Department of Medicine – Box Hill Hospital
THE AUSTRALIAN CENTRE FOR BLOOD DISEASES
Novel Therapies in the Treatment of Cancer
Supervisors: Dr A Dear, Dr R Medcalf
Email: anthony.dear@med.monash.edu.au
Location: Box Hill Hospital
Cancer represents one of the most common causes of death in western society. Much effort has been expended on developing new therapies for the treatment of cancer, a consequence of an increasing incidence of the disease together with the development of resistance of many cancers to conventional chemotherapy.
Our research efforts have concentrated on the design and testing of new cancer treatments in an attempt to overcome the current limitations of conventional cancer treatments. We have been interested in identifying molecules which have the capacity to inhibit enzyme systems thought to be integral to the spread of cancer cells from the site of primary tumour growth to distant regions of the body, a process referred to as metastasis. The metastatic process is responsible for the vast majority of the morbidity and mortality associated with cancer. We have identified several molecules with the potential to inhibit the metastatic process and we are currently in the process of testing these molecules in vitro and generating animal models to assess the anti-metastatic activity of these agents in vivo.
This project will involve both in vitro testing of new agents in assays developed in the lab, with the potential to identify new actions of these compounds, together with involvement in establishing and monitoring the effects of compounds in animal models. Students interested in understanding the process of new drug discovery together with the testing and application of drug discovery programs will find the project particularly attractive. This project will compliment much of the pre-clinical material students have been exposed to and in addition will augment much of the information provided in the clinical years ahead.
Regulation of Gene Expression in the Plasminogen Activating System
Supervisors: Dr R Medcalf
Email: robert.medcalf@med.monash.edu.au
Location: Box Hill Hospital
Broad scope of laboratory: Our laboratory is interested in understanding the mechanisms that govern gene expression in human cells. The genes that we study encode the members of a proteolytic enzyme system known as the plasminogen activating system. The end product of this system is a powerful protease called plasmin. Plasmin can degrade a number of other proteins, including most proteins of the extracellular matrix. It is also very important in the removal of blood clots ("fibrinolysis") from the circulation and facilitates cell movement. Indeed, cancer cells harness this enzyme system to enable them to metastasize to other organs. The important role the plasminogen activating system plays in these (and many other) processes has fuelled much interest to understand how its individual components are regulated.
Components of the plasminogen activating system:
Plasmin is formed when its inactive precursor, plasminogen is cleaved by one of two proteases known as tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). These activating enzymes are themselves regulated by specific inhibitors known as plasminogen activator inhibitor type 1 (PAI-1) and PAI-2. Other components exist that allow plasmin generation to occur at specific sites and on the surface of some cells. This in part is achieved by cell-bound receptors for either t-PA or u-PA. To make matters even more intricate, these receptors can associate with other cell bound proteins to activate intracellular signalling processes which can in turn change the cells behaviour. An outline of this system is provided in the figure below.
Our laboratory has a number of concurrent projects that address various aspects of the regulation of expression of essentially all components of the plasminogen activating system. One of the projects available addresses the biology of the inhibitor PAI-2. PAI-2 is expressed in many cell types, but is mainly found in monocytes, macrophages and in trophoblast cells of the placenta. It is an unusual protease inhibitor, mainly because it exists predominantly as an intracellular protein. Its target protease (u-PA) is present in the extracellular space or on the cell surface, bound to its receptor. There has been much speculation that PAI-2 plays an intracellular role in addition to its role as a protease inhibitor. We have designed a project that will address the functional role of PAI-2 in monocytes.
Another project addresses the role of tissue-type plasminogen activator in the central nervous system. Although t-PA is well known for its ability to remove blood clots from the circulation, it is now well established that t-PA plays a critical role in the central nervous system. We are interested to determine the mechanisms underlying expression of t-PA in the brain.
Projects available:
The intracellular role of PAI-2 in THP-1 monocytes
Supervisors: Dr Robert Medcalf and Dr Hong Yu
Email: robert.medcalf@med.monash.edu.au /
Location: Box Hill Hospital
We have available a monocyte cell line (THP-1 cells) that contains a unique defect in the production of PAI-2. We determined the molecular basis of this mutation during the course of a BSc. (Hons) project in 1999 and a paper describing this was published in Thrombosis and Haemostasis (Katsikis et al., 2000). Unlike all other monocytic cells available, THP-1 cells cannot make a functional PAI-2 protein. We have taken the opportunity offered by THP-1 cells to assess the intracellular role of PAI-2 by expressing an active PAI-2 in these cells. We observed that the THP-1 cells proliferation rates were significantly reduced and they did not respond to classical triggers of differentiation. We recently completed a microarray analysis of these PAI-2 expressing THP-1 cells and have identified a number of genes that have been either induced or down-regulated as a consequence of PAI-2 expression. The aim of the project is to assess some of these target genes regulated by PAI-2 to determine their role in monocyte proliferation and differentiation. Methods: Cell culture, Northern and Western blotting, PCR, DNA preparation and manipulation.
Studies on the regulation of the human tissue-type plasminogen activator gene in vivo using transgenic mice
Supervisor: Dr Robert Medcalf and Gabriel Liberatore
Email: robert.medcalf@med.monash.edu.au
Location: Box Hill Hospital
Tissue-type plasminogen activator (t-PA) is an important serine protease that is involved in the removal of blood clots from the circulation as well as the turnover of the extracellular matrix. More recently however, t-PA has been shown to be expressed at high levels in the brain where it plays a crucial role in the development of memory, visual processing, and neurodegeneration.
Our laboratory has been studying the transcriptional regulation of the t-PA gene in vitro and we have identified one particular control element within the t-PA gene promoter that may play a role in the regulation of the t-PA gene in the central nervous system. The protein binding to this element is suspected to belong to the NFAT family of transcription factors. The project available is to verify the role of NFAT in the regulation of t-PA gene expression in the murine brain and in vitro using a number of neural cell systems.
Signal Transduction in Platelets
Supervisors: Dr Yuping Yuan, Dr S Jackson
Email: yuping.yuan@med.monash.edu.au
Location: Box Hill Hospital
Platelet adhesion to the site of vessel wall damage is essential for normal haemostasis and thrombosis. This process is mediated by the interaction between the subendothelial protein, von Willebrand Factor (vWf), and the platelet surface receptors, glycoprotein Ib/V/IX and integrin aIIbb3,. Exaggerated adhesion can result in life threatening heart attacks and strokes, which represent the major cause of death in developed countries. On the other hand, defects in platelet adhesion can lead to severe bleeding disorders.
Our current research aims to improve our understanding of the regulation of platelet adhesion, particularly by intracellular signalling proteins and pathways. Using cellular, molecular and biochemical approaches, we have discovered critical roles for multiple signalling proteins including protein tyrosine kinases, protein kinase C and the protease, calpain, in regulating platelet adhesion.
Our recent studies have established for the first time the ability of GP Ib/V/IX to induce calcium (Ca2+) mobilisation from intracellular stores, as a necessary event for platelet cytoskeletal remodelling and integrin _IIb_3 activation. We are currently investigating, at a molecular and structural level, the important signalling events linking the vWf-GPIb/IX interaction to intracellular Ca2+ mobilisation. These studies will significantly enhance our current understanding of the mechanisms regulating platelet activation and may help identify key signalling molecules or pathways as novel antithrombotic targets.
The Dynamic Cytoskeleton – A Role in Haemostasis and Thrombosis
Supervisor: Dr S Schoenwaelder and Shaun Jackson
Email: simone.schoenwaelder@med.monash.edu.au
Location: Box Hill Hospital
Platelets are small specialised blood cells, which play a key role in clot formation and the arrest of bleeding (Haemostasis). Under normal conditions, platelets circulate throughout the blood stream without interacting with the vessel wall endothelium. However, upon vessel wall damage, platelets are recruited to exposed subendothelial proteins, where they adhere, spread and form a thrombus that effectively seals off the site of injury and prevents further blood loss. Many of the changes platelets undergo in response to vessel injury are driven by reorganisation of their cytoskeleton. Remodelling of the platelet cytoskeleton leads to changes in cell shape and behaviour, which in turn affect the ability of these cells to attach to an injured vessel wall and stop bleeding. We are working towards a better understanding of the mechanisms that regulate cytoskeletal reorganisation, so that we may more effectively regulate platelet function in vivo.
In many cell types, the cytoskeleton is modulated by the Rho family of small GTPases. Recent studies from our laboratory have demonstrated that RhoA plays an important role in platelet adhesion and thrombus growth under physiological flow conditions. The exact mechanism by which RhoA elicits this regulatory effect is unclear, however, it is possible that shear-induced conformational changes in platelet receptors and/or subendothelial proteins may lead to increases in the activation of platelet integrin aIIbb3, cytosolic calcium and the activation of RhoA. We are currently investigating the potential mechanisms mediating this shear-dependent regulation of platelet adhesion by RhoA. These studies will provide further understanding about cytoskeletal rearrangement, assisting the development of drugs targeting prevention of thrombus formation and other clotting related disorders.
Structure and Functional Interactions of Platelet Surface Receptors
Supervisor: Teresa Domagala and Dr Shaun Jackson
Email: teresa.domagala@med.monash.edu.au
Location: Box Hill Hospital
Platelets are specialised blood cells that play a critical role in blood clotting. For these cells to initiate the formation of a blood clot, they must first stick to the blood vessel wall at the site of injury. They do this through the action of specific platelet receptors. The interaction between the platelet glycoprotein (GP)Ib/V/IX receptor complex and von Willebrand factor (vWf) exposed on the injured vessel wall is of particular significance in this process. Once GP Ib/V/IX adheres to vWf, changes are initiated inside the platelet that allow it to stick firmly, change its shape and go on to form a firm clot that can ‘plug’ the area of injury and stop bleeding. The importance of GPIb/V/IX in the clotting process is highlighted in certain rare bleeding disorders in which they are abnormal or absent.
GPIb/V/IX has ‘external domains’ that are involved in sticking to the blood vessel and other platelets as well as ‘internal domains’ that bind to proteins inside the platelet. We are currently investigating the role of specific structural domains in the interaction of this receptor with intracellular structural and signalling proteins. Current studies in transfected cell lines will be complemented by in vitro protein binding studies using purified recombinant proteins. These studies will help to further define receptor interactions that regulate platelet adhesion and activation and contribute to our overall understanding of the processes that lead to normal (haemostasis) and pathological (thrombosis, a causative agent of heart attack and stroke) blood clot formation.
Haematopoiesis – The role of haemopoietic serpins in cell growth and differentiation
Supervisor: Dr P Coughlin
Email: paul.coughlin@med.monash.edu.au
Location: Box Hill Hospital
The blood of a normal person contains three main components – red cells, white cells and platelets which are made within the bone marrow. All mature cells are generated from a small group of primitive "stem cells". It is now thought that leukaemia, and related bone marrow diseases, are caused by malfunction of the stem cells, often as a result of faulty genes.
We are seeking new insights into the origins of leukaemia by investigating the genes which are normally active in these blood precursors. One such gene, serpin2A, produces a protein which we have found in the nucleus of "stem cells". The nucleus is a special compartment where genes are kept and controlled. In order to understand the biology of this unusual serpin we are analysing its molecular interactions with target proteins using the yeast-2-hybrid system. We are also examining its effects on growth and differentiation in model cell lines.
Our hypothesis is that serpin2A is a component of the mechanism which regulates copying of genes and may also control the way cells grow and divide. Our studies link serpin2A to several other proteins in the nucleus which take part in these control mechanisms. We are investigating the way that these proteins interact with each other to co-ordinate the development of primitive blood cells. Our results so far suggest new and exciting roles for this serpin which require further detailed study. We believe this will lead to new insights into the causes of blood diseases and ultimately give rise to safer and more effective treatments.
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