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Liver disease and carcinogenesis
Current research projects
Project: Urea synthesis defect resulting from OTC deficiency
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.
Project: Characterising wild type LPCs, Spf-ash LPCs and LPCs generated from mESC
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.
Project: Understanding the cellular dynamics that underlie the conversion of a non-tumorigenic LPC line into a tumorigenic line
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.
Project: Understanding interactions between LPCs and inflammatory cells in the liver
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.