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Con a induced liver injury: a new model to study hepatocyte proliferation
87 HEPATOCELLULAR
CARCINOMA,
LIVER REGENERATION,
LARGE SCALE SCREENING OF HBV-DNA INTEGRATION SITES INTO HUMAN LIVER TUMOR DNA: EVIDENCE FOR SERCA 1 HBV MEDIATED CISACTIVATION. 2
G. G-Y 4. J. Wm
2
B. CAURIQiY_ 1
3
PATERLINI~.IINsERM u370,
‘Genethon and 3Pasteur Institute, Paris, France.4Second Department of Surgery, Chiba University School of Medicine, Chiba 260, Japan. ‘University of Witwatersrand, Johannesbourg, South Africa. The impact of cis activation on HBV related liver cancers is a controversial issue. According to the current view, it would be a rare event in humans, in contrast with WHV related liver tumors in woodchucks. We have therefore applied a PCR based large survey approach to explore this field. Methods: we have screened 42 HCCs from different geographical areas by using an original HBV-Alu PCR method(l) to amplify viral/celJular DNA junctions (VCJ). Amplified DNA was analysed by sequencing, data bank analysis, irradiation hybrid test, RT-PCR. &&@: with X specific primers we have isolated and sequenced 16 VCJ. Chromosomal localization through irradiation hybrids analysis has been obtained in 7 and is ongoing in 9 Data bank analysis of a group of 5 VCJ has shown, in one tumor, HBVDNA integration into the 3rd exon of SERCAl gene. An hybrid viral/SERCAl RNA was detected in the tumorous tissue. Cloning and sequencing analyses showed that HBV integration takes place “in frame” with the SERCA 1 gene and potentially leads to a predicted hybrid HBV/SERCAI protein. SERCA 1 RNA is not detectable in the corresponding non tumorous tissue, normal liver and HepG2 cells. SERCA proteins pump Ca2+ from cytosol to endoplasmic reticulum; they are likely to be involved in cell growth through regulation of intracellular calcium concentrations. Our work shows that 1) cis activation is not a rare event in HBV related liver carcinogenesis 2) analysis of HBV integration sites can be used to discover new genes potentially involved in cell proliferation. Minami, et al. Genomics 29: 403-408, 1995.
MOLECULAR
BASES OF X INDUCED
CELL CYCLE ARREST AND APOPTOSIS C Brdcbpt ._ INSERM U370, Institut Necker, Paris, France.
It is debated whether X might induce or not cell apoptosis and which are the mechanisms involved. It is also unclear what would be the actual impact for the viral cycle. We have adressed this issue by using transient transfections, to avoid phenotypic selection of stable clones, in two cellular cell types’ transformed human Chang cells and immortalised, TGF alpha expressing, mouse aML hepatocytes. Results: I X induces both Gl arre~r anA apoptosis: In both Chang and aML cells, X expression inhibited colony outgrowth formation. Detailed analysis in Chang cells showed a complete Cl cell cycle arrest. In Chang and, to a much lower rate, in aML cells, X expression induced in a second step apoptosis, shown by morphological analysis, FACS, and DNA laddering. Gl arrest and apoptosis were not dependent on p53 status and Xlproteasome interaction since no endogenous ~53 could be detected even after treatment with the proteasome inhibitor ALLN, and cell cycle effects were not abolished. 2. ~53 und X show synergistic effects: Ectopic expression of ~53 in Chang cells induced Cl arrest and apoptosis to the same extent as X, leading to p21 and p27 CKI accumulation Cotnnsfection of X and ~53 led to a 2-3 fold increase in p21 and p27 and mdm2 promoter transcriptional activation; ~53. induced apoptosis and cell cycle arrest were amplified to the same extent. X induced an increase in p53 level which was not due to its stabilisation, as shown by metabolic labelling. Conclusions: 1. The major cell cycle effect of X is a Gl block. Depending on the cell type and its differentiation, X can also induce apoptosis. 2. These effects do not depend on but are patentiated by ~53. p21 and p27 are effectors of the p53-mediated cell cycle effects of X. 3. X induced Gl arrest and apoptosis do not depend on Xlproteasome interactions. We propose that a main effect of X accumulation might actually be to halt liver cells progressloo and maintain their differentiated status by both ~53. dependent and -independent mechanisms; cell proliferation and transformation would then sensitize these cells to apoptosis.
APOPTOSIS
CON A INDUCED LIVER INJURY: A NEW MODEL TO STUDY HEPATOCYTE PROLIFERATION C Trautwein_lAkemann. NP Malek. J PlOmpe. G Tieas’. MP __-Manns Gastroenterology and Hepatology, Medical Hochschule Hannover. Pharmacology and Toxicology, University of Erlangen, FRG’. Concanavalin A (Con A) injection into mice leads to immune mediated liver injury. After moderate Con A doses liver function is completely reconstituted. Here we studied whether hepatoycte proliferation does occur and investigated TNF- and lL6-dependent pathways which might be involved in this process. 2 hours after Con A injection maximal TNFn serum levels were found, while maximal IL6 concentrations were measured after 4 to 6 hours. The rise in aminotransferases and DNA fragmentation started at the 4 hours time points and maximal levels were evident after 8 hours. BrdU staining and nuclear Cyclin A as markers of DNA synthesis were first detected in the nucleus of hepatocytes after 24 hours and were most prominent after 48 hours. An increase in the TNF-dependenl nuclear expression of C/EBP(IILAP was detected after 1 hour while an increase in RNA expression was evident only after 4 hours. C/EBP[j/LAP expression returned to normal values belore progression into S-phase. The changes in C/EBPP/LAP regulation could be inhibited by anti-TNF blocking experiments. The activation of IL6 dependent intracellular pathways was studied through Stat3. Nuclear expression and DNA binding of STAT3 to its target sequence increased for up to 8 hours. Besides STAT3 also STAT1 binds to the sarne DNA sequence as found by supershift experiments. In Ihe time course gel shift experiments, DNA-binding of the apoptosis-related STAT1 started earlier than DNA-binding of STAT3 which regulates hepalocyte proliferation. However, the subsequent decrease in DNA-binding of both factors was comparable. Our study shows that Con A induced liver injury induces hepatocyte proliferation. TNF and IL6 dependent signals trigger nuclear events which regulate proliferation. Thus Con A induced liver injury - closely resembling liver injury in human diseases like autoimmune and viral hepatitis _ can also be used as a new model to study liver regeneration.
EXPRESSIONOF A TRUNCATED gCATENIN mRNA IN RUMAN LIVER CANCER CELLS, A POSSIRLE MARKER OF NEOPLASIA
‘Istituto di Biolo&
ddl oncologia_ l_Jnivcr& di PaicmKI, Italy; ncattcdm universita di Palctmo, Italy.
di Mcdieina Intern&
The B-catti protein is involved both in controlling cell adhesion and in signaling patways. In normal cells &catenin level is maintencd low by rapid degrad&n of the f& molecules. Deletions in menin gene, resulting in cytopl6unic stab&4tion of the mutant B-catcniQ have been recently rcportcdinmdanomaandingastriccarcinomaccllhncs.In~c carcinomacdlsthe~ofatruncatedB~~rnduaionof ceucelladhesivencapcontn’butingtothercquisitonofiIlvasivepotentid. Wherras,itlItl&ttotllaC&St&ili&~QlirtaSSO&tCdwiththCL&l transcription faaor in a complex capable of activating gene(s) involved io the control of cell growth. We have rsceatly shown that the human hepIuocareinomacelllineHepG2qresscshighlevelofa~cdj3eateninproteinofabwt75LDa,tog*hawithesmall~ofthewild type 92 kDe protein (1). This result rises the possii that tnmeation reaeculedeletioninthe&catarinmRNAIntbisshrdy,we~theBCatcninmRNAbythe~ tmnseriptasc PCR method. Usiq speci6c primcrscov>heallmolccukofthe~-cateninmRNAwcfoundthttt primers located ill the most 5’ rqion of the molecule tlmpiisied two products,oneofacpectcdBize~mothaabwt3oObpsmrlla.Authe remai&gpfutofthcmolcculerc&tednormal.Ourre8ultssbowfbrtbe 6rsttimetltatinhumanlivercanccreeUsemolec&f~shnilarto that described for @ric-.&irc&na and nn&nomacdls,euuldcontriie to loss of cell growth control during bmmr+c&. 1. Montaitoetal.,He@o1ogy(1997),26(4)pt2:5641\ ThisahldywassuppoltcdinpartbytbcAlRc.
CARCINOMA,
LIVER REGENERATION,
LARGE SCALE SCREENING OF HBV-DNA INTEGRATION SITES INTO HUMAN LIVER TUMOR DNA: EVIDENCE FOR SERCA 1 HBV MEDIATED CISACTIVATION. 2
G. G-Y 4. J. Wm
2
B. CAURIQiY_ 1
3
PATERLINI~.IINsERM u370,
‘Genethon and 3Pasteur Institute, Paris, France.4Second Department of Surgery, Chiba University School of Medicine, Chiba 260, Japan. ‘University of Witwatersrand, Johannesbourg, South Africa. The impact of cis activation on HBV related liver cancers is a controversial issue. According to the current view, it would be a rare event in humans, in contrast with WHV related liver tumors in woodchucks. We have therefore applied a PCR based large survey approach to explore this field. Methods: we have screened 42 HCCs from different geographical areas by using an original HBV-Alu PCR method(l) to amplify viral/celJular DNA junctions (VCJ). Amplified DNA was analysed by sequencing, data bank analysis, irradiation hybrid test, RT-PCR. &&@: with X specific primers we have isolated and sequenced 16 VCJ. Chromosomal localization through irradiation hybrids analysis has been obtained in 7 and is ongoing in 9 Data bank analysis of a group of 5 VCJ has shown, in one tumor, HBVDNA integration into the 3rd exon of SERCAl gene. An hybrid viral/SERCAl RNA was detected in the tumorous tissue. Cloning and sequencing analyses showed that HBV integration takes place “in frame” with the SERCA 1 gene and potentially leads to a predicted hybrid HBV/SERCAI protein. SERCA 1 RNA is not detectable in the corresponding non tumorous tissue, normal liver and HepG2 cells. SERCA proteins pump Ca2+ from cytosol to endoplasmic reticulum; they are likely to be involved in cell growth through regulation of intracellular calcium concentrations. Our work shows that 1) cis activation is not a rare event in HBV related liver carcinogenesis 2) analysis of HBV integration sites can be used to discover new genes potentially involved in cell proliferation. Minami, et al. Genomics 29: 403-408, 1995.
MOLECULAR
BASES OF X INDUCED
CELL CYCLE ARREST AND APOPTOSIS C Brdcbpt ._ INSERM U370, Institut Necker, Paris, France.
It is debated whether X might induce or not cell apoptosis and which are the mechanisms involved. It is also unclear what would be the actual impact for the viral cycle. We have adressed this issue by using transient transfections, to avoid phenotypic selection of stable clones, in two cellular cell types’ transformed human Chang cells and immortalised, TGF alpha expressing, mouse aML hepatocytes. Results: I X induces both Gl arre~r anA apoptosis: In both Chang and aML cells, X expression inhibited colony outgrowth formation. Detailed analysis in Chang cells showed a complete Cl cell cycle arrest. In Chang and, to a much lower rate, in aML cells, X expression induced in a second step apoptosis, shown by morphological analysis, FACS, and DNA laddering. Gl arrest and apoptosis were not dependent on p53 status and Xlproteasome interaction since no endogenous ~53 could be detected even after treatment with the proteasome inhibitor ALLN, and cell cycle effects were not abolished. 2. ~53 und X show synergistic effects: Ectopic expression of ~53 in Chang cells induced Cl arrest and apoptosis to the same extent as X, leading to p21 and p27 CKI accumulation Cotnnsfection of X and ~53 led to a 2-3 fold increase in p21 and p27 and mdm2 promoter transcriptional activation; ~53. induced apoptosis and cell cycle arrest were amplified to the same extent. X induced an increase in p53 level which was not due to its stabilisation, as shown by metabolic labelling. Conclusions: 1. The major cell cycle effect of X is a Gl block. Depending on the cell type and its differentiation, X can also induce apoptosis. 2. These effects do not depend on but are patentiated by ~53. p21 and p27 are effectors of the p53-mediated cell cycle effects of X. 3. X induced Gl arrest and apoptosis do not depend on Xlproteasome interactions. We propose that a main effect of X accumulation might actually be to halt liver cells progressloo and maintain their differentiated status by both ~53. dependent and -independent mechanisms; cell proliferation and transformation would then sensitize these cells to apoptosis.
APOPTOSIS
CON A INDUCED LIVER INJURY: A NEW MODEL TO STUDY HEPATOCYTE PROLIFERATION C Trautwein_lAkemann. NP Malek. J PlOmpe. G Tieas’. MP __-Manns Gastroenterology and Hepatology, Medical Hochschule Hannover. Pharmacology and Toxicology, University of Erlangen, FRG’. Concanavalin A (Con A) injection into mice leads to immune mediated liver injury. After moderate Con A doses liver function is completely reconstituted. Here we studied whether hepatoycte proliferation does occur and investigated TNF- and lL6-dependent pathways which might be involved in this process. 2 hours after Con A injection maximal TNFn serum levels were found, while maximal IL6 concentrations were measured after 4 to 6 hours. The rise in aminotransferases and DNA fragmentation started at the 4 hours time points and maximal levels were evident after 8 hours. BrdU staining and nuclear Cyclin A as markers of DNA synthesis were first detected in the nucleus of hepatocytes after 24 hours and were most prominent after 48 hours. An increase in the TNF-dependenl nuclear expression of C/EBP(IILAP was detected after 1 hour while an increase in RNA expression was evident only after 4 hours. C/EBP[j/LAP expression returned to normal values belore progression into S-phase. The changes in C/EBPP/LAP regulation could be inhibited by anti-TNF blocking experiments. The activation of IL6 dependent intracellular pathways was studied through Stat3. Nuclear expression and DNA binding of STAT3 to its target sequence increased for up to 8 hours. Besides STAT3 also STAT1 binds to the sarne DNA sequence as found by supershift experiments. In Ihe time course gel shift experiments, DNA-binding of the apoptosis-related STAT1 started earlier than DNA-binding of STAT3 which regulates hepalocyte proliferation. However, the subsequent decrease in DNA-binding of both factors was comparable. Our study shows that Con A induced liver injury induces hepatocyte proliferation. TNF and IL6 dependent signals trigger nuclear events which regulate proliferation. Thus Con A induced liver injury - closely resembling liver injury in human diseases like autoimmune and viral hepatitis _ can also be used as a new model to study liver regeneration.
EXPRESSIONOF A TRUNCATED gCATENIN mRNA IN RUMAN LIVER CANCER CELLS, A POSSIRLE MARKER OF NEOPLASIA
‘Istituto di Biolo&
ddl oncologia_ l_Jnivcr& di PaicmKI, Italy; ncattcdm universita di Palctmo, Italy.
di Mcdieina Intern&
The B-catti protein is involved both in controlling cell adhesion and in signaling patways. In normal cells &catenin level is maintencd low by rapid degrad&n of the f& molecules. Deletions in menin gene, resulting in cytopl6unic stab&4tion of the mutant B-catcniQ have been recently rcportcdinmdanomaandingastriccarcinomaccllhncs.In~c carcinomacdlsthe~ofatruncatedB~~rnduaionof ceucelladhesivencapcontn’butingtothercquisitonofiIlvasivepotentid. Wherras,itlItl&ttotllaC&St&ili&~QlirtaSSO&tCdwiththCL&l transcription faaor in a complex capable of activating gene(s) involved io the control of cell growth. We have rsceatly shown that the human hepIuocareinomacelllineHepG2qresscshighlevelofa~cdj3eateninproteinofabwt75LDa,tog*hawithesmall~ofthewild type 92 kDe protein (1). This result rises the possii that tnmeation reaeculedeletioninthe&catarinmRNAIntbisshrdy,we~theBCatcninmRNAbythe~ tmnseriptasc PCR method. Usiq speci6c primcrscov>heallmolccukofthe~-cateninmRNAwcfoundthttt primers located ill the most 5’ rqion of the molecule tlmpiisied two products,oneofacpectcdBize~mothaabwt3oObpsmrlla.Authe remai&gpfutofthcmolcculerc&tednormal.Ourre8ultssbowfbrtbe 6rsttimetltatinhumanlivercanccreeUsemolec&f~shnilarto that described for @ric-.&irc&na and nn&nomacdls,euuldcontriie to loss of cell growth control during bmmr+c&. 1. Montaitoetal.,He@o1ogy(1997),26(4)pt2:5641\ ThisahldywassuppoltcdinpartbytbcAlRc.