841 Integrin-stimulated cell scattering is regulated by extracellular signal-regulated kinase (ERK) pathways in highly metastatic hepatocellular carcinoma cells

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

AASLD ABSTRACTS

and p73. The p73 gene is transcribed from two promoters, P1 and P2, that direct the expression of the TA (transactivafion competent, proapoptofic and anti-proliferative) and the DN (transactivafion deficient, anti-apoptotic and pro-proliferative) p73 proteins, respectively. We have previously demonstrated that, in response to a non apoptotic DNA damage the P2-p73 promoter is transcriptionally activated in a p53-dependent manner, leading to the expression and accumulation of DN-p73 proteins, that in turn selectively downregulate their own promoter as well as the p21 promoter to ensure the cell-cyde reentry of cells that have repaired their DNA damage. We show here that fiver DN-p73 mRNA levels increase in chronic hepatitis and cirrhosis and correlate with hepatocytes loss and proliferation. Using small interfering RNAs specific for DN-p73 to selectively abrogate its function, we found that DN-p73 is required for TNF-alpha induced pr liferafion of hepatocytes and that, in this context DN-p73 expression is controlled by NFkB. To study the regulation of DN-p73 expression at the transcriptional level we doned the DN-p73 alternative promoter (P2p73Pr). Differently from the Plp73 promoter, that directs the expression of TA-p73 proteins and is regulated by E2F1, the P2p73 promoter is activated by TNF-alpha through NFkB sites and downregulated by IFN-alpha in hepatocarcinoma cell lines and in immortalized Met Murine Hepatocytes D3 (MMH-D3) cells. Functional studies showed that DN-p73 counteracts both p53 and TA-p73-induced growth arrest and apoptosis in MMH-D3 cells. In conclusion we have identified a role of DN-p73 variants in controlling hepatocytes growth in the context of an inflammatory liver environment. Disclosures: Clara Balsano - No relationships to disclose Antonio Costanzo - No relationships to disclose Marcella Fulco - No relationships to disclose Massimo Levrero - No relationships to disclose Francesca Moretti - No relationships to disclose Emanuele Palescandolo - No relationships to disclose Stefania Vossio - No relationships to disclose

841

INTEGRIN-STIMULATED CELL SCATTERING IS REGULATED BY E X T R A C E L L U L A R SIGNAL-REGULATED KINASE (ERK) PATHWAYS IN HIGHLY METASTATIC HEPATOCELLULAR CARCINOMA CELLS. Nobuyuki Honma,

Takuya Genda, Yasunobu Matsuda, Takafumi Ichida, Yutaka Aoyagi, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Background: Metastasis to other parts of the liver through ~ r n o r cell dispersal via the portal vein is a common fea~tre of h u m a n hepatocellular carcinoma (HCC). This so-called intrahepatic metastasis is one of the main causes of recurrence after initial treatment and the poor prognosis of HCC patients. A h u m a n HCC cell line, KYN-2, shows vascular invasion and intrahepatic metastasis mimicking the clinical behavior of h u m a n HCC when orthotopically implanted into the liver of SCID mice (Hepatology, 30, 1027). Therefore KYN-2 is thought to provide a good model for studying the molecular basis of invasion and metastsis of h u m a n HCC. Recently we have shown that integrinmediated cell-substratum adhesion induces E-cadherin inactivation and cell scattering in KYN-2 cells (Lab. Invest. 80, 387). Since loss of cell-cell contact and gain of cell motility is thought to be a critical step in the process of cancer cell invasion and metastasis, the present study was performed to analyze the integrin-elicited intracellular signals involved in KYN-2 cell scattering. Method: KYN-2 cells were kindly provided by Dr M. Kojiro (Kurume University, Kurume, Japan). Extracellular signal-regulated kinase (ERK) aCdvity was examined by immunoblot analysis using anti-phospho-ERK antibody. To prevent ERK kinase (MEK) activation, the cells were treated with 25/.dVI PD98059. The cells were transfected with a constitutively active MEK1 mutant ($218/222D) by the lipofection method, and Geneticin-resistant stable dones were isolated and analyzed. Results: In vitro cultures of KYN-2 cells formed compact cell aggregates in suspension but lost their tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. We found that ERK was highly activated by integrin-mediated cell-substratum adhesion, and addition of an inhibitory monodonal antibody against /31 integrin suppressed both ERK phosphorylation and cell scattering. Upon treatment with PD98059, phospho-ERK was decreased and fight intercel-

