SUBCELLULAR-LOCALIZATION OF PROTEASOMES IN APOPTOTIC LUNG-TUMOR CELLSAND PERSISTENCE AS COMPARED TO INTERMEDIATE FILAMENTS

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 mu M olomoucine, a specific cycl in-dependent kinase inhibitor, The morphological changes characteristi c for apoptotic cells were visible: the cells reduced in size, the chr omatin condensed and the membranes became convoluted, As the process c ontinued, the nuclei became fragmented, and the cells broke up into cy toplasmic vesicles and apoptotic bodies. Immunocytochemically, apoptot ic cells were detected by the ability to bind annexin V at their surfa ce. During tile initial stages of apoptosis, proteasomes were present in the nucleus as well as in the cytoplasm. Upon increased chromatin c ondensation, nuclear proteasomes were found predominantly surrounding rounding the chromatin, while the chromatin itself remained devoid of staining, That tire proteasomes persisted relatively long in the apopt otic cells was shown by immunoblotting of non-denaturing gels, which i ndicated that both 20S and 26S proteasomes were present in apoptotic c ells. In immunofluorescence microscopy the proteasome fluorescence int ensity of apoptotic cells seemed higher than that of non-apoptotic cel ls. These differences in intensity were even more pronounced after Tri ton X-100 extraction, Flow cytometry revealed that the absolute le ref s of proteasome staining in cells were decreased after Triton X-100 ex traction, However, no differences in staining levels were detected bet ween apoptotic and non-apoptotic cells. A relative increase of proteas ome concentration through cell shrinkage or a concentration in certain cell compartments may be the origin of the apparently increased signa l that was seen in immunofluorescence microscopy. Furthermore, proteas omes were clearly detectable in the apoptotic bodies and cytoplasmic v esicles 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 from cytometry. The proteasome content was retained only partially in the cells after Triton X-100 extraction, while the intermediate filaments were not de tectable anymore in the apoptotic cells.