1 - TD Pancrease to Liver Flashcards
What are AR42-B13 cells?
Pancreatic cells from azaserine-treated rats expressing exocrine and neuroendocrine properties
Treatment with what agent can trigger AR42-B13 TD from P-H fate?
Dexamethasone - synthetic glycocorticoid
Tosh’s study set out to delve deeper into TD mechanisms, why did they chose P-L TD?
Hepatic foci appear in rat or human pancreas in repsonse to:
- Copper depletion
- Post transplantation
- Over expression of kaeratinocyte growth factor in the pancreas of transgenic mice
- Is observed naturally in primates
THEREFORE already knew this was a process that did occur, making it a good starting point for understanding TD
What are the 8 liver markers that Tosh’s study chose to look for to prove TD had occured?
- Transferrin
- alpha1-antitrypsin
- Transthyretin
- Glucose-6-phosphate
- alpha-fetoprotein
- Cytokeratin 8
- P-Glycoprotein
- Penol Sulphotransferase
What was oberved over 3 weeks following treatment of AR42J-B13 cells with 10nM Dex? (amylase and albumin detection only)
D5 = increase amylase expression but a sub-population become progressively flattened and dont express amylase
2Wk = Sub-population extremely flattened (epithelium-like) and a loss of amylase expression
3Wk = Strong albumin expression in flattened morphology cells confirms TD to Liver fate has occurred
What was observed over 3 week following 10nm Dex treatment? (all 8 liver markers) and what does this imply about TD?
- Cells initially lose P-phenotype (loss of amylase)
- Begin to express H-genes produced in fetal liver (G6P, Transferring)
- These same cells then later express larger amounts of adult liver proteins (albumin, P-glucoprotein)
Which all implies that TD is a progressive process - involving a co-ordinated programme of gene activation
What three features were seen in the dex treated cells?
Extensive ER
Structures resembling bile canaliculi
Polyploidy (multi-nucleiate cells, typical of hepatocytes)
What effect did a dex cocnentration >10nM have on cell growth rate?
Significant decrease
6 down to 3 doublings per week
One of the experiments saw them test 1um Dex Vs 1uM Dex + 10ng/ml oncostatin M over 2 weeks. What were the results of this experiment when looking at the % of the population expressing liver markers G6P and albumin?
Dex only:
- 25% G6P +VE
- 6% Albumin +VE
Dex + oncostatin:
- 89% G6P +VE
- 64% Albumin +ve
Give me some liver genes proven to be expressed in Dex treated cultures
Gluconeogenic enzymes (G6P)
Fibrinogen family (alpha-fibrinogen)
Phase 1 metabolism (CYP3A1)
Phase II metabolism (Bilirubin UGT)
Ammonia detoxification (Arginase)
What functions were the induced hepatocytes able to do that are similar to normal liver cells?
- Synthesise and secrete albumin
- Synthesise lipid
- Metabolise drugs
- Replicates hepatitis B virus
How did they prove lineage relationship? (how did they prove that the AR42J-B13 cells did become hepatocytes)
- Labelled AR42J-B13 cells with GFP attached to Elastase promotor (one only expressed in pancreatic cells)
- Added dex and allowed TD to occur, see the following:
> Some GFP +ve cells co-express liver protein G6P = immature liver cells
> Some GFP -ve but express G6P = mature liver cells
BUT all of this proves that the liver cells are arising directly from pancreatic cells
When trying to understand the molecular mechanism of TD, what did they discover in relation to HNFalpha?
It begins to translocate into the nucleus @D5 post treatment with 1um Dex
What is C/EBDbeta and is it seen in AR42J-B13 cells?
A TF expressed during the differentiation of adipose tissue and liver
Therefore, isnt seen in AR42J-B13 cells
When investigating the molecular mechanism of TD, what did they discover in relation to C/EBDbeta expression and what did it correlate with?
It’s expression in AR42J-B13 cells was induced 3D post dex treatment
These same cells also showed decreased amylase and increased G6P expression after 2 weeks
Expression continued to increase over 2wks
