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Metabolic basis of bile acid-induced changes in DNA synthesis during liver regeneration
$83
Hepatocellular Carcinoma
P2C9/226]
I P2 c91224 J V A R I A N T LIVER ESTROGEN RECEPTOR (ER) IN PATIENTS WITH H E P A T O C E L L U L A R CARCINOMA (HCC) M A Y CAUSE UNRESPONSIVENESS TO ANT[ESTROGENS
TRANSFORMING GROWTH FACTOR BETA1 (TGF-BETAI) IN HUMAN HEPATOCELLULAR CARCINOMA (HCC). COMPARISON WITH CIRRHOTIC AND NORMAL LIVER. L
L.Carneilini, A.Cn'ottola~ P.Buttafoco,A.DuFani I.Ferretti, F.Manen~. EVilla. Chair o f Gastroenterology, University o f Modena, Italy. Increased levels o f liver ERs have been bflen demonstrated in HCC; therefore anti-estrogens have been used as adjuvants in advanced HCC. However, this palliation therapy has been unsatisfactory.As it has been shown in mammary cancers unresponsive to tamoxifen, ERs may have abnormal hormone-binding domains as a result o f an alternatively spliced transcript; we therefore investigated the neoplastic (T) and non-neoplