A Natural L-Arginine Analog, L-Canavanine-Induced Apoptosis is Suppressed by Protein Tyrosine Kinase p56lck in Human Acute Leukemia Jurkat T Cells

인체 급성백혈병 Jurkat T 세포에 있어서 L-canavanine에 의해 유도되는 세포자살기전에 미치는 단백질 티로신 키나아제 p56lck의 저해 효과

  • Park, Hae-Sun (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Jun, Do-Youn (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Woo, Hyun-Ju (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Rue, Seok-Woo (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Kim, Sang-Kook (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Kim, Kyung-Min (School of Applied Ecological Resources, College of Ecology and Environmental Sciences, Kyungpook National University) ;
  • Park, Wan (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Moon, Byung-Jo (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Kim, Young-Ho (School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University)
  • 박해선 (경북대학교 생명과학부) ;
  • 전도연 (경북대학교 생명과학부) ;
  • 우현주 (경북대학교 생명과학부) ;
  • 류석우 (경북대학교 생명과학부) ;
  • 김경민 (대구경북대학교 상주캠퍼스, 생태자원응용학부) ;
  • 김상국 (경북대학교 생명과학부) ;
  • 박완 (경북대학교 생명과학부) ;
  • 문병조 (경북대학교 생명과학부) ;
  • 김영호 (경북대학교 생명과학부)
  • Published : 2009.11.30


To elucidate further the antitumor effects of a natural L-arginine analogue, L-canavanine, the mechanism underlying apoptogenic activity of L-canavanine and its modulation by protein tyrosine kinase $p56^{lck}$ was investigated in human Jurkat T cells. When the cells were treated with 1.25 to 2.5 mM L-canavanine for 36 h, several apoptotic events including mitochondrial membrane potential (${\Delta\Psi}m$) loss, activation of caspase-9, -3, -8, and -7, poly (ADP-ribose) polymerase (PARP) degradation, and DNA fragmentation were induced without alteration in the levels of Fas or FasL. These apoptotic changes were more significant in $p56^{lck}$-deficient Jurkat clone JCaM1.6 than in $p56^{lck}$-positive Jurkat clone E6.1. The L-canavanine-induced apoptosis observed in $p56^{lck}$-deficient JCaM1.6 cells was significantly reduced by introducing $p56^{lck}$ gene into JCaM1.6 cells by stable transfection. Treatment of JCaM1.6/lck cells with L-canavanine caused a transient 1.6-fold increase in the kinase activity of $p56^{lck}$. Both FADD-positive wild-type Jurkat T cell clone A3 and FADD-deficient Jurkat T cell clone I2.1 exhibited a similar susceptibility to the cytotoxicity of L-canavanine, excluding involvement of Fas/FasL system in triggering L-canavanine-induced apoptosis. The L-canavanine-induced apoptotic sub-$G_1$ peak and activation of caspase-3, -8, and -7 were abrogated by pan-caspase inhibitor (z-VAD-fmk), whereas L-canavanine-induced activation of caspase-9 was not affected. These results demonstrated that L-canavanine caused apoptosis of Jurkat T cells via the loss of ${\Delta\Psi}m$, and the activation of caspase-9, -3, -8, and -7, leading to PARP degradation, and that the $p56^{lck}$ kinase attenuated the ${\Delta\Psi}m$ loss and activation of caspases, and thus contributed as a negative regulator to L-canavanine-induced apoptosis.


