Roscovitine-induced up-regulation of p53AIP1 protein precedes the onset of apoptosis in human MCF-7 breast cancer cells.
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.
Wesierska-Gadek
Józefa
J
Cell Cycle Regulation Group, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8 a, A-1090 Vienna, Austria. Jozefa.Gadek-Wesierski@meduniwien.ac.at
Gueorguieva
Marieta
M
Horky
Marcel
M
D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001943:Breast Neoplasms / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011506:Proteins / Q000235:genetics*; D011687:Purines / Q000633:toxicity*; D000077546:Roscovitine
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C414742:P53AIP1 protein, human; D011506:Proteins; D011687:Purines; D000077546:Roscovitine; D010768:Phosphoserine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases