Subcellular localization of proteasomes in apoptotic lung tumor cells and persistence as compared to intermediate...

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.