  1. Bae, M. A., D. Y. Jun, K. M. Kim, S. K. Kim, J. S. Chun, D. Taub, W. Park B. J. Moon, and Y. H. Kim. 2005. Protein tyrosine kinases, $p56^{lck}$ and $p59^{fyn}$, and MAP kinase JNK1 provide an early signal required for upregulation of Fas ligand expression in aburatubolactam C-induced apoptosis of human Jurkat T cells. Journal of Microbiology and Biotechnology 15, 756-766
  2. Chen, Y. and P. W. Piper. 1995. Consequences of the overexpression of ubiquitin in yeast: elevated tolerances of osmostress, ethanol and canavanine, yet reduced tolerances of cadmium, arsenite and paromomycin. Biochimica et Biaphysica Acta 1268, 59-64
  3. Ding, Y, Y. Matsukawa, N. OhtaniFujita, D. Kato, S. Dao, T. Fujii, Y. Naito, T. Yoshikawa, T. Sakai, and G. A. Rosenthal. 1999. Growth inhibition of A549 human lung adenocarcinoma cells by L-canavanine is associated with p21/WAF1 induction. Japanese Journal of Cancer Research 90, 69-74
  4. Friesen, C., I. Herr, P. H. Krammer, and K. M. Debatin. 1996. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells. Nature Medicine 2, 574-578
  5. Green, M. H. and F. J. Ward. 1983. Enhancement of human tumor cell killing by L-canavanine in combination with gamma radiation. Cancer Research 43, 4180-4182
  6. Green, M. H., T. L. Brooks, J. Mendelsohn, and S. B. Howell. 1980. Antitumor activity of L-canavanine against L1210 murine leukemia. Cancer Research 40, 535-537
  7. Hannun, Y. A. 1997. Apoptosis and dilemma of cancer chemotheraphy. Blood 89, 1845-1853
  8. Jang, M. H., D. Y. Jun, S. W. Rue, K. H. Han, W. Park, and Y. H. Kim. 2002. Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jukat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL. Biochemical and Biophysical Research Communications 295, 283-288
  9. Jun, D. Y., J. S. Kim, H. S. Park, C. R. Han, Z. Fang, M. H. Woo, I. K. Rhee, and Y. H. Kim. 2007. Apoptogenic activity of Zanthoxylum schinifolium toward human acute leukemia Jurkat T cells is associated with ER stress-mediated caspase-8 activation that stimulates mitochondria-dependent or -independent caspase cascade. Carcinogenesis 28, 1303-1313
  10. Jun, D. Y., H. S. Park, J. S. Kim, J. S. Kim, W. Park, B. H. Song, H. S. Kim, D. Taub, and Y. H. Kim. 2008. 17$\alpha$-estradiol arrests cell cycle progression at $G_{2}$/M and induces apoptotic cell death in human acute leukemia Jurkat T cells. Toxicology and Applied Pharmacology 231, 401-412
  11. Juo, P., M. S. Woo, C. J. Kuo, P. Signorelli, H. P. Biemann, Y. A. Hannun, and J. Blenis. 1999. F ADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth and Differentiation 10, 797-804
  12. Kim, Y. H., M. J. Buchholz, and A. A. Nordin. 1993. Murine T-lymphocyte proliferation induced by interleukin 2 correlates with a transient increase in $p56^{lck}$ kinase activity and the tyrosine phosphorylation of a 97-kDa protein. Proceedings of National Academy of Sciences USA 90, 3187-3191
  13. Mengoli, M., N. Bangi, G. Luccarini, V. N. Ronchi, and D. Serafini-Fracassini. 1989. Daucus carota cell cultures: polyamines and effect of polyamine biosynthesis inhibitors in the preembryogenic phase and different embryo stages. Journal of Plant Physiology 134, 389-394
  14. Momoi, T. 2004. Caspases involved in ER stress-mediated cell death. Journal of Chemical Neuroanatomy 28, 101-105
  15. Muller, M., S. Strand, H. Hug, E. M. Heinemann, H. Walczak, W. J. Hofmann, W. Stremmel P. H. Krammer, and P. R. Galle. 1997. Drug-induced apoptotsis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. Journal of Clinical Investigation 99, 403-413
  16. Nagarkatti, N., and B. A. Davis. 2003. Tamoxifen induces apoptosis in $Fas^{+}$ tumor cells by upregulating the expression of Fas ligand. Cancer Chemotherapy and Pharmacology 51, 284-290
  17. Robertson, A. T., R. C. Bates, and E. R. Stout. 1984. Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine. Journal of General Virology 65, 1497-1505
  18. Rosenthal, G. 1998. The protective action of a higher plant toxic product. Bioscience 38, 104-108
  19. Rosenthal, G. A. 1977. The biological effects and mode of action of L-canavanine, a structural analog of L-canavanine. Quarterly Review of Biology 52, 155-178
  20. RosenthaL G. A., J. -M. Reichart, and J. A. Hoffman. 1989. L-canavanine incorporation into vitellogenin and macromolecular conformation. Journal of Biological Chemistry 264, 13693-13696
  21. Schwartz, M., A. Altman, Y. Cohen, and T. Arzee. 1997. Inhibition of polyamine biosynthesis by L-canavanine and its effect on meristematic activity, growth, and development of Zea mays roots. Israel Journal of Plant Sciences 45, 23-30
  22. Seu, J. H., B. H. Song, and C. B. Yu. 1973. Studies on the inhibitory substance of yeast growth: effect on the nitrogen uptake. Korean Journal of Applied Microbiology and Bioengineering 1, 3-11
  23. Slee, E. A., H. Zhu, S. C. Chow, M. MacFarlane, D. W. Nicholson, and G. M. Cohen. 1996. Benzyloxycarbonyl-ValAla-Asp (OMe) fluoromethylketone (z-VAD-fmk) inhibits apoptosis by blocking the processing CPP32. Biochemical Journal 315, 21-24
  24. Swaffar, D. S. and C. Y. Ang. 1999. Growth inhibitory effect of L-canavanine against MIA PaCa-2 pancreatic cancer cells is not due to conversion to its toxic metabolite canaline. Anticancer Drugs 10, 113-118
  25. Swaffar, D. S., C. Y. Ang, P. B. Desai, G. A. Rosenthal, D. A. Thomas, P. A. Crooks, and W. J. John. 1995. Combination therapy with 5-fluoruracil and L-canavanine: in vitro and in vivo studies. Anticancer Drugs 6, 586-593
  26. Thomas, D. A., G. A. Rosenthal, D. V. Gold, and K. M. Dickey. 1986. Growth inhibition of a rat colon tumor by L-canavanine. Cancer Research 46, 2898-2903
  27. Tsirigotis, M., M. Zhang, R. K. Chiu, B. G. Wouters, and D. A. Gray. 2001. Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents. Journal of Biological Chemistry 276, 46073-46078
  28. Twiddy, D. and K. Cain. 2007. Caspase-9 cleavage, do you need it? Biochemical Journal 405, e1-e2
  29. Veillette, A., I. D. Horak, E. M. Horak, M. A. Bookman, and J. B. Bolen. 1988. Alterations of the lymphocyte-specific protein tyrosine kinase ($p56^{lck}$) during T-cell activation. Molecular and Cellular Biology 8, 4353-4361
  30. Worthen, D. R., L. Chien, C. P. Tsuboi, X. Y. Mu, M. M. Bartik, and P. A. Crooks. 1998. L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines. Cancer Letter 132, 229-239
  31. Zamzami, N., P. Marchetti, M. Caste do, C. Zanin, J. L. Vayssiere, P. X. Petit, and G. Kroemer. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. Journal of Experimental Medicine 181, 1661-1672
  32. Zou, H., R. Yang, J. Hao, J. Wang, C. Sun, S. W. Fesik, J. C. Wu, K. J. Tomaselli, and R. C. Armstrong. 2003. Regulation of the Apaf-1/ caspase-9 apoptosome by caspase-3 and XIAP. Journal of Biological Chemistry 278, 8091-8098