I-tEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 499A

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EFFECT OF CIGLITAZONE, A PPAR-GAMMA AGONIST, ON THE PRO- HFERATION OF HUMAN HEPATOMA CELLS IN VITRO. Kazuhiko Nakai, Victor Wu, University of California, San Francisco, CA

Background: PPAR-gamma is a nuclear receptor which functions as a ligand-acti- vated transcription factor. PPAR-gamma agonists have been shown to inhibit the proliferation of muhiple types of cancer cell lines (breast, lung. colon, prostate, liposarcoma) in vitro. PPAR-gamma expression and activity have not been charac- terized in liver cancer cell lines. Objectives: To investigate the expression of PPAR- gamma in HUH-7 and HepG2 human hepatoma cell lines. To investigate the expres- sion of PPAR-gamma in human hepatoma tissues. To investigate the effect of ciglitazone (a PPAR-gamma agonist) on the growth of HUH-7 and HepG2 cells in vitro. Methods: RT-PCR and immunocytochemistry were used to assay for expres- sion of PPAR-gamma. In cell culture experiments, HUH-7 and HepG2 cells were groven in serum-free media. Cells were counted 24 to 72 hours after addition of ciglitazone or vehicle. Results: PPAR-gamma mRNA expression was detected in HUH-7 and HepG2 cell lines using RT-PCR with PPAR-gamma specific primers. PPAR-gamma protein expression was detected in HUH-7 and HepG2 cells using immuuocytochemistry with a PPAR-gamma-specific antibody (Santa Cruz). PPAR- gamma mRNA expression was detected in five paired sets of human hepatomas and non-tumorous liver tissues. The level of PPAR-gamma mRNA expression in hepa- tomas and non-tumorous tissues was comparabIe. At a concentration of 5 uM, ciglitazone reproducibly inhibited HUH-7 cell proliferation, whereas cells were not affected by WY-14643 (a PPAR-alpha agonist) at a concentration of 10 uM. For HUH-7 cells, ciglitazone induced rapid cell death at concentrations of 10 uM or greater. Ciglitazone reproducibly inhibited HepG2 cell proliferation at a concentra- t:Lon of 10 uM. HepG2 cells were also not affected by WY-14643. Death of HepG2 cells was induced by ciglttazone at concentrations of 20 uM or greater. Conclusions: PPAR-gamma is expressed in human hepatoma ceil lines HUH-7 and HepG2, as well as in human hepatoma tissues. At pharmacologically relevant concentrations, cigli- tazone inhibits the proliferation of HUH-7 and HepG2 cells in vitro. Further inves- tigation of PPAR-gamma agonists as potential therapy for liver cancer is warranted.

Cell number, as percent of control

Compound Vehicle Ciglitazone luM Ciglitazone 5 uM Ciglitazone 10 uM Ciglitazone 20 uM WY14643 10 uM

HUH-? ceils HepG2 cells Time from addition (hrs) Time from addition (hrs) 24 48 72 24 48 72 100 100 100 t00 I00 100

100::t:11 100+18 97-+16 100±7 102_+1 97-+8 50±3 34±7 17+-5 101±8 100_+1 60+-8

0 0 0 57+_4 4~5 7+1 0 0 0 1+1 0 0

106_+27 98_+14 113_+7 I02+8 I05±7 1034-_7

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HEPATIC ADENOMAS: ANALYSIS OF SEX STEROID RECEPTOR STA- TUS AND THE W N T SIGNALING PATHWAY. Michael Torbenson, Jae- Hyuk Lee, Michael Choti, Wesley Gage, Susan Abraham, Elizabeth Montgom- ery, John Boitnott, Tsung-Teh Wu, Johns Hopkins Hospital, Baltimore, MD

Hepatic adenomas are strongly linked to excess hormonal exposure, but little else is known about their pathogenesis. The Wnt signaling pathway, which is activated in both hepatocellular carcinomas and hepatoblastomas, has not been studied in hepatic adenomas. 15 hepatic adenomas were studied by im- munohistochemistry for estrogen, progesterone, and androgen receptors (ER, PR, AR) and correlated with the results of immunostaining for jS-catenin. Direct sequencing was performed to look for mutations in key proteins in- volved in the Writ signaling pathway: exon 3 of/3-catenin encompassing the glycogen synthase kinase 3/3 (GSK-3]3) phosphorylation region and the muta- tional cluster region of the adenomatosis polyposis coli protein (APC). Anal- ysis for loss of heterozyosity (LOH) at chromosome 5q was also performed. Immunostaining for both ER and PR was present in 11/15 (73%) of the ade- nomas and staining with one hormone receptor was positively associated with staining for the other receptor. AR positivity was present in 3/15 cases. Nuclear accumulation of jB-catenin was present in 7/15 (46%) of adenomas, indicating activation of the Wnt signaling pathway. However, no/3-catenin mutations, no APC mutations in the mutational duster region, and no 5q LOH were detected. Two APC polymorphisms of unknown significance were seen. No clear asso- ciation between/3-catenin nuclear accumulation and hormone receptor posi- tivity was discerned. Thus,activation of the Wnt signaling pathway appears to be important in a subset of hepatic adenomas, but does not result from ~-cate- inn or APC mutations and does not appear to be directly linked to hormonal receptor status.

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HHM: A N E W DOMINANT INHIBITORY HLH PROTEIN WHICH IS AS- SOCIATED WITH LIVER CARCINOGENESIS. Shuji Terai, Isao Sakaida, Hitoshi Shirahashi, Kouicbi Uchida, Naoki Yamamoto, Koji Miyamoto, Dept of Molecular Science & Applied Med (Gastroenterology & Hepatology), Yamaguchi Univ Sch of Medicine, Ube, Yamaguchi Japan; Snorri S Thorgeirs- son, Lab of Experimental Carcinogenesis, Natl Cancer Inst, NIH, Bethesda, MD; Kiwamu Okita, Dept of Molecular Science & Applied Med (Gastroenter- ology & Hepatology), Yamaguchi Univ Sch of Medicine, Ube, Yamaguchi Ja- pan

The helix-loop-helix (HLH) family of transcriptional regulatory proteins are key regulators in numerous developmental process. We had identified a novel dominant inhibitory HLH protein, human homologue of Maid (HHM) from human fetal liver cDNA library (Hepatology 2000,32;357-366). HHM is ex- pressed at high level in fetal liver as well as transiently during hepatic stem cell activation in 2-acetylaminofluorene/ Partial hepatectomy model (AAF/PH) model. HHM also inhibited the luciferase gene activation induced by HNF4 promoter. These results suggest that HHM is a novel type of dominant inhib- itory HLH protein associatedwith liver development. Two other groups inde- pendently identified same gene with HHM. They had named the gene, DIP1 and GCtP(Exp. Cell. Res. 2000, 257;22-32,JBC 2000,275;20942-20948). DIP1 might bind with cyclin D1. GCIP bind with Grap2 which is an adaptor protein identified as Gab-1 docking protein. But the precise function of this protein HHM/GCIP/DIP1 had not yet identified. AIM: The aim of this study is to evaluate the possible involvement of HHM in liver carcinogenesis. Method: We also analyzed the interaction of HHM with cyclin D 1 by immunoprecipitaion using COS1 cell. We have analyzed HHM mRNA expression in choline deft- cient-L-amino acids administration rat model (CDAA model) by in situ hy- bridization. We have raised a HHM rabbit polyclonal antibody, Anti-HHM-C, that recoginized HHM(aa.347-360). Nine cases of early stage human hepato- cellular carcinoma (HCC) (diameter under 15 mm), and 14 cases of advanced E[CC (over 15 mm) were analyzed. A minimum labeling index of 10% of cancer cells was required to count as a positive sample. Results: Immunoprecipitaion analysis showed that HHM could bind to cyclinD 1. HHM mRNA expression is specifically high in basophilic hepatocyte loci in CDAA model. In the cases of advanced HCC, HHM positive staining are 92%. In the cases of small HCC, 77% cases are shown as a positive (Well and, moderate differentiated HCC were 75% positive,whereas poorly differentiated HCC were 100% positive). We could not detect staining of HHM in liver cirrhosis sample. Conclusion: HHM might regulate cell differentiation and proliferation by binding various kinds of proteins such as CyclinD 1. The expression of HHM was very high in human HCC and foci in CDAA model. These results suggest that HHM may play a role in liver carcinogenesis.

