- Email: [email protected]
358 HCC invasiveness induced by SCCA is associated with nuclear β-catenin accumulation
Posters
132
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DRAMATIC ANTITUMORAL EFFECT OF METRONOMIC DOSING OF SIROLIMUS IN COMBINATION WITH CHEMOTHERAPY TO TREAT HEPATOCELLULAR CARCINOMA: INHIBITION OF ANGIOGENESIS AND REGULATION OF P21WAF1/CIP1
A.C. Piguet 1, D. Semele 1, M. Lederrnann 1, E Theilet2, C. Stoupis 2, J-E Dufour 1. ;Department of Clinical Pharmacology, University of Bern,
Bern, Switzerland," 2Department of Radiology, University Hospital of Bern, Bern, Switzerland Hepatocollular carcinoma is increasing in incidence and bears a poor prognosis. Malignant hepatocytes are resistant to conventional chemotherapy. This might be modified by using anti-angiogenic metronomic therapy which targets proliferating endothelial cells or by reducing the expression of p21Waf1/Cip1. Chemotherapy induces the expression o f p21Wafl/Cip1 favoring cell survival. Abolition of this response sensitizes cells to chenlotherapentic agents. Our aim was to investigate the effects of sirolimus on these parameters. Methods: Single 2ram 3 cubes of subcutaneously grown Morris Hepatoma cells (MH) were implanted into livers of 40 ACI rats. Five days later rats were randomized to sirolinms (2 m~day/kg p.o.), pegylated doxombicin (first injection 4.5mg/kg i.v., then i mg/kg i.v./4 days), both or no treatmont. Tumoral growth was assessed by MRI, vascularisation by CD31 immunostaining. Sensitivity of MH and primary rat aortic endothelial cells (EC) to CCI-779 (sirolimus prodrug, 1-10ngjml) and doxombicin (10 100 ng/ml) was measured by MTT, thymidine incorporation and tube formation assay. Expression of p21Wafl/Cip 1 and pho sporylation o f 4EBP 1 was determined by Western blotting. Data are presented as Meand-S D. Results: Tumor sizes were 1538d-884mrn 3 controls, 894±93mrn 3 sirolimus, 749±114ram 3 pegylated doxorubicin, 284± 105 m m 3 combination (p<0.01), growth rate was also significantly reduced with the combination. Phosphorylation of 4E-BP1 was decreased in the tumors of animals receiving sirolimus (roTOR inhibition). Blood levels were 1 ng/ml sirolimus and 1 ug/ml doxorubicin. Tunloral microvessel density decreased significantly by 38% with sirolimus. Compared to controls, tube formation assay decreased to 24% with CCI-779 + doxorubicin vs. 55% with CCI-779 and 73% with doxorubicin (p <0.05). CCI-779 inhibited thymidine incorporation in EC by 61% and MTT assay in MH cells by 20%. In both MH and EC, the combination of CCI-779+doxorubicin significantly decreased cell viability (MTT) compared to monotherapy; an increased inhibition of thymidine incorporation occurred only in EC. Doxorubicin increased the expression of p21Wafl/Cip 1 in MH and more in F£;. This response was completely blocked in both cell types by CCI-779. Conclusions: In an orthotopic hepatocellular carcinoma model sirolimus shows antiangiogenic properties. Moreover, sirolimus enhances the antitumoral effect of chemotherapy by preventing the increase of p21Wafl/Cip 1 in response to DNA damage. These findings provide a molecular rationale for testing sirolimus in the treatment of hepatocellular carcinoma.
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TELOMERE SHORTENING CORRELATES WITH ANEUPLOIDY OF CHROMOSOME 8 IN PRIMARY HEPATOCELLULAR CARCINOMA
R.R. Plentzl, B. Schlegelberger 2, P. Flemming3, M.P. Manns I , K.L. Rudolph I , L. Wilkens 2,3. 1Department of Gastroenterology,
Hepatology and Endocrinology, Medical School of Hannovez Hannovec Germany; 2Institute for Cell and Molecular Pathology, Germany; 3Department of Pathology, Germany Introduction: Hepatocellular carcinoma (HCC) is a common malignant tumor world-wide. It is well known that chromosomal instability leads to an increase of aneuploidy in human HCC with an increased rate o f chromosomal aberrations. In our recent study we combined for the first time the analysis of hepatocellular telomere length by cell type specific
telomere length measurement with an analysis on the copy number of chromosome 8 using a centrornere-specific probe. Patients and Methods: We chose 15 biopsies of aneuploid HCC and 5 touch prints of 5 cadaver livers without liver cirrhosis or HCC. The patients' age was between 47-84. For histology and cytology the samples were paraffin embedded and stained with hernotoxylin and eosin. Hepatocyte specific analysis of telomere length and the measurement of centromere-specific probe for chromosome 8 were both performed by quantitative fluorescence in situ hybridization (qFISH). Results: For the HCC samples the mean number of chromosome 8 per nucleus was 3.25 to 7.5 (range of individual nuclei: 3-15). The mean telornere fluorescence intensity (TFI) was 81 to 389 (range of individual nuclei: 32-657). Comparative analysis of telornere length and chromosome 8 copy numbers showed sig'nificanfly shorter telomers in hepatocytes of HCC with increased chromosome 8 copy-number. There was a correlation between shorter telomeres and increasing chromosome 8 copy number. However, above the level of 5 copies of chromosome 8 per nuclei no further shortening of telomeres was found indicating that telomeres had reached a critically short length. Compared to tile group of HCC no aneuploidy o f chromosome 8 was found in tile control samples, which had significantly longer telomeres compared to the HCC. Discussion: Our study ~ves first experimental evidence for a correlation of telomere shortening in HCC and increased copy numbers of chromosome 8 - a hallmark of cytogentic abnormality. According to the telomere hypothesis of cancer iniiflation telomere shortening results in loss of telomere capping function leading to chromosomal fusions and fusion bridge breakage cycles. In addition, our data on HCC indicate that telomere shortening is not only linked to random genomic instability but leads to specific cytogentic changes reflecting the cytogenfic fingerprint of a tumor type.
~HCC INVASIVENESS INDUCED BY SCCA IS ASSOCIATED WITH NUCLEAR I~-CATENIN ACCUMULATION S. Quarta I , L. Vidalino I , M.G. Ruvoletto I , M. Della Barbera 2, E Calabrese 2, M. Valente 2, M. Merino 3, L. Beneduce 3, A. Gatta I , G. Fassina3, R Pontisso 1 . : Clinica Medica 5, Department of Clinical and
Experimental Medicine, University of Padova, Padova, Italy," 2Department of Pathology, University of Padova, Padova, Italy," 3Xeptagen S.RA., Pozzuoli (NA), Italy Background: The molecular changes and mechanisms that re,gflate the development and progression of HCC are still unclear. A variety of cell-cell and cell-substrate adhesion complexes have been described in mammalian tissues which are often aberrantly regulated in malignant cells. To date, the potential involvement in migration and invasion of Squamous cell carcinoma antigen (SCCA), a serpin overexpressed in HCC, has not yet been investigated. Aim: To analyze the role of SCCA on cell adhesion system and to investigate its potential motogenic activity in vitro. Materials a n d Methods: Morpholo~cal and ultrastructural characteristics of cultured HepG2 cells stably transfected with a plasmid vector carrying tile human SCCA1 gone were analyzed by transmission electron microscopy and compared with untransfected HepG2 cells. The expression of pivotal molecules of the cell adhesion system, E-cadherin and f5-catenin, was analyzed by Western blot, while their cellular distribution was evaluated by immunofluorescence analysis. The potential SCCA motogenic activity was investigated by 2 4 h incubation of epithelial cells (VERO) with increasing amounts of exogenous SCCA1. Results: Ultrastructural analysis showed that HepG2 expressing human SCCA1 had a remarkable decrease of desmosomal junctions and microvilli, widening o f intercellular spaces, compared with control cells, indicating a lack of intercellular complexes. The down-regulation of cell adhesion system was associated with a reorganization of cytoscheleton complexes, documented by initial changes of intermediate filaments.
Category 4d." Molecular and Cellular Biology." Liver Regeneration and Experimental Oncology Moreover, HepG2/SCCA appeared quite undifferentiated, with ovoidal nuclei rich in euchrornatin, all morphological alterations typical ofinvasive metastatic cells. Western blot analysis showed 56% reduction of E-cadherin in HepG2/SCCA cells, while by immunofluorescence a nuclear accumulation, in particular of [3-catenin, was detected, beside its cytoplasmic distribution, observed in untransfected cells. The effect of SCCA on modulation of cell to cell adhesion and motility system was also supported by the progressive scatter activity in VERO cells, induced by increasing concentrations of exogenous SCCA in a dose-dependent manner. Conclusion: The observed findings suggest that SCCA induces morphological and ultrastructural changes typical of progression and invasion of neoplastic cells. Aberrant nuclear accumulation of [3-catenin, assodated with SCCA overexpression, plays a crucial role in malignant cell behaviour.
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OVEREXPRESSION OF TGFa AND EGF RECEPTOR, A KEY EVENT IN LIVER CARCINOGENESIS, IS INDUCED BY HYPOXIA SPECIFICALLY IN HEPATOCY-rES
E. Schiffer 1, O. R o s m o r d u c 1,2, C. R e y 1 , V Barbu 1 , C. Housset1.
IINSERM Unitd 402, Facultg de Mgdecine Saint-Antoine, UPMC, Paris, France," 2Service d'H@atologie, Hdpital Saint-Antoine, Paris, France TGF0~ is a mitogenic t'actor for hepatocyte and a ligand of the EGF receptor (EGF-R). TGF0~ has tile ability to promote liver carcinogenesis since TGF(x transgenic mice constantly develop hepatocelhilar carcinoma. TGFa is also overexpressed in regenerative nodules of the cirrhotic liver but the mechanism of this expression is poorly known. Because local hypoxia is a constant feature of cirrhotic livers and hypoxia may induce TGF(x and FA3F-R expressions, tile aim of this study was to determine whether the TGF0~/EGF-R pathway may be affected by hypoxia in liver cells. Methods: Cell isolates were prepared from normal Wistar rats. Liver myofibroblasts were obtained in culture by activation of hepatic stellate cells HSC (HSC), and by outgrowth of peribiliary myofibroblasts from bile duct segments. Hepatocytes, Kupffer cells and liver myofibroblasts in culture were submitted to hypoxia during 4 24 hours. Hypoxia was achieved using a catalytic system, which reduces oxygen concenwation to less than 1% within 30 minutes. Tile absence of toxicity was verified by measuring lactate dehydrogenase release. Vascular endothelial growth factor (VEGF) served as a hypoxia-inducible control gene. Gene expressions were assessed by real-time RT-PCR. Results are expressed as a number of transcripts ×10?/p~g 18S RNA. Results: Under normoxia, the expression of TGFo, was significantly higher in hepatocytes than in non-parenchymal cells under study (~l.7-fold). EGF-R transcripts were also more abundant in hepatocytes than in myofibroblasts (~3-fold) or in Kupffer cells (~22-fold). Hypoxia induced arl increase in VEGF mRNA to a similar extent in all cell types. By contrast, hypoxia caused an increase in TGF0t Wanscripts mafitly in hepatocytes (112±7 vs 32±2 under normoxia), also but to a lesser extent in peribiliary myofibroblasts (35±5 vs 17±4), but not in HSC-derived myofibroblasts nor in Kl~pffer cells. An increase in EGF-R expression was induced by hypoxia also predominantly in hepatocytes (125±12 vs 44±6), and to a much lesser extent in other cell types. Conclusion: These results demonstrate that hypoxia induces TGFa and FA3F-R overexpression in hepatocytes and, thereby, might act as a promoting event in liver carcinogenesis upon cirrhotic liver.
133
~VEGF AS A POTENT STIMULATOR OF LIVER REGENERATION AFTER PARTIAL HEPATECTOMY
M. Bockhorn2, E Damrnann2, D. Prokofiev2, M. Goralsld 1,2, P GNnewald 1, J.E Schlaak I , A. Frilling2, C.E. Broelsch2 . 1Department
of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany; 2Department of Surgery, University Hospital of Essen, Essen, Germany Background and aims: After partial hepatectomy (PH), clinical outcome, morbidity and mortality of the patients depend on an efficient regeneration of the liver. Angiogenesis is essential for the regeneration of the liver and Vascular Endothelial Growth Factor (VEGF) belongs to the most potent angiogenic factors. The aims of the study were to determine the effects of exogenous VEGF administration in rats after 2/3 hepatecomy on angiogenesis by intravital microscopy, on regeneration by imrnunohistochemistry and on expression of angiogenic genes by cDNA arrays. Methods: Adult male Lewis rats (n = 5/group) were subjected to 70% PH and splitin 3 groups. Group A (VEGF), group B (anti-VEGF), andgroup C (Control) were administered VEGF (100ng/~tl), anti-VEGF (4pg/pl) or NaC1 i.v. at Oh, 36h, and 96h. At Oh, 24h, 48h, 72h, 120h, 168h postoperatively 5 rats each underwent intmvital microscopy of the exposed liver remnant. Recorded parameters were: Vessel density (VD), vessel diameter (VDi) and vessel flow (VF). Liver regeneration was monitored by measuring liver body weight ratio (LBR) and immunohistochemically by proliferating cell nuclear antigen (Ki-67). Subseqllenfly, specimens from regenerating liver were harvested, and angiogenic gene expression profiles was determined by an inhouse cDNA macroarry including 70 genes involved in liver regeneration. Results: In VEGF treated rats (group A), VD was significantly increased compared to group B or C (p <0.05). VDi was significantly increased through 24 72 h in group A (p < 0.05) compared to group A and C. In the VEGF treated animals, LBR was significantly higher after 48 h, 72 h and 120 h (p < 0.025). PCNA imrnunostaining showed a significantly higher labelling index of hepatocytes at 24 h after PH with 82% compared to the control groups (3%). cDNA macroarrays showed a complex moditlation of genes involved in liver regeneration. Conclusion: Exogenous VEGF admfilistration leads to increased anglogenesis, which subsequently results in faster liver regeneration. This effect can be blocked by anti-VEGE Therefore, VEGF treatment may provide a novel strategy for optimization of liver regeneration in patients after PH.
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EFFECTS OF HEPATITIS B VIRUS REPLICATION ON OXIDATIVE STRESS, GENE EXPRESSION AND INDUCTION OF GENETIC CHANGES IN AN INDUCIBLE CELL CULTURE MODEL OF HEPAD38 CELLS
T. Severi I , C.H. Ying 2, J. Verrneesch3, M. Zeegers I , A. Van Lommel 4, R. Servaes I , J. Neyts 2, J. Fevery I , J.E van Peltt . ILabo Hepatology, Univ
Hospital Gasthuisberg, Leuven, Belgium," 2Labo Nrology, REGA Institute, Fact Medicine, Leuuen, Belgium," SDepartrnent of Human Genetics, Uniu Hospital Gasthuisberg, Leuven, Belgium," 4Department of Pathology, Uniu Hospital Gasthuisberg, Leuuen, Belgium We investigated whether HBV replication in liver cells can give rise to genetic changes in the hepatocytes cell as a mechanism contributing to malign transformation and HCC development. We used the model of HepAD38 cell that contain the entire genome of HBV and where the virus replicates under control of a tetracycline-regxtlated promotor. We determined in these cells parameters of oxidative stress [malondialdehyde (MDA), ghitathione (GSHtot) and oxidized glutathione (GSSG)] arid the rate of cell growth. Karyotyping was performed on cells at the start of the experiment and at passage 6 and 18 of cells with no virus production or cells that produced HBV Twenty-four hours after induction of HBV replication MDA and GSHtot had increased but returned to starting levels
132
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DRAMATIC ANTITUMORAL EFFECT OF METRONOMIC DOSING OF SIROLIMUS IN COMBINATION WITH CHEMOTHERAPY TO TREAT HEPATOCELLULAR CARCINOMA: INHIBITION OF ANGIOGENESIS AND REGULATION OF P21WAF1/CIP1
A.C. Piguet 1, D. Semele 1, M. Lederrnann 1, E Theilet2, C. Stoupis 2, J-E Dufour 1. ;Department of Clinical Pharmacology, University of Bern,
Bern, Switzerland," 2Department of Radiology, University Hospital of Bern, Bern, Switzerland Hepatocollular carcinoma is increasing in incidence and bears a poor prognosis. Malignant hepatocytes are resistant to conventional chemotherapy. This might be modified by using anti-angiogenic metronomic therapy which targets proliferating endothelial cells or by reducing the expression of p21Waf1/Cip1. Chemotherapy induces the expression o f p21Wafl/Cip1 favoring cell survival. Abolition of this response sensitizes cells to chenlotherapentic agents. Our aim was to investigate the effects of sirolimus on these parameters. Methods: Single 2ram 3 cubes of subcutaneously grown Morris Hepatoma cells (MH) were implanted into livers of 40 ACI rats. Five days later rats were randomized to sirolinms (2 m~day/kg p.o.), pegylated doxombicin (first injection 4.5mg/kg i.v., then i mg/kg i.v./4 days), both or no treatmont. Tumoral growth was assessed by MRI, vascularisation by CD31 immunostaining. Sensitivity of MH and primary rat aortic endothelial cells (EC) to CCI-779 (sirolimus prodrug, 1-10ngjml) and doxombicin (10 100 ng/ml) was measured by MTT, thymidine incorporation and tube formation assay. Expression of p21Wafl/Cip 1 and pho sporylation o f 4EBP 1 was determined by Western blotting. Data are presented as Meand-S D. Results: Tumor sizes were 1538d-884mrn 3 controls, 894±93mrn 3 sirolimus, 749±114ram 3 pegylated doxorubicin, 284± 105 m m 3 combination (p<0.01), growth rate was also significantly reduced with the combination. Phosphorylation of 4E-BP1 was decreased in the tumors of animals receiving sirolimus (roTOR inhibition). Blood levels were 1 ng/ml sirolimus and 1 ug/ml doxorubicin. Tunloral microvessel density decreased significantly by 38% with sirolimus. Compared to controls, tube formation assay decreased to 24% with CCI-779 + doxorubicin vs. 55% with CCI-779 and 73% with doxorubicin (p <0.05). CCI-779 inhibited thymidine incorporation in EC by 61% and MTT assay in MH cells by 20%. In both MH and EC, the combination of CCI-779+doxorubicin significantly decreased cell viability (MTT) compared to monotherapy; an increased inhibition of thymidine incorporation occurred only in EC. Doxorubicin increased the expression of p21Wafl/Cip 1 in MH and more in F£;. This response was completely blocked in both cell types by CCI-779. Conclusions: In an orthotopic hepatocellular carcinoma model sirolimus shows antiangiogenic properties. Moreover, sirolimus enhances the antitumoral effect of chemotherapy by preventing the increase of p21Wafl/Cip 1 in response to DNA damage. These findings provide a molecular rationale for testing sirolimus in the treatment of hepatocellular carcinoma.
•]
TELOMERE SHORTENING CORRELATES WITH ANEUPLOIDY OF CHROMOSOME 8 IN PRIMARY HEPATOCELLULAR CARCINOMA
R.R. Plentzl, B. Schlegelberger 2, P. Flemming3, M.P. Manns I , K.L. Rudolph I , L. Wilkens 2,3. 1Department of Gastroenterology,
Hepatology and Endocrinology, Medical School of Hannovez Hannovec Germany; 2Institute for Cell and Molecular Pathology, Germany; 3Department of Pathology, Germany Introduction: Hepatocellular carcinoma (HCC) is a common malignant tumor world-wide. It is well known that chromosomal instability leads to an increase of aneuploidy in human HCC with an increased rate o f chromosomal aberrations. In our recent study we combined for the first time the analysis of hepatocellular telomere length by cell type specific
telomere length measurement with an analysis on the copy number of chromosome 8 using a centrornere-specific probe. Patients and Methods: We chose 15 biopsies of aneuploid HCC and 5 touch prints of 5 cadaver livers without liver cirrhosis or HCC. The patients' age was between 47-84. For histology and cytology the samples were paraffin embedded and stained with hernotoxylin and eosin. Hepatocyte specific analysis of telomere length and the measurement of centromere-specific probe for chromosome 8 were both performed by quantitative fluorescence in situ hybridization (qFISH). Results: For the HCC samples the mean number of chromosome 8 per nucleus was 3.25 to 7.5 (range of individual nuclei: 3-15). The mean telornere fluorescence intensity (TFI) was 81 to 389 (range of individual nuclei: 32-657). Comparative analysis of telornere length and chromosome 8 copy numbers showed sig'nificanfly shorter telomers in hepatocytes of HCC with increased chromosome 8 copy-number. There was a correlation between shorter telomeres and increasing chromosome 8 copy number. However, above the level of 5 copies of chromosome 8 per nuclei no further shortening of telomeres was found indicating that telomeres had reached a critically short length. Compared to tile group of HCC no aneuploidy o f chromosome 8 was found in tile control samples, which had significantly longer telomeres compared to the HCC. Discussion: Our study ~ves first experimental evidence for a correlation of telomere shortening in HCC and increased copy numbers of chromosome 8 - a hallmark of cytogentic abnormality. According to the telomere hypothesis of cancer iniiflation telomere shortening results in loss of telomere capping function leading to chromosomal fusions and fusion bridge breakage cycles. In addition, our data on HCC indicate that telomere shortening is not only linked to random genomic instability but leads to specific cytogentic changes reflecting the cytogenfic fingerprint of a tumor type.
~HCC INVASIVENESS INDUCED BY SCCA IS ASSOCIATED WITH NUCLEAR I~-CATENIN ACCUMULATION S. Quarta I , L. Vidalino I , M.G. Ruvoletto I , M. Della Barbera 2, E Calabrese 2, M. Valente 2, M. Merino 3, L. Beneduce 3, A. Gatta I , G. Fassina3, R Pontisso 1 . : Clinica Medica 5, Department of Clinical and
Experimental Medicine, University of Padova, Padova, Italy," 2Department of Pathology, University of Padova, Padova, Italy," 3Xeptagen S.RA., Pozzuoli (NA), Italy Background: The molecular changes and mechanisms that re,gflate the development and progression of HCC are still unclear. A variety of cell-cell and cell-substrate adhesion complexes have been described in mammalian tissues which are often aberrantly regulated in malignant cells. To date, the potential involvement in migration and invasion of Squamous cell carcinoma antigen (SCCA), a serpin overexpressed in HCC, has not yet been investigated. Aim: To analyze the role of SCCA on cell adhesion system and to investigate its potential motogenic activity in vitro. Materials a n d Methods: Morpholo~cal and ultrastructural characteristics of cultured HepG2 cells stably transfected with a plasmid vector carrying tile human SCCA1 gone were analyzed by transmission electron microscopy and compared with untransfected HepG2 cells. The expression of pivotal molecules of the cell adhesion system, E-cadherin and f5-catenin, was analyzed by Western blot, while their cellular distribution was evaluated by immunofluorescence analysis. The potential SCCA motogenic activity was investigated by 2 4 h incubation of epithelial cells (VERO) with increasing amounts of exogenous SCCA1. Results: Ultrastructural analysis showed that HepG2 expressing human SCCA1 had a remarkable decrease of desmosomal junctions and microvilli, widening o f intercellular spaces, compared with control cells, indicating a lack of intercellular complexes. The down-regulation of cell adhesion system was associated with a reorganization of cytoscheleton complexes, documented by initial changes of intermediate filaments.
Category 4d." Molecular and Cellular Biology." Liver Regeneration and Experimental Oncology Moreover, HepG2/SCCA appeared quite undifferentiated, with ovoidal nuclei rich in euchrornatin, all morphological alterations typical ofinvasive metastatic cells. Western blot analysis showed 56% reduction of E-cadherin in HepG2/SCCA cells, while by immunofluorescence a nuclear accumulation, in particular of [3-catenin, was detected, beside its cytoplasmic distribution, observed in untransfected cells. The effect of SCCA on modulation of cell to cell adhesion and motility system was also supported by the progressive scatter activity in VERO cells, induced by increasing concentrations of exogenous SCCA in a dose-dependent manner. Conclusion: The observed findings suggest that SCCA induces morphological and ultrastructural changes typical of progression and invasion of neoplastic cells. Aberrant nuclear accumulation of [3-catenin, assodated with SCCA overexpression, plays a crucial role in malignant cell behaviour.
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OVEREXPRESSION OF TGFa AND EGF RECEPTOR, A KEY EVENT IN LIVER CARCINOGENESIS, IS INDUCED BY HYPOXIA SPECIFICALLY IN HEPATOCY-rES
E. Schiffer 1, O. R o s m o r d u c 1,2, C. R e y 1 , V Barbu 1 , C. Housset1.
IINSERM Unitd 402, Facultg de Mgdecine Saint-Antoine, UPMC, Paris, France," 2Service d'H@atologie, Hdpital Saint-Antoine, Paris, France TGF0~ is a mitogenic t'actor for hepatocyte and a ligand of the EGF receptor (EGF-R). TGF0~ has tile ability to promote liver carcinogenesis since TGF(x transgenic mice constantly develop hepatocelhilar carcinoma. TGFa is also overexpressed in regenerative nodules of the cirrhotic liver but the mechanism of this expression is poorly known. Because local hypoxia is a constant feature of cirrhotic livers and hypoxia may induce TGF(x and FA3F-R expressions, tile aim of this study was to determine whether the TGF0~/EGF-R pathway may be affected by hypoxia in liver cells. Methods: Cell isolates were prepared from normal Wistar rats. Liver myofibroblasts were obtained in culture by activation of hepatic stellate cells HSC (HSC), and by outgrowth of peribiliary myofibroblasts from bile duct segments. Hepatocytes, Kupffer cells and liver myofibroblasts in culture were submitted to hypoxia during 4 24 hours. Hypoxia was achieved using a catalytic system, which reduces oxygen concenwation to less than 1% within 30 minutes. Tile absence of toxicity was verified by measuring lactate dehydrogenase release. Vascular endothelial growth factor (VEGF) served as a hypoxia-inducible control gene. Gene expressions were assessed by real-time RT-PCR. Results are expressed as a number of transcripts ×10?/p~g 18S RNA. Results: Under normoxia, the expression of TGFo, was significantly higher in hepatocytes than in non-parenchymal cells under study (~l.7-fold). EGF-R transcripts were also more abundant in hepatocytes than in myofibroblasts (~3-fold) or in Kupffer cells (~22-fold). Hypoxia induced arl increase in VEGF mRNA to a similar extent in all cell types. By contrast, hypoxia caused an increase in TGF0t Wanscripts mafitly in hepatocytes (112±7 vs 32±2 under normoxia), also but to a lesser extent in peribiliary myofibroblasts (35±5 vs 17±4), but not in HSC-derived myofibroblasts nor in Kl~pffer cells. An increase in EGF-R expression was induced by hypoxia also predominantly in hepatocytes (125±12 vs 44±6), and to a much lesser extent in other cell types. Conclusion: These results demonstrate that hypoxia induces TGFa and FA3F-R overexpression in hepatocytes and, thereby, might act as a promoting event in liver carcinogenesis upon cirrhotic liver.
133
~VEGF AS A POTENT STIMULATOR OF LIVER REGENERATION AFTER PARTIAL HEPATECTOMY
M. Bockhorn2, E Damrnann2, D. Prokofiev2, M. Goralsld 1,2, P GNnewald 1, J.E Schlaak I , A. Frilling2, C.E. Broelsch2 . 1Department
of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany; 2Department of Surgery, University Hospital of Essen, Essen, Germany Background and aims: After partial hepatectomy (PH), clinical outcome, morbidity and mortality of the patients depend on an efficient regeneration of the liver. Angiogenesis is essential for the regeneration of the liver and Vascular Endothelial Growth Factor (VEGF) belongs to the most potent angiogenic factors. The aims of the study were to determine the effects of exogenous VEGF administration in rats after 2/3 hepatecomy on angiogenesis by intravital microscopy, on regeneration by imrnunohistochemistry and on expression of angiogenic genes by cDNA arrays. Methods: Adult male Lewis rats (n = 5/group) were subjected to 70% PH and splitin 3 groups. Group A (VEGF), group B (anti-VEGF), andgroup C (Control) were administered VEGF (100ng/~tl), anti-VEGF (4pg/pl) or NaC1 i.v. at Oh, 36h, and 96h. At Oh, 24h, 48h, 72h, 120h, 168h postoperatively 5 rats each underwent intmvital microscopy of the exposed liver remnant. Recorded parameters were: Vessel density (VD), vessel diameter (VDi) and vessel flow (VF). Liver regeneration was monitored by measuring liver body weight ratio (LBR) and immunohistochemically by proliferating cell nuclear antigen (Ki-67). Subseqllenfly, specimens from regenerating liver were harvested, and angiogenic gene expression profiles was determined by an inhouse cDNA macroarry including 70 genes involved in liver regeneration. Results: In VEGF treated rats (group A), VD was significantly increased compared to group B or C (p <0.05). VDi was significantly increased through 24 72 h in group A (p < 0.05) compared to group A and C. In the VEGF treated animals, LBR was significantly higher after 48 h, 72 h and 120 h (p < 0.025). PCNA imrnunostaining showed a significantly higher labelling index of hepatocytes at 24 h after PH with 82% compared to the control groups (3%). cDNA macroarrays showed a complex moditlation of genes involved in liver regeneration. Conclusion: Exogenous VEGF admfilistration leads to increased anglogenesis, which subsequently results in faster liver regeneration. This effect can be blocked by anti-VEGE Therefore, VEGF treatment may provide a novel strategy for optimization of liver regeneration in patients after PH.
•
EFFECTS OF HEPATITIS B VIRUS REPLICATION ON OXIDATIVE STRESS, GENE EXPRESSION AND INDUCTION OF GENETIC CHANGES IN AN INDUCIBLE CELL CULTURE MODEL OF HEPAD38 CELLS
T. Severi I , C.H. Ying 2, J. Verrneesch3, M. Zeegers I , A. Van Lommel 4, R. Servaes I , J. Neyts 2, J. Fevery I , J.E van Peltt . ILabo Hepatology, Univ
Hospital Gasthuisberg, Leuven, Belgium," 2Labo Nrology, REGA Institute, Fact Medicine, Leuuen, Belgium," SDepartrnent of Human Genetics, Uniu Hospital Gasthuisberg, Leuven, Belgium," 4Department of Pathology, Uniu Hospital Gasthuisberg, Leuuen, Belgium We investigated whether HBV replication in liver cells can give rise to genetic changes in the hepatocytes cell as a mechanism contributing to malign transformation and HCC development. We used the model of HepAD38 cell that contain the entire genome of HBV and where the virus replicates under control of a tetracycline-regxtlated promotor. We determined in these cells parameters of oxidative stress [malondialdehyde (MDA), ghitathione (GSHtot) and oxidized glutathione (GSSG)] arid the rate of cell growth. Karyotyping was performed on cells at the start of the experiment and at passage 6 and 18 of cells with no virus production or cells that produced HBV Twenty-four hours after induction of HBV replication MDA and GSHtot had increased but returned to starting levels