Influence of Environmental Conditions on c-Jun N-terminal Kinase Mediated Apoptosis of HL60 Cells by Anti-Cancer Drugs

  • Hur, Eun-Hye (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Kang, Mun-Jung (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Kim, Sung-Doo (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Lim, Sung-Nam (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Kim, Dae-Young (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Lee, Jung-Hee (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Lee, Kyoo-Hyung (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Lee, Je-Hwan (Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine)
  • Published : 2010.01.31


Activation of JNK has long been associated with the apoptotic response induced by various anti-cancer drugs including doxorubicin, vinblastine, and etoposide. In this study, we examined and compared patterns of apoptosis and JNK activation according to three different anti-cancer drugs (daunorubicin, vinblastine, and etoposide) and two different sources of HL60 cells (Jackson Laboratory and ATCC). HL60 cells from Jackson Laboratory (HL60/RPMI) were maintained in RPMI 1640 containing 5% fetal bovine serum and those from ATCC (HL60/IMDM) in IMDM containing 20% fetal bovine serum as to each manufacture's guideline. In general, HL60/RPMI cells were more sensitive to anti-cancer drugs compared to HL60/IMDM cells, demonstrated by the XTT and flow cytometric analyses. Apoptotic pathways after treatment with anti-cancer drugs seemed to be different between HL60/RPMI (daunorubicin and etoposide, caspase 3 dependent, but caspase 8 or 9 independent; vinblastine, caspase 3 independent) and HL60/IMDM (caspase 3 and caspase 9 dependent). The expression of apoptotic protein, BID, was consistent with caspase 3 activation. Immunoblotting of phospho-JNK and JNK kinase assay showed JNK activation by all three anti-cancer drugs in HL60/RPMI, while JNK activation was observed only in vinblastine-treated cells in HL60/IMDM. Our study results suggest that in vitro environmental conditions have a significant influence on JNK mediated apoptosis of HL60 cells by anti-cancer drugs and in vitro culture conditions are important factors in JNK or possibly other MAPK related studies.


