酪氨酸激酶和磷酸酶及蛋白激酶C在As_2O_3调控NB4细胞和皮层神经元凋亡中的作用

Effects of protein tyrosine kinase, protein tyrosine phosphatase and protein kinase C on the apoptosis of arsenic trioxide treated NB4 cells and human cortex neurons

目的 研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶及蛋白激酶C(PKC)在三氧化二砷 (As2O3 )调控白血病细胞和人脑皮层神经元凋亡中的作用 ,观察As2 O3 对人脑皮层神经元和白血病细胞的胞浆游离钙 ([Ca2 + ]i)浓度的影响。方法 用Fluo 3/AM荧光探针标记人白血病细胞和人脑皮层神经元[Ca2 + ]i,激光共聚焦显微镜实时测定不同浓度As2 O3 干预后 [Ca2 + ]i的变化并观察蛋白酪氨酸激酶和磷酸酶抑制剂对 [Ca2 + ]i变化的影响。磷基转移法测定细胞膜和胞浆的PKC活性。琼脂糖凝胶电泳法观察细胞DNA的片段化。结果 1μmol /L的As2 O3 使NB4细胞的 [Ca2 + ]i明显增高 ,而对人脑皮层神经元 [Ca2 + ]i影响不明显。 2 μmol /L以上的As2 O3 引起两种细胞 [Ca2 + ]i增高 ,此作用被磷酸酶抑制剂钒酸盐呈浓度依赖性促进 ,被蛋白酪氨酸激酶抑制剂金雀异黄素呈浓度依赖性抑制 ,2 ,5 ,10μmol/L钒酸盐作用 2 4 0s时NB4细胞的 [Ca2 + ]i总增加率分别为 (6 .5± 2 .3) % ,(2 1.7± 2 .1) %和 (4 9.2± 2 .5 ) % ;人脑皮层神经元为 (6 .7± 2 .1) % ,(19.4± 2 .5 ) %和 (5 2 .3± 2 .7) % ;2 ,5 ,10 μmol/L金雀异黄素作用 2 4 0s时NB4细胞的总抑制率分别为 (6 .7± 2 .9) % ,(2 5 .6± 2 .5 ) %和 (5 2 .2± 3.5 ) % ;

Objective To investigate the effects of protein tyrosine kinase(PTK), protein tyrosine phosphatase (PTP) and protein kinase C (PKC)on apoptosis and observe the changes of cytosolic calcium ([Ca^(2+)]i) of arsenic trioxide (As_(2)O_(3)) treated human leukemia cells NB4 and cortex neurons. Methods [Ca^(2+)]i of NB4 cells and cortex neurons was probed with Fluo-3/AM, its changes were assayed with laser confocal microscopy in real-time after As_(2)O_(3) treatment at different concentrations, the effects of PTK and PTP and the activation of PKC on these changes with confocal microscopy and phosphorus radioisotope assay. DNA ladders of NB4 cells and cortex neurons after exposed to As_(2)O_(3) were observed. Results As_(2)O_(3 ) at 1 μmol/L could remarkably increase the [Ca^(2+)]i of NB4 cells but had no effects on neurons. Vanadate, a kind of PTP inhibitor, could promote the increase of [Ca^(2+)]i treated by 2,5,10 μmol/L As_(2)O_(3 ) in a dose-dependent manner.The mean total increase rates at 240 seconds after exposed to As_(2)O_(3) at different concentrations were ((6.5±)2.3)%, (21.7±2.1) %, (49.2±2.5)% for NB4 cells, and (6.7±2.1)%,(19.4±2.5) %, ((52.3)±2.7)% for cortex neurons, respectively. Genistein, a kind of PTK inhibitor, could decrease the increase of [Ca^(2+)]i treated by 2,5,10 μmol/L As_(2)O_(3 ) in a dose-dependent maner.The mean total inhibited rates at 240 seconds after As_(2)O_(3) treatment at different concentrations were (6.7±(2.9))%, (25.6±(2.5) %,) ((52.2)±3.5)% for NB4 cells, and (7.8±3.1)%,(18.1±2.8)%, (51.3±3.3)% for cortex neurons, respectively. The activation of PKC began to increase as exposed to As_(2)O_(3) at 1μmol / L for 3 h,and kept rising continuously in NB4 cells and at 24 h DNA ladders emerged. However, none of the above results was found in human cortex neurons,but when exposed to 2 μmol / L As_(2)O_(3), the activation of PKC and DNA ladders did emerge in neurons. Conclusions The phosphorylation and dephosphorylation of PTK and PTP participated in nonspecific apoptosis signal transduction pathway related to As_(2)O_(3), and accompanied with PKC activation. The [Ca^(2+)]i elevation was closely related to increased PKC activation. There existed difference in dose tolerances to As_(2)O_(3) between NB4 cell and cortex neurons.