beta-Catenin is critical for early postnatal liver growth.

The Wnt/beta-catenin pathway plays an important role in embryonic liver development, morphogenesis, and organogenesis. Here, we report on the activation of beta-catenin during early postnatal liver growth. Modulation of beta-catenin expression was studied in CD-1 mice livers over a time course of 0 to 30 postnatal days (PD) and 3 mo. Increases in total and active beta-catenin were observed in developing livers from PD 5 to 20. A concomitant increase in the beta-catenin-transcription factor (TCF) complex along with nuclear and cytoplasmic beta-catenin was also evident, which coincided with ongoing hepatocyte proliferation by PCNA immunohistochemistry. This activation of beta-catenin was multifactorial, including cyclical inhibition of glycogen synthase kinase-3beta, suppression of casein kinase-IIalpha, and a transient increase in beta-catenin gene expression. Coprecipitation experiments revealed the formation of the beta-catenin-cadherin complex at PD 5, whereas adequate beta-catenin-c-Met complex at the hepatocyte membrane did not form until PD 20, which might be contributing to the free beta-catenin pool during early postnatal growth. Furthermore, beta-catenin liver-specific knockout mice exhibited smaller livers at PD 30, secondary to diminished hepatocyte proliferation. These data indicate that the activation of beta-catenin is critical for early postnatal liver growth and development.

R-Etodolac decreases beta-catenin levels along with survival and proliferation of hepatoma cells.

BACKGROUND/AIMS: Inhibition of hepatoma cells by cyclooxygenase (COX)-2-dependent and -independent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, a COX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drug Etodolac, which lacks COX-inhibitory activity, on the Wnt/beta-catenin pathway and human hepatoma cells. METHODS: Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/beta-catenin pathway components, and downstream target gene expression. RESULTS: Celecoxib at high doses affected beta-catenin protein by inducing its degradation via GSK3beta and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated beta-catenin protein secondary to decrease in its gene expression and post-translationally through GSK3beta activation. In addition, increased beta-catenin-E-cadherin was also observed at the membrane. An associated inhibition of beta-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutamine synthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident. CONCLUSIONS: The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (at physiological doses) on HCC cells were accompanied by the down-regulation of beta-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer.

Wnt'er in liver: expression of Wnt and frizzled genes in mouse.

UNLABELLED: The Wnt signaling pathway is essential for a wide array of developmental and physiological processes. Wnts are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing beta-catenin nuclear translocation and activation. Although beta-catenin has been shown to be critical in liver biology, the expression of the 19 Wnt and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of Wnt and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 Wnt, 10 frizzled receptors genes, and secreted Frizzled-related protein-1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 Wnts and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 Wnt and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 Wnt genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although 12 Wnt and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 Wnt and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 Wnt, seven Fz, and Fzb genes were expressed in SECs. CONCLUSION: These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways.

Activation of Wnt/beta-catenin pathway during hepatocyte growth factor-induced hepatomegaly in mice.

Hepatocyte growth factor (HGF) and beta-catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that beta-catenin forms a complex with c-Met (HGF receptor) that undergoes dissociation because of beta-catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized beta-catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF-induced hepatomegaly is mediated, at least in part, by activation of the Wnt/beta-catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via beta-catenin activation in the liver in 1- and 4-week studies. The mechanisms of beta-catenin activation in the 1-week study included loss of c-Met-beta-catenin association as well as canonical beta-catenin activation, leading to its nuclear translocation. In the 4-week study, beta-catenin activation was observed via canonical mechanisms, whereas the c-Met-beta-catenin complex remained unchanged. In both studies there was an associated increase in the E-cadherin-beta-catenin association at the membrane. In addition, we generated liver-specific beta-catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these beta-catenin conditionally null mice. In conclusion, beta-catenin- and HGF-mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma.

Mouse fetal liver cells in artificial capillary beds in three-dimensional four-compartment bioreactors.

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone metabolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for alpha-fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering.

Serum-free cryopreservation of porcine hepatocytes.

The use of porcine hepatocytes in xenotransplantation, bioartificial liver support or pharmacological approaches demands serum-free cryopreservation protocols yielding high quality, viable, functional hepatocytes. Here, primary porcine hepatocytes were frozen without serum in liquid nitrogen by the use of a computer-assisted freezing device. After thawing, more than 90% of the initial hepatocytes were lost, in part because of damage to genomic DNA. When cryoprotectants were used, the loss was lowered to 70% of the initial cell number; 90% of the remaining cells excluded trypan blue indicating a high degree of viability. Cells were seeded serum-free onto collagen-coated plastic dishes to determine proliferation and retainment of specific functions representing prominent features of hepatocytes in vivo. Whereas no cells adhered to the substratum effectively in conventional culture medium, the addition of conditioned medium derived from hepatic non-parenchymal cells improved attachment. Cells proliferated, retained hepatocyte-specific functions, such as urea production and cytochrome P450 activity, and expressed liver-specific genes to levels observed in non-cryopreserved hepatocytes. Thus, serum-free cryopreserved primary porcine hepatocytes may serve as a valid source of cells for downstream applications. The cells seem to function adequately when an appropriate environment is chosen for recovery after cryopreservation, an ultimate demand for the clinical application of human hepatocytes.

Genotoxicological characterisation of complex mixtures. Genotoxic effects of a complex mixture of perhalogenated hydrocarbons.

Toxicological and genotoxicological investigation of complex mixtures is one of the main focus of the recent research in toxicology. Testing complex mixtures present a formidable scientific problem since most recently available toxicological data has been obtained from single substance studies and is not simply transferable to mixtures of chemicals. Although there are no special strategies and standard protocols available for determining toxic and genotoxic effects of complex mixtures, the fundamental concepts of evaluation are the same as those for single substances. The focus of interest of the submitted paper is the genotoxicological characterisation of a complex mixture of mostly perhalogenated hydrocarbons which is generated as a waste product from the plasma etching process in the semiconductor industry. By use of several in vitro test systems (comet assay and micronucleus test), the clastogenic potency of the mixture was tested in various human cell types (lymphocytes and normal bronchial epithelial cells) and in rat hepatocytes. Results demonstrated that the complex perhalogenated hydrocarbons mixture causes DNA single-strand breaks and micronuclei formation, and direct concentration-to-effect correlations were proved in all experiments. The presence of an external metabolising system (S9 mix from rat hepatocytes) in human cell culture systems did not cause any change of the observed effects when compared to experiments performed in the absence of the S9 mix. Therefore, we conclude that the mixture acts as direct genotoxicant and that there is no detoxification by the external enzyme system.Further, convincing and reproducible results of the in vitro comet assay and the micronucleus assay in primary human cell cultures indicated these tests may be utilized for the genotoxicological analyses of complex mixtures with concern to human health hazard.