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Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Ueberham E, Lindner R, Kamprad M, Hiemann R, Hilger N, Woithe B, Mahn D, Cross M, Sack U, Gebhardt R, Arendt T, Ueberham U - J. Cell. Mol. Med. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Leipzig, Medical Faculty, Leipzig, Germany.

ABSTRACT
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.

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Expression of cell cycle proteins in liver extracts of ptetp16INK4aTALAP-2 mice and histological features of livers of p16INK4a expressing mice. (A) Photomicrographs of liver slides. P16INK4a protein was detected in hepatocytes of induced ptetp16INK4aTALAP-2 mice with an anti-human p16INK4a antibody (C-20) (A1, brown colour). In livers of male p16INK4a expressing mice many of enlarged hepatocytes were observed (A1-A6, black arrows). These hepatocytes do not express p16INK4a (A1) but express liver region-specific proteins like glutamine synthetase (A2) and cytochrome-P450 IIE (A5) representing pericentrally specific enzymes and carbamoyl phosphate synthase (A3), and E-cadherin (A6) being marker proteins for periportal and midzonal hepatocytes and also store glycogen (A4, pink colour).Proteins were detected with primary antibodies and immunocomplexes were visualized by DAB staining (brown). Bars represent 50 μm; CV, central vein; PV, portal vein (B, C) Western blots with antibodies against cell cycle proteins in liver extracts of induced (minus Dox) respectively switched off (+Dox) ptetp16INK4aTALAP-2 mice.
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fig04: Expression of cell cycle proteins in liver extracts of ptetp16INK4aTALAP-2 mice and histological features of livers of p16INK4a expressing mice. (A) Photomicrographs of liver slides. P16INK4a protein was detected in hepatocytes of induced ptetp16INK4aTALAP-2 mice with an anti-human p16INK4a antibody (C-20) (A1, brown colour). In livers of male p16INK4a expressing mice many of enlarged hepatocytes were observed (A1-A6, black arrows). These hepatocytes do not express p16INK4a (A1) but express liver region-specific proteins like glutamine synthetase (A2) and cytochrome-P450 IIE (A5) representing pericentrally specific enzymes and carbamoyl phosphate synthase (A3), and E-cadherin (A6) being marker proteins for periportal and midzonal hepatocytes and also store glycogen (A4, pink colour).Proteins were detected with primary antibodies and immunocomplexes were visualized by DAB staining (brown). Bars represent 50 μm; CV, central vein; PV, portal vein (B, C) Western blots with antibodies against cell cycle proteins in liver extracts of induced (minus Dox) respectively switched off (+Dox) ptetp16INK4aTALAP-2 mice.

Mentions: Immunocytochemical studies revealed transgenic human p16INK4a protein to be localized to both the cytoplasm and the nuclei of expressing cells (Fig.1C) similar to results described for other cell cycle proteins (for review see [32]). The expression of trans-genic p16INK4a did not result in any gross phenotypical changes either in liver or other organs. However, distinct changes were found at the histological level. The hepatocyte nuclei of p16INK4a expressing male mice were both fewer in number and larger in size than those of the controls (Fig. 2), while p16INK4a expressing female mice accumulated fat in hepato-cytes and rarely possessed hypertrophic hepato-cytes. All subsequent experiments were performed using male mice only. Flow cytometric analysis of propidium iodide stained hepatocytes [33] subsequently confirmed that p16INK4a expressing male mice had an increased proportion of hepatocytes with ploidy > 4N (Fig. 3A and B). Interestingly, the hypertrophic hepatocytes did not themselves express detectable levels of transgenic p16INK4a (Fig. 4A1). However, they clearly did demonstrate metabolic activity appropriate to their environment, producing enzymes of nitrogen metabolism (including glutamine synthetase, Fig. 4A2) if localized in the pericentral region of liver lobulus, and carbamoyl phosphate synthetase (Fig. 4A3) if located periportally or midzonally. The hypertrophic hepatocytes also accumulated glycogen in a manner comparable to normal hepatocytes (Fig. 4A4) andexpressed both the pericentral-specific detoxification enzyme P450 IIE1 (Fig. 4A5) and E-cadherin (Fig.4A6) which is an additional marker of periportal hepatocytes [34].


Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Ueberham E, Lindner R, Kamprad M, Hiemann R, Hilger N, Woithe B, Mahn D, Cross M, Sack U, Gebhardt R, Arendt T, Ueberham U - J. Cell. Mol. Med. (2007)

Expression of cell cycle proteins in liver extracts of ptetp16INK4aTALAP-2 mice and histological features of livers of p16INK4a expressing mice. (A) Photomicrographs of liver slides. P16INK4a protein was detected in hepatocytes of induced ptetp16INK4aTALAP-2 mice with an anti-human p16INK4a antibody (C-20) (A1, brown colour). In livers of male p16INK4a expressing mice many of enlarged hepatocytes were observed (A1-A6, black arrows). These hepatocytes do not express p16INK4a (A1) but express liver region-specific proteins like glutamine synthetase (A2) and cytochrome-P450 IIE (A5) representing pericentrally specific enzymes and carbamoyl phosphate synthase (A3), and E-cadherin (A6) being marker proteins for periportal and midzonal hepatocytes and also store glycogen (A4, pink colour).Proteins were detected with primary antibodies and immunocomplexes were visualized by DAB staining (brown). Bars represent 50 μm; CV, central vein; PV, portal vein (B, C) Western blots with antibodies against cell cycle proteins in liver extracts of induced (minus Dox) respectively switched off (+Dox) ptetp16INK4aTALAP-2 mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822548&req=5

fig04: Expression of cell cycle proteins in liver extracts of ptetp16INK4aTALAP-2 mice and histological features of livers of p16INK4a expressing mice. (A) Photomicrographs of liver slides. P16INK4a protein was detected in hepatocytes of induced ptetp16INK4aTALAP-2 mice with an anti-human p16INK4a antibody (C-20) (A1, brown colour). In livers of male p16INK4a expressing mice many of enlarged hepatocytes were observed (A1-A6, black arrows). These hepatocytes do not express p16INK4a (A1) but express liver region-specific proteins like glutamine synthetase (A2) and cytochrome-P450 IIE (A5) representing pericentrally specific enzymes and carbamoyl phosphate synthase (A3), and E-cadherin (A6) being marker proteins for periportal and midzonal hepatocytes and also store glycogen (A4, pink colour).Proteins were detected with primary antibodies and immunocomplexes were visualized by DAB staining (brown). Bars represent 50 μm; CV, central vein; PV, portal vein (B, C) Western blots with antibodies against cell cycle proteins in liver extracts of induced (minus Dox) respectively switched off (+Dox) ptetp16INK4aTALAP-2 mice.
Mentions: Immunocytochemical studies revealed transgenic human p16INK4a protein to be localized to both the cytoplasm and the nuclei of expressing cells (Fig.1C) similar to results described for other cell cycle proteins (for review see [32]). The expression of trans-genic p16INK4a did not result in any gross phenotypical changes either in liver or other organs. However, distinct changes were found at the histological level. The hepatocyte nuclei of p16INK4a expressing male mice were both fewer in number and larger in size than those of the controls (Fig. 2), while p16INK4a expressing female mice accumulated fat in hepato-cytes and rarely possessed hypertrophic hepato-cytes. All subsequent experiments were performed using male mice only. Flow cytometric analysis of propidium iodide stained hepatocytes [33] subsequently confirmed that p16INK4a expressing male mice had an increased proportion of hepatocytes with ploidy > 4N (Fig. 3A and B). Interestingly, the hypertrophic hepatocytes did not themselves express detectable levels of transgenic p16INK4a (Fig. 4A1). However, they clearly did demonstrate metabolic activity appropriate to their environment, producing enzymes of nitrogen metabolism (including glutamine synthetase, Fig. 4A2) if localized in the pericentral region of liver lobulus, and carbamoyl phosphate synthetase (Fig. 4A3) if located periportally or midzonally. The hypertrophic hepatocytes also accumulated glycogen in a manner comparable to normal hepatocytes (Fig. 4A4) andexpressed both the pericentral-specific detoxification enzyme P450 IIE1 (Fig. 4A5) and E-cadherin (Fig.4A6) which is an additional marker of periportal hepatocytes [34].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Leipzig, Medical Faculty, Leipzig, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus