Journal of Cancer Research and Clinical Oncology

, Volume 134, Issue 2, pp 245–253 | Cite as

Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells: the role of ATM-Chk2-Cdc25A and ATM-p53-p21-pathways

  • Leander Gaul
  • Sonja Mandl-Weber
  • Philipp Baumann
  • Bertold Emmerich
  • Ralf Schmidmaier
Original Paper

Abstract

Purpose

Multiple myeloma is a fatal hematological disease caused by malignant transformation of plasma cells. Bendamustine has been proven to be a potent alternative to melphalan in phase 3 studies, yet its molecular mode of action is still poorly understood.

Methods

The four-myeloma cell lines NCI-H929, OPM-2, RPMI-8226, and U266 were cultured in vitro. Apoptosis was measured by flow cytometry after annexin V FITC and propidium iodide staining. Cell cycle distribution of cells was determined by DNA staining with propidium iodide. Intracellular levels of (phosphorylated) proteins were determined by western blot.

Results

We show that bendamustine induces apoptosis with an IC50 of 35–65 μg/ml and with cleavage of caspase 3. Incubation with 10–30 μg/ml results in G2 cell cycle arrest in all four-cell lines. The primary DNA-damage signaling kinases ATM and Chk2, but not ATR and Chk1, are activated. The Chk2 substrate Cdc25A phosphatase is degraded and Cdc2 is inhibited by inhibitory phosphorylation of Tyr15 accompanied by increased cyclin B levels. Additionally, p53 activation occurs as phosphorylation of Ser15, the phosphorylation site for ATM. p53 promotes Cdc2 inhibition by upregulation of p21. Targeting of p38 MAPK by the selective inhibitor SB202190 significantly increases bendamustine induced apoptosis. Additionally, SB202190 completely abrogates G2 cell cycle arrest.

Conclusion

Bendamustine induces ATM-Chk2-Cdc2-mediated G2 arrest and p53 mediated apoptosis. Inhibition of p38 MAPK augments apoptosis and abrogates G2 arrest and can be considered as a new therapeutic strategy in combination with bendamustine.

Keywords

Multiple myeloma Bendamustine Cell cycle Ataxia telangiectasia mutated protein Checkpoint kinase 2 

