Handrick R, Rudner J, Müller I, Eibl H, Belka C, Jendrossek V
Department of Radiation Oncology, University of Tübingen, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany.
Biochem Pharmacol. 2005 Sep 15;70(6):837-50. doi: 10.1016/j.bcp.2005.06.021.
The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2. Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood. To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/DeltaTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria. The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.
合成磷脂衍生物芥酸磷胆碱（ErPC）是人类肿瘤细胞系中一种有效的凋亡诱导剂。这种靶向细胞膜的药物通过一条被Bcl-2过表达所抑制的线粒体途径，独立于死亡受体信号传导诱导凋亡。在细胞内，Bcl-2存在于线粒体、内质网（ER）和细胞核的膜中。然而，其在不同细胞器中的亚细胞定位对抵抗凋亡的保护作用的重要性尚未完全了解。为了研究定位于特定亚细胞区室的Bcl-2对其抵抗ErPC诱导凋亡的保护作用的影响，利用具有修饰膜锚定的Bcl-2突变体，将Bcl-2的表达导向Jurkat T淋巴瘤细胞的线粒体外膜或内质网。ActA的线粒体插入序列将Bcl-2导向线粒体（Bcl-2/MT），细胞色素b5的内质网特异性序列将其导向内质网（Bcl-2/ER）。此外，还使用了表达野生型Bcl-2（Bcl-2/WT）或缺乏跨膜结构域的突变体（Bcl-2/DeltaTM）的Jurkat细胞。虽然Bcl-2仅在线粒体膜或内质网上的限制性表达强烈干扰了ErPC诱导的线粒体损伤和凋亡，但胞质中的Bcl-2/DeltaTM仅表现出降低的保护作用。因此，Bcl-2的膜定位是抵抗ErPC诱导凋亡的实质性保护的先决条件。为了有效长期抑制ErPC诱导的凋亡，Bcl-2必须同时存在于内质网和线粒体这两个区室的膜中。内质网靶向的Bcl-2干扰ErPC诱导的线粒体损伤这一发现表明内质网参与了线粒体上游的凋亡信号传导以及两个区室之间的相互作用。
