人M-07e白血病细胞凋亡过程中激活Caspase-3引起的Bcl-2 蛋白酶解与Lynp53/56激酶失活相关

摘要

The growth of M-07e human megakaryocytie leukemia cells is strictly dependent on GM-CSF. In M-07e cells, the GM-CSF receptor (GM-CSF R) is composed of two subunits: a low affinity α subunit and a phosphorylated β subunit, which is constitutively linked to lyn53/56 protein tyrosine kinase. In this study, The role of lyn kinase in regulating TGF-β 1-induced apoptosis in M-07e cells was examined. The removal of rhGM-CSF from the culture medium resulted in down-regulation of lyn kinase activity, followed by growth inhibition and programmed cell death. Apoptosis of M-07e cells was accompanied with a massive cleavage of Bcl-2 and Bax proteins into shortened fragments with molecular mass of 22 kD and 18 kD, respectively. Using specific inhibitors, the cleavage of Bcl-2, but not Bax, was found to be processed through activated caspase-3 (CPP32), which is abundantly expressed in M-07e cells. TGF-β 1 inhibited rhGM-CSF-stimulated cell growth and promoted apoptosis in M-07e cells with a pattern identical to that induced by rhGM-CSF depletion, which included massive cleavage of both Bcl-2 and Bax proteins and inactivation of lyn kinase activity. TGF-β 1 did not affect the levels of lyn protein or the β-subunit, neither did it block the interaction between these two components. Also, TGF-β 1 treatment did not diminish the expression of the α subunit in M-07e cells. Our results showed that TGF-β 1 inhibits cell proliferation and promotes apoptosis in M-07e cells by inactivating the GM-CSF R-associated lyn kinase activity. Further, This study showed that Bcl-2 cleavage by activated CPP32 is a naturally occurring event associated with apoptosis, which is under the regulation of lyn kinase activation.%人巨核白血病细胞系M-07e的生长严格依赖于GM-CSF.在M-07e细胞,GM-CSF受体(GM-CSF R)由两个亚基所组成:低亲合力的配体特异的α亚基和一个磷酸化的β亚基,后者与lynp53/56酪氨酸蛋白激酶固定相连.本研究检测了lyn激酶在调节TGF-β 1诱导的M-07e细胞凋亡过程中的作用.从培养液中去除rhGM-CSF首先导致了lyn激酶活性受抑,接着发生细胞生长受阻和凋亡.M-07e细胞凋亡过程中伴随有大量Bcl-2和Bax蛋白分别被酶解为22 kD和18 kD的较小片段.应用特异性的抑制剂,发现上述Bcl-2蛋白的变化是循激活的caspase-3(CPP32)途径发生的,后者在M-07e细胞中大量表达.Bax蛋白变化的机制尚不清楚.TGF-β 1对rhGM-CSF刺激的细胞生长具有抑制作用,促进M-07e细胞凋亡的机制与去除rhGM-CSF所致相同,包括大量Bcl-2和Bax蛋白被特异酶解和lyn激酶失活.TGF-β 1并不影响lyn蛋白和β链的表达水平,也不阻止这两个信号传导元件的相互作用.研究结果表明,TGF-β 1通过抑制GM-CSF R相关的lyn激酶活性而抑制M-07e细胞生长并促进凋亡发生.本实验还表明激活的CPP32对Bcl-2蛋白的酶解是与细胞凋亡发生相关的一个自然过程,处于lyn激酶活性的调控之下.