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Mitochondrial benzodiazepine receptor system in hepatocellular carcinoma: a possible mechanism of resistance to death during cell proliferation
POStt7.S
88
1 P/CO4/005
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HEPATOCYTE GROWTH FACTOR (HGF) STIMULATES PROLIFERATION OF HEPGZ CELLS: ROLE OF THE TRANSCRITION COMPLEX. N. Malekkiani. A. Brinpuier, D Bemuau. Medecine Xavier Bichat, Paris, France.
JNSERM
U327,
THE AP-1
Faculte
FAS AND FAS-LIGAND EXPRESSION CARCINOMA AND ADJACENT LIVER
IN HBPATGCELLULAR PARENCHYMA: WES
CANCER INDUCE SUICIDE IN THE PERITUMORAL CELLS? de
While the proliferative effect of HGF on normal hepatocytes is well established. its role on the proliferation of transformed cell is less clear. The aims of our study were 1) to clarify the effect of HGF on the proliferation of human transformed hepatocytes, the HepG2 cell line 2) to determine whether the AP- I complex is induced in response to this growth factor, and 3) to investigate whether AP-I induction exerts an effect on the proliferative response. Cells were plated at Ix10’/cm2 and treated with long/ml HGF. The proliferative response was evaluated by measuring the incorporation of ‘HThymidine into DNA, and by appreciating the number of cells by the crystal violet technique. A 2-fold increase of the incorporation of ‘H-Thymidine and a I.7-fold increase of the cell number were observed 48h and 72h, respectively, after HGF treatment. Electrophoretic mobility shift assay with oligonucleotide probes containg either a consensus AP-I site or a CRE site (another AP-I binding site), showed increased binding of the nuclear proteins to the AP-I and CRE sites at 2h and 4h, respectively, after HGF treatment. The binding activity to both probes remained elevated at 24h. Supershift analysis identified ‘JunD and Fral proteins in the AP-l-binding complexes, while only JunD was detected in the CRE-binding dimers. We next compared the functional consequences of increased DNA binding by transfecting cells with TRE-tK-CAT or CRE-tK-LUC plasmids. While no increase in CAT activity was detected after HGF treatment of TRE-tK-CATtransfected cells, a 1.7- fold increase of LUC activity was observed in CREtK-LUC-transfected cells. Finally co-transfection of cells with a CRE-tKLUC and TAM 67 (a strong functional AP-I inhibitor), drastically decreased both basal and HGF-stimulated LUC expressions. These results demonstrate that HGF activates the proliferation of HepG2 cells. This effect might result from stimulation of the AP-1 complex, which is able to induce CREdependent genes.
T. Van Q~~JIQLLB.. Laboratory for Histo-and Cytochemisuy, University of Leuven, Leuven, Belgium Fas (Apo-1, CD95) mediates apoptosis in response to agonistic antibodies or Fas-ligaud (Fas-L) binding. Fas is a member of the nerve growth factor (NGF)/tumour necrosis factor (TNF) receptor superfamily, which is involved in cell growth signaling. Since some of these receprors (NGFB, TNFR, CD40) have opposing functions, namely both apoptosis and pmliferation, we investigated expression of Fas, Fas-L and proliferation marker Ki67, using specific monoclonal antibodies on frozen tissue. 25 hepatocellular carcinomas (HCC) and the adjacent parenchyma, and 5 normal livers were studied. In normal livers, Fas was faintly expressed on membranes of hepatocytes and bile duct cells. Fas-L surprisingly showed granular immunoreactivity in the cytoplasm of occasional hepatocytes. In the hepatocytes immediately ti@mt to HCC, a very strong co-expression of Fas and Fas-L was a constant finding: hepatocytes showed a strong membrane immunoreactivity for Fas, while the same cells showed strong granular cytoplasmic positivity for Fas-L. In contrast, within the tumours, Fas expression did not correlate with Fas-L expression. Fas-L immunoreactivity was weakly present in a minority of cells. Fas expression varied from almost negative to a strong honey-comb pattern of immunoreactivity. Tumours with a high proliferation index had a stronger Fas expression than tumours with a lower proliferation index. Double immune-staining revealed coexpression of Fas and Ki67 in tumour cells. In conclusion, hepatocytes adjacent to HCC show high co-expression of Fas and Fas-L. suggesting their ability to induce apoptosis in an auto- or pdlacrine way. In contrast, within HCC, Fas-L immunoreactivity is focal and weak and is discrepant from Fas immunoreactivity, suggesting only minor chance of Fas-Fas-L mediated apoptosis. Within HCC, Fas shows a striking relationship with the proliferation index, suggesting that Fas-mediated signaling may be involved in proliferation as well as induction of apoptosis. Fas-L expression in peritumoral hepatocytes might result in a paracrine way from cytokine(s) produced by tumor cells, in analogy with paracrine TGF-B-mediated peritumoral apoptosis (Gressner AM et al. J Hepatol 1997;26; 1079-1092). ROSkams
1 P/C&l/O06] POLYMORPHIC
GENOMIC
FINGERPRINTING
OF SYNCHRONOUS
HEPATOCELLULAR CANCERS IDENTIFIED BY ARBITRARILY PRIMED-POLYMERASE CHAIN REACTION (AP-PCR) Y Sirtvatanauksorn’ ‘. V Strivatanauksorn’. S Bhattacharva’. K Savage’. J Rees’. AP Dhillon’. NR Lcmomc’. RCN W~ll~an~son’, BR Da\ idson’ Dept of Surgery’ & Htstopathology’. Royal Free Hospital School of Medtcine. Dept of Surgery’ & ICRF Oncolog! Unit’. Han~mcrsmnh Hospnal. London. UK Hepatoccllular Carctnoma multifocal It has usually
(HCC) tn cirrhotic been postulated that
ltvcr arc the smaller
frcqucntlv ..satellitc”
tumour nodules represent intrahepatic metastases from the larger “primal” Icsionts). but no direct contirmntion of this concept has prcv tousl! hccn cstabltshcd WC proposed lo assess the dcgrcc of gcnomic hctcrogcncit! tn such Icstons. using AP-PCR. This is a new but wcllvahdated technique that has not been previously used to assess genomtc relauonship bclwecn synchronous HCC nodules. Fifty parafftn-zmbeddcd HCC spccmiens wcrc sampled from I3 cirrhotic csplant livers rcmoccd at orthotop~c liver transplantatton. Using microdissection technique. tumour ttssues were selected and phenol extraction of genomtc DNA was performed. Two dtfferent arbitrary primers were utilised to amplify the genomic DNA of each sample and run on polyacrylamide gel cleclrophorcsis WC gcncrntcd spcctfic and reproducible gcnomic DNA fmgcrprinttngs. w htch wcrc h~rghly polymorphic bctwccn drffcrcnt nodnlcs Marked genomic hclcrogcncuy was noted amongst the nodules studied There are not any nodules had identical electrophorettc patterns. In addition. cv’cn small sntcllitc nodules surrounding large primary tumour were found to have dtstinct genonuc patterns. AP-PCR IS a highly spccttic technique and effctive approach for analysmg the fractton of the genome with ampliticatton m multiple HCC nodules. Highly polymorphtc genomic tingerprmting in synchronous HCCs has implications for therapy. Lesions with diffcrcnt gcnomic composilions may respond diffcrcntly to cytotosic agents or growth/angiogenests-inhibitors and the current concepts regarding the desirable c~tcm of rcscction may require evaluation.
MITOCHONDRIAL BENZODIAZEPINE RECEPTOR SYSTEM IN HEPATOCELLULAR CARCINOMA: A POSSIBLE MECHANISM OF RESISTANCE TO DEATH DURING CELL PROLIFERATION. I. V n urini* Arrieo”. j$&@. *Cattedra di Semeiotica e Metodologia Medica, “Dip. Scienze Farmaceutiche, Universita di Modena; “Dip.Trapianti d’organo, Universita di Geneva; “Dip. di Neurologia, Universita di Milan0 (Monza); Italy. Mitochondrial benzodiazepine receptors (MBRs) trigger intracellular metabolic events and has been associated with cell proliferation. Their endogenous ligands are the diazepam binding inhibitor (DBI), which contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. Cholesterol is essential for biosynthetic processes and for steroid synthesis. Protoporphyrin IX (PPIX), which regulates heme synthesis and protects mitochondria against oxygen radical damages is viewed as a ligand for MBRs. The densities of MBRs and the levels of DBI and PPIX were tested in tumoral and non-tumoral liver tissue (NTLT) of patients with hepatocellular carcinoma (HCC, N. 10). Moreover the concentrations of cholesterol (C) and of DBI were assayed in the plasma of patients with HCC and compared with those of uncomplicated liver cirrhosis. The characteristics of MBRs were studied on mitochondrial preparations by binding assay using [‘H]PK I 1195. PPIX was assayed by HPLC. DBI was assayed by RIA. The densities of MBRs increased 4 to 7 fold in HCC in comparison to NTLT (p < 0.0001). PPIX and DBI-LI in HCC tissues were significantly lower than in NTLT (p < 0.05 and p < 0.01, respectively). In serum C and DBI were higher in HCC (N. 23) when compared with cirrhotic patients (N. 73). These data demonstrated that in HCC patients there is an increased availability of the substrate for steroid synthesis, associated with an increased functional status of MBRs in HCC tissue. This suggest that in HCC tissue there is an increased steroidogenesis and a resistance of tumoral cells against radical damage.
