Liver nonparenchymal cells and sera from liver failure patients stimulate transdifferentiation of bone marrow cells into hepatocytes

4

Category

Methods: HCC was staged according to the Okuda, BCLC, CLIP, French classifications and estrogen receptor status in 96 patients. The analysis of survival was performed by the Kaplan-Mayer test and was made for each classification system and ER. A comparison between classifications was made by univariate and multivariate analysis. Results: Among the clinical classification systems, only the CLIP was able to identify patient’ populations with good, intermediate and poor prognosis. At multivariate analysis the ER classification was shown to be the best predictive classification for survival of patients with HCC (p=O.OOOO). This difference was due to a better allocation of patients with ominous prognosis (variant ER) having nevertheless good clinical score. Conclusion: The evaluation of the presence of wild type or variant ER transcripts in the tumor is the best predictor of survival in patients with HCC. Its accuracy in discriminating patients with good or unfavorable prognosis is significantly greater than that of the commonly used scoring systems for the staging of HCC. [l] Hepatology 2000, 32; 233-238

I 360

ESSENTIAL

107

we investigated the effects of liver nonparenchymal cells (NPCs) and sera from liver failure patients (HSLF) on the in vitro transdifferentiation of murine BMCs into hepatocytes. Methods: Liver NPCs from wild type mice, and 5-azacytidine-treated BMCs from green fluorescence protein transgenic mice, were cocultured in medium containing HSLF in combination with several cytokines. Hepatocyte-specific gene expression in BMCs was identified by immunocytochemistry and RT-PCR. Results: Bone marrow cell-derived hepatocyte-like colonies appeared after several days of coculture in medium containing HSLF, oncostatin M (OSM) and hepatocyte growth factor (HGF). These colonies expressed hepatocytespecific genes such as albumin and cytokeratin 8. Transdifferentiation was enhanced by 5-azacytidine treatment, and by HSLF, OSM and HGF. It did not take place when the BMCs were separated from the NPCs in a dual chamber dish, or cultured with other mesenchymal cells. Conclusions: Direct interaction of murine BMCs with liver NPCs, as well as soluble factors in the HSLF, strongly stimulate transdifferentiation into hepatocytes.

ROLE FOR BCL-XL AS AN ANTIAPOPTOTIC

FACTOR IN TRAIL MEDIATED

APOPTOSIS

OF HEPATOCYTES

I 362

DURING VIRAL INFECTION

SMALL INTERFERING

RNA (SIRNA) AND ANTISENSE

OLIGONUCLEOTIDES

(ASO) FOR REVERSION

CHEMOTHERAPY

RESISTANCE

OF

IN HEPATOMA

CELLS

M. Waltemathe, L. Zender, S. Huetker, B. Mundt, M.P. Manns, F. Kuehnel, S. Kubicka. Department Of Gastroenterology, Hepatology,

L. Zender, S. Huetker, M.P. Manns, F. Kuehnel, S. Kubicka.

And Endocrinology,

Of Gastroenterology,

Hepatology,

Hannoves

Germany

Medical School Hamoves

Hannoves

Germany

NFkB is a strong antiapoptotic factor in the TNFa-pathway, but the role of NFkB signalling in FasL and TRAILmediated apoptosis in the liver is unknown. We used AdikBa to inhibit NFkB activity in the liver of mice and subsequently treated the animals with AdTRAIL, AdFasL or AdGFP In contrast to FasL-mediated apoptosis, TRAILmediated apoptosis was strongly enhanced by NFkB-inhibition. To investigate the mechanism of NFkB dependent sensitisation of TRAIL-mediated apoptosis we analysed the livers after AdikBa administration compared to the AdlacZ control. Western blot experiments showed constant protein expression of the TRAIL decoy receptors DcRl and DcR2, but upregulation of TRAILreceptor DR.5 after inhibition of NFkB. However, DRSmRNA was not upregulated in quantitative RT-PCR analysis, indicating posttranscriptional mechanisms in NFkB dependent DR.?-regulation. According to this result we observed no transcriptional activation of the DR.5 promotor by AdikBa in luciferase assays. We revealed no significant regulation of c-FLIP and BCL2, but we found a 20.fold upregulation of bcl-xl eight hours after adenoviral infection, which was almost completely abolished after inhibition of NFkB by AdikBa. To specifically block bcl-xl expression without inhibition of NFkB activity we used RNA-interference. Hepatocytes were highly susceptible for AdTRAIL-mediated apoptosis after systemic application of siRNA-bcl-xl compared to a control siRNA. Inhibition of bcl-xl by RNAinterference in hepatoma cells led to sensitisation of AdTRAII-, but not of AdFasL-mediated apoptosis, confirming the specific antiapoptotic role of bcl-xl in AdTRAII-mediated apoptosis. Our results describe bcl-xl as the most important NFkB dependent protective factor in TRAILmediated apoptosis of hepatocytes.

I 361

LIVER NONPARENCHYMAL

Tokyo, Japan;

Graduate

School Of Medicine

2Department

An important protein that mediates resistance to chemotherapy in hepatocellular carcinomas is bcl-xl. Is was the aim of our study to evaluate the inhibition of bcl-xl expression by antisense oligonucleotides and siRNA in the treatment of hepatocellular carcinomas. Hepatoma cell lines were transfected with bcl-xl AS0 or siRNA. As a control nonsense AS0 and siRNAs were used. Inhibition of bcl-xl expression was investigated by western blotting and immunofluorescence. Unspecific toxicity and specific sensitization for chemotherapyand adenovirus~53 mediated apoptosis were determined microscopically and by histone ELISA in hepatoma cell lines. Bcl-xl expression was significantly more downregulated by siRNA-bcl-xl (10.20% expression level) compared to ASO-bcl-xl (30.40% expression level). Inhibition of bcl-xl expression results in strongly enhanced susceptibility of hepatoma cell lines to chemotherapyand p53-mediated apoptosis. Interestingly, not only ~53 mutated hepatoma cell lines (Huh7 and Hep3B) but also ~53 wild type hepatoma cells (HepG2) became sensitive for p53-mediated apoptosis after inhibition of bcl-xl expression. The sensitization of hepatoma cells to chemotherapyand p53-mediated apoptosis was greater after siRNA-bcl-xl treatment compared to ASO-bcl-xl, while nonspecific toxicity of siRNA was significantly lower. Consequently the therapeutic efficacy of siRNA-bcl-xl is superior to ASO-bcl-xl. Our results demonstrate that the sensitivity of hepatoma cells to chemotherapyand p53-mediated apoptosis can be effectively enhanced by inhibition of bcl-xl expression through specific siRNA. Since ASOBc12 already showed clinical efficacy in chemotherapy of patients with melanomas, the improved inhibition of gene expression by siRNA may be an attractive perspective for the treatment of hepatocellular carcinomas.

TRANSDIFFERENTIATION

CELLS INTO HEPATOCYTES

S. Yamazaki, K. Miki, K. Hasegawa, M. Sata, T. Takayama, M. Makuuchi. ‘Third Department Of Surgey, Nihon University School Of Medicine,

Department

Medical School

CELLS AND SERA FROM LIVER

FAILURE PATIENTS STIMULATE OF BONE MARROW

Hannoves

And Endocrinology,

Of Cardiovascular

Medicine,

University Of Tokyo, Tokyo, Japan

Background/Aim: The plasticity of bone marrow cells (BMCs) is shown by their ability to differentiate into mesenchymal as well as endodermal and ectodermal lineages. Transdifferentiation of BMCs into hepatocytes has also been demonstrated, both in vitro and in viva. In the present study

I

363

ISOLATION

AND IDENTIFICATION

IN MURINE

HEPATOCELLULAR

OF CYCLIN El ISOFORMS CARCINOMAS

(HCC)

N.-H. Zschemisch, C. Liedtke, R. Halter, .I. Borlak, M.P. Manns, C. Trautwein. ’Gastroenterology, Hepatology And Endocrinology, Medical School Hannoves Medical Biotechnology, Reserach,

Drug Research

Hannoves

Fraunhofer

Germany;

2Drug Research And

Institute Of Toxicology And Aerosol

And Clinical Inhalation,

Hannoves

Cyclin El is involved in controlling GlIS-phase progression cell cycle. Earlier results indicated that Cyclin El misregulation

Germany

during the correlates