1 Summary

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The aim of this thesis was to investigate the role of the pro-apoptotic BH3-only protein Bim, at the endoplasmic reticulum (ER) and the mitochondria and additionally to reveal the apoptotic signalling pathway of this Bcl-2 family member.

For this purpose, a full length human myc-tagged Bim cDNA was cloned into an adenoviral vector, which allows for the conditional expression of the transgene under the control of a Tet-Off-system. Two different kinds of Bim isoforms were used for these investigations. One was BimL, the long version of the Bim protein, which is bound to the motor dynein complex of the microtubule and the other one was BimS, the short splicing variant, which is localized in the cytosol.

The enforced expression of each of these two isoforms in the prostate cancer cell line DU145, showed the capability of BimL and BimS to induce apoptosis via either Bak or Bax. Further, expression of both Bim isoforms in DU145 cells, stably expressing GFP-Bax or GFP-Bak, confirmed that Bim induces redistribution and clustering of Bax and Bak. Also, both DU145-Bax and DU145-Bak cells showed breakdown of the mitochondrial membrane potential upon overexpression of either Bim isoforms. This effect was also observed in DU145 cells overexpression the anti-apoptotic protein Bcl-2 at the ER. However, targeting Bcl-2 to the mitochondria gave these cells partial resistance to Bim-induced mitochondrial permeabilization. These findings indicated the employment of the intrinsic apoptotic pathway for cell death induction upon Bim signalling. Nevertheless, expression of Bcl-2 at the mitochondria partially suppressed Bim-induced apoptosis whereas Bcl-2 was targeted to the ER entirely prevented cell death induction by Bim underlining the importance of the ER in the Bim-mediated cell death pathway. To examine the events at the ER in the Bim pathway, the calcium effluxes from the endoplasmic reticulum into the cytosol were assessed. Increased cytosolic calcium levels could be detected upon BimS expression and preceded activation of the mitochondria. Further, Western blot analysis showed an upregulation of ER stress proteins upon Bim expression. Bim-induced DNA-fragmentation was accompanied by cytochrome c release form the mitochondria and the processing and activation of caspase-9, -3 and -8. Cleavage products of these caspases were detected by Western blot analysis and their activation were shown by binding of FITC-labelled substrates against the indicated caspases. In addition, the complete inhibition of Bim-induced cell death by a pan caspase inhibitor revealed that caspases are crucial for the execution of apoptosis. Inhibition of caspase-8 by a specific inhibitor did not lead to the breakdown of the mitochondrial membrane potential upon BimS overexpression suggesting that capase-8 takes an important place upstream of the mitochondria.

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In conclusion, Bim induces the mitochondrial apoptotic pathway and, in parallel, triggers ER stress. It seems that Bim mediates cell death through the interaction of the mitochondria, the ER and caspase activation. Induction of a secondary mitochondrial activation by an ER-mitochondria cross-talk leads to the amplification of the apoptotic death signal.


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