567A

lular contact was recovered, resulting in formation of cohesive colonies even by the adherent cells. Immunocytochemical examination demonstrated accumulation of adherence junction components, such as E-cadherin, catenin and aCdn filaments, in zones of intercellular contact in the presence of PD98059. On the other hand, constitutively active MEK1 transfectants showed a flattened phenotype with broad lamellipodia and increased cell moffiity. ImmunoreaCdvity of adherence junction components in the zones of intercellular contact was lower in these transfectants than in the parent cells or mock transfectants. Condusion: These observations indicate that the MEK-ERK pathway regulates integrin-stimulated cell scattering in KYN-2 cells, and suggest its important role in invasion and metastasis of human HCC. Disclosures: Yutaka Aoyagi - No relationships to disclose Takuya Genda - No relationships to disclose Nobuyuki H o n m a - No relationships to disclose Takafumi Ichida - No relationships to disclose Yasunobu Matsuda - No relationships to disclose

842 ROLE OF H E M E OXYGENASE-1 IN ALCOHOL MEDIATED

CYTOKINE REGULATION. Yvonne Drechsler, Laszlo Romics Jr,

Weibo Li, Gyongyi Szabo, University of Massachusetts Medical School, Worcester, MA Chronic alcohol use can lead to liver disease and altered i m m u n e activation. Both acute and chronic alcohol use affect inflammatory pathways and induce immunoregulatory cytokines. Acute alcohol has b e e n shown to inhibit lipopolysaccharide (LPS) mediated induction of inflammatory cytokines including tumor necrosis factor a (TNFa) both in vitro and in vivo. We reported that alcohol can augment LPS induced upregulation of the immunoregulatory cytokine interleukin-10 (IL-10) in h u m a n monocytes. Recent studies suggested a possible role of h e m e oxygenase-1 (HO-1), a stress inducible protein with potential anti-inflammatory effect, in the regulation of IL-10. Here we hypothesized that intracellular events induced by acute alcohol may involve regulation of HO-1 that in turn regulate IL-10 production. To investigate if the effects of alcohol are mediated by HO-1, h u m a n monocytes, mouse macrophages and mouse splenocytes were stimulated with LPS (0.1 ug/ml), LPS and the HO-1 inhibitor zinc-protoporphyrine (ZnPP, 20 tLM) or the HO-1 activator cobalt-protoporphyrine (CoPP, 50 tLM), with and without alcohol (25 mM). Here we show that LPS-induced HO-1 enzyme activity in h u m a n monocytes was further increased by alcohol after in vitro stimulation (p<0.05). Alcohol induced augmentation of IL-10 production could be abrogated by the HO-1 inhibitor, ZnPP (p<0.01). Consistent with the involvement of HO-1 in IL-10 induction, the HO-1 activator (CoPP) a u g m e n t e d IL-10 levels significantly (p<0.05). In mouse macrophages and splenocytes alcohol also upregulated LPS-induced IL-10 production. ZnPP, again, abrogated IL-10 induction, whereas CoPP increased IL-10 levels. Consistent with the findings in h u m a n monocytes alcohol downregulated TNFa in mouse macrophages and splenocytes. ZnPP pretreatment upregulated TNFa levels that may reflect loss of IL-10 mediated TNFa inhibition, as elevated IL-10 levels have b e e n shown to downregulate TNFa. To validate the in vitro results in an animal model, C57BL/6 mice were injected intraperiteonally with LPS (0.5/~g/g body weight in 200 td saline), CoPP (50 tLM), ZnPP (50 tLM) with and without pretreatment with alcohol (200 td of 20% (v/v)). In vivo, alcohol upregulated CoPP-induced HO-1 activity in splenocytes. In serum and splenocytes, LPS-induced IL-10 release was also a u g m e n t e d by alcohol administration. Consistent with the in vitro findings, alcohol downregulated LPS-induced serum TNFa levels. In summary, our results show a possible role for HO-1 in augmentation of IL-10 production by alcohol and in alcohol mediated antiinflammatory regulation after in vitro and in vivo stimulation of h u m a n monocytes, mouse splenocytes and macrophages. Disclosures: Yvonne Drechsler - No relationships to disclose Weibo Li - No relationships to disclose Laszlo Romics Jr - No relationships to disclose Gyongyi Szabo - No relationships to disclose