Modulation of Ara-C Cytotoxicity by Coadministration with Antisignalling Drugs in HL60 and Ara-C-Resistant HL60/Ara-C Cells

  • A. Freund
  • A. Gescher
  • J. Boos
Conference paper
Part of the Haematology and Blood Transfusion / Hämatologie und Bluttransfusion book series (HAEMATOLOGY, volume 39)


Ara-C (cytosine arabinoside) is one of the most effective drugs in the treatment of acute myeloid leukemia (AML). Ara-C is phosphorylated to Ara-CTP (cytosine arabinoside triphosphate) which inhibits DNA polymerase and induces DNA strand breaks after incorporation into DNA. There is increasing evidence that ara-C also affects some cellular signal transduction pathways; it induces transcription/expression of c-fos, c-jun, NF kappaB, protein kinase C δ, mitogen-activated protein kinase and cyclin E and downregulates cyclin-dependent-kinase-2 and retinoblastoma protein phosphorylation. These events may contribute to ara-C cytotoxicity. The antisignalling drugs all-trans retinoic acid (ATRA), staurosporine, quercetin and bryostatin-1 have recently been shown to enhance ara-C cytotoxicity.

We chose 7 different antisignalling agents [ATRA and the kinase inhibitors quercetin, genistein, CGP 52411, tyrphostin A48, nordihydroguaiaretic acid (NDGA) and staurosporine] and compared their ara-C sensitising potencies with those of hydroxyurea (HU), arabinosyl-2-fluoroadenine (F-Ara-A) and 2-chlorodeoxyadenosine (2-CdA), agents currently in use as “second line treatment” in resistant and relapsed AML. Cytotoxicity was assessed by the tetrazolium (MTT) assay in HL60 cells and a newly derived ara-C-resistant subline (HL60/ara-C) in at least three separate experiments. Supraadditive cytotoxicity was found for combinations containing ATRA, CGP 52411, tyrphostin A48 and HU in both cell lines, for 2-CdA, staurosporine and NDGA in HL60, and for F-Ara-A in HL60/ara-C cells. Quercetin and genistein did not sensitise cells against ara-C.

To elucidate the mechanism of sensitisation in HL60 cells we studied the influence of the modulators of ara-C cytotoxicity on cellular markers of apoptosis. After 4h-coincubation we measured cell size (volume), DNA loss by flow cytometry (sub-G1 peak) and DNA fragmentation by conventional gel electrophoresis. Tyrphostin A48, NDGA, ATRA and HU increased ara-C-induced apoptosis, whereas staurosporine did not affect it. CGP 52411 decreased the effect of ara-C on apoptotic indicators after 4h-, but no longer after 12h-coincubation.

The results suggest that antisignalling drugs such as ATRA, CGP 52411, tyrphostin A48, staurosporine and NDGA may be efficacious alternatives to the already clinically applied ara-C modulators. Among the clinically used ara-C modulators, HU sensitised more potently against ara-C than F-Ara-A and 2-CdA.


