Marcelli M, Marani M, Li X, Sturgis L, Haidacher S J, Trial J A, Mannucci R, Nicoletti I, Denner L
Departments of Medicine, Molecular and Cellular Biology, Veterans Administration Medical Center and Baylor College of Medicine, Houston, Texas 77030, USA.
Prostate. 2000 Mar 1;42(4):260-73. doi: 10.1002/(sici)1097-0045(20000301)42:4<260::aid-pros3>3.0.co;2-0.
The goal of this work was to identify mechanisms for the inability of metastatic prostate cancer cells to engage the apoptotic pathway following hormonal or cytotoxic therapy.
Genotypically diverse cell lines isolated from patients with metastatic disease were used.
The LNCaP and TsuPr(1) lines exhibited quintessential apoptotic features in response to the pleiotropic apoptotic inducer staurosporine (STS): rapid cytochrome c translocation to the cytosol, proteolytic processing and catalytic activation of caspase-3 and -7, proteolytic inactivation of the death substrates DNA fragmentation factor (DFF) and poly-ADP-ribose polymerase (PARP), and TUNEL-positive polyfragmented nuclei. In contrast, DU-145 and PC-3 cells exhibited few, if any, of these features, while appearing necrotic by confocal microscopy. The presence of caspase-3 and -7 without proteolytic processing suggested that the apoptotic blockade was upstream of executioner caspases in these resistant cell lines. To identify the locus of this block, Western blot analysis of cytochrome c subcellular localization and of pro- and antiapoptotic Bcl-2 family members was performed, and suggested that heterogeneous expression of these proteins might be the underlying mechanism for apoptotic resistance to STS in these cell lines. Thus, the absence of the proapoptotic Bax in DU-145 cells indicated a mechanism for apoptotic resistance of these cells. Similarly, decreased Bax expression during STS treatment, coupled with overexpression of the antiapoptotic Bcl-x(L) and inability to translocate cytochrome c to the cytosol, provided a mechanism for the insensitivity of PC-3 cells.
These observations suggest that activation of the apoptotic machinery in metastatic prostate cancer cell lines may be determined by expression levels of Bcl-2 family members, by the ability of cytochrome c to translocate to the cytosol, and by the ability of the caspase pathway to react in response to activation of the mitochondrial phase.
本研究旨在确定转移性前列腺癌细胞在激素或细胞毒性治疗后无法启动凋亡途径的机制。
使用从转移性疾病患者中分离出的基因类型多样的细胞系。
LNCaP和TsuPr(1)细胞系对多效性凋亡诱导剂星形孢菌素（STS）表现出典型的凋亡特征：细胞色素c迅速转移至细胞质，半胱天冬酶-3和-7进行蛋白水解加工并被催化激活，死亡底物DNA片段化因子（DFF）和聚ADP核糖聚合酶（PARP）发生蛋白水解失活，以及TUNEL阳性的多核碎裂。相比之下，DU-145和PC-3细胞几乎没有这些特征，通过共聚焦显微镜观察显示为坏死。半胱天冬酶-3和-7存在但未进行蛋白水解加工，表明在这些耐药细胞系中凋亡阻断发生在执行半胱天冬酶的上游。为了确定该阻断的位点，对细胞色素c亚细胞定位以及促凋亡和抗凋亡Bcl-2家族成员进行了蛋白质印迹分析，结果表明这些蛋白质的异质性表达可能是这些细胞系对STS凋亡抵抗的潜在机制。因此，DU-145细胞中促凋亡蛋白Bax的缺失表明了这些细胞凋亡抵抗的一种机制。同样，STS处理期间Bax表达降低，同时抗凋亡蛋白Bcl-x(L)过表达且细胞色素c无法转移至细胞质，这为PC-3细胞的不敏感性提供了一种机制。
这些观察结果表明，转移性前列腺癌细胞系中凋亡机制的激活可能由Bcl-2家族成员的表达水平、细胞色素c转移至细胞质的能力以及半胱天冬酶途径对线粒体阶段激活作出反应的能力所决定。
