915 Expression of TNF-related apoptosis inducing ligand in human hepatocellular carcinoma

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AASLD ABSTRACTS

913

FAMILIAL LIVER ADENOMATOSIS ASSOCIATED WITH HEPATOCYTE NUCLEAR FACTOR 1 ALPHA INACTIVATION. Yannick Bacq, H6pital Trousseau, Tours, France;

mmanuel Jacquemin, H6pital de Bic~tre, Le Kremlin Bic~tre, France; Charles Balabaud, H6pita! de Bordeaux, Bordeaux, France; mmanuelle Jeannot, INS~ RM U434, ~ PH-Fondah'on Jean Dausset, Paris, France; B~atfice Scotto, H6pital Trousseau, Tours, France; Sophie Branchereau, H6pital de Bic~tre, Le Kremlin Bic~tre, France; Christophe Laurent, H6pital de Bordeaux, Bordeaux, France; Pascal Bourlier, H6pital Trousseau, Tours, France; Daniel Pariente, H6pital de Bic~tre, Le Kremlin Bic~tre, France; Anne de Muret, H6pital Trousseau, Tours, France; Monique Fabre, H6pital de Bic~tre, Le Kremlin Bic~tre, France; Paulette Bioulac-Sage, H6pital de Bordeaux, Bordeaux, France; Jessica Zucman-Rossi, INS~ RM U434, ~ PHFondah'on Jean Dausset, Paris, France Background and Aims: Germline mutations in the hepatocyte nuclear factor I alpha (TCF1/HNF1) are associated with maturityonset diabetes of the young type 3 (MODY3) and somatic biallelic inactivations of the gene are found in hepatocellular adenomas and liver adenomatosis (Nature genetics, 2002;32:312-315). This study investigated cosegregation of TCF1/HNF1 germline mutations with diabetes and liver adenomatosis in two families. Methods: Two patients suffering from liver adenomatosis were screened for TCF1/HNF1 germline and somatic mutations. Subsequenfiy, we screened 9 relatives in the two i n d e p e n d e n t families for diabetes, hepatocellular adenomas and TCF1/HNF1 germline mutations. Results: In family A, a father and his son presented with an intra-peritoneal hemorrhagic rupture of a liver adenomatosis without diabetes. A heterozygous R229X germline mutation was identified in TCF1/HNF1 in the father and his son, and also in his second 27-year-old son without hepatocellular adenomas. In family B, a diagnosis of liver adenomatosis was made fortuitously in a 14-year-old girl. A heterozygous G55fsX57 germline mutation in TCF1/HNF1 was identified in this patient, in her diabetic father and her two sisters. Systematic exploration with ultrasonography and magnetic resonance imaging revealed liver adenomatosis in the two sisters. Somatic inactivation of the second TCF1/HNF1 allele was found in liver tumors in both families. Conclusion: This study describes familial liver adenomatosis and shows their association with germline TCF1/HNF1 mutations in adults and children. It also highlights the importance of a screening for hepatocellular adenomas, diabetes and TCF1/HNF1 germline mutations in relatives of patients with liver adenomatosis. Finally, prevalence of liver adenomatosis remains to be evaluated in MODY3 subjects. Disclosures: Yannick Bacq - No relationships to disclose Charles Balabaud - No relationships to disclose Paulette Bioulac-Sage - No relationships to disclose Pascal Bourlier - No relationships to disclose Sophie Branchereau - No relationships to disclose Anne de Muret - No relationships to disclose Monique Fabre - No relationships to disclose Emmanuel Jacquemin - No relationships to disclose Emmanueile Jeannot - No relationships to disclose Christophe Laurent - No relationships to disclose Daniel Pariente - No relationships to disclose B6atrice Scotto - No relationships to disclose Jessica Zucman-Rossi - No relationships to disclose

914

HEPARANASE EXPRESSION DURING NORMAL LIVER DEVELOPMENT AND LIVER REGENERATION FOLLOWING PARTIAL HEPATECTOMY. Om't Goldshmidt, Rita Yeikilis, Melia

Paizi, Israel Vlodavsky, Gadi Spira, Technion - Israel Institute of Technology, Haifa, Israel Heparan sulfate proteoglycans (HSPG) are a major component of the extracellular matrix (ECM), playing an essential role in maintaining the integrity and functional state of tissues and organs. Cleavage of HSPGs may thus influence normal and pathological processes including architectural changes, cell migration and re-

HEPATOLOGY, October 2003

sponse to heparin-binding growth factors sequestered by the ECM. Heparanase is a mammalian endoglycosidase capable of a specific cleavage of heparan sulfate chains (HS). Our study points for a possible involvement of heparanase during normal liver development and liver regeneration following partial hepatectomy. Utilizing in situ hybridization, immunohistochemistry and Western blotting we have shown that heparanase mRNA and protein are notably expressed in the embryonic normal liver (18 and 22 weeks) while absence from the mature healthy liver (human, or rat). In a 39 week h u m a n embryo liver, only Kupffer cells were stained as compared to strong cellular staining of hepatocytes in the liver of the 18 and 22 weeks embryos. In the regenerating rat liver, a peak of heparanase expression appears as early as12 hours post surgery. A second prominent peak was evident at day seven (168 hours). Heparanase cellular distribution alternates during the regenerative phase from homogenous cytoplasmic staining (till 96 hours) to a polar granular pattern adjacent to the cell membrane, at 168 hours post hepatectomy. In order to assess other ECM degrading enzymes previously demonstrated to be involved in liver regeneration a gelatin zymography of MMP-2 and MMP-9 was used. Based on our results we propose, for the first time, that heparanase may play an important role in liver development and regeneration. Disclosures: Orit Goldshmidt - No relationships to disclose Melia Paizi - No relationships to disclose Gadi Spira - No relationships to disclose Israel Vlodavsky - No relationships to disclose Rita Yeikilis - No relationships to disclose

915 EXPRESSION

O F T N F - R E L A T E D A P O P T O S I S INDUCING LIGAND IN HUMAN HEPATOCELLULAR CARCINOMA.

Katsuya Shiraki, Takenari Yamanaka, Hiroshi Okano, Tomoyuki Kawakita, Yukiko Saitou, Yumi Yamaguchi, Kazushi Sugimoto, Kazumoto Murata, Takeshi Nakano, 1st Medicine, Mie University, Tsu, Japan Backgroud:TNF-related apoptosis-inducing ligand (TRAIL), as well as Fas ligand, plays a pivotal role in lymphocyte cytotoxicity and the maintenance of immunological homeostasis in various tissues, but its physiological role in immune evasion of cancer ceils remains unknown. We have previous shown strong resistance to TRAIL-induced cytotoxicity in h u m a n hepatocellular carcinomas (HCCs). However, the TRAIL expression and its function in HCC was not well elucidated. Therefore, the current study investigates the expression of TRAIL in HCCs. Methods: mRNA and protein expression of TRAIL were examined by RT-PCR, western blotting, immunohistochemical staining and flow cytometry in HCC cell lines and HCC tissues. TRAIL expression was analyzed using HCC ceils treated with doxorubicin, other chemotherapeutic drugs and various cytokines. Function of TRAIL was analyzed using co-culture experiments with Jurkat ceils which is sensitive to TRAIL. Results:We found that three HCC ceils, HepG2, Hep3B and Huh7 ceils, constitutively express TRAIL mRNA and protein, as detected by RT-PCR and Western blotting. Four of 10 h u m a n HCC tissues also demonstrated positive staining for TRAIL, whereas non-tumor tissues showed little detectable staining. TRAIL located in both cytoplasm and membrane. TRAIL expression on tumor ceils was also detected by flow cytometry and furthermore was dramatically induced after the addition of doxorubicin, a chemotherapeutic agent, or NF-KB activating cytokine stimulation with TNF-cY, IL-1/3 or IL-18 in all HCC cell lines examined. This upregulation of TRAIL was also confirmed by Western blotting. In addition, this expression was induced principally via the NF-KB activation pathway, since IKB transfection significantly reduced TRAIL expression. In addition, the expressed TRAIL was functional. The TRAIL on HCC ceils induced apoptosis in Jurkat ceils that are sensitive to TRAIL-mediated apoptosis, and this process was specifically inhibited by recombinant TRAIL-receptors:Fc which binds to TRAIL. This inhibitory effect was stronger than that using anti-FasL antibody. Conclusions; TRAIL expressed on the surface of HCC ceils by cytokines

HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

or cytostatic drugs might contribute to an alternative mechanism that enables tumors to evade immune surveillance by inducing apoptosis of activated h u m a n lymphocytes. Disclosures: Tomoyuki Kawakita - No relationships to disclose Kazumoto Murata - No relationships to disclose Takeshi Nakano - No relationships to disclose Hiroshi Okano - No relationships to disclose Yukiko Saitou - No relationships to disclose Katsuya Shiraki - No relationships to disclose Kazushi Sugimoto - No relationships to disclose Yumi Yamaguchi - No relationships to disclose Takenari Yamanaka - No relationships to disclose

916

SYSTEMIC ADMINISTRATION OF AN ADENOVIRUS ENCODING A TRUNCATED, SOLUBLE DOMINANT NEGATIVE VEGF (FLK-1/KDR) RECEPTOR IN A MURINE TUMOR MODEL FOR LIVER METASTASIS OF COLORECTAL CANCER. Volker Schmitz, Tobias Hilbert, Christian

Dzienisowicz, ~ sther RaskopJ:, Christian Rabe, Tilman Sauerbruch, Wolfgang H Caselmann, University Hospital Bonn, Bonn, Germany Introduction: It has been demonstrated recently that the systemic administration of an adenovirus expressing a truncated, soluble VEGF-receptor (AdsFlk-1) inhibited the tumor growth in a subcutaneous model for colorectal cancer (CRC) in mice. In this study we evaluated the effects of a systemic administration of AdsFlk-1 in a murine tumor model for liver metastases of CRC. Method: Protein expression and secretion of sFLK-1 into cell supernatant were determined by ELISA technique. Hepatic metastases of CRC were established by intrahepatic injection of 105 CT26 tumor cells. AdsFLK-1 or AdlacZ control were injected intravenously and intrahepatic tumors were measured after laparotomy 10 days later. Hepatic toxicity was evaluated by ALTdetermination. Results: Tumor cells infected with AdsFlk-1 secreted sFLK-1 into cell supernatant in vitro. All animals showed tumor take and 7/7 control animals had a progressive tumor disease. In contrast 43 % (3/7) of AdsFlk-1 treated mice showed a stable or decreasing tumor burden. Two weeks after tumor cell implantation the mean tumor volume reached 452 m m 3 and 1080 m m 3 in the AdsFlk-1 and AdlacZ control group, respectively. Mean ALT-levels were about 3fold elevated in the AdsFlk group. Conclusion: The data show that the systemic injection of an antagonistically acting sFlk-1 construct inhibited the growth of liver metastasis in this CRC tumor model in about 40 % of the animals. Disclosures: Wolfgang H Caselmann - No relationships to disclose Christian Dzienisowicz - No relationships to disclose Tobias Hilbert - No relationships to disclose Christian Rabe - No relationships to disclose Esther Raskopf - No relationships to disclose Tilman Sauerbruch - No relationships to disclose Volker Schmitz - No relationships to disclose

917

WITHDRAWN

918

CONSENSUS INTERFERON (INTERFERON-ALPHACON1) I N H I B I T S THE GROWTH OF LIVER CANCER CELL LINES IN VITRO AND IN VIVO. Hirohisa Yano, Toru Hisaka, Seiya

Momosaki, Sachiko Ogasawara, Naoyo Nishida, Yumi Takemoto, Sakiko Kojiro, ~ riko Nitta, Masamichi Kojiro, Kurume University School of Medicine, Kurume, Japan [AIM] Interferon (IFN)-alpha has b e e n reported to decrease the frequency of hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-related liver cirrhosis patients; however, the mechanisms by which IFN-alpha suppresses liver cancer development have not been clarified. Consensus IFN (IFN-alphaConl) is a synthetic

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nonnaturally occurring type I IFN derived by scanning the most frequently observed amino acid in each corresponding position. IFN-alphaConl has b e e n shown to have greater anti-viral and anti-proliferative activities than all other type I IFNs. In the present study, we investigated anti-proliferative effects of IFNalphaConl on liver cancer cells in vitro and in vivo. [Materials and Methods] Thirteen h u m a n liver cancer cell lines (11 HCC cell lines and 2 combined hepatocellular and cholangiocarcinoma cell lines), which were originally established and characterized in out laboratory, were used in the in vitro study. Two HCC cell lines (KIM-1 and HAK-1B) were used in the in vivo study. (1) Cells were cultured with 4-1024 IU/ml IFN-alphaConl (Advaferon(~), Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan) or without IFN-alphaConl (control); after 1, 2, 3, or 4 days of culture, morphological observation and MTr-cell growth assay were carried out. (2) HCC cells (1.0 x 107 cells/site) were transplanted subcutaneously into the back of nude mice. After tumor formation was confirmed, three different doses of IFN-alphaConl (0.01, 0.1, 1 /~g/mouse/day, n - 1 0 in each group) or PBS alone (control, n - 1 0 ) was injected subcutaneously for consecutive 14 days. The lowest dose of IFN-alphaConl (0.01/~g) is equivalent to the clinical dose (1.8 x 107 IU) used for HCV-related chronic hepatitis. Tumor measurements were done every 2 days, and tumor volume (ram 3) was estimated by using "length x (width)2 x 0.5." On the next day of the final injection, the tumor in each mouse was resected and processed for histological examination. Apoptotic cell numbers in the tumors were examined on the hematoxylin-eosin-stained sections. Also, the number of blood vessels in and around the tumors was examined. [Results] (1) IFN-alphaConl dose- and time-dependently inhibited the growth of all liver cancer cell lines in vitro, but the sensitivity of cell lines to IFN-alphaConl was varied. On Day 4, relative viable cell numbers of 7 cell lines decreased to 50% or lower w h e n 1024 IU/ml IFN-alphaConl was added to the cultures. Morphological characteristics of apoptosis were observed in all cell lines cultured with IFN-alphaCon% (2) Subcutaneous IFNalphaConl injection dose- and time-dependently suppressed the HCC tumor growth in nude mice. On the next day of the final IFN-alphaConl injection, the tumor volumes of mice received 0.01, 0.1, and 1 /~g/mouse of IFN-alphaConl decreased to about 60%, 25%, and 10%, respectively, of control tumor volumes in the case of KIM-1. Similar results were obtained in the case of HAK1B, except that the tumors disappeared in all mice received 1 /~g/mouse of IFN-alphaConl. Apoptotic cell numbers in the tumors of mice received IFN-alphaConl increased with an increase of IFN-alphaConl dose and were significantly higher than that of control mice. The number of blood vessels in and around the tumors of mice received IFN-alphaConl decreased with an increase of IFN-alphaConl dose and was significantly lower than that of control mice. Conclusions: IFN-alphaConl dose- and time-dependently inhibits the growth of liver cancer cell lines in vitro and in vivo. Because even clinical dose of IFN-alphaConl for HCV-related chronic hepatitis treatment suppressed HCC growth in nude mice, it is suggested that IFN-alphaConl may inhibit the growth of clinically undetectable HCC cells by directly inducing apoptosis of HCC cells and by indirectly suppressing tumor angiogenesis and prevent or delay the development of HCC in patients with chronic viral liver diseases. Disclosures: Toru Hisaka - No relationships to disclose Masamichi Kojiro - No relationships to disclose Sakiko Kojiro - No relationships to disclose Seiya Momosaki - No relationships to disclose Naoyo Nishida - No relationships to disclose Eriko Nitta - No relationships to disclose Sachiko Ogasawara - No relationships to disclose Yumi Takemoto - No relationships to disclose Hirohisa Yano - No relationships to disclose