Browsing by Person "Daubrawa, Merle"

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Aktivierung eines neuartigen Apoptose-Signalweges durch den Proteinkinaseinhibitor Staurosporin
(2009) Daubrawa, Merle; Graeve, Lutz
The protein kinase inhibitor staurosporine induces apoptosis via the activation of the intrinsic pathway. First staurosporine was described as a specific PKC inhibitor. Today it is known as a broad range kinase inhibitor and is used as a potent apoptosis inductor. However, the mechanism of the apoptotic effect remains elusive. Furthermore, staurosporine obviously exhibit the potential to eliminate chemotherapy resistant tumors by the induction of a novel intrinsic apoptotic signaling pathway. Different derivatives of staurosporine, e.g. UCN-01, PKC-412 or Enzastaurin are already tested in clinical trials phase I-II for cancer therapy. In the present work it could be shown that overexpression of Bcl-2 does not impede the caspase-dependent induction of apoptosis in J16- and JE6.1-Jurkat T-lymphocytes or in DT40 B-lymphocytes following staurosporine treatment . After generation of apaf-1 -/- DT40 cells it was demonstrated that staurosporine induces apoptosis despite the absence of Apaf-1 and therefore independently of the apoptosome. Together with the generated caspase-9 -/- DT40 cells, caspase-9 was identified as the central effector protein of both staurosporine-induced apoptotic pathways. The involvement of published and putative caspase-9 kinases could not be confirmed by the usage of specific inhibitors. Using phospho-mimicking and phospho-deficient caspase-9 variants, S183 could be identified as an essential phosphorylation site during staurosporine-induced apoptosis. In addition, after treatment with anticancer drugs apoptosome formation was blocked by an N-terminal tag of caspase-9. However, this tag could not prevent staurosporine-induced apoptosis. In further studies the potential role of cathepsines for this novel apoptosis signaling pathway could be analysed by their specific inhibition. In order to investigate the involvement of multiple kinases in this novel apoptotic signaling pathway, combination experiments with specific inhibitors of the respective kinases should be accomplished. Further investigations should clarify whether the influence of S183 on staurosporine-induced apoptosis is based on conformational alteration or on phosphorylation of caspase-9. The generation of additional caspase-9 variants including deltaCARD-caspase-9 or non-cleavable caspase-9 could lead to a deeper understanding of the role of caspase-9 for staurosporine-induced apoptosis. For this purpose caspase-9 -/- DT40 cells and cells reconstituted with different caspase-9 variants could be employed. The phosphorylation pattern of caspase-9 could be determined by mass spectrometric analysis. Xenograft or chorio allantois membrane models were used to investigate if the staurosporine derivative UCN-01 is also able to induce this novel apoptosis signaling pathway in vivo. The identification of both the mechanisms and the effector proteins of this staurosporine-induced apoptotic signaling pathway should provide the opportunity to develop novel agents for the elimination of chemotherapy-resistant tumors.