The new murine hepatic 3A cell line responds to stress stimuli by activating an efficient Unfolded Protein Response (UPR).

In the present study we have investigated the properties of a novel cell line (3A cells) obtained from the liver of 14.5days post coitum (dpc) wild-type mouse embryo. 3A cells morphology was characterized by fluorescent localization of F-actin and β-catenin. The expression of specific genes and proteins essential to liver function in these cells was comparable or even more efficient then in the differentiated hepatocytic cell line MMH-D6. 3A cells also showed the capability to excrete molecules in extracellular spaces resembling functional bile canaliculi, glycogen storage activity and the ability to control retinol-binding protein 4 secretion in response to retinol deprivation. Their response to the exogenous stress stimulus induced by tunicamycin was analysed by PCR Pathway Array containing 84 genes involved in the Unfolded Protein Response (UPR). 3A cells were shown to activate the UPR following a typical stressful event, indicating that this cellular model could be further exploited to investigate hepatic proteins secretion and specific reaction to different injuries

MMH Cells: An In Vitro Model For The Study Of Retinol- Binding Protein Regulated Secretion.

The untransformed stable cell line Met murine hepatocytes (MMH), generated from liver explants of transgenic mice expressing a constitutively active truncated form of the human hepatocyte growth factor receptor (cyto-Met), represents an innovative tool for in vitro studies of liver function. In the present report, we show that the MMH-D3 line isolated from the liver of a 3-day-old mouse is a useful model to investigate the regulation of the synthesis and secretion of retinol-binding protein (RBP) by retinol (vitamin A alcohol). Experiments with Northern blot hybridization, metabolic labeling of cellular proteins followed by immunoprecipitation, and Western blot analysis demonstrated that, similarly to the in vivo situation, in MMH-D3 cells the presence of retinol does not affect transcriptional and translational rate of the REP gene but is essential for regulating the secretion rate of the protein. Unlike HepG2 human hepatocarcinoma cells used thus far in studies of retinoid metabolism, including the synthesis and secretion of REP, vitamin A deficiency causes, in MMH-D3 cells, the inhibition of REP secretion and the protein accumulation in the cell, whereas retinol repletion promptly results in REP secretion. This model will be very useful in future studies on vitamin A distribution in the organism

Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest

MMH CELLS: AN IN VITRO MODEL FOR THE STUDY OF RETINOL-BINDING PROTEIN REGULATED SECRETION

The untransformed stable cell line Met murine hepatocytes (MMH), generated from liver explants of transgenic mice expressing a constitutively active truncated form of the human hepatocyte growth factor receptor (cyto-Met), represents an innovative tool for in vitro studies of liver function. In the present report, we show that the MMH-D3 line isolated from the liver of a 3-day-old mouse is a useful model to investigate the regulation of the synthesis and secretion of retinol-binding protein (RBP) by retinol (vitamin A alcohol). Experiments with Northern blot hybridization, metabolic labeling of cellular proteins followed by immunoprecipitation, and Western blot analysis demonstrated that, similarly to the in vivo situation, in MMH-D3 cells the presence of retinol does not affect transcriptional and translational rate of the REP gene but is essential for regulating the secretion rate of the protein. Unlike HepG2 human hepatocarcinoma cells used thus far in studies of retinoid metabolism, including the synthesis and secretion of REP, vitamin A deficiency causes, in MMH-D3 cells, the inhibition of REP secretion and the protein accumulation in the cell, whereas retinol repletion promptly results in REP secretion. This model will be very useful in future studies on vitamin A distribution in the organism

Increased expression of cellular retinol-binding protein 1 in laryngeal squamous cell carcinoma

PURPOSE: To investigate the genomic alterations in larynx carcinomas (LaCa) tissues and its prognostics values in predicting survival. METHODS: To analyse the aberrations in the genome of LaCa patients, we used array comparative genomic hybridization in 19 human laryngeal tumour samples. DNA samples were also subjected to detect human papillomavirus (HPV) sequences by polymerase chain reaction (PCR). Copy number gain was confirmed by real-time PCR. The cellular retinol-binding protein 1 (CRBP-1) gene expression was also confirmed by immunohistochemistry assay on LaCa tissues. To identify prognostic feature, CRBP-1 gene gain was correlated to patient survival. RESULTS: The most common gains were detected for CRBP-1 and EGFR genes, while DNA lost in RAF-1 gene. Immunohistochemistry assay was revealed strong expression of CRBP1 protein in those cases with CRBP-1 gene gain. The CRBP-1 gene gain and its expression correlated significantly with survival (P = 0.003). Cox regression analysis indicated that CRBP-1 expression level was a factor of survival (P = 0.008). HPV sequences were detected in 42% of the samples, and did not show any relationship with specific gene alterations. CONCLUSION: Our data shows that CRBP-1 gene gain can be determined by immunohistochemistry on routinely processed tissue specimens, and could support as a potential novel marker for long-term survival in laryngeal squamous cell carcinoma