Segregation of nucleolar components coincides with caspase-3 activation incisplatin-treated HeLa cells

We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell d eath and TUNEL staining. We used different light microscopic nucleolar stai ning methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyr ophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 ho urs. Staining with Toluidine Blue and Methyl-green Pyronine revealed segreg ated nucleoli 12 hours after the treatment with cisplatin, TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of n ucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is execute d in two phases. During an early apoptotic stage beginning 4.5 hours post t reatment an activation of caspase-9 and caspase-3 was observed. This was ac companied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) . The caspase-9 activation seems to be mediated by recruitment by the activ ating factor Apaf-1 because the increased accumulation of Apaf-1 and cytoch rome C in cytosol preceded the generation of mature caspase-9 form. A secon d phase of apoptosis occurring between 10 and 15 hours post treatment was c haracterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23. In conclusion, remarkable segregation of nucleolar argyrophilic proteins, n ucleolar RNA and a simultaneous activation of the cascade of caspases marke dly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby s ubstantiating the hypothesis that the nucleolus is a preferred target for c aspase-3-dependent proteolysis in cisplatin-treated HeLa cells.