Machiels B M, Henfling M E, Schutte B, van Engeland M, Broers J L, Ramaekers F C
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht/The Netherlands.
Eur J Cell Biol. 1996 Jul;70(3):250-9.
We have studied the subcellular localization and expression levels of proteasomes during apoptosis in a lung cancer cell line. Apoptosis was induced by exposing the cells to 200 microM olomoucine, a specific cyclin-dependent kinase inhibitor. The morphological changes characteristic for apoptotic cells were visible: the cells reduced in size, the chromatin condensed and the membranes became convoluted. As the process continued, the nuclei became fragmented, and the cells broke up into cytoplasmic vesicles and apoptotic bodies. Immunocytochemically, apoptotic cells were detected by the ability to bind annexin V at their surface. During the initial stages of apoptosis, proteasomes were present in the nucleus as well as in the cytoplasm. Upon increased chromatin condensation, nuclear proteasomes were found predominantly surrounding the chromatin, while the chromatin itself remained devoid of staining. That the proteasomes persisted relatively long in the apoptotic cells was shown by immunoblotting of non-denaturing gels, which indicated that both 20S and 26S proteasomes were present in apoptotic cells. In immunofluoresence microscopy the proteasome fluorescence intensity of apoptotic cells seemed higher than that of non-apoptotic cells. These differences in intensity were even more pronounced after Triton X-100 extraction. Flow cytometry revealed that the absolute levels of proteasome staining in cells were decreased after Triton X-100 extraction. However, no differences in staining levels were detected between apoptotic and non-apoptotic cells. A relative increase of proteasome concentration through cell shrinkage or a concentration in certain cell compartments may be the origin of the apparently increased signal that was seen in immunofluorescence microscopy. Furthermore, proteasomes were clearly detectable in the apoptotic bodies and cytoplasmic vesicles at the time immunocytochemical reactivity for cytokeratins and lamins had diminished to a large extent. Immunoblotting of denaturing polyacrylamide gels confirmed the results obtained by flow cytometry. The proteasome content was retained only partially in the cells after Triton X-100 extraction, while the intermediate filaments were not detectable anymore in the apoptotic cells.
我们研究了肺癌细胞系凋亡过程中蛋白酶体的亚细胞定位和表达水平。通过将细胞暴露于200微摩尔的olomoucine（一种特异性细胞周期蛋白依赖性激酶抑制剂）来诱导凋亡。凋亡细胞特有的形态变化可见：细胞体积减小，染色质浓缩，细胞膜变得卷曲。随着过程的持续，细胞核碎片化，细胞分裂成细胞质小泡和凋亡小体。免疫细胞化学检测显示，凋亡细胞可通过其表面结合膜联蛋白V的能力来检测。在凋亡的初始阶段，蛋白酶体存在于细胞核和细胞质中。随着染色质浓缩增加，核蛋白酶体主要围绕染色质存在，而染色质本身仍无染色。非变性凝胶免疫印迹显示，蛋白酶体在凋亡细胞中持续存在相对较长时间，这表明凋亡细胞中同时存在20S和26S蛋白酶体。在免疫荧光显微镜下，凋亡细胞的蛋白酶体荧光强度似乎高于非凋亡细胞。经Triton X-100提取后，这些强度差异更加明显。流式细胞术显示，Triton X-100提取后细胞中蛋白酶体染色的绝对水平降低。然而，凋亡细胞和非凋亡细胞之间未检测到染色水平的差异。免疫荧光显微镜下观察到的明显增加的信号可能源于细胞收缩导致的蛋白酶体浓度相对增加或某些细胞区室中的浓度增加。此外，当细胞角蛋白和核纤层蛋白的免疫细胞化学反应在很大程度上减弱时，在凋亡小体和细胞质小泡中仍可清楚地检测到蛋白酶体。变性聚丙烯酰胺凝胶免疫印迹证实了流式细胞术获得的结果。Triton X-100提取后，细胞中仅部分保留了蛋白酶体含量，而凋亡细胞中不再能检测到中间丝。
