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CURRENT RESEARCH PROJECTS

Project

Urea synthesis defect resulting from OTC deficiency

Project

Urea synthesis defect resulting from OTC deficiency

Project Outline

Ornithine transcarbamylase (OTC) is a urea cycle enzyme that is mutated in individuals with a metabolic disorder - OTC deficiency. The consequence of accumulating ammonia affects many tissues, and the liver in particular is damaged. The condition affects young children with neurologic consequences, hence liver organ transplant is necessary to treat those severely affected. Cell therapy using normal hepatocytes may also be possible, but hepatocytes are difficult to maintain and store; and once transplanted may not survive for very long. In contrast LPCs are robust and have the added advantage of long-term survival and the ability to proliferate and continue to generate hepatocytes in situ means they have the potential to confer sustained benefits following transplant.
We have access to the Spf-ash mouse model of human OTC deficiency through our collaboration with Professor Ian Alexander of the Children’s’ Medical Research Institute in Sydney. This group also has expertise in ESC and iPSC technology and this allows us to test our LPC lines and LPCs generated from ESCs and iPSCs in these mice.

Projects on offer follow from recent progress we have made:
We have generated LPC lines from the Spf-ash mouse. These have to be extensively characterised to confirm their LPC status and their ability to differentiate into hepatocytes and cholangiocytes.
We have preliminary evidence to suggest that mESC maintained in culture medium that supports LPCs can assume an LPC phenotype.

Research area
Liver disease

Laboratory
Liver disease and carcinogenesis

Contact
Professor George Yeoh
[email protected]

Project

Characterising wild type LPCs, Spf-ash LPCs and LPCs generated from mESC

Project

Characterising wild type LPCs, Spf-ash LPCs and LPCs generated from mESC

Project Outline

This project will assess the LPC status of the respective cell lines; first by validating their epithelial status (EpCAM and Ecad positive) and eliminating the possibility that they are mesenchymal (vimentin negative). Then the cells will be assessed for expression of LPC markers (CK19, M2PK, A6). Next, their ability to differentiate into hepatocytes and cholangiocytes will be determined following differentiation in culture as monolayers and in a 3D matrix (matrigel). Finally we will test their tumorigenic status by assessing their ability to grow as colonies in soft agar and confirm this by transplant into immune-deficient mice. Characterisation of the cells will involve immunohistochemical methods, qPCR and Western Blotting on fixed cells, mRNA and protein extracted from cultured cells.
What makes LPCs become cancerous?
LPC lines have been established from p53 -/- and p53 +/+ mice. LPC lines from both mice grow in soft agar and produce tumours when injected subcutaneously into nude mice; some do not. We are defining the differences between these cell lines at the molecular and cellular level to identify features which are causative and those which are consequential in terms of cancer. Specifically we are documenting chromosomal changes and focusing on oncogene candidates identified by gene profiling. Two anti-apoptotic genes IAP and Yap are prime suspects and their expression at the mRNA level (through qPCR) and protein level (by Western Blot) are increased in a range of cell lines correlating with tumorigenesis during culture. In contrast, expression of tumour suppressor proteins from the INK4a/ARF locus, p16INK4a and Arf respectively is lost. Current studies follow changes in LPCs as they are passaged and progressively become tumorigenic. We also document changes in expression of p53, p16 and Arf to determine if changes in their expression are causal or consequential to the process of transformation.

Research area
Liver cancer

Laboratory
Liver disease and carcinogenesis

Contact
Professor George Yeoh
[email protected]

Project

Understanding the cellular dynamics that underlie the conversion of a non-tumorigenic LPC line into a tumorigenic line

Project

Understanding the cellular dynamics that underlie the conversion of a non-tumorigenic LPC line into a tumorigenic line

Project Outline

When a non-tumorigenic (NT) cell line is passaged extensively by sub-culture it can become tumorigenic (T). The NT line does not produce colonies in soft agar and is therefore incapable of non-adherent growth and it does not produce tumours when transplanted subcutaneously in immune-deficient (nude) mice in contrast to the T line. There are two possible mechanisms by which the transformed cells can arise. he first (upper row in figure) proposes that T cells are present in the original culture and their numbers increase with each passage until there are sufficient numbers to produce a positive result by agar or nude mouse assay. The second mechanism (lower row in figure) suggests that there are no T cells in the initial culture, but some NT cells progressively acquire mutations with passaging and eventually a T cell arises and takes over the culture. The alternate mechanisms are depicted in the following figure.
Approach: The two mechanisms will show very different outcomes if progressive passages of an original NT cell culture is tested for its ability to generate colonies. If the first mechanism is correct, there will be increasing numbers of colonies in agar with passage; in addition, the colonies will have similar characteristics in terms of growth and pattern of gene expression. If the second mechanism is correct, there will not be increasing numbers of colonies with passage, but they should appear suddenly. If individual cells are changing differently and some are able to generate colonies, then the colonies should have different characteristics. The ability of colonies to grow will be assessed using the Cellavista instrument that is capable of continuously monitoring the growth of cells cultured in 96-well plates. The characteristics of the colonies will be determined in respect of the oncogenes and tumour suppressor genes that we have identified are differentially expressed when we compare NT and T cell lines.

Research area
Liver cancer

Laboratory
Liver disease and carcinogenesis

Contact
Professor George Yeoh
[email protected]

Project

Understanding interactions between LPCs and inflammatory cells in the liver

Project

Understanding interactions between LPCs and inflammatory cells in the liver

Project Outline

The increase in LPC numbers in both human and mouse liver disease pathology is accompanied by increased macrophage numbers. Using the choline-deficient, ethionine-supplemented murine liver disease model, we have now shown that monocyte-derived macrophage expression of the LPC mitogen, TNFa, initiates the increase in LPC numbers. CX3CR1(fractalkine receptor) expression is also increased in chronic liver disease, and we have recently found that CX3CR1 regulates production of TNFa together with the induction of LPC proliferation.
Our future studies will include determining whether macrophages can modulate ongoing LPC proliferation during chronic liver injury as well as the mechanisms by which the monocytes are recruited into the liver. We will also assess if it is possible to modulate liver production of TNFa by up- or down-regulating the function of CX3CR1 with its ligand, CX3CL1 (fractalkine), or blocking antibody, respectively.

Research area
Liver cancer

Laboratory
Liver disease and carcinogenesis

Contact
Professor George Yeoh
[email protected]

CURRENT STUDENT PROJECTS

Student Project

Understanding the cellular and molecular basis of liver progenitor cell transformation to cancer

Student Project

Understanding the cellular and molecular basis of liver progenitor cell transformation to cancer

Project Outline

Our laboratory has generated many liver progenitor cell lines that can be propagated and directed to differentiate into cholangiocytes and hepatocytes. The main thrust of the lab is to generate cells that can be used for cell therapy for transplant to alleviate symptoms related to liver pathology. Our lab, as well as our collaborators which have received these cell lines have found that following extensive passaging, some lines transform and become cancerous. When we compare the pattern of gene expression between the tumorigenic and non-tumorigenic LPCs, there are several oncogenes that are up-regulated, and several tumour suppressor genes that are down-regulated. In particular, we have focussed our attention on transcripts of p53 and the INK/Arf locus.
There are two possible mechanisms to explain the progression of tumorigenicity that we observe with passaging the cells. One model (1) proposes that at the beginning, extremely rare tumorigenic LPCs are present and they increase in number with passaging. The alternate model (2) hypothesises that no tumorigenic cells are present initially, but these accumulate mutations progressively and ultimately tumorigenic LPCs appear in extensively passaged cultures. The diagram below contrasts these models. The aim of the project is to determine which model applies to the transformation of LPCs during culture.  Two approaches will be used. First the transformed cells will be selected by virtue of their ability to grow in soft agar. The size and growth properties of the colonies will be determined. The colonies will be expanded and expression of p53, INK and Arf will be determined at the level of transcript abundance by qPCR.

Chief supervisor
Professor George Yeoh

Project suitable for
Honours

Essential qualifications
BSc with major in Biochemistry, Molecular Biology or Genetics

Start date
Semester 1 or Semester 2