A Natural L-Arginine Analog, L-Canavanine-Induced Apoptosis is Suppressedby Protein Tyrosine Kinase p56 lck in Human Acute Leukemia Jurkat T Cells

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 (Δψm) 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-G1 peak and activation of caspase-3, -8, and -7 were abrogated by pan-caspase inhibitor (z-VADfmk), 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 Δψm, and the activation of caspase-9, -3, -8, and -7, leading to PARP degradation, and that the p56 lck kinase attenuated the Δψm loss and activation of caspases, and thus contributed as a negative regulator to L-canavanine-induced apoptosis.