References

  1. Abraham RT (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 15:2177–2196PubMedCrossRefGoogle Scholar
  2. Agner J, Falck J, Lukas J, Bartek J (2005) Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway. Exp Cell Res 302:162–169PubMedCrossRefGoogle Scholar
  3. Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF et al (1996) A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 335:91–97PubMedCrossRefGoogle Scholar
  4. Bode AM, Dong Z (2006) The enigmatic effects of caffeine in cell cycle and cancer. Cancer Lett 247:26–39PubMedCrossRefGoogle Scholar
  5. Bozko P, Sabisz M, Larsen AK, Skladanowski A (2005) Cross-talk between DNA damage and cell survival checkpoints during G2 and mitosis: pharmacologic implications. Mol Cancer Ther 4:2016–2025PubMedCrossRefGoogle Scholar
  6. Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, Basrur V, Potapova O et al (2001) Initiation of a G2/M checkpoint after ultraviolet radiation requires P38 kinase. Nature 411:102–107PubMedCrossRefGoogle Scholar
  7. Buscemi G, Perego P, Carenini N, Nakanishi M, Chessa L, Chen J et al (2004) Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks. Oncogene 23:7691–7700PubMedCrossRefGoogle Scholar
  8. Busino L, Chiesa M, Draetta GF, Donzelli M (2004) Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis. Oncogene 23:2050–2056PubMedCrossRefGoogle Scholar
  9. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K et al (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281:1677–1679PubMedCrossRefGoogle Scholar
  10. Chaturvedi P, Eng WK, Zhu Y, Mattern MR, Mishra R, Hurle MR et al (1999) Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Oncogene 18:4047–4054PubMedCrossRefGoogle Scholar
  11. Child JA, Morgan GJ, Davies FE, Owen RG, Bell SE, Hawkins K et al (2003) Medical Research Council Adult Leukaemia Working Party. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 348:1875–1883PubMedCrossRefGoogle Scholar
  12. Damia G, Broggini M (2004) Cell cycle checkpoint proteins and cellular response to treatment by anticancer agents. Cell cycle 3:46–50PubMedGoogle Scholar
  13. Falk J, Mailand N, Syljuasen RG, Bartek J, Lukas J (2001) The ATM-Chk2-cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 410:842–847CrossRefGoogle Scholar
  14. Garner AP, Weston CR, Todd DE, Balmanno K, Cook SJ (2002) Delta MEKK3:ER* activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint. Oncogene 21:8089–8104PubMedCrossRefGoogle Scholar
  15. Heider A, Niederle N (2001) Efficacy and toxicity of bendamustine in patients with relapsed low-grade Non Hodgkin’s lymphomas. Anticancer Drugs 12:725–729PubMedCrossRefGoogle Scholar
  16. Herold M, Schulze A, Niederwieser D, Franke A, Fricke HJ, Richter P et al. for the East German Study Group Hematology and Oncology (OSHO) (2006). Bendamustine, vincristine and prednisone (BOP) versus cyclophosphamide, vincristine and prednisone (COP) in advanced indolent non-Hodgkin`s lymphoma and mantle cell lymphoma: results of a randomised phase 3 trial (OSHO# 19). J Cancer Res Clin Oncol 132:105–112Google Scholar
  17. Hideshima T, Akiyama M, Hayashi T, Richardson P, Schlossman R, Chauhan D et al (2003) Targeting p38 MAPK inhibits multiple myeloma cell growth in the bone marrow milieu. Blood 101:703–705PubMedCrossRefGoogle Scholar
  18. Hideshima T, Podar K, Chauhan D, Ishitsuka K, Mitsiades C, Tai YT et al (2004) P38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells. Oncogene 23:8766–8776PubMedCrossRefGoogle Scholar
  19. Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H et al (2000) DNA-damage induced activation o P53 by the checkpoint kinase Chk2. Science 287:1824–1827PubMedCrossRefGoogle Scholar
  20. Hoffken K, Merkle K, Schonfelder M, Anger G, Brandtner M, Ridwelski K, Seeber S (1998) Bendamustine as salvage treatment in patients with advanced progressive breast cancer: a phase II study. J Cancer Res Clin Oncol 124:627–632PubMedCrossRefGoogle Scholar
  21. Kath R, Blumenstengel K, Fricke HJ, Hoffken K (2001) Bendamustine monotherapy in advanced and refractory chronic lymphocytic leukemia. J Cancer Res Clin Oncol 127:48–54PubMedCrossRefGoogle Scholar
  22. Konstantinov SM, Kostovski A, Topashka-Ancheva M, Genova M, Berger MR (2002) Cytotoxic efficacy of bendamustine in human leukemia and breast cancer cell lines. J Cancer Res Clin Oncol 128:271–278PubMedCrossRefGoogle Scholar
  23. Knop S, Straka C, Haen M, Schwedes R, Hebart H, Einsele H (2005) The eficiacy and toxicity of bendamustine in recurrent multiple myeloma after high-dose chemotherapy. Haematologica 90:1287–1288PubMedGoogle Scholar
  24. Leoni L, Bailey B, Reifert J, Niemeyer C, Bendall H, Dauffenbach L, Kerfoot C (2003) SDX-105 (Bendamustine), a clinically active antineoplastic agent possesses a unique mechanism of action. Blood 102(11):534-IIGoogle Scholar
  25. Lissitchkov T, Arnaudov G, Peytchev D, Merkle Kh (2006) Phase-I/II study to evaluate dose limiting toxicity, maximum tolerated dose, and tolerability of bendamustine HCl in pre-treated patients with B-chronic lymphocytic leukaemia (Binet stages B and C) requiring therapy. J Cancer Res Clin Oncol 132:99–104PubMedCrossRefGoogle Scholar
  26. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K et al (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint. Genes Dev 14:1448–1459PubMedCrossRefGoogle Scholar
