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Józefa Wsierska-Gdek¹, Marieta Gueorguieva¹ and Marcel Horky¹

¹ Cell Cycle Regulation Group, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria and ² Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Requests for reprints: Józefa Wsierska-Gdek Cell Cycle Regulation Group, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8 a, A-1090 Vienna, Austria. Phone: 43-1-4277-65247; Fax: 43-1-4277-65194. E-mail: Jozefa.Gadek-Wesierski{at}meduniwien.ac.at

We reported recently that roscovitine arrested human MCF-7 cancer cells at G₂-M phase of the cell cycle and concomitantly induced apoptosis. After roscovitine treatment, the level of wild-type p53 protein strongly increased and p53 was accumulated in the nucleus. Here, we raised the question of which pathway would be involved in roscovitine-induced apoptosis in MCF-7 cells, which are known to be caspase-3-deficient, and whether roscovitine-mediated activation of p53 protein might positively affect the execution of cell death. Roscovitine induced a depolarization of mitochondrial potential beginning at 6 hours posttreatment as evidenced by changes in J-aggregate formation and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Interestingly, roscovitine stimulated a site-specific phosphorylation of wild-type p53 protein in a time-dependent manner. p53 protein was specifically phosphorylated at Ser46. P-Ser46-activated wild-type p53 tumor suppressor up-regulated p53AIP1 protein, its downstream target known to mediate the depolarization of mitochondria. The onset of phosphorylation of p53 at Ser46 preceded the up-regulation of p53AIP1 protein and the depolarization of mitochondrial potential. We compared the kinetics of roscovitine-mediated p53 activation between caspase-3-deficient parental MCF-7 cells and cells reconstituted with caspase-3. The kinetics and the extent of p53 protein activation in caspase-3-proficient cells differed from those observed in caspase-3-deficient parental cells. Remarkably, roscovitine failed to induce phosphorylation at Ser46 in caspase-3-reconstituted MCF-7 cells. Our results indicate that, depending on the status of caspase-3 in MCF-7 cells, different apoptotic pathways were initialized.

Key Words: G₂ cell cycle arrest • up-regulation of p53AIP1 • depolarization of mitochondria • caspase-3 reconstitution of MCF-7 cells • J-aggregates • p53 half-life

Grant support: Jubileumsfonds from Oesterreichische Nationalbank (grant no. 10364).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/ 9/04; revised 10/ 4/04; accepted 11/ 8/04.