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Sensitivity and Resistance to Therapy

Amifostine (WR-2721) selective protection against melphalan genotoxicity

Leukemia volume 14, pages 1642–1651 (2000)Cite this article

Abstract

Amifostine (WR-2721) is an aminothiol compound dephosphorylated at the tissue site by alkaline phosphatase to the active metabolite, which is able to inactivate electrophilic substances and scavenge free radicals. Amifostine effects against melphalan-induced DNA strand breaks were studied in normal human white blood cells (WBC) and K562 leukemic cells using the single cell gel electrophoresis (SCGE) or Comet assay, a reported method for measuring DNA damage in individual cells. Prior to treatment (1 h, 37°C) with increasing doses of melphalan, with or without S9, the cells were treated (15 min, 37°C) with a control medium or amifostine (3 mg/ml). Treatment of normal and leukemic cells with melphalan induced a dose-dependent ‘comet formation’. Melphalan-induced DNA damage follows a normal distribution in WBC. On the other hand, in K562, a significant proportion of undamaged cells remains even with doses at which mean DNA damage is serious. Pre-treatment with WR-2721 protects WBC, but not K562, against the genotoxic effect of melphalan. Amifostine might even strengthen the action of the antiblastic drug against K562 cells. S9 addition appears to enhance melphalan effectiveness. SCGE appears as a suitable primary screening method for in vitro and in vivostudies on drug–DNA interactions and their modulations by endogenous/exogenous factors. Leukemia (2000) 14, 1642–1651.

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References

  1. Glover DJ, Fox KR, Weiler C, Kligerman MM, Turrisi A, Glick JH . Clinical trials of WR-2721 prior to alkylating agent chemotherapy and radiotherapy Pharmacol Ther 1988 39: 3–7

    Article  CAS  Google Scholar 

  2. Smoluk GD, Fahey RC, Calabro-Jones PM, Aguilera JA, Ward JF . Radioprotection of cells in culture by WR-2721 and derivatives: form of the drug responsible for protection Cancer Res 1988 48: 3641–3647

    CAS  PubMed  Google Scholar 

  3. Littlefield LG, Joiner EE, Colyer SP, Frome EL . Concentration dependent protection against X-ray induced chromosome aberrations in human lymphocytes by the aminothiol WR-1065 Radiat Res 1993 133: 88–94

    Article  CAS  Google Scholar 

  4. Clark LS, Albertini RJ, Nicklas JA . The aminothiol WR-1065 protects T lymphocytes from ionizing radiation-induced deletions of the HPRT gene Cancer Epidemiol Biomarkers Prev 1997 6: 1033–1037

    CAS  PubMed  Google Scholar 

  5. Wasserman TH, Phillips TL, Ross G, Kane LJ . Differential protection against cytotoxic chemotherapeutic effects on bone marrow CFUs by WR-2721 Cancer Clin Trials 1981 4: 3–6

    CAS  PubMed  Google Scholar 

  6. Nagy B, Grdina DJ . Protective effects of 2-((Aminopropyl) Amino) ethanethiol against bleomycin and nitrogen mustard-induced mutagenicity in V-79 cells Int J Radiat Oncol Biol Phys 1986 12: 1475–1478

    Article  CAS  Google Scholar 

  7. van der Wilt CL, van Laar JAM, Gyergyay F, Smid K, Peters GJ . Biochemical modification of toxicity and the anti-tumour effect of 5-fluorouracil and cisplatinum by WR-2721 in mice Eur J Cancer 1992 28A: 2017–2024

    Article  CAS  Google Scholar 

  8. Lenoble M . Amifostine: applications actuelles et perspectives en cytoprotection Bull Cancer 1996 83: 773–787

    CAS  PubMed  Google Scholar 

  9. Foster-Nora JA, Siden R . Amifostine for protection from antineoplastic drug toxicity Am J Health Syst Pharm 1997 54: 787–800

    CAS  PubMed  Google Scholar 

  10. Taylor CW, Wang LM, List AF, Fernandes D, Paine-Murrieta GD, Johnson CS, Capizzi RL . Amifostine protects normal tissue from paclitaxel toxicity while cytotoxicity against tumour cells is maintained Eur J Cancer 1997 33: 1693–1698

    Article  CAS  Google Scholar 

  11. Chvetzoff G, Bonnotte B, Chauffert B . Anticancer chemotherapy. Prevention of toxicity Presse Med 1998 27: 2106–2112

    CAS  PubMed  Google Scholar 

  12. Kurbacher CM, Mallmann PK . Chemoprotection in anticancer therapy: the emerging role of Amifostine (WR-2721) Anticancer Res 1998 18: 2203–2210

    CAS  PubMed  Google Scholar 

  13. Pierelli L, Scambia G, Fattorossi A, Bonanno G, Battaglia A, Perillo A, Menichella G, Panici PB, Leone G, Mancuso S . In vitro effect of amifostine on haematopoietic progenitors exposed to carboplatin and non-alkylating antineoplatic drugs: haematoprotection acts as a drug-specific progenitor rescue Br J Cancer 1998 78: 1024–1029

    Article  CAS  Google Scholar 

  14. Millar JL, McElwain TJ, Clutterbuck RD, Wist EA . The modification of melphalan toxicity in tumor bearing mice by 1-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR-2721) Am J Clin Oncol 1982 5: 321–328

    Article  CAS  Google Scholar 

  15. Calabro-Jones PM, Fahey RC, Smoluk GD, Ward JF . Alkaline phosphatase promotes radioprotection and accumulation of WR-1065 in V79-171 cells incubated in medium containing WR-2721 Int J Radiat Biol Relat Stud Phys Chem Med 1985 47: 23–27

    Article  CAS  Google Scholar 

  16. Peiper RO, Erickson LC . DNA adenine adducts induced by nitrogen mustards and their role in transcription termination in vitro Carcinogenesis 1990 11: 1739–1746

    Article  Google Scholar 

  17. Adams SP, Laws GM, Storer RD, DeLuca JG, Nichols WW . Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms Mutat Res 1996 368: 235–248

    Article  CAS  Google Scholar 

  18. Bauer GB, Povirk LF . Specificity and kinetics of interstrand and intrastrand bifunctional alkylation by nitrogen mustards at a G–G–C sequence Nucleic Acids Res 1997 25: 1211–1218

    Article  CAS  Google Scholar 

  19. Benedict WF, Baker MS, Haroun L, Choi E, Ames BN . Mutagenicity of cancer chemotherapeutic agents in the Salmonella/microsome test Cancer Res 1977 37: 2209–2213

    CAS  PubMed  Google Scholar 

  20. Sanderson BJ, Shield AJ . Mutagenic damage to mammalian cells by therapeutic alkylating agents Mutat Res 1996 355: 41–57

    Article  Google Scholar 

  21. Russel LB, Hunsicker PR, Shelby MD . Melphalan, a second chemical for which specific-locus mutation induction in the mouse is maximum in early spermatids Mutat Res 1992 283: 151–158

    Article  Google Scholar 

  22. Generoso WM, Witt KL, Cain KT, Hughes L, Cacheiro NLA, Lockhart AC, Shelby MD . Dominant lethal and heritable translocation tests with chlorambucil and melphalan in male mice Mutat Res 1995 345: 167–180

    Article  CAS  Google Scholar 

  23. Clement JJ, Johnson RK . Influence of WR 2721 on the efficacy of radiotherapy and chemotherapy of murine tumors Int J Radiat Oncol Biol Phys 1982 8: 539–542

    Article  CAS  Google Scholar 

  24. Wist EA . Effect of radioprotector WR 2721 on the response of metastatic Lewis lung carcinoma colonies to alkylating agents Acta Radiol Oncol 1985 24: 259–262