88
1 P/CO4/005
pii%zzq
]
HEPATOCYTE GROWTH FACTOR (HGF) STIMULATES PROLIFERATION OF HEPGZ CELLS: ROLE OF THE TRANSCRITION COMPLEX. N. Malekkiani. A. Brinpuier, D Bemuau. Medecine Xavier Bichat, Paris, France.
JNSERM
U327,
THE AP-1
Faculte
FAS AND FAS-LIGAND EXPRESSION CARCINOMA AND ADJACENT LIVER
IN HBPATGCELLULAR PARENCHYMA: WES
CANCER INDUCE SUICIDE IN THE PERITUMORAL CELLS? de
While the proliferative effect of HGF on normal hepatocytes is well established. its role on the proliferation of transformed cell is less clear. The aims of our study were 1) to clarify the effect of HGF on the proliferation of human transformed hepatocytes, the HepG2 cell line 2) to determine whether the AP- I complex is induced in response to this growth factor, and 3) to investigate whether AP-I induction exerts an effect on the proliferative response. Cells were plated at Ix10’/cm2 and treated with long/ml HGF. The proliferative response was evaluated by measuring the incorporation of ‘HThymidine into DNA, and by appreciating the number of cells by the crystal violet technique. A 2-fold increase of the incorporation of ‘H-Thymidine and a I.7-fold increase of the cell number were observed 48h and 72h, respectively, after HGF treatment. Electrophoretic mobility shift assay with oligonucleotide probes containg either a consensus AP-I site or a CRE site (another AP-I binding site), showed increased binding of the nuclear proteins to the AP-I and CRE sites at 2h and 4h, respectively, after HGF treatment. The binding activity to both probes remained elevated at 24h. Supershift analysis identified ‘JunD and Fral proteins in the AP-l-binding complexes, while only JunD was detected in the CRE-binding dimers. We next compared the functional consequences of increased DNA binding by transfecting cells with TRE-tK-CAT or CRE-tK-LUC plasmids. While no increase in CAT activity was detected after HGF treatment of TRE-tK-CATtransfected cells, a 1.7- fold increase of LUC activity was observed in CREtK-LUC-transfected cells. Finally co-transfection of cells with a CRE-tKLUC and TAM 67 (a strong functional AP-I inhibitor), drastically decreased both basal and HGF-stimulated LUC expressions. These results demonstrate that HGF activates the proliferation of HepG2 cells. This effect might result from stimulation of the AP-1 complex, which is able to induce CREdependent genes.
T. Van Q~~JIQLLB.. Laboratory for Histo-and Cytochemisuy, University of Leuven, Leuven, Belgium Fas (Apo-1, CD95) mediates apoptosis in response to agonistic antibodies or Fas-ligaud (Fas-L) binding. Fas is a member of the nerve growth factor (NGF)/tumour necrosis factor (TNF) receptor superfamily, which is involved in cell growth signaling. Since some of these receprors (NGFB, TNFR, CD40) have opposing functions, namely both apoptosis and pmliferation, we investigated expression of Fas, Fas-L and proliferation marker Ki67, using specific monoclonal antibodies on frozen tissue. 25 hepatocellular carcinomas (HCC) and the adjacent parenchyma, and 5 normal livers were studied. In normal livers, Fas was faintly expressed on membranes of hepatocytes and bile duct cells. Fas-L surprisingly showed granular immunoreactivity in the cytoplasm of occasional hepatocytes. In the hepatocytes immediately ti@mt to HCC, a very strong co-expression of Fas and Fas-L was a constant finding: hepatocytes showed a strong membrane immunoreactivity for Fas, while the same cells showed strong granular cytoplasmic positivity for Fas-L. In contrast, within the tumours, Fas expression did not correlate with Fas-L expression. Fas-L immunoreactivity was weakly present in a minority of cells. Fas expression varied from almost negative to a strong honey-comb pattern of immunoreactivity. Tumours with a high proliferation index had a stronger Fas expression than tumours with a lower proliferation index. Double immune-staining revealed coexpression of Fas and Ki67 in tumour cells. In conclusion, hepatocytes adjacent to HCC show high co-expression of Fas and Fas-L. suggesting their ability to induce apoptosis in an auto- or pdlacrine way. In contrast, within HCC, Fas-L immunoreactivity is focal and weak and is discrepant from Fas immunoreactivity, suggesting only minor chance of Fas-Fas-L mediated apoptosis. Within HCC, Fas shows a striking relationship with the proliferation index, suggesting that Fas-mediated signaling may be involved in proliferation as well as induction of apoptosis. Fas-L expression in peritumoral hepatocytes might result in a paracrine way from cytokine(s) produced by tumor cells, in analogy with paracrine TGF-B-mediated peritumoral apoptosis (Gressner AM et al. J Hepatol 1997;26; 1079-1092). ROSkams
