olive PL, Banath JP (1993) Detection of DNA double-strand breaks through the cell cycle after exposure to X-rays, bleomycin, etoposide and 125IdUrd. Int J Radiat Biol 64:349–358

    Article  PubMed  CAS  Google Scholar 

  4. Moneef MA, Sherwood BT, Bowman KJ, Kockelbergh RC, Symonds RP, Steward WP, Mellon JK, Jones GD (2003) Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity. Br J Cancer 89:2271–2276

    Article  PubMed  CAS  Google Scholar 

  5. Woods JA, Young AJ, Gilmore IT, Morris ABilton RF (1997) Measurement of menadione-mediated DNA damage in human lymphocytes using the comet assay. Free Radic Res 26:113–124

    Article  PubMed  CAS  Google Scholar 

  6. Braybrooke JP, Houlbrook S, Crawley JE, Propper DJ, O’Byrne KJ, Stratford IJ, Harris AL, Shuker DE, Talbot DC (2000) Evaluation of the alkaline comet assay and urinary 3-methyladenine excretion for monitoring DNA damage in melanoma patients treated with dacarbazine and tamoxifen. Cancer Chemother Pharmacol 45:111–119

    Article  PubMed  CAS  Google Scholar 

  7. Tice RR, Strauss GH, Peters WP (1992) High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single cell gel electrophoresis assay. Mutat Res 271:101–113

    PubMed  CAS  Google Scholar 

  8. Vaghef H, Nygren P, Edling C, Bergh J, Hellman B (1997) Alkaline single-cell gel electrophoresis and human biomonitoring for genotoxicity: a pilot study on breast cancer patients undergoing chemotherapy including cyclophosphamide. Mutat Res 395:127–138

    PubMed  CAS  Google Scholar 

  9. 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

    PubMed  CAS  Google Scholar 

  10. Collins A, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S, Fillion L, Panayiotidis M, Raslova K, Vaughan N (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen 30:139–146

    Article  PubMed  CAS  Google Scholar 

  11. Belpaeme K, Cooreman K, Kirsch-Volders M (1998) Development and validation of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish. Mutat Res 415:167–184

    PubMed  CAS  Google Scholar 

  12. De Boeck M, Touil N, De Visscher G, Vande PA, Kirsch-Volders M (2000) Validation and implementation of an internal standard in comet assay analysis. Mutat Res 469:181–197

    PubMed  Google Scholar 

  13. Suggitt M, Fearnley J, Swaine DJ, Volpato M, Phillips RM, Bibby MC, Loadman PM, Anderson D (2003) Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action. Teratog Carcinog Mutagen Suppl 2:13–29

    Article  CAS  Google Scholar 

  14. Ward TH, Butler J, Shahbakhti H, Richards JT (1997) Comet assay studies on the activation of two diaziridinylbenzoquinones in K562 cells. Biochem Pharmacol 53:1115–1121

    Article  PubMed  CAS  Google Scholar 

  15. Friedmann B, Caplin M, Hartley JA, Hochhauser D (2004) Modulation of DNA repair in vitro after treatment with chemotherapeutic agents by the epidermal growth factor receptor inhibitor gefitinib (ZD1839). Clin Cancer Res 10:6476–6486

    Article  PubMed  CAS  Google Scholar 

  16. Dehn DL, Winski SL, Ross D (2004) Development of a new isogenic cell-xenograft system for evaluation of NAD(P)H:quinone oxidoreductase-directed antitumor quinones: evaluation of the activity of RH1. Clin Cancer Res 10:3147–3155

    Article  PubMed  CAS  Google Scholar 

  17. Winski SL, Hargreaves RH, Butler J, Ross D (1998) A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent. Clin Cancer Res 4:3083–3088

    PubMed  CAS  Google Scholar 

  18. Loadman PM, Phillips RM, Lim LE, Bibby MC (2000) Pharmacological properties of a new aziridinylbenzoquinone, RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), in mice. Biochem Pharmacol 59:831–837

    Article  PubMed  CAS  Google Scholar 

  19. Cummings J, Ritchie A, Butler J, Ward TH, Langdon S (2003) Activity profile of the novel aziridinylbenzoquinones MeDZQ and RH1 in human tumour xenografts. Anticancer Res 23:3979–3983

