Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with Rab25 expression in ovarian cancer

The small GTPase Rab25 has been functionally linked to tumour progression and aggressiveness in ovarian cancer and promotes invasion in three-dimensional environments. This type of migration has been shown to require the expression of the hypoxia-inducible factor 1 alpha (HIF-1α). In this report we demonstrate that Rab25 regulates HIF-1α protein expression in an oxygen independent manner in a panel of cancer cell lines. Regulation of HIF-1α protein expression by Rab25 did not require transcriptional upregulation, but was dependent on de novo protein synthesis through the Erbb2/ERK1/2 and p70S6K/mTOR pathways. Rab25 expression induced HIF-1 transcriptional activity, increased cisplatin resistance, and conferred intraperitoneal growth to the A2780 cell line in immunocompromised mice. Targeting HIF1 activity by silencing HIF-1β re-sensitised cells to cisplatin in vitro and reduced tumour formation of A2780-Rab25 expressing cells in vivo in a mouse ovarian peritoneal carcinomatosis model. Similar effects on cisplatin resistance in vitro and intraperitoneal tumourigenesis in vivo were obtained after HIF1b knockdown in the ovarian cancer cell line SKOV3, which expresses endogenous Rab25 and HIF-1α at atmospheric oxygen concentrations. Our results suggest that Rab25 tumourigenic potential and chemoresistance relies on HIF1 activity in aggressive and metastatic ovarian cancer. Targeting HIF-1 activity may potentially be effective either alone or in combination with standard chemotherapy for aggressive metastatic ovarian cancer

Routledge Handbook of Southeast Asian Development

This Handbook traces the uneven experiences that have accompanied development in Southeast Asia. The region is often considered to be a development success story; however, it is increasingly recognized that growth underpinning this development has been accompanied by patterns of inequality, violence, environmental degradation and cultural loss. In 30 chapters, written by established and emerging experts of the region, the Handbook examines development encounters through four thematic sections: • Approaching Southeast Asian development, • Institutions and economies of development, • People and development and • Environment and development. The authors draw from national or sub-national case studies to consider regional scale processes of development – tracing the uneven distribution of costs, risks and benefits. Core themes include the ongoing neoliberalization of development, issues of social and environmental justice and questions of agency and empowerment. This important reference work provides rich insights into the diverse impacts of current patterns of development and in doing so raises questions and challenges for realizing more equitable alternatives. It will be of value to students and scholars of Asian Studies, Development Studies, Human Geography, Political Ecology and Asian Politics

Post Transcriptional Regulation of HSV Gene Expression and Studies of the Mutagenic Properties of HSV-1

During herpes simplex virus (HSV) infection expression of the different classes of viral genes is co-ordinately regulated and sequentially ordered. The three major classes of genes - immediate-early (IE), early and late can be separated on the basis of the kinetics of their expression and requirements for ongoing DNA synthesis. At the core of this regulation are three of the IE proteins IE110, IE175 and IE63. Both IE175 and IE63 are essential for viral growth and IE110 while non-essential does confer growth advantage in cell culture. While much is known about transcriptional control of gene expression and the interplay of these three IE proteins, little is known of any post- transcriptional regulatory mechanisms that may be employed by the virus. Recent interest has focused on this area and the aim of this study has been the investigation of a virus induced factor, termed LPF (late processing factor), which was previously shown to selectively increase the 3' processing efficiency of the herpes simplex virus type-2 (HSV-2) UL38 late poly(A) site. In this study an in vitro 3' processing assay was used to examine the 3' processing efficiencies of a selection of herpes simplex virus type-1 (HSV-1) poly(A) sites from the three temporal classes of genes. It was demonstrated that LPF selectively increased processing at the poly(A) sites of two late HSV-1 genes while having no effect on the processing efficiencies of another four poly(A) sites from the IE and early classes of genes. In addition both LPF responsive poly(A) sites were shown to be inherently less efficient 3' processing sites that the non-responsive sites. No common factor, which could be responsible for the reduced efficiency of these two sites, was identified by sequence or two dimensional structural analysis. Examination of the protein binding properties of each of the test poly(A) site RNAs revealed that three protein bands A, B and C were consistently bound to the poly(A) site RNAs. These proteins were of similar size to components of the 3' processing complex required for efficient cleavage and polyadenylation of precursor mRNAs. In addition the level of protein binding was shown to be increased by HSV infection. As stated previously the HSV-1 IE63 gene is essential for viral growth and it has been shown by a number of studies to be required for late gene expression. There is evidence that it can exert this influence at both the transcriptional and post- transcriptional levels and also that it may or may not influence late gene expression via an effect on DNA synthesis. In this study IE63 has been shown to be required for LPF activity and the expression of selected true-late genes (UL38, UL44 and US 11). IE63 also appears to be required for the efficient expression of three additional genes, two early genes UL29 and UL42 components of the viral DNA synthesis machinery and the IE gene lEllO. The evidence presented here supports a central role for IE63 in the regulation of gene expession. At least part of the mechanism of this regulation is at the post-transcriptional level mediated by its ability to increase the processing efficiency of selected poly(A) sites, with specific deficiencies in individual poly(A) sites making them targets for regulation. It is clear this is not the whole story and that IE63 may exert an additional influence via regulation of viral DNA synthesis. HSV-1 and HSV-2 can transform mammalian cells to a tumourigenic phenotype and it has been proposed that transformation occurs via a hit-and-run mechanism since the continued presence of viral DNA or proteins is not required for the maintenance of the transformed state. One way in which such a hit-and-run mechanism might operate is by increasing the frequency of mutations and there is evidence that HSV-1 can act as a mutagen. The aim of the second part of this study was to identify the properties of HSV-1 which could induce such mutations using a mutagenesis assay based on the shuttle vector pZ189. This study was the continuation of work initiated in the Institute by P. Clarke and the first step was to increase the efficiency of transformant recovery in the assay. To this end each aspect - plasmid preparation, transfection, infection and tranformation was optimized in turn and transformant recovery increased 3 to 5-fold. Using this assay HSV-1 infection was shown to increase the mutation frequency by 2.5-fold, however this was not considered to be significantly different from the spontaneous mutation frequency and further use of the assay to determine the mutagenic properties of HSV-1 was not pursued

Studies on the AROM Pentafunctional Enzyme in Saccharomyces cerevisiae and Aspergillus nidulans

Aromatic amino acids are synthesised via the shikimate pathway. AROM is a pentafunctional enzyme which catalyses the five central steps of the shikimate pathway and is found in fungi and yeast. This thesis describes the purification and characterisation of AROM from overexpressing strains of Saccharomyces cerevisiae and Aspergillus nidulans. S. cerevisiae AROM was purified 30-fold with a 10% yield from a yeast overexpression strain which exploited the ubiquitin-fusion system of yeast. The purified protein was found to possess all five enzyme activities in a similar ratio to that observed in crude extract and had a subunit molecular weight of 175kDa. The main S. cerevisiae protein was shown to have several minor, lower molecular weight contaminants following SDS PAGE and three of these were found to cross-react with anti-AROM antibodies raised against Neurospora crassa AROM. The peak AROM fraction eluted from gel filtration chromatography was found to be composed of two proteins which were separable by native PAGE and both of which were shown to have shikimate DH activity. The poor recovery and multiple protein bands suggested that during the AROM preparation limited proteolysis was occurring despite a number of anti-proteinase measures. No means of eliminating limited proteolysis during S. cerevisiae AROM isolation were found and purification studies were carried out on the AROM of A. nidulans in the hope that proteolysis might not be as problematic in this species. A rapid procedure for the purification of A. nidulans AROM from the overexpresion strain A. nidulans 1314 has been developed which results in 13-fold purification and a 9% yield. The subunit molecular weight was estimated at 175kDa and the native molecular weight suggests that the protein is a dimer. The N-terminal DHQ synthase activity in the AROM protein was found to be severly deficient in both crude extract and the purified protein from A. nidulans 1314. This was independently attributed to the introduction of a missense muta tion in this region of the polypeptide. A preliminary limited proteolysis study was carried out on AROM purified from A. nidulans 1314 which suggested that the proteolysis pattern is complicated and which show that the DHQase and shikimate DH enzyme activities are least susceptible to limited proteolysis