Izeradjene Kamel, Douglas Leslie, Delaney Addison B, Houghton Janet A
Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Cancer Res. 2004 Nov 1;64(21):8036-44. doi: 10.1158/0008-5472.CAN-04-0762.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor-mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIalpha isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIalpha plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）可诱导多种恶性细胞系发生凋亡，与正常细胞形成对比，但不同细胞系的反应存在显著异质性。死亡受体介导的凋亡可能会被多种不同机制减弱，包括基于磷酸化的信号通路。我们已经证明，酪蛋白激酶I可减弱TRAIL诱导源自结肠腺癌（HT29和HCT8）以及小儿横纹肌肉瘤（JR1）的人细胞系发生凋亡。在HT29、HCT8和JR1细胞中，CKI-7对酪蛋白激酶I（CKI）磷酸化事件的抑制作用在暴露于TRAIL后显著增加了凋亡，而单独的TRAIL处理并不会诱导凋亡。CKI抑制作用增强了TRAIL处理后Fas相关死亡结构域和procaspase-8向死亡诱导信号复合物的募集，并增强了procaspase-8在死亡诱导信号复合物处的切割。在进一步研究的HT29细胞中，当CKI-7与TRAIL联合使用时，caspase-8、caspase-3、Bid以及caspase底物聚（ADP-核糖）聚合酶会迅速被切割。在存在CKI抑制剂的情况下，Bcl-2、Bcl-xL或突变型DN-Fas相关死亡结构域的过表达可保护HT29细胞免受TRAIL诱导的凋亡。此外，TRAIL与CKI-7联合使用促进了细胞色素c、Smac/DIABLO、HtrA2/Omi和AIF从线粒体的释放，并下调了XIAP和c-IAP1的表达。针对CKI的小发夹RNA显示，CKIα亚型对抑制TRAIL诱导的凋亡有显著作用。这些发现表明，CKIα通过在死亡诱导信号复合物水平产生磷酸化位点发挥抗凋亡作用，从而赋予对TRAIL介导的caspase切割的抗性。
