The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.

Malignant pleural mesothelioma (MPM) is an aggressive, treatment-resistant tumour. Cisplatin (cis-diamminedichloroplatinum (II)) is the best single-agent chemotherapy for MPM, but platinum-based combination therapies give the best overall response rates. However, cisplatin use is limited by resistance and severe side effects. This thesis has increased the knowledge concerning cisplatin-induced cell death in MPM by describing a novel potential therapeutic target, and three novel phenotypes of cisplatin-resistance in a human MPM cell line (P31) and its cisplatin-resistant sub-line (P31res1.2). The novel potential therapeutic target, and one of the novel phenotypes, was cisplatin-resistant pro-apoptotic BH3-only proteins. In the P31 cells, cisplatin transiently increased pro-apoptotic BH3-only proteins during 6 h of exposure. This response was almost completely abrogated in the P31res1.2 cells. De-regulated caspase activity and activation was the second novel phenotype identified. The P31res1.2 cells had earlier, possibly mitochondria-independent, caspase-3 activation, increased basal caspase-3 activity and increased basal cleavage of caspase-8 and -9. Despite these differences, 6-h equitoxic cisplatin exposures rendered 50-60% of the cells apoptotic in both cell lines. The third novel phenotype was abrogated Na+K+2Cl--cotransporter (NKCC1) activity. Although NKCC1 activity was dispensable for cisplatin-induced apoptosis, balanced potassium transport activity was essential for P31 cell survival. Finally, the survival signalling protein Protein Kinase B (PKB or Akt) isoforms α and γ were constitutively activated in a PI3K-independent manner in P31 cells. In the P31res1.2 cells, PKBα and γ activities were increased, and there was PI3K-dependent activation of PKBβ. However, this increase in PKB isoform activity was not strongly associated to the cisplatin-resistance of the P31res1.2 cells.

Ziel der vorliegenden Arbeit war es, die Rolle des pro-apoptotischen Proteins Bim am endoplasmatischen Retikulum (ER) und an den Mitochondrien zu untersuchen. Für diese Untersuchungen wurden zwei Isoformen von Bim verwendet, zum einen BimL, welches an den Motor Dynein Komplex gebunden ist, zum anderen BimS, welches im Zytosol lokalisiert ist. Um eine konditionale Expression von Bim zu erreichen, wurde Myc-markierte humane cDNA unter der Kontrolle des Tet-Off Systems in einen adenoviralen Vector kloniert. Eine Überexpression von BimL und BimS induzierte in der Prostatakarzinomzelllinie DU145 Bax- und Bak-abhängigen apoptotischen Zelltod. Eine Überexpression des anti-apoptotischen Proteins Bcl-2 lokalisiert am ER zeigte eine vollständige Hemmung der Bim-induzierten Apoptose, was die Wichtigkeit des ER unterstreicht. Überexpression von Bcl-2 an den Mitochondrien führte eine partielle Hemmung herbei. Bim Expression induzierte Bax- und Bak-abhängig den Zusammenbruch des mitochondrialen Membranpotentials. Dieses wurde ebenso in mit am ER lokalisiertem Bcl-2 Zellen beobachtet. Bcl-2 lokalisiert an den Mitochondrien verminderte dagegen mitochondriale Permeabilisation. Proteinanalysen zeigten eine Hochregulierung von ER-Stress Proteinen nach Bim Überexpression. Zusätzlich wurde Cytochrom c Freisetzung aus den Mitochondrien und Aktivierung von Caspase-9, -3 und -8 beobachtet. Mit einem Breitband-Caspase Hemmer konnte der Bim-induzierte Zelltod vollständig gehemmt werden, was zeigt, dass Caspasen essentiell sind. Zusammenfassend kann gesagt werden, dass Bim, parallel zum mitochondrialen Signalweg, ER-Stress auslöst und, dass Bim eine effektive Apoptose durch die Interaktion des ER und der Mitochondrien induziert. / 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. 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 Bim isoforms were used for these investigations. One was BimL, which is bound to the motor dynein complex of the microtubule and the other one was BimS, 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. Also, Bax- and Bak-dependent breakdown of the mitochondrial membrane potential upon overexpression of either Bim isoforms was measured. This effect was also observed in cells overexpression the anti-apoptotic protein Bcl-2 at the ER. However, targeting Bcl-2 to the mitochondria partially inhibited Bim-induced mitochondrial permeabilization. These findings indicated the execution of the intrinsic apoptotic pathway upon Bim signalling. Nevertheless, expression of Bcl-2 at the mitochondria partially suppressed Bim-induced apoptosis whereas ER-targeted Bcl-2 entirely prevented cell death induction by Bim underlining the importance of the ER. Further, an upregulation of ER stress proteins upon Bim expression was seen. Cytochrome c release form the mitochondria and activation of caspase-9, -3 and -8 was observed. In addition, the complete inhibition of Bim-induced cell death by a pan caspase inhibitor revealed that caspases are crucial. 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 and the ER. The ER-mitochondria cross-talk leads to the amplification of the apoptotic death signal.