Malignant mesothelioma (MM) is an aggressive and highly chemo-resistant tumour of the mesothelium. Asbestos is indicated as the environmental factor most commonly associated with mesothelioma. The chemo-resistance is possibly due to impaired apoptotic mechanisms since it is known most chemotherapeutic drugs act via apoptosis. Murine MM cell lines that have been derived from tumours induced by inoculation of crocidolite asbestos into mice provide a suitable model, since both phenotypic and biological properties are closely similar to the human disease. Four murine MM cell lines were used in this study, namely ABl, AB12, AC29 and AC34. The aim of this study was to determine susceptibility of those MM cell lines to induction of apoptosis and the expression of key molecules in those cells. Many apoptosis-related genes are known, expression of some of these genes in four murine MM cell lines were investigated in this study. Gene expression was determined using conventional reverse transcription PCR (RT-PCR) and quantitated using real-time RT-PCR with SYBR-Green I detection on a Rotor-Gene 2000 (Corbett Research, N.S.W., Australia). Gene expression data was normalised against the most stable housekeeping genes as determined by the geNonn software. Susceptibility of the four murine MM cell lines to apoptosis induction was determined using cisplatin or TNF-a or IFN-y at different concentrations and at different times in the case of cisplatin. Apoptosis was assessed by a DNA laddering assay. Conventional RT-PCR results showed that all four murine MM cell lines expressed DR5, Bax, Bcl-xL, FLIP-L, c-Myc and caspase-3. Fas mRNA was detected in all cell lines except AC29. Neither FasL nor Bcl-2 was expressed in the four murine MM cell lines. / Quantitation of gene expression showed that there were significant differences in Fas, DRS, Bax, Bcl-xL, c-Myc, FLIP-L and caspase-3 mRNA levels across the cell lines (P<0.05). Absence of Fas receptor in AC29 may play a role in the immunogenicity of this cell line. DNA laddering results indicated that cisplatin induced apoptosis in a dose- and time- dependent manner in those MM cell lines. Susceptibility as reflected by the minimum apoptosis-inducing dose varied among the cell lines from 1 pdml (AB12) to 10 & n l (AC34). Susceptibility to TNF-a or IFN-y also varied among the cell lines where AB12 was sensitive while AC29 was resistant to those cytokines. The AC29 and AB1 cell lines were used to examine cisplatin or TNF-a induced genes related to apoptosis, expression of Fas, caspase-3, Bax and Bcl-xL mRNA was determined using real-time RT-PCR after 6 and 24 hours induction with cisplatin or TNF-a. The results demonstrated that caspase-3 and Bax were up-regulated whereas Bcl-xL was down-regulated in AC29 after 6 hours treatment with cisplatin. Although only Bcl-xL was down-regulated in ABl after 6 hours treatment with cisplatin, the down regulation was more pronounced than in AC29. TNF-a induction of gene expression showed that Bcl-xl decreased and Fas was up-regulated significantly in AB1 after 6 hours whereas only Bcl-xL was down-regulated in AC29 after 6 hours and continued to decrease until 24 hours. The differences in gene expression changes were noticed not only between cell lines but also between two inducer agents. There were several significant results of this study. Firstly, gene expression in murine MM was seen to parallel those that have previously been described for the human disease. / Therefore murine MM is likely to be a useful in in vivo model for future studies targeting apoptotic molecules. Secondly, a number of genes were not previously examined in MM were characterized in the murine model. Finally, differences in basal and induced gene expression between cell lines and inducing agents were characterized which should be followed in further studies at the protein level (eg caspase-3 activity).

Malignant mesothelioma (MM) is an aggressive tumour, which is highly associated with previous asbestos exposure and is resistant to most conventional anticancer therapies. Previous studies have used a mouse model of to 01 p effective approaches to induction of anti-tumour immunity using modification of tumour cells by the introduction of genetic constructs expressing genes such as that for B7-1 so that tumour growth can be inhibited in vivo. Transfectant clones, AC29 B7-7 and AC29 B7-6, which showed equal levels of expression of B7-1 but were markedly different in tumorigenicity were assessed using suppression subtractive hybridization (SSH) in order to isolate transcripts which may have been differentially expressed in the two clones. SSH allowed isolation of a number of cDNAs which were apparently differentially expressed in the cell lines. These required characterisation in order to determine their possible relevance to tumorigenicity. Two cDNAs designated as 7-7-76 and 7-7-43 had been isolated previously and the aim of this project was to characterise these cDNAs by sequencing, searching for their homology relationships and investigating gene expression profiles. Preliminary searches revealed that clone 7-7-43 had homology to cyclin-dependent kinase regulatory subunit 1 which plays a role in the cell cycle. On the other hand, clone 77-76 showed only homology to an EST of hypertension related protein and therefore, further investigation was required to obtain the identity of clone 7-7-76. The first part of this project was to in investigate and evaluate gene expression on clone 7-7-43, using both relative RT-PCR and Northern blotting.' In the second part of this project, a more intense study of clone 7-7-76 was conducted. Clone 7-7-76 was investigated for its homology relationships and its gene expression profile. / Results obtained from relative RT-PCR suggested no difference in the expression of the either eDNA clone (7-7-43 and 7-7-76) between the MM clones AC29 B7-6 and AC29 B7-7, the cells used to derive these clones by SSH. Therefore, it was concluded that neither clone 7-7-43 nor 7-7-76 was differentially expressed in MM cells of differing immuno enicit RACE was employed in order to derive a longer sequence of clone 7-7-76 and the newly derived sequence of 7-7-76 was again used to search for homologies using a wider range of sequences for human and other species. These investigations on clone 7-7-76 showed it to correspond to the sequence of human mitofusin 2 which is involved in determining mitochondrial morphology The results determined in this project suggest that clones 7-7-43 and 7-7-76 are not differentially expressed in the range of MM cell lines tested. The data have however highlighted the potential of the SSH technique to easily derive cDNA clones worthy of investigation, but underline the possibility of false positive clones being isolated. The need for an efficient, accurate screening procedure such as real-time PCR is acknowledged.

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.

Cancer is a leading cause of death in the western world and is increasing in frequency world-wide. Although diagnosis, treatment and therapeutic approaches to cancer have improved, many types of cancer are still lethal due to the lack of radical treatment. One of the fatal neoplastic disease types with poor prognosis is represented by malignant mesothelioma (MM). MM is characterised by very high mortality rate and limited therapeutic options. The etiology of the disease is mainly associated with exposure to asbestos fibres. The incidence of MM is increasing in many countries. The search for novel molecular targets, anti-cancer strategies and drugs, which would considerably improve the treatment is of great importance. Certain new drugs, especially those with specific molecular targets, show high selectivity in their action to cancer cells, and have considerably increased the cure rate in some types of cancer. Mitochondria have recently emerged as a very promising target for anti-cancer agents. A group of compounds with anti-cancer activity that induce apoptosis by way of mitochondrial destabilisation, termed 'mitocans', have been a recent focus of research. Several mitocans have been shown to selectively induce apoptosis in cancer cells and suppress the growth of many types of carcinomas in...