Malignant mesothelioma (MM) is an aggressive malignancy of the pleural and peritoneal surfaces. Australia has the highest reported national incidence of mesothelioma in the world, and rates are increasing (Leigh et al., 2002). The clinical outcome for patients with this disease is extremely poor, with median survival of 9 to 12 months (Rizzo et al., 2001; Carbone et al., 2002). The latest developments in chemotherapy, radiotherapy and radical surgery have done little to improve the overall survival rate (Kindler 2000; Zellos et al., 2002). New approaches to therapy are thus required (Nowak et al., 2002). Cancer therapy using conditionally replicative adenoviruses (CRAds) and attenuated measles virus (vaccine strain MV-Edm) are novel and promising approaches to cancer treatment. CRAds strategy relies on selective viral replication in tumour cells but not normal cells. Major efforts have been directed toward achieving selective replication by the deletion of viral functions dispensable in tumour cells or by the regulation of viral genes with tumour-specific promoters (Alemany et al., 2000). However, the major clinical limitation of viral therapy has been lack of efficacy rather than safety concerns. In this study, I constructed CRAds in which tumour-specific promoter for Flt-1 (vascular endothelial growth factor receptor) control the essential E1 gene expression, and evaluated the cell-killing efficacy and specificity of CRAds driven by VEGF and Flt-1 promoters in the number of established mesothelioma cell lines and actual primary tumour cells from patients. CRAds with either VEGF or flt-1 promoters showed a strong killeg effect on mesothelioma cells. Co-delivery of CRAds with MMP-9 (matrix metalloproteinase-9) was assessed to determine whether therapeutic efficacy could be improved by reducing tumourassociated fibrosis thereby enhancing viral spread through a tumour mass. Combined therapy did result in greater suppression of tumour growth in vivo. I also identified an immuno-competent murine model of mesothelioma that was permissive for adenoviral replication. Combined viral therapy with immunotherapy (FGK45, an anti-CD40 antibody) in this model resulted in greater effect than Adwt or FGK45 alone and in greatest survival. I evaluated the capacity of MV-Edm to infect human mesothelioma cells to form syncytia, and lead to apoptosis and cell death. I also assessed the mode of death by analysis of markers of apoptosis including caspase-3. In vivo study showed that MVEdm- GFP transduction could be detected in human xenografts in immune deficient mice. Further studies to evaluate the mechanisms and efficacy of anti-tumour immune stimulation induced by tumour cell killing with CRAds and MV-Edm will be discussed in this study. MV-Edm has good killing effect on mesothelioma cells in vitro. In summary the work presented herein provide new insights into stratgies to improve viral therapies for mesothelioma. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342596 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

Malignant mesothelioma (MM) is an aggressive malignancy that originates from mesothelial cells and is highly resistant to conventional forms of anti-cancer therapy. Defects in apoptotic pathways are believed to play a major role in determining resistance to chemotherapy. The characterization of these pathways in mesothelioma is essential in order to develop more effective therapies. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that regulate apoptosis and have been implicated in the resistance of malignant cells. There is evidence that upregulation of specific IAP molecules can influence tumour progression and response to chemotherapy. In this study we examined the apoptotic signalling in MM cells and the potential role of IAPs in both cell proliferation and chemosensitivity. We examined expression of six IAP genes or isoforms in both malignant and normal mesothelial cells. Results demonstrated that XIAP, IAP-1, IAP-2, survivin and Bruce were expressed in all four MM cell lines and four primary mesothelial cultures. There was no evidence for differential expression of these genes between MM and mesothelial cultures. Livin expression was detected in only one MM cell line. Various aspects of apoptotic signalling pathways in response to the chemotherapeutic drug cisplatin were also analysed including: a) the mitochondrial integrity, b) caspase activation, c) cell viability and d) phosphatidylserine translocation. In order to further characterize the role of IAPs, the transcriptional regulation of these genes in response to cisplatin was investigated using real-time RT-PCR. The results of these experiments indicated that there was no significant regulation of IAPs at the transcriptional level in the cells examined during cisplatin-induced apoptosis. Overall the data was consistent with cisplatin inducing apoptosis in MM cells via intrinsic signalling pathways in a dose dependent manner. Regulation of IAP expression was not seen at the RNA transcription level as has been described in other tumour types but may occur through protein posttranslational events. In order to further investigate IAP function we performed analyses of two IAPs which had previously been proposed as having a role in mesothelioma: XIAP and survivin. Protocols for RNAi knockdown at the protein expression level were established. Although the data indicated significant reduction in protein expression, the effects on cell survival after treatment with cisplatin were moderate. These studies were then extended to other molecules that are known to interact with and modulate the function of IAPs. We characterized the expression of the proteins: XAF1, HTRA2, ARTS in MM cells. RT-PCR data showed that HTRA2 and XAF1 genes were expressed in MM cell lines, however we did not see expression of ARTS. On the basis of recently published data we examined the XAF1 splice variants expressed in MM cell lines by sequence determination and PCR screening.