  1. Benjamin, C. W., Hiebsch, R. R. and Jones, D. A. (1998). Caspase activation in MCF7 cells responding to etoposide treatment. Mol. Pharmacol. 53, 446-450.
  2. Boldt, S., Weidle, U. H. and Kolch, W. (2002). The role of MAPK pathways in the action of chemotherapeutic drugs. Carcinogenesis. 23, 1831-1838.
  3. Brantley-Finley, C., Lyle, C. S., Du, L., Goodwin, M. E., Hall, T., Szwedo, D., Kaushal, G. P. and Chambers, T. C. (2003). The JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide. Biochem. Pharmacol. 66, 459-469.
  4. Choi, J. H. and Lee, K. T. (2009). Costunolide-induced apoptosis in human leukemia cells: involvement of c-jun N-terminal kinase activation. Biol. Pharm. Bull. 32, 1803-1808.
  5. Cripe, L. D., Gelfanov, V. M., Smith, E. A., Spigel, D. R., Phillips, C. A., Gabig, T. G., Jung, S. H., Fyffe, J., Hartman, A. D., Kneebone, P., Mercola, D., Burgess, G. S. and Boswell, H. S. (2002). Role for c-jun N-terminal kinase in treatmentrefractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation. Leukemia 16, 799-812.
  6. Cui, J., Zhang, M., Zhang, Y.Q. & Xu, Z. H. (2007). JNK pathway: diseases and therapeutic potential. Acta Pharmacol. Sin. 28, 601-608.
  7. Davis, R. J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103, 239-252.
  8. Dhanasekaran, D. N. and Reddy, E. P. (2008). JNK signaling in apoptosis. Oncogene 27, 6245-6251.
  9. Do, J. H., Oh, S. H., Song, E. J., Chung, J. S., Kang, C. D. and Lee, E. Y. (2007). Treatment outcome of multidrug resistance related mRNA expression and c-jun-N-terminal kinase activity in patients with acute myeloid leukemia. Korean J. Lab. Med. 27, 229-236.
  10. Fan, M. and Chambers, T. C. (2001). Role of mitogen-activated protein kinases in the response of tumor cells to chemotherapy. Drug Resist. Updat. 4, 253-267.
  11. Fan, M., Goodwin, M. E., Birrer, M. J. & Chambers, T. C. (2001). The c-Jun NH(2)-terminal protein kinase/AP-1 pathway is required for efficient apoptosis induced by vinblastine. Cancer Res. 61, 4450-4458.
  12. Gamen, S., Anel, A., Lasierra, P., Alava, M. A., Martinez-Lorenzo, M. J., Pineiro, A. and Naval, J. (1997). Doxorubicininduced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way. FEBS Lett. 417, 360-364.
  13. Gerlier, D. and Thomasset, N. (1986). Use of MTT colorimetric assay to measure cell activation. J. Immunol. Methods 94, 57-63.
  14. Herr, I. and Debatin, K.M. (2001). Cellular stress response and apoptosis in cancer therapy. Blood 98, 2603-2614.
  15. Johnson, G. L. and Nakamura, K. (2007). The c-jun kinase/stress- activated pathway: regulation, function and role in human disease. Biochim. Biophys. Acta 1773, 1341-1348.
  16. Jost, L. M., Kirkwood, J. M. and Whiteside, T. L. (1992). Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J. Immunol. Methods 147, 153-165.
  17. Kolomeichuk, S. N., Bene, A., Upreti, M., Dennis, R. A., Lyle, C. S., Rajasekaran, M. & Chambers, T. C. (2008). Induction of apoptosis by vinblastine via c-Jun autoamplification and p53-independent down-regulation of p21WAF1/CIP1. Mol. Pharmacol. 73, 128-136.
  18. Krishan, A. (1975). Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell. Biol. 66, 188-193.
  19. Lagadinou, E. D., Ziros, P. G., Tsopra, O. A., Dimas, K., Kokkinou, D., Thanopoulou, E., Karakantza, M., Pantazis, P., Spyridonidis, A. and Zoumbos, N. C. (2008). c-Jun N-terminal kinase activation failure is a new mechanism of anthracycline resistance in acute myeloid leukemia. Leukemia 22, 1899-1908.
  20. Lavrik, I. N., Golks, A. and Krammer, P. H. (2005). Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115, 2665-2672.
  21. Lin, A. and Dibling, B. (2002). The true face of JNK activation in apoptosis. Aging Cell 1, 112-116.
  22. Liu, J. and Lin, A. (2005). Role of JNK activation in apoptosis: a double-edged sword. Cell Res. 15, 36-42.
  23. Nishina, H., Wada, T. and Katada, T. (2004). Physiological roles of SAPK/JNK signaling pathway. J. Biochem. 136, 123-126.
  24. Osborn, M.T. and Chambers, T.C. (1996). Role of the stressactivated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs. J. Biol. Chem. 271, 30950-30955.
  25. Panaretakis, T., Laane, E., Pokrovskaja, K., Bjorklund, A. C., Moustakas, A., Zhivotovsky, B., Heyman, M., Shoshan, M. C. and Grander, D. (2005). Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis. Mol. Biol. Cell. 16, 3821-3831.
  26. Philchenkov, A. (2004). Caspases: potential targets for regulating cell death. J. Cell. Mol. Med. 8, 432-444.
  27. Potapova, O., Gorospe, M., Dougherty, R. H., Dean, N. M., Gaarde, W. A. and Holbrook, N. J. (2000). Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner. Mol. Cell. Biol. 20, 1713-1722.
  28. Potapova, O., Haghighi, A., Bost, F., Liu, C., Birrer, M. J., Gjerset, R. and Mercola, D. (1997). The Jun kinase/stressactivated protein kinase pathway functions to regulate DNA repair and inhibition of the pathway sensitizes tumor cells to cisplatin. J. Biol. Chem. 272, 14041-14044.
  29. Sampath, D., Cortes, J., Estrov, Z., Du, M., Shi, Z., Andreeff, M., Gandhi, V. and Plunkett, W. (2006). Pharmacodynamics of cytarabine alone and in combination with 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial. Blood 107, 2517-2524.
  30. Sampath, D. and Plunkett, W. (2000). The role of c-Jun kinase in the apoptotic response to nucleoside analogue-induced DNA damage. Cancer Res. 60, 6408-6415.
  31. Stadheim, T.A., Saluta, G.R. and Kucera, G.L. (2000). Role of c-Jun N-terminal kinase/p38 stress signaling in 1-beta-Darabinofuranosylcytosine-induced apoptosis. Biochem. Pharmacol. 59, 407-418.
  32. Tashiro, E., Simizu, S., Takada, M., Umezawa, K. and Imoto, M. (1998). Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung carcinoma Ms-1 cells. Jpn. J. Cancer Res. 89, 940-946.
  33. Turnbull, K. J., Brown, B. L. and Dobson, P. R. (1999). Caspase-3-like activity is necessary but not sufficient for daunorubicin-induced apoptosis in Jurkat human lymphoblastic leukemia cells. Leukemia 13, 1056-1061.
  34. Weston, C. R. and Davis, R. J. (2007). The JNK signal transduction pathway. Curr. Opin. Cell. Biol. 19, 142-149.
  35. Wisdom, R., Johnson, R. S. and Moore, C. (1999). c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 18, 188-197.
  36. Yu, R., Shtil, A. A., Tan, T. H., Roninson, I. B. and Kong, A. N. (1996). Adriamycin activates c-jun N-terminal kinase in human leukemia cells: a relevance to apoptosis. Cancer Lett. 107, 73-81.

Cited by

  1. Protective effect of triphlorethol-A against ultraviolet B-mediated damage of human keratinocytes vol.106, 2012,