1 P/C&l/O06] POLYMORPHIC
GENOMIC
FINGERPRINTING
OF SYNCHRONOUS
HEPATOCELLULAR CANCERS IDENTIFIED BY ARBITRARILY PRIMED-POLYMERASE CHAIN REACTION (AP-PCR) Y Sirtvatanauksorn’ ‘. V Strivatanauksorn’. S Bhattacharva’. K Savage’. J Rees’. AP Dhillon’. NR Lcmomc’. RCN W~ll~an~son’, BR Da\ idson’ Dept of Surgery’ & Htstopathology’. Royal Free Hospital School of Medtcine. Dept of Surgery’ & ICRF Oncolog! Unit’. Han~mcrsmnh Hospnal. London. UK Hepatoccllular Carctnoma multifocal It has usually
(HCC) tn cirrhotic been postulated that
ltvcr arc the smaller
frcqucntlv ..satellitc”
tumour nodules represent intrahepatic metastases from the larger “primal” Icsionts). but no direct contirmntion of this concept has prcv tousl! hccn cstabltshcd WC proposed lo assess the dcgrcc of gcnomic hctcrogcncit! tn such Icstons. using AP-PCR. This is a new but wcllvahdated technique that has not been previously used to assess genomtc relauonship bclwecn synchronous HCC nodules. Fifty parafftn-zmbeddcd HCC spccmiens wcrc sampled from I3 cirrhotic csplant livers rcmoccd at orthotop~c liver transplantatton. Using microdissection technique. tumour ttssues were selected and phenol extraction of genomtc DNA was performed. Two dtfferent arbitrary primers were utilised to amplify the genomic DNA of each sample and run on polyacrylamide gel cleclrophorcsis WC gcncrntcd spcctfic and reproducible gcnomic DNA fmgcrprinttngs. w htch wcrc h~rghly polymorphic bctwccn drffcrcnt nodnlcs Marked genomic hclcrogcncuy was noted amongst the nodules studied There are not any nodules had identical electrophorettc patterns. In addition. cv’cn small sntcllitc nodules surrounding large primary tumour were found to have dtstinct genonuc patterns. AP-PCR IS a highly spccttic technique and effctive approach for analysmg the fractton of the genome with ampliticatton m multiple HCC nodules. Highly polymorphtc genomic tingerprmting in synchronous HCCs has implications for therapy. Lesions with diffcrcnt gcnomic composilions may respond diffcrcntly to cytotosic agents or growth/angiogenests-inhibitors and the current concepts regarding the desirable c~tcm of rcscction may require evaluation.
MITOCHONDRIAL BENZODIAZEPINE RECEPTOR SYSTEM IN HEPATOCELLULAR CARCINOMA: A POSSIBLE MECHANISM OF RESISTANCE TO DEATH DURING CELL PROLIFERATION. I. V n urini* Arrieo”. j$&@. *Cattedra di Semeiotica e Metodologia Medica, “Dip. Scienze Farmaceutiche, Universita di Modena; “Dip.Trapianti d’organo, Universita di Geneva; “Dip. di Neurologia, Universita di Milan0 (Monza); Italy. Mitochondrial benzodiazepine receptors (MBRs) trigger intracellular metabolic events and has been associated with cell proliferation. Their endogenous ligands are the diazepam binding inhibitor (DBI), which contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. Cholesterol is essential for biosynthetic processes and for steroid synthesis. Protoporphyrin IX (PPIX), which regulates heme synthesis and protects mitochondria against oxygen radical damages is viewed as a ligand for MBRs. The densities of MBRs and the levels of DBI and PPIX were tested in tumoral and non-tumoral liver tissue (NTLT) of patients with hepatocellular carcinoma (HCC, N. 10). Moreover the concentrations of cholesterol (C) and of DBI were assayed in the plasma of patients with HCC and compared with those of uncomplicated liver cirrhosis. The characteristics of MBRs were studied on mitochondrial preparations by binding assay using [‘H]PK I 1195. PPIX was assayed by HPLC. DBI was assayed by RIA. The densities of MBRs increased 4 to 7 fold in HCC in comparison to NTLT (p < 0.0001). PPIX and DBI-LI in HCC tissues were significantly lower than in NTLT (p < 0.05 and p < 0.01, respectively). In serum C and DBI were higher in HCC (N. 23) when compared with cirrhotic patients (N. 73). These data demonstrated that in HCC patients there is an increased availability of the substrate for steroid synthesis, associated with an increased functional status of MBRs in HCC tissue. This suggest that in HCC tissue there is an increased steroidogenesis and a resistance of tumoral cells against radical damage.