靶向激活蛋白激酶PKR诱导白血病K562细胞凋亡

Targeted activation of protein kinase PKR induced apoptosis of leukemia K562 cells

目的:采用双链RNA能激活细胞内蛋白激酶PKR引起细胞凋亡的策略,研究重组慢性粒细胞白血病独特的bcr/ablb3a2型外源反义RNA诱导白血病K562细胞的凋亡及其作用机制。方法:用含bcr/abl融合基因序列40bp的反转录病毒载体RV-40AS作用于白血病K562细胞,以光学显微镜、电子显微镜、FCM法和DNA梯状条带法检测细胞凋亡情况,化学比色法检测caspase-8和caspase-9活性变化,RT-PCR法检测Bax、Bcl-2 mRNA表达变化,并以Western印迹法检测双链RNA依赖性蛋白激酶(double-stranded RNA-dependent protein kinase,PKR)、磷酸化PKR(p-PKR)、Bax和Bcl-2的蛋白变化。结果:RV-40AS作用K562细胞组在光学显微镜下出现胞体皱缩、染色质浓集以及折光性减弱,电子显微镜下可见细胞质电子密度增大、核固缩和出现凋亡小体;FCM法检测到凋亡细胞占(22.70±1.42)%[未处理组(3.24±0.66)%],DNA电泳出现典型的梯状条带;caspase-8和caspase-9活性升高,PKR蛋白无明显变化,但p-PKR水平升高;Bax的mRNA和蛋白表达均升高,而Bcl-2 mRNA和蛋白表达水平没有明显变化。对照组和PKR抑制剂处理组均未发生上述改变。结论:bcr/abl反义RNA反转录病毒载体RV-40AS可特异性激活PKR,从而诱导白血病K562细胞发生凋亡,其可能的机制是PKR活化激活了凋亡反应的上游分子caspase-8,并通过上调Bax表达水平、降低Bcl-2/Bax的比值,导致线粒体膜稳定性被破坏,促使凋亡小体形成和caspase-9活化,最终激活其他凋亡分子使细胞发生凋亡。

Objective：To study the activated protein kinase PKR inducing apoptosis by using double-stranded RNA （dsRNA） and investigate the apoptosis-inducing effect of bcr/abl b3a2 antisense RNA on chronic myeloid leukemia （CML） K562 cells and the action mechanism. Methods： K562 cells were transfected with 40 bp recombinant retroviral vector （ RV-40AS ） expressing bcr/abl fusion sequence. The cell apoptosis was observed under optical microscope and electron microscope and detected by flow cytometry and DNA ladder test. The activities of caspase-8 and caspase-9 were also assayed by colorimetry. RT-PCR was used to determine the mRNA expressions of Bax and Bcl-2. Expressions of double-stranded RNA-dependent protein kinase （PKR） , phosphorylated PKR, Bax, and Bel-2 were detected by Western blotting. Results： RV-40AS-treated K562 cells showed the characteristic apoptotic morphological changes such as shrinkage of cell bodies, chromatin condensation, and lower light refraction. Electroscopy indicated increased electronic density in cytoplasm, pyknosis, and appearance of apoptotic bodies. FCM suggested that apoptosis occurred in （22.70 ± 1.42 ） % cells in the treatment group and in （ 3.24±0.66 ） % cells in the untreated group. The DNA ladder test showed the typical ladder of DNA fragmentation. Caspase-8 and caspase-9 activities were increased. Protein PKR had no significant change, however, p-PKR significantly increased. The mRNA and protein levels of Bax were elevated, whereas those of Bcl-2 had no apparent changes. The above mentioned changes were not observed in the control group and PKR inhibitor treatment group. Conclusion：These data indicate that retroviral vector （RV-40AS） containing bcr/abl antisense RNA activated protein kinase PKR specifically, thereby inducing apoptosis of K562 cells. The mechanism may be explained by that activation of PKR stimulated apoptosis upstream molecule caspase-8, damaged the stability of mitochondrial membrane, induced the formation of apoptotic bodies, and activated caspase-9 through upregulation of Bax expression and down- regulation of Bcl-2/Bax ratio. The signaling cascades finally activated other apoptosis-related molecules and caused apoptosis.