    PubMed  CAS  Google Scholar 

  20. Kim JY, Patterson AV, Stratford IJ, Hendry JH (2004) The importance of DT-diaphorase and hypoxia in the cytotoxicity of RH1 in human breast and non-small cell lung cancer cell lines. Anticancer Drugs 15:71–77

    Article  PubMed  CAS  Google Scholar 

  21. Ward TH, Danson S, McGown AT, Ranson M, Coe NA, Jayson GC, Cummings J, Hargreaves RH, Butler J (2005) Preclinical evaluation of the pharmacodynamic properties of 2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone. Clin Cancer Res 11:2695–2701

    Article  PubMed  CAS  Google Scholar 

  22. Tudor G, Alley M, Nelson CM, Huang R, Covell DG, Gutierrez P, Sausville EA (2005) Cytotoxicity of RH1: NAD(P)H:quinone acceptor oxidoreductase (NQO1)-independent oxidative stress and apoptosis induction. Anticancer Drugs 16:381–391

    Article  PubMed  CAS  Google Scholar 

  23. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221

    Article  PubMed  CAS  Google Scholar 

  24. Moller P (2006) The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures. Basic Clin Pharmacol Toxicol 98:336–345

    Article  PubMed  Google Scholar 

  25. Rosengren NFaB (2001) Directive 2001/20/EC of the European Parliment and of the Council of 4 April 2001 on the approximation of the laws, regulations and administrative provisions of the Member States relating to the implementation of good clinical practice in the conduct of clinical trials on medicinal products for human use. Official J Eur Communities L121:34–44

    Google Scholar 

  26. Rothenberg ML, Carbone DP, Johnson DH (2003) Improving the evaluation of new cancer treatments: challenges and opportunities. Nat Rev Cancer 3:303–309

    Article  PubMed  CAS  Google Scholar 

  27. Woude GF, Kelloff GJ, Ruddon RW, Koo HM, Sigman CC, Barrett JC, Day RW, Dicker AP, Kerbel RS, Parkinson DR, Slichenmyer WJ (2004) Reanalysis of cancer drugs: old drugs, new tricks. Clin Cancer Res 10:3897–3907

    Article  PubMed  Google Scholar 

  28. Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A (2000) Bioanalytical method validation–a revisit with a decade of progress. Pharm Res 17:1551–1557

    Article  PubMed  CAS  Google Scholar 

  29. Miller KJ, Bowsher RR, Celniker A, Gibbons J, Gupta S, Lee JW, Swanson SJ, Smith WC, Weiner RS (2001) Workshop on bioanalytical methods validation for macromolecules: summary report. Pharm Res 18:1373–1383

    Article  PubMed  CAS  Google Scholar 

  30. Collins AR, Dusinska M, Gedik CM, Stetina R (1996) Oxidative damage to DNA: do we have a reliable biomarker? Environ Health Perspect 104(suppl 3):465–469

    Google Scholar 

  31. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat Res 122:86–94

    Article  PubMed  CAS  Google Scholar 

  32. Oppitz U, Schulte S, Stopper H, Baier K, Muller M, Wulf J, Schakowski R, Flentje M (2002) In vitro radiosensitivity measured in lymphocytes and fibroblasts by colony formation and comet assay: comparison with clinical acute reactions to radiotherapy in breast cancer patients. Int J Radiat Biol 78:611–616

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported and funded by Cancer Research UK.

Author information

Authors and Affiliations

  1. Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Paterson Laboratories, Wilmslow Road, Manchester, M20 4BX, UK

    S. Danson, O. Denneny, J. Cummings & T. H. Ward

  2. Cancer Research UK Department of Medical Oncology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

    M. Ranson

Authors
  1. S. Danson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Ranson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. O. Denneny
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Cummings
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. H. Ward
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. H. Ward.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Danson, S., Ranson, M., Denneny, O. et al. Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial. Cancer Chemother Pharmacol 60, 851–861 (2007). https://doi.org/10.1007/s00280-007-0432-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-007-0432-9

Keywords

Search

Navigation