295 Nuclear steroid receptors modulate cellular trafficking of ursodeoxycholic acid in primary rat hepatocytes

295 Nuclear steroid receptors modulate cellular trafficking of ursodeoxycholic acid in primary rat hepatocytes

Category 4a." Molecular and Cellular Biology." Cell Cycle Control/Apoptosis the expression of Eg'r-1 in human liver cells and the sig-nalling pathways...

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Category 4a." Molecular and Cellular Biology." Cell Cycle Control/Apoptosis the expression of Eg'r-1 in human liver cells and the sig-nalling pathways involved. PHH were isolated by 2-step collagenase perfusion of human liver tissue. The influence of HGF and EGF on the expression of Eg'r-1 was tested in PHH as well as in human hepatoma cells. Treatment of PHH with HGF or EGF in concentrations comparable to those found in tile sera of patients with liver failure, resulted in a significant upregulation of Eg'r-1 mRNA and Eg'r-1 protein in PHH derived from different donors starting at 30min of incubation. The same applied for the human hepatoma cell lines Hep3B, HepG2 and Huh7. Treatment of PHH and hepatoma cells with HGF or EGF led to an activation of the anti-apoptotic sigualling pathways PI3K/Akt and MEK/ERK. Inhibition of MEK1 by applying the inhibitor PD98059 resulted in a complete inhibition of Egr-1 induction in human hepatoma cells. In PHH blockage of file PI3-kinase through the inhibitor LY294002 partially inhibited Egr-1 induction. In addition, blockage of MEK1 led to sensitization of human hepatoma cells towards chemotherapeutic drug-induced apoptosis. Blockage of PI3K led to an increase in the sensitivity of PHH towards CD95 (APO-1/Fas)-mediated apoptosis. Due to their anti-apoptotic effects g-rowth factors such as HGF might be used for the treatment of liver diseases. Transcription thctors such as Egr-1 nfight contribute to file anti-apoptotic effects of HGF and EGF on human liver cells.

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NUCLEAR STEROID RECEPTORS MODULATE CELLULAR TRAFFICKING OF URSODEOXYCHOLIC ACID IN PRIMARY RAT HEPATOCYTES

S. Sohl l'e, J.D. Amaral 1, R.E. Castro 1, R.M. Rarnalho 1, B.Z Kren2, C.J. Steere'3, C.M.E Rodrigues 1. 1Centro de Patogdnese Molecular,

Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal," 2Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA," 3Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN, USA Background and Aims: Ursodeoxycholic acid (UDCA) inhibits apoptosis in hepatocytes after diverse toxic stimuli. It regulates classical mitochondrial pathways either by directly" stabilizing mitochondrial membranes or modulating tile expression of specific upstream targets. In addition, as a cholesterol-derived molecule, UDCA may regulate transcription of several genes by interacting with nuclear steroid receptors (NSR). In this study, we investigated the potential role of giuencortienid (GR) and mineralocorticoid (MR) receptors in tile anti-apoptotic function of UDCA. Results: Primary rat hepatocy'tes were treated with 1 nM TGF-[51 for 24h, in tile presence or absence of UDCA. Morphologic characteristics of apoptosis after Hoechst staining confirmed that UDCA siguificanfly prevents TGF-[51-induced hepatocyte apoptosis (p <0.05). In contrast, when short interference RNA (siRNA) was used to specifically inhibit NSR expression, the protective effect of UDCA was significantly decreased (p < 0.05). Irnmunoprecipitation assays showed that UDCA promotes GR/hsp90 dissociation, while confocal microscopy after wansfection with GFP-GR plasmids confirmed subsequent GR nuclear translocation. However, when a C-terminal deleted form of GFP-GR was used, UDCA no longer induced NSR translocation. Surprisingly, in cotvansfection experiments with a GR response element-reporter construct and GR or MR overexpression plasmids, UDCA did not induce NSR transactivation after TGF-[51 exposure. Finally, using an NBD-UDCA fluorescent molecule (kindly provided by A. Ho~lann, University of San Diego), the bile acid appeared diffuse in the cytosol but was aggregated in the nuclens of hepatocytes. Nuclear traffic!ring occurred through a NSR-dependent mechanism as determined by siRNA assays. Conclusions: These data further clarify the anti-apoptotic mechanism(s) of UDCA, and suggest that NSR are crucial for tile nuclear translocation of this bile acid for inhibiting apoptosis. Supported, in part, by POCTI/BCI/44929/02 from FCT, Portugal.

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T R A N S C R I P T I O N A L MAPPING ON C H R O M O S O M E 19q INDICATED CONSISTENT DOWN-REGULATIONS OF ATF5 IN ASSOCIATION WITH PROGRESSION OF HUMAN HEPATOCELLULAR C A R C I N O M A

N. Wong 1, K.Y-Y. Chan 1, J.A. Squire 3, P.F. Macgregor4, B. Beheshti 3, M. Albert4, P.B-S. Lai 2 . 1Department of Anatomical and Cellular

Pathology, The Chinese University of Hong Kong, Hong Kong, China," 2Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China," SDepartments of Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada," 4Microarray Centre, Clinical Genomics Centre, University Health Network, Toronto, ON, Canada Spectral Karyotyping analysis on human hepatocellular carcinoma (HCC) has indicated previously undescribed frequent chromosome 19q translocations (Wong et al., Hepatology 2000). Tile non-random rearrangements involving regional 19q13 found have also been described in many solid turnouts. In an effort to characterize file differential genes expression in tile proximity of 19q13, we performed mapping study using a cDNA microarray that contains annotated eDNA clones along the length of chromosome 19q at an average interval o f - 8 0 kb. A strikingly high and consistent down-regulation of tile ATF5 gene (cyclic-AMP-dependent Activating Transcriptional Factor 5) at 19q13.33 was suggested in 9 HCC cell lines examined (fold reductions ranged from 2.5 to 91). The array-derived observation was confirmed by quantitative RT-PCR, which further pinpointed to a repressed ATF5 expression in relation to advanced metastatic T3/T4 HCC tumors (p < 0.01). A growth inhibitory effect on cell survival was also demonstrated from the stable transfection of ATF5 into non-expressing HCC cell lines. Several lines of evidence have indicated the ATF family of genes to be involved in cell adhesion, cancer cell invasion, apoptosis and signalling pathways, although little is known about the function of ATF5. Our finding supports a tumor suppressive role of ATF5 in the progression of HCC, and provide basis for further downstream characterizations of ATF5 in the HCC oncogenesis.

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TARGETING XIAP OR SURVIVIN BY SIRNAS SENSITIZES HCC CELLS TO TRAIL- AND CHEMOTHERAPEUTIC AGENTS-INDUCED CELL DEATH

Y. Yamaguchi, K. Shiraki, H. Fuke, T. Inoue, K. Miyashita, Y. "~amanaka, Y. Saitou, K. Sugimoto. First Department of Internal Medicine, Mie

University School of Medicine, Mie, Japan Background: hthibition of apoptosis leads to tumorgenesis and resistance to therapy. The inhibitors of apoptosis proteins (IAPs) regulate apoptosis by preventing the action of tile central execution phase ofapoptosis through direct inhibition of the effector caspase-3 and/or caspase-7. IAPs are expressed in a variety of human cancers. We found that approximately 70 88% ofhepatocellular carcinoma (HCC) tissues showed strong staining for XL'kP or survivin, whereas nontumor tissues showed little detectable staining by immunohistochernistry. Furthermore, we found that survivin promotes cell proliferation in HCC cells. Thus, targeting LAPs may be a promising strategy, but it is not well elucidated in HCC. Aims: We investigated whether downregulation of LAPs (XIAP or survivin) by small interfering RNA (siRNA) sensitizes HCC cells to TNF-related apoptosis-inducing ligand (TRAIL)- or chemotherapeutic agents-induced cell death. Methods: We selected oligonucleotides 588-609 (siRNA-XIAP) as the target sequence of XIAP (ac.-no.U45880) and nucleotides 282-303 (siRNA-survivin) as the target sequence of survivin (ac.-no.U75285) that had no significant homology to any known human mRNA in the databases. A single pulse of siRNAs was administered to the two HCC cell lines (SK-Hepl or HLE) by transfection with Yransmessenger Transfect Regent (Qiagen Inc., CA), according to manufacturer instructions. XIAP