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HEPATOCELLULAR CARCINOMA CELL LINES DOWN-REGULATE EX- PRESSION OF MMP-2 BY HUMAN HEPATIC MYOFIBROBLATS: AN IGF-1 MEDIATED PROCESS. Jean-Frederic Blanc, Christ~le Bisson, Groupe de recherche pour l'Etude du Foie INSERM E9917 Universit~ Bordeaux 2, Bordeaux France; Francis Frankenne, Agnes Noel, Laboratoire de biologic des Tumeurs et du Developpement Universit¢ de liege Sart Tilman, Liege Belgium; Jean Rosenbaum, Groupe de recherche pour l'Etude du Foie INSERM E9917 Universit~ Bordeaux 2, Bordeaux France; Jean-Michel Foidart, Laboratoire de biologic des Tumeurs et du Developpement Universit~ de liege Sart Tilman, Liege Belgium

MMPs are a family of proteases that degrade extracellnlar matrix components. Over expression of MMP-2 (or gelatinase A) is a frequent event in hepatocel- lular carcinoma (HCC) where in situ studies show that its expression is appar- ently restricted to stromal myofibroblasts. MMP-2 is considered to play a key role in tumor progression and metastasis. In this work; we studied the pattern of expression of MMP-2 by human HCC cell lines and human hepatic myofi- broblasts (MF), and the regulation of its expression in a model of co-culture between HCC cells and MF. MMP-2 production was studied by zymography and quantitative RT-PCR. We also studied the expression of MT1-MMP (an enzyme involved in activation of pro-MMP-2) by Western-Blot and RT-PCR and of TIMP-2 (an inhibitor of MMP-2) by ELISA and RT-PCR. IGF-1 secre- tion was measured by radioimmunoassay. Recombinant IGF-1 and antibody against IGF-1 receptor were used to determine the role of IGF-1 in the regu- lation of MMP-2 production. MF abundantly expressed MMP-2 and MT1- MMP and secreted high levels of TIMP-2. The HCC cell lines tested (HepG2, HUH7, Hep3B) did not express MMP-2 and expressed only weakly TIMP-2 and MT1-MMP. In co-culture, no modulation of MT1-MMP and TIMP-2 expres- sion was ever observed. Activation of pro-MMP-2 was minimal in every con- dition tested. Co-culture of MF with HCC cell lines led to a strong decrease in MMP-2 activity and mRNA expression by MF. No modifications of MF cell numbers were observed as assessed by measuring vimentin mRNA by RT-PCR. The effect of the co-culture was partly reproduced by HCC cells conditioned media, indicating that it was mediated by a soluble factor. HepG2, but not HuH7 or Hep3B, secreted high amounts of IGF-1. The down-regulation of MMP-2 in co-cultures with HepG2 cells was blocked by a monoclonal antibody against the IGF-1 receptor and was mimicked by recombinant IGF-1. In con- clusion, HCC cell lines down-regulate the production of MMP-2 by MF which represent the main source of this protease. In the case of HepG2, this effect is mainly mediated by IGF-1 whereas other as yet unidentified mediators are likely involved for the other HCC cell lines.