    Article  CAS  Google Scholar 

  25. Adamson PC, Balis FM, Belasco JE, Lange B, Berg SL, Blaney SM, Craig C, Poplack DG . A phase I trial of amifostine (WR-2721) and melphalan in children with refractory cancer Cancer Res 1995 55: 4069–4072

    CAS  PubMed  Google Scholar 

  26. Singh NP, McCoy MT, Tice RR, Schneider EL . A simple technique for quantitation of low levels of DNA damage in individual cells Exp Cell Res 1988 175: 184–191

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Rydberg B, Johanson KJ . Estimation of DNA strand breaks in single mammalian cells. In: Hanawalt EC, Friedberg EC, Fox CF (eds) DNA Repair Mechanisms Academic Press: New York 1978 465–468

    Chapter  Google Scholar 

  29. Kohn KW, Ewig RAG, Erikson LC, Zwelling LA . Measurement of strand breaks and crosslinking by alkaline elution. In: Friedberg EC, Hanawalt PC (eds) DNA Repair, a Laboratory Manual of Research Procedures Marcel Dekker: New York 1981 pp 379–401

    Google Scholar 

  30. Olive PL, Chan APS, Cu CS . Comparison between the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks and cross-links Cancer Res 1988 48: 6444–6449

    CAS  PubMed  Google Scholar 

  31. Sasaki YF, Fujikawa K, Ishida K, Kawamura N, Nishikawa Y, Ohta S, Satoh M, Madarame H, Ueno S, Susa N, Matsusaka N, Tsuda S . The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and US NTP Mutat Res 1999 440: 1–18

    Article  CAS  Google Scholar 

  32. Albertini RJ, Nicklas JA, O’Neill JP . Future research directions for evaluating human genetic and cancer risk from environmental exposures Environ Health Perspect 1996 104: 503–510

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Kirsh-Volders M, Lauwerys R, Lison D . Evaluation of three methods for the detection of DNA single strand breaks in human lymphocytes: alkaline elution, nick translation and single-cell gel electrophoresis J Toxicol Environ Health 1996 47: 409–422

    Article  Google Scholar 

  34. Mc Kelvey-Martin VJ, Green MHL, Schmezer P, Pool-Zobel BL, De Meo MP, Collins A . The single cell gel electrophoresis assay (Comet assay): a European review Mutat Res 1993 288: 47–63

    Article  CAS  Google Scholar 

  35. Mattii L, Barale R, Petrini M . Use of the comet test in the evaluation of multidrug resistance of human cell lines Leukemia 1998 12: 627–632

    Article  CAS  Google Scholar 

  36. Godard T, Deslandes E, Lebailly P, Vigreux C, Sichel F, Poul JM, Gauduchon P . Early detection of staurosporine induced apoptosis by comet and annexin V assays Histochem Cell Biol 1999 112: 155–161

    Article  CAS  Google Scholar 

  37. Qiao L, Hanif R, Sphicas E, Shiff SJ, Rigas B . Effect of aspirin on induction of apoptosis in HT-29 human colon adenocarcinoma cells Biochem Pharmacol 1998 55: 53–64

    Article  CAS  Google Scholar 

  38. Aravindan GR, Bjordahl J, Jost LK, Evenson DP . Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis Exp Cell Res 1997 236: 231–237

    Article  CAS  Google Scholar 

  39. Wagner ED, Rayburn AL, Anderson D, Plewa MJ . Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells Mutagenesis 1998 13: 81–84

    Article  CAS  Google Scholar 

  40. Godard T, Deslandes E, Lebailly P, Vigreux C, Poulain L, Sichel F, Poul JM, Gauduchon P . Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis Cytometry 1999 36: 117–122

    Article  CAS  Google Scholar 

  41. Olive PL, Frazer G, Banath JP . Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay Radiat Res 1993 136: 130–136

    Article  CAS  Google Scholar 

  42. Hartmann A, Speit G . Genotoxic effects of chemicals in the single cell gel (SCG) test with human blood cells in relation to the induction of sister-chromatid exchange (SCE) Mutat Res 1995 345: 49–56

    Article  Google Scholar 

  43. Frenzilli G, Betti C, Davini T, Desideri M, Fornai E, Giannessi L, Maggiorelli F, Paoletti P, Barale R . Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis Mutat Res 1997 375: 117–123

    Article  CAS  Google Scholar 

  44. Van Goethem F, Lison D, Kirsch-Volders M . Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt-tungsten carbide Mutat Res 1997 392: 31–43

    Article  CAS  Google Scholar 

  45. Malcolmson AM, Davies G, Hanson JA, Deeley JO, Gaffney CC, McGregor AD, Kerby IJ . Determination of radiation-induced damage in lymphocytes using the micronucleus and microgel electrophoresis ‘Comet’ assays Eur J Cancer 1995 31A: 2320–2323

    Article  CAS  Google Scholar 

  46. Carrano AV, Natarajan AT . Considerations for population monitoring using cytogenetic techniques Mutat Res 1988 204: 379–406

    Article  CAS  Google Scholar 

  47. Slamenová D, Gabelová A, Ruzeková L, Chalupa I, Horvathová E, Farkasová T, Bozsakyová E, Stetina R . Detection of MNNG-induced DNA lesions in mammalian cells; validation of Comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations Mutat Res 1997 383: 243–252

    Article  Google Scholar 

  48. Cebulska-Wasilewska A, Nowak D, Niedzwiedz W, Anderson D . Correlations between DNA and cytogenetic damage induced after chemical treatment and radiation Mutat Res 1998 421: 83–91

    Article  CAS  Google Scholar 

  49. Maron DM, Ames BN . Revised method for the Salmonella mutagenicity test Mutat Res 1983 113: 173–215

    Article  CAS  Google Scholar 

  50. Gorczyca W, Bigman K, Mittelman A . Induction of DNA strand breaks associated apoptosis during treatment of leukemia Leukemia 1993 7: 659–670

    CAS  PubMed  Google Scholar 

  51. Shpall EJ, Stemmer SM, Hami L, Franklin WA, Shaw L, Bonner HS, Bearman SI, Peters WP, Bast RC, McCulloch W . Amifostine (WR-2721) shortens the engraftment period of 4-hydroxycyclophosphamide-purged bone marrow in breast cancer patients receiving high-dose chemotherapy with autologous bone marrow support Blood 1994 83: 3132–3137

    CAS  PubMed  Google Scholar 

  52. Douay L, Hu C, Giarratana MC, Bouchet S, Conlon J, Capizzi RL, Gorin NC . Amifostine improves the antileukemic therapeutic index of mafosfamide: implications for bone marrow purging Blood 1995 86: 2849–2855

    CAS  PubMed  Google Scholar 

  53. Miyamae Y, Zaiken K, Ohara K, Mine Y, Sasaki YF . Detection of DNA lesions induced by chemical mutagens by the single cell gel electrophoresis (Comet) assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens Mutat Res 1998 415: 229–235

    Article  CAS  Google Scholar 

  54. Rappaport SM, Yeowell-O’Connell K . Protein adducts as dosimeters of human exposure to styrene, styrene-7,8-oxide, and benzene Toxicol Lett 1999 108: 117–126

    Article  CAS  Google Scholar 

  55. Holz O, Jörres R, Kästner A, Magnussen H . Differences in basal and induced DNA single-strand breaks between human peripheral monocytes and lymphocytes Mutat Res 1995 332: 55–62

    Article  CAS  Google Scholar 

  56. Digue L, Orsière T, De Méo M, Mattéi MG, Depetris D, Duffaud F, Favre R, Botta A . Evaluation of the genotoxic activity of Paclitaxel by the in vitro micronucleus test in combination with fluorescent in situ hybridization of a DNA centromeric probe and the alkaline single cell gel electrophoresis technique (Comet Assay) in human T-lymphocytes Environ Mol Mutagen 1999 34: 269–278

    Article  CAS  Google Scholar 

  57. Olive PL . DNA damage and repair in individual cells: applications of comet assay in radiobiology Int J Radiat Biol 1999 75: 395–405

    Article  CAS  Google Scholar 

  58. Aoyama T, Gonzales FJ, Gelboin HV . Human cDNA-expressed cytochrome P450 IA2: mutagen activation and substrate specificity Mol Carcinog 1989 2: 192–198

    Article  CAS  Google Scholar 

  59. Gut I, Nedelcheva V, Stopka P, Vodicka P, Galboin HV, Ingelman-Sundberg M . The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives Arch Toxicol 1996 71: 45–56

    Article  CAS  Google Scholar 

  60. Ono S, Hatanaka T, Hotta H, Satoh T, Gonzales FJ, Tsutsui M . Specificity of substrate and inhibitor probes for cytochrome P450s: evolution of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes Xenobiotica 1996 26: 681–693

    Article  CAS  Google Scholar 

  61. Baron JM, Zwadlo-Klarwasser G, Jugert F, Hamann W, Rübben A, Mukhtar H, Merk HF . Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets Biochem Pharmacol 1998 56: 1105–1110

    Article  CAS  Google Scholar 

  62. Olive PL, Banáth JP, Durand RE . Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the ‘Comet’ assay Radiat Res 1990 122: 86–94

    Article  CAS  Google Scholar 

  63. Thomale J, Seiler F, Müller MR, Seeber S, Rajewsky MF . Repair of O6-alkylguanines in nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level Br J Cancer 1994 69: 698–705

    Article  CAS  Google Scholar 

  64. Olive PL, Banáth JP, Durand RE . Detection of subpopulations resistant to DNA-damaging agents in spheroids and murine tumours Mutat Res 1997 375: 157–165

    Article  CAS  Google Scholar 

  65. Šalagovič J, Maes A, Van Gorp U, Verschaeve L, Kalina I . The cell cycle positions influence DNA migration as measured with the alkaline comet assay in stimulated human lymphocytes Folia Biol (Praha) 1997 43: 79–82

    Google Scholar 

  66. Villani P, Altavista PL, Castaldi L, Leter G, Cordelli E . Analysis of DNA oxidative damage related to cell proliferation Mutat Res 2000 464: 229–237

    Article  CAS  Google Scholar 

  67. Fortini P, Raspaglio G, Falchi M, Dogliotti E . Analysis of DNA alkylation damage and repair in mammalian cells by the Comet assay Mutagenesis 1996 11: 169–175

    Article  CAS  Google Scholar 

  68. Hartmann A, Pfuhler S, Dennog C, Germadnik D, Pilger A, Speit G . Exercise-induced DNA effects in human leukocytes are not accompanied by increased formation of 8-hydroxy-2′-deoxyguanosine or induction of micronuclei Free Rad Biol Med 1998 24: 245–251

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by grants from ‘Ministero dell’Università e della Ricerca Scientifica e Tecnologica’ (MURST – 40% & 60%), and ‘Associazione Italiana per la Ricerca sul Cancro’ (AIRC, Milano). ER is a recipient of an AIRC fellowship.

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  1. Istituto di Genetica Università di Parma, Italy

    A Buschini, E Anceschi, P Poli & C Rossi

  2. Dipartimento di Ematologia, Università di Parma, Italy

    C Carlo-Stella, E Regazzi & V Rizzoli

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Buschini, A., Anceschi, E., Carlo-Stella, C. et al. Amifostine (WR-2721) selective protection against melphalan genotoxicity. Leukemia 14, 1642–1651 (2000). https://doi.org/10.1038/sj.leu.2401877

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