182 Lack of caspase-8 expression in murine hepatocellular carcinomas is associated with hypermethylation at SP1 binding sites in the murine caspase-8 promoter

242A

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180 INHIBITION OF NUCLEAR FACTOR-KB AND PHOSPHATIDYLINOSITOL 3-KINASE/AKT IS ESSENTIAL FOR MASSIVE HEPATOCYTE APOPTOSIS INDUCED BY TUMOR NECROSIS FACTOR ¢~ IN MICE. Motoaki Imose,

Masahito Nagaki, Takafumi Naiki, Yosuke Osawa, Gifu University School of Medicine, Gifu City, Japan; David A Brenner, University of North Carolina, Chapel Hill, NC; Takahiko Asano, Hideki Hayashi, Motohiro Imao, Shinji Takai, Kiminori Kimura, Tomohiro Kato, Hisataka Moriwaki, Gifu University School of Medicine, Gifu City, Japan Background/Aims: Tumor necrosis factor (TNF)-c~ itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF-cd TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through activation of nuclear factor (NF)-KB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-m Here, we focused on the roles of NF-KB and the phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt. Methods: TNF-c~ was administered into BALB/c mice after the treatment with an adenovirus expressing a mutant from Ad5IKB,the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, or the both.Liver injury was assessed biochemically and histologically.The expression of Bcl-2 family m e m b e r s and caspase activity were examined. Results: DNA gel shifts showed that the NF-KB activation induced by TNF-c~ was equivalent in livers treated or untreated with wortmannin, indicating that NF-KB activation was i n d e p e n d e n t of PI3K/Akt in the liver.Either suppression of NF-KB or Akt showed a slight increase in transaminase levels and focal liver cell death after 0.5/~gTNF-c~ administration. On the other h a n g in mice pretreated with both Ad5IKB and wortmannin, 0.5/~gTNF-c~ administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. After TNF-cYinjection, a marked increase in the number of apoptotic cells, determined by TUNELassy, was observed in the livers of mice pretreated with wortmannin and Ad5IKB.The combination of Ad5IKB, wortmannin, and TNF-c~ markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-c~-induced hepatocyte apoptosis is d e p e n d e n t on an intrinsic cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-KB and PI3K/Akt pathways had no effect on the expression of the antiapoptotic molecules BcI-XL,McI-1 and Bfl-1 or that of the proapoptotic molecules BOX, Bad and Bid in the presence or absence of TNF-cY,as determined by Rnase protection assay and Western blot analysis. Conclusions: We demonstrated that TNF-c~ -induced NF-KB or constitutively activated Akt signaling,which were mutually independent, was vital for the escape from TNF-c~-induced massive liver cell death and lethlity in mice, due, at least in part, to liver failure.Both NF-KB and PI3K/Akt - d e p e n d e n t protective actions may occur upstream of caspase-9. Disclosures: Takahiko Asano - No relationships to disclose David A Brenner - No relationships to disclose Hideki Hayashi - No relationships to disclose Motohiro Imao - No relationships to disclose Motoaki Imose - No relationships to disclose Tomohiro Kato - No relationships to disclose Kiminori Kimura - No relationships to disclose Hisataka Moriwaki - No relationships to disclose Masahito Nagaki - No relationships to disclose Takafumi Naiki - No relationships to disclose Yosuke Osawa - No relationships to disclose Shinji Takai - No relationships to disclose

HEPATOLOGY, October 2003

181

FOXA1 AND FOXA2 ARE REQUIRED FOR THE SPECIFICATION OF THE LIVER. Joshua R Friedman, Catherine S

Lee, Klaus H Kaestner, University of Pennsylvania, Philadelphia, PA The development of the mouse liver begins with the differentiation of hepatoblasts from the ventral foregut e n d o d e r m on embryonic day (E) 8.0-8.5. The endodermal factors responsible for the initiation of hepatoblast formation are unknown. Foxal and Foxa2 are forkhead box transciption factors which are expressed in the ventral foregut e n d o d e r m during the period of hepatoblast differentiation. They are also known to regulate the expression of a variety of liver-specific genes in the adult liver. While these features suggest that Foxal and Foxa2 may be crucial regulators of hepatoblast differentiation, targeted deletion of these genes individually does not affect liver development. To overcome possible functional redundancy between Foxal and Foxa2, we generated mice which are deficient for Foxal and Foxa2 throughout the definitive endoderm. These embryos die during gestation between E9.5-10.5. Double mutant embryos examined at E8.5-9.5 are smaller than control littermates. No hepatic primordium is evident in these embryos, and expression of the early hepatic marker AFP is lost. Furthermore, the double mutant embryos have enlarged hearts, pericardial edema, and hypoplasia of the myocardial cell layer. These results define Foxal and Foxa2 as the earliest factors involved in the specification of the liver and suggest that inductive signaling from the primordial liver is required for proper heart formation. Disclosures: Joshua R Friedman - No relationships to disclose Klaus H Kaestner - No relationships to disclose Catherine S Lee - No relationships to disclose

182

LACK OF CASPASE-8 EXPRESSION IN MURINE HEPATOCELLULAR CARCINOMAS IS ASSOCIATED WITH HYPERMETHYLATION AT SP1 BINDING SITES IN THE MURINE CASPASE-8 PROMOTER. Christian Liedtke, Nils-Holger

Zschemisch, Anne Cohrs, Medical School of Hannover, Hannover, Germany; Juergen Borlack, Fraunhofer Institute of Toxicology and xperimental Medicine, Hanover, Germany; Michael P Manns, Christian Trautwein, Medical School of Hannover, Hannover, Germany Hepatocytes are hypersensitive against Fas-induced apoptosis in vivo. Caspase-8 is the most upstream caspase essential for triggering Fas-induced apoptosis. We recently characterised the human caspase-8 promoter and found that caspase-8 can be upregulated via p53. As during carcinogenesis p53 mutations become selected frequently, we were interested to study the role and expression of caspase-8 in hepatocellular carcinomas. For our analysis we used a recently described transgenic HCC mouse model (c-myc and IgEGF transgenes, respectively). These animals develop hepatocellular carcinomas between 6 and 12 months of age. We isolated HCC tumor nodes from c-myc, IgEGF and double transgenic mice, respectively and subjected t h e m to specific RTPCR for murine caspase-8. Interestingly, 11 out of 24 (46%) of the tumors investigated did not show any caspase-8 transcription. We next isolated HCC-derived genomic DNA and sequenced the caspase-8 promoter. Sequence comparison to sequences obtained from normal liver tissue and the wt caspase-8 region revealed no differences in the tumor-derived caspase-8 promoter. Additionally, luciferase promoter analysis of the tumor-derived sequences did not show any differences in promoter activity compared to the wt region in transient transfection experiments. To test whether CpG methylation of the promoter contributes to caspase-8 silencing we next established an methylation assay for the caspase-8 promoter using an PCR approach and the methylation sensitive restriction enzymes AciI and HpaII, respectively. These experiments resulted in the identification of at least five hypermethylated sites in the caspase-8 promoters derived from hepatocellular carcinomas. These data were supported by a computer analysis predicting CpG Islands within this region. Interestingly, two of the hypermethylated sites within tumor-derived

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

AASLD ABSTRACTS

promoters co-localized with binding sites for the transcription factor SP1, which we recently reported as indispensable for basal promoter activity in the h u m a n caspase-8 promoter. In further experiments we now demonstrate by gel retardation analysis and luciferase reporter assays that the SP1 sites in the murine promoter are also important for mediating basal promoter activity. Finally we simulated the methylation pattern of the tumor derived caspase-8 promoters in vitro by treating promoter-luciferase constructs with CpG methylase resulting in a dramatic reduction of caspase-8 promoter activity. In summary, our results demonstrate that caspase-8 expression is absent in nearly 50% of murine HCCs derived from c-myc-or IgEGF-transgenic mice and suggest that caspase-8 downregulation might be the result of hypermethlafion of SP1 binding sites in its promoter. Disclosures: Juergen Borlack - No relationships to disclose Anne Cohrs - No relationships to disclose Christian Liedtke - No relationships to disclose Michael P M a n n s - No relationships to disclose Christian Trautwein - No relationships to disclose Nils-Holger Zschemisch - No relationships to disclose

183

THE INDUCTION OF THE E2F GENES DURING HEPATIC CELL CYCLE PROGRESSION IS DEPENDENT ON C/EBPBETA. Brian Larris, Jessica Garces, Linda~ Greenbaum,

University of Pennsylvania, Philadelphia, PA Liver regeneration after partial hepatectomy is one of the best in vivo models of synchronized exit from GO and entry into the cell cycle. We have investigated the role of the E2F transcription factors during this process, as these factors have b e e n shown to control the expression of a large n u m b e r of genes involved in DNA replication and cell cycle progression. In addition, the E2F proteins play a critical role in the regulation of cellular growth by their interaction with the retinoblastoma tumor suppressor protein and related pocket proteins p107 and p130. In the quiescent liver, E2F1 mRNA is 28 fold more a b u n d a n t than E2F2 and more t h a n 300 more a b u n d a n t t h a n E2F3 as determined by real time PCR. After partial hepatectomy, E2F1, E2F2 levels are induced 26 and 32 fold, respectively, with peak levels reached at 40 hours post-hepatectomy, which is the time point w h e n most hepatocytes have entered S-phase. Given recent evidence that CCAAT enhancer binding protein alpha can repress E2F regulated genes in the regenerating liver (Iakova, Ceil 113:495), and our previous data demonstrating a decrease in the C/EBPalpha (anti-proliferative) to C/EBPbeta (pro-proliferative) ratio following partial hepatectomy (Greenbaum, JCI, 102:996) we hypothesized that the dramatic induction of E2F1 and E2F2 transcription during liver regeneration might be caused, at least in part by release of C/EBPalpha-E2F repressive complexes. By electrophoretic mobility shift assay with antibodies specific to C/EBPalpha we could indeed demonstrate binding to an E2F binding site by C/EBPalpha in nuclear extracts from quiescent and early G1 phase control livers, with an absence of C/EBPalpha binding in E2F complexes beginning 18h (mid-G1) posthepatectomy. In contrast to control livers, C/EBPalpha binding was detected in E2F complexes at time points extending to at least 24h posthepatectomy in nuclear extracts from C/EBPbeta -/- livers. The activation of b o t h E2F1 and E2F2 gene transcription following partial hepatectomy is reduced by 50% in C/EBPbeta -/- mice, suggesting that one potential functional consequence of the decreased C/EBPalpha:beta ratio posthepatectomy is the relief of C/EBPalpha mediated-repression of E2F regulated cell cycle associated genes during liver regeneration. Disclosures: Jessica Garces - No relationships to disclose Linda E G r e e n b a u m - No relationships to disclose Brian Larris - No relationships to disclose

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184 URSODEOXYCHOLIC ACID PREVENTS TGF-/31-INDUCED APOPTOSIS T H R O U G H MODULATION OF THE GLUCOCORTICOID RECEPTOR IN PRIMARY RAT HEPATOCYTES. Susana Sola, Rui~ Castro, University of Lisbon,

Faculty of Pharmacy, Lisbon, Portugal; Betsy T Kren, Clifford J Steer, University of Minnesota Medical School, Minneapolis, MN; Cecilia M P Rodrigues, University of Lisbon, Faculty of Pharmacy, Lisbon, Portugal We have recently shown that ursodeoxycholic acid (UDCA) prevents TGF-/31-induced hepatocyte apoptosis, in part, by modulating E2F-1 transcriptional activation. Moreover, several reports have demonstrated the inhibitory effects of glucocorticoids against apoptosis in various systems. In the absence of glucocorticoid ligand, UDCA can activate the glucocorticoid receptor (GCR), thus potentially regulating a n u m b e r of targets such as E2F-1. In this study we investigated the potential role of the GCR during TGF-/31-induced hepatocyte apoptosis; and identified additional antiapoptotic targets for UDCA. Primary rat hepatocytes were treated with 1 n M TGF-/31 for 12, 24, 36 and 48 h _+ 100/~M UDCA or its taurine conjugate TUDCA. We determined b o t h nuclear translocation and total protein levels of GCR by western blot analysis, while its transcript expression was assessed by RTPCR. The role of GCR in the antiapoptotic mechanisms of UDCA was evaluated using interference RNA (RNAi) technology. The effect of post-transcriptional gene silencing was assessed by immunoblotting and caspase-3 like activation. The results showed that incubation with TGF-/31 induced a significant decrease of gcr mRNA expression in primary rat hepatocytes (p < 0.05), consistent with a - 60% reduction in its protein levels at 48 h (p < 0.01). The loss of GCR with TGF-/31 resulted in a marked reduction of b o t h nuclear and cytosolic fractions (p < 0.05). Notably, pre-treatment with either UDCA or TUDCA resulted in a 3- and 4-fold increase of GCR nuclear translocation, respectively, w h e n compared with TGF-/31 alone (p < 0.05). Moreover, w h e n RNAi was used to specifically inhibit GCR synthesis, UDCA no longer protected hepatocytes against TGF-/31-induced apoptosis. In fact, the protective effect of UDCA reduced TGF-/31-induced caspase-3 like activation from 65% to < 10% w h e n GCR function was blocked. Finally, E2F-1 transcriptional activation with TGF-/31 normally inhibited by UDCA, was increased with this bile acid after GCR interference. In conclusion, these results demonstrate an additional pathway for the protective m e c h a n i s m of UDCA that involves the nuclear receptor GCR as a key factor. Further, the transcription factor E2F-1 appears to be a potent target for the UDCA-induced GCR activation. Disclosures: Rui E Castro - No relationships to disclose Betsy T Kren - No relationships to disclose Cecilia M P Rodrigues - No relationships to disclose Susana Sola - No relationships to disclose Clifford J Steer - No relationships to disclose