Acute Myeloid Leukemia HL60 Cell Cytosine Arabinoside Human Myeloid Leukemia Cell Relapse Acute Myeloid Leukemia 
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  1. 1.
    Kufe D, Spriggs D, Egan EM, Munroe D (1984) Relationship among ara-CTP pools, formation of (ara-C) DNA, and cytotoxicity of human leukemic cells. Blood 64: 54–58PubMedGoogle Scholar
  2. 2.
    Plunkett W, Iacoboni S, Keating MJ (1986) Cellular pharmacology and optimal therapeutic concentrations of 1–13-D-arabinofuranosylcytosine 5’-triphosphate in leukemic blasts during treatment of refractory leukemia with high-dose 1ß-D-arabinofuranosylcytosine. Scand J Haematol 34 (Suppl. 44): 51–59Google Scholar
  3. 3.
    Boos J, Hohenlöchter B, Schulze-Westhoff P, Schiller M, Zimmermann M, Creutzig U, Ritter J, Jürgens H (1996) Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia. Med Ped Oncol 26: 397–404CrossRefGoogle Scholar
  4. 4.
    Zühlsdorf M, Vormoor J, Boos J: Cytosine arabinoside resistance in childhood leukemia. Submitted.Google Scholar
  5. 5.
    Gandhi V, Estey E, Keating MJ, Plunkett W (1993) Fludarabine potentiates metabolism of cytarabine in patients with acute myelogenous leukemia. J Clin Oncol 11: 116–124PubMedGoogle Scholar
  6. 6.
    Gandhi V, Estey E, Keating MJ, Chucrallah A, Plunkett W (1996) Chlorodeoxyadenosine and arabinosylcytosine in patients with acute myelogenous leukemia: pharmacokinetic, pharmacodynamic, and molecular interactions. Blood 87: 256–264PubMedGoogle Scholar
  7. 7.
    Lishner M, Curtis JE, Minkin S, McCulloch EA: Interaction between retinoic acid and cytosine arabinoside affecting the blast cells of acute myeloblastic leukemia. Leukemia 3, 1989: 784–788PubMedGoogle Scholar
  8. 8.
    Yang GS, Minden MD, McCulloch EA (1993) Influence of schedule on regulated sensitivity of AML blasts to cytosine arabinoside. Leukemia 7: 1012–1019PubMedGoogle Scholar
  9. 9.
    Teofili L, Pie relli L, lovino MS, Leone G, Scambia G, de Vincenzo R, Benedetti-Panici P, Menichella G, Macri E, Piantello M, Ranelletti FO, Larocca LM (1992) The combination of quercetin and cytosine arabinoside synergistically inhibits leukemic cell growth. Leukemia Res 16: 497–503Google Scholar
  10. 10.
    Grant S, Turner AJ, Bartimole TM, Nelms PA, Joe VC, Jarvis WD (1994) Modulation of 1-[3-Darabinofuranosyl] cytosine-induced apoptosis in human myeloid leukemia cells by staurosporine and other pharmacological inhibitors of protein kinase C. Oncology Res 6: 87–99Google Scholar
  11. 11.
    Grant S, Jarvis WD, Swerdlow PS, Turner AJ, Traylor RS, Wallace HJ, Lin PS, Pettit GR, Gewirtz DA (1992) Potentiation of the activity of 113-D-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: Association with enhanced fragmentation of mature DNA. Cancer Res 52: 6270–6278PubMedGoogle Scholar
  12. 12.
    Jarvis WD, Povirk LF, Turner AJ, Traylor RS, Gewirtz DA, Pettit GR, Grant S (1994) Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[ß-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem Pharmacol 47: 839–852PubMedGoogle Scholar
  13. 13.
    Brach MA, Herrmann F, Kufe DW (1992) Activation of the AP-1 transcription factor by arabinofuranosylcytosine in myeloid leukemia cells. Blood 79: 728–734PubMedGoogle Scholar
  14. 14.
    Brach MA, Kharbanda SM, Herrmann F, Kufe DW (1992) Activation of the transcription factor KB in human KG-1 myeloid leukemia cells treated with 1-ß-D-arabinofuranosylcytosine. Mol Pharmacol 41: 60–63PubMedGoogle Scholar
  15. 15.
    Henschler R, Brennscheidt U, Mertelsmann R, Herrmann F (1991) Induction of c-jun expression in the myeloid leukemia cell line KG-1 by 1ß-D-arabinofuranosylcytosine. Mol Pharmacol 39: 171–176PubMedGoogle Scholar
  16. 16.
    Kharbanda S, Emoto Y, Kisaki H, Saleem A, Kufe D (1994) 1-ß-D-arabinofuranosylcytosine activates serine/threonine protein kinases and cjun gene expression in phorbol ester-resistant myeloid leukemia cells. Mol Pharmacol 46: 67–72Google Scholar
  17. 17.
    Emoto Y, Kisaki H, Manome Y, Kharbanda S, Kufe D (1996) Activation of protein kinase Cd in human myeloid leukemia cells treated with 1-(3D-arabinofuranosylcytosine. Blood 87: 1990–1996PubMedGoogle Scholar
  18. 18.
    Yuan ZM, Kharbanda S, Kufe D (1995) 1–0-Darabinofuranosylcytosine activates tyrosine phosphorylation of p34cdc2 and its association with the Src-like p56/p53’ kinase in human myeloid leukemia cells. Biochem 34: 1058–1063Google Scholar
  19. 19.
    Dou QP, An B, Yu C (1995) Failure to dephosphorylate retinoblastoma protein in drug-resistant cells. Biochem Biophys Res Corn 214: 771–780CrossRefGoogle Scholar
  20. 20.
    Dou QP, Lui WYL (1995) Activation of cyclin E-dependent kinase by DNA damage signals during apoptosis. Cancer Res 55: 5222–5225PubMedGoogle Scholar
  21. 21.
    Traxler P, Lydon N (1995) Recent advances in protein tyrosine kinase inhibitors. Drugs of the Future 20: 1261–1274Google Scholar
  22. 22.
