PKC and Raf-1 inhibition-related apoptotic signalling in N2a cells.
In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS(10) ) or by inhibition PI3-K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf-1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10 - or WM-induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL-2-dependent mode of cell death after both treatments. Furthermore, in typical 'gain of function' experiments, cells with overexpression of permanently active Raf-1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf-1/BAD112, as well as PI3-K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross-talk between these two pathways at a level upstream from Raf-1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3-K is able to induce apoptosis.
Bronisz
Agnieszka
A
Department of Neurochemistry, Laboratory of Molecular Neurophatology, Medical Research Centre Polish Academy of Science, Pawińskiego, Warsaw, Poland.
Gajkowska
Barbara
B
Domańska-Janik
Krystyna
K
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001790:Blood Physiological Phenomena; D002352:Carrier Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D000077191:Wortmannin; D051025:bcl-Associated Death Protein
D000730:Androstadienes; C494820:Bad protein, mouse; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D051025:bcl-Associated Death Protein; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019311:Staurosporine; D000077191:Wortmannin