LIVER DISEASE AND CARCINOGENESIS

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.

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, p16^INK4a 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.

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.

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.

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.