    Gazit A, Yaish P, Gilon C, Levitzki A (1989) Tyrphostins: synthesis and biological activity of protein tyrosine kinase inhibitors. J Med Chem 32: 2344–2352PubMedCrossRefGoogle Scholar
  23. 23.
    Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinase. JBC 262: 5592–5595Google Scholar
  24. 24.
    Meggio F, Donella Deana A, Ruzzene M, Brunati AM, Cesaro L, Guerra B, Meyer T, Mett H, Fabbro D, Furet P, Dobrowolska B, Pinna LA (1995) Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur J Biochem 234: 317–322PubMedCrossRefGoogle Scholar
  25. 25.
    Domin J, Higgins T, Rozengurt E (1994) Preferential inhibition of platelet-derived growth-factor ( PDGF)-stimulated DNA synthesis and protein tyrosine phosphorylation by nordihydroguaiaretic acid. JBC 269: 8260–8267Google Scholar
  26. 26.
    Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, Tsai S, Metgar R, Aulakh G, Ting R, Ruscetti F, Gallo R (1979) Characterization of the continuous differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54: 713PubMedGoogle Scholar
  27. 27.
    Bhalla K, Nayak R, Grant S (1984) Isolation and characterization of a deoxycytidinekinase-deficient human promyelocytic leukemic cell line highly resistant to 1-(3-D-arabinofuranosylcytosine. Cancer Res 44: 5029–5037PubMedGoogle Scholar
  28. 28.
    Mosmann T: Rapid colorimetric assay for cellular growth and survival (1983) Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63Google Scholar
  29. 29.
    Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47: 936–942PubMedGoogle Scholar
  30. 30.
    Habtemariam S (1995) Catechols and quercetin reduce MTT through iron ions: a possible artefact in cell viability assays. Phytother Res 9: 603–605CrossRefGoogle Scholar
  31. 31.
    Pagliacci MC, Spinozzi F, Migliorati G, Fumi G, Smacchia M, Grignani F, Riccardi C, Nicoletti I (1993) Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival. Eur J Cancer 29A: 1573–1577CrossRefGoogle Scholar
  32. 32.
    Gorczyka W, Gong J, Ardelt B, Traganos F, Darzynkievicz Z (1992) The cell cycle related changes in susceptibility of HL60 cells to apoptosis induced by antitumour agents. Cancer Res 52: 6270–6278Google Scholar
  33. 33.
    Sorenson CM, Bertrand R, Pommier Y (1990) Apoptosis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin. J Nat Cancer Inst 82: 749–755PubMedCrossRefGoogle Scholar
  34. 34.
    Ormerod MG (1990) Flow cytometry–a practical approach ( Ormerod ). Oxford University Press: pp 69–87Google Scholar
  35. 35.
    Bhalla K, Swerdlow P, Grant S (1991) Effects of thymidine and hydroxyurea on the metabolism and cytotoxicity of 1-ß-D-arabinofuranosylcytosine in highly resistant human leukemia cells. Blood 78: 2937–44PubMedGoogle Scholar
  36. 36.
    Donehower RC (1992) An overview of the clinical experience with hydroxyurea. Sem Oncol 19: 11–19Google Scholar
  37. 37.
    Chen LL, Gansbacher B, Gilboa E, Taetle R, Oval J, Hibbs MS, Huang CK, Clawson ML, Bilgrami S, Schlessinger J et al. (1993) Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoid acid-induced granulocytic differentiation. Cell Growth Differ 4: 769–776PubMedGoogle Scholar
  38. 38.
    Gillis JC, Goa KL (1995) Tretinoin. A review of its pharmacodynamic and pharmacokinetic properties and use in the management of acute promyelocytic leukemia. Drugs 50: 897–923PubMedCrossRefGoogle Scholar
  39. 39.
    Curtis JE, Messner HA, Minden MD, Tritchler DL, McCulloch EA (1989) Improved maintenance therapy for acute myelogenous leukemia (AML) using a retinoic acid ( RA) containing regimen. Proc Am Assoc Cancer Res 30: 60–66Google Scholar
  40. 40.
    Venditti A, Stasi R, Masi M, Del Poeta G, Cox C, Franchi A, Piccioni D, Bruno A, Coppetelli U, Tribalto M, Papa G (1993) All-trans retinoic acid plus low doses of cytarabine for the treatment of “poor-risk” acute myeloid leukemias. Ann Hematol 66: 59–60PubMedCrossRefGoogle Scholar
  41. 41.
    Hu ZB, Yang GS, Miyamoto N, Minden MD, McCulloch EA (1995) Mechanism of cytosine arabinoside toxicity to the blast cells of acute myeloblastic leukemia: involvement of free radicals. Leukemia 9: 789–798PubMedGoogle Scholar
  42. 42.
    Hu Z-B, Minden MD, McCulloch EA (1995) Direct evidence for the participation of bd-2 in the regulation by retinoic acid of the ara-C sensitivity of leukemic stem cells. Leukemia 9: 1667–1673PubMedGoogle Scholar
  43. 43.
    Beerenbaum MC (1989) What is synergy? Pharmacol Rev 41: 93–141Google Scholar
  44. 44.
    Bouffard DY, Momparler RL (1995) Comparison of the induction of apoptosis in human leukemic cell lines by 2 ’,2’-difluorodeoxycytidine (gemcitabine) and cytosine arabinoside. Leukemia Res 19: 849–856CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • A. Freund
    • 1
  • A. Gescher
    • 2
  • J. Boos
    • 1
  1. 1.Dept. of Pediatric OncologyUniversity of MünsterGermany
  2. 2.Medical Research Council Toxicology UnitUniversity of LeicesterUK

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