  27. Matsuoka S, Huang M, Elledge SJ (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282:1893–1897PubMedCrossRefGoogle Scholar
  28. Mailand N, Falck J, Lukas C, Syljuåsen RG, Welcker M, Bartek J et al (2000) Rapid destruction of human Cdc25A in response to DNA damage. Science 288:1425–1429PubMedCrossRefGoogle Scholar
  29. Maity A, Hwang A, Janss A, Phillips P, McKenna WG, Muschel RJ (1996) Delayed cyclin B1 expression during the G2 arrest following DNA damage. Oncogene 13:1647–1657PubMedGoogle Scholar
  30. Miyakoda M, Suzuki K, Kodama S, Watanabe M (2002) Activation of ATM and phosphorylation of p53 by heat shock. Oncogene 21:1090–1096PubMedCrossRefGoogle Scholar
  31. Myeloma Trialists’ Collaborative Group (1998) Combination chemotherapy versus melphalan plus prednisone as treatment for multiple myeloma: an overview of 6,633 patients from 27 randomized trials. J Clin Oncol 16:3832–3842Google Scholar
  32. Navas TA, Nguyen AN, Hideshima T, Reddy M, Ma JY, Haghnazari E et al (2006) Inhibition of p38alpha MAPK enhances proteasome inhibitor-induced apoptosis of myeloma cells by modulating Hsp27, Bcl-X(L), Mcl-1 and p53 levels in vitro and inhibits tumor growth in vivo. Leukemia 20:1017–1027PubMedCrossRefGoogle Scholar
  33. Nguyen AN, Stebbins EG, Henson M, O’Young G, Choi SJ, Quon D et al (2006) Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation. Exp Cell Res 312:1909–1923PubMedCrossRefGoogle Scholar
  34. Nurse P (1990) Universal control mechanism regulating onset of M-phase. Nature 344:503–508PubMedCrossRefGoogle Scholar
  35. O’Driscoll M, Ruiz-Perez VL, Woods CG, Jeggo PA, Goodship JA (2003) A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet 33:497–501PubMedCrossRefGoogle Scholar
  36. Pedraza-Alva G, Koulnis M, Charland C, Thornton T, Clements JL, Schlissel MS et al (2006) Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint. EMBO J 25:763–773PubMedCrossRefGoogle Scholar
  37. Palumbo A, Bringhen S, Petrucci MT, Musto P, Rossini F, Nunzi M et al (2004) Intermediate-dose melphalan improves survival of myeloma patients aged 50 to 70: results of a randomized controlled trial. Blood 104:3052–3057PubMedCrossRefGoogle Scholar
  38. Ponisch W, Mitrou PS, Merkle K, Herold M, Assmann M, Wilhelm G et al (2006) Treatment of bendamustine and prednisolone in patients with newly diagnosed multiple myeloma results in superior complete response rate, prolonged time to treatment failure and improved quality of life compared to treatment with Melphalan and Prednisone—a randomised phase 3 study of the East German Study group of Hematology and Oncology (OSHO). J Cancer Res Clin Oncol 132:205–212PubMedCrossRefGoogle Scholar
  39. Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ (1997) Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277:1497–1501PubMedCrossRefGoogle Scholar
  40. Sarkaria JN, Busby EC, Tibbets RS, Roos P, Taya Y, Karnitz LM et al (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59:4375–4382PubMedGoogle Scholar
  41. Strumberg D, Harstrick A, Doll K, Hoffmann B, Seeber S (1996) Bendamustine hydrochloride activity against doxorubicin-resistant human breast carcinoma cell lines. Anticancer Drugs 7:415–421PubMedCrossRefGoogle Scholar
  42. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY et al (1999) A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 13:152–157PubMedGoogle Scholar
  43. Turenne GA, Paul P, Laflair L, Price BD (2001) Activation of p53 transcriptional activity requires ATM’s kinase domain and multiple N-terminal serine residues of p53. Oncogene 20:5100–5110PubMedCrossRefGoogle Scholar
  44. von Minckwitz G, Chernozemsky I, Sirakova L, Chilingirov P, Souchon R, Marschner N et al (2005) Bendamustine prolongs progression-free survival in metastatic breast cancer (MBC): a phase III prospective, randomized, multicenter trial of bendamustine hydrochloride, methotrexate and 5-fluorouracil (BMF) versus cyclophosphamide, methotrexate and 5-fluorouracil (CMF) as first-line treatment of MBC. Anticancer Drugs 16:871–877CrossRefGoogle Scholar
  45. Wang S, Yang J, Qian J, Wezeman M, Kwak LW, Yi Q (2006) Tumor evasion of the immune system: inhibiting p38 MAPK signaling restores the function of dendritic cells in multiple myeloma. Blood 107:2432–2439PubMedCrossRefGoogle Scholar
  46. Yamane K, Taylor K, Kinsella TJ (2004) Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway. Biochem Biophys Res Commun 318:297–302PubMedCrossRefGoogle Scholar
  47. Yan T, Desai AB, Jacobberger JW, Sramkoski RM, Loh T, Kinsella TJ (2004) CHK1 and CHK2 are differentially involved in mismatch repair-mediated 6-thioguanine-induced cell cycle checkpoint responses. Mol Cancer Ther 3:1147–1157PubMedGoogle Scholar
  48. Zulkowski K, Kath R, Semrau R, Merkle K, Hoffken K (2002) Regression of brain metastases from breast carcinoma after chemotherapy with bendamustine. J Cancer Res Clin Oncol 128:111–113PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Leander Gaul
    • 1
  • Sonja Mandl-Weber
    • 1
  • Philipp Baumann
    • 1
  • Bertold Emmerich
    • 1
  • Ralf Schmidmaier
    • 1
  1. 1.Department of Haematology and OncologyKlinikum der Universität München, Medizinische Klinik InnenstadtMunichGermany

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