Induction of apoptosis in human cervix carcinoma cells during therapy by cisplatin

J Wesierska-Gadek PhD a, V Kotala, PhD b, S Kuchtickova, PhD b and M. Horky MD b

aInstitute of Cancer Research, University of Vienna, VIENNA, Austria b Institute of Pathophysiology, Masaryk University, BRNO, Czech Republik

AIMS: The aim of the therapy of human malignancies is the inhibition of cell proliferation and/or induction of apoptosis. We studied the kinetics of the morphological and biochemical changes in HeLa cells during chemotherapy by cisplatin. METHODS: Morphological changes of the nucleoli were studies using specific staining methods that allow to visualize not only nucleolar proteins but also nucleolar RNA. Apoptosis was evaluated by scoring of cells exhibiting changes characteristic for early and late apoptosis as determined by Hoechst 33258 staining and by examination of TUNEL and activated caspase-3 positivity. Expression and intracellular distribution of distinct proteins was analysed by immunoblotting and immunocytochemistry, respectively. RESULTS: We observed segregation of nucleolar components preceding onset of apoptosis in cisplatin treated HeLa cells. Monitoring of the biochemical changes revealed that activation of distinct caspases and degradation of their downstream effector proteins is executed in two phases. During an early apoptotic stage beginning 4,5 hours post- treatment, a release of cytochrome C, formation of apoptosome as well as activation of caspase9 and caspase-3 was observed. This was accompanied by cleavage of poly(ADP-ribose)polymerase-1 (PARP-1). A second phase of apoptosis occurring between 10 and 15 hours post-treatment was characterized by degradation of other nuclear and nucleolar proteins such as nuclear lamins, topoisomerase I and B23. CONCLUSIONS: Remarkable segregation of nucleoli coinciding with the activation of caspase-3 and its translocation into the nucleus substantiates the hypothesis that the nucleolus is a preferred effector target for caspase-3 mediated protein degradation during execution of apoptosis in HeLa cells.

KEY WORDS: nucleolar segregation, activated caspases, HeLa cells, apoptosome, PARP-1 cleavage, cytochrome C release.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Apoptosis - Molecular Mechanisms.