Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.
Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a).Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes.However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required.
Affiliation: Institute of Biochemistry, University of Leipzig, Medical Faculty, Leipzig, Germany.
Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a). Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli. We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.
- Cell Proliferation/drug effects*
- Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism*
- Growth Substances/pharmacology*
- Peptides, Cyclic/pharmacology*
- Stem Cells/drug effects*/physiology*
- Cells, Cultured
- Crosses, Genetic
- Green Fluorescent Proteins/metabolism
- Mice, Inbred C57BL
- Mice, Inbred DBA
- Mice, Transgenic
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fig07: Liver-specific stimulation of proliferation by nodularin.Mice were treated with nodularin thrice weekly and killed next day after the last injection (NDLN) or 4 weeks after the last injection (NDLN+). (A) Cyclin D1 mRNA levels were determined by real time RT-PCR. Differences between untreated p16INK4a expressing and control mice, between controls and NDLN-treated controls (NDLN), and between nodularin treated controls that were killed 4 weeks after NDLN treatment were statistically significant (P < 0.05, Student's t-test). The number of different animals tested was: p16(untreated) = 7, control(untreated) = 5, p16(NDLN) = 8, control(NDLN) = 12, p16(NDLN+) = 21, control (NDLN+) = 12.(B) in situ immunodetection of PCNA (B1, B2), A6 antigen (B3, B4) and E-Cadherin (B5, B6) in nodularin treated mice.80-90% of hepatocytes nuclei of control mice were immunostained with the PCNA antibody (brown colour, B2). In p16INK4a expressing mice predominantly small cells with oval nuclei were positively stained with the PCNA antibody (white arrow) and some of the hypertrophic hepatocytes (B1, black arrows), but less intensive than hepatocytes in control mice (B2, black arrows). The anti-A6 antibody identifies these cells as facultative liver stem cells, oval cells, forming ductular structures (B3, white polygon). These bipotent facultative stem cells of liver express the A6 antigen like cells of the biliary system (cholangiocytes, B3, B4, black polygon). A common feature of epithelial cells, hepatocytes, cholangiocytes and oval cells, in liver is the expression of E-cadherin.On the histological level E-cadherin is detected in periportal hepatocytes (B6, black arrowhead) biliary ductuli (B5, B6, black polygon) and oval cells (B5, white polygons), whereas the latter form the same ductular structures detected with the A6 antibody (B3).(C) Nodularin treated p16INK4a expressing mice (C1, C2) develop p16INK4a negative loci (C2) consisting of accumulating hypertrophic hepa-tocytes. Some of the hypertrophic hepatocytes were PCNA positive (C1, brown). The black lines mark the two foci in the PCNA stained liver slide (C1). (D) Oval cells were isolated from nodularin treated p16INK4a expressing mice by two-step collagenase perfusion and subsequently density gradient centrifugation. A6-positive cells (red colour) of passage 0 (D1) growing clonally were trypsinated and cultured to passage 10 (D2) without loss of A6 antigen. In higher passages (D3, passage 16) a loss or down-regulation of A6 expression was observed.Representative photomicrographs of OVUE265 are displayed. Bar represents 50 μm.
The induction of p16NK4a expression resulted in associated changes in the expression of a number of other genes involved in cell cycle control. Specifically, the levels of p27Kip1, cyclin E and CDK6 protein were decreased, while those of cyclin D1 and CDK4 proteins were increased as determined by western blot analysis (Fig. 4B and C). Differences between p16INK4a expressing mice and controls on transcriptional level, measured by microarray analysis were only marginal (data available at http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-1264). Despite the only slight increase of cyclin D1 expression in microarray analysis it was also determined by quantitative RT-PCR (Fig. 7A) to be significantly increased at the level of mRNA, whereas CDK4 mRNA levels quantified also with RT-PCR were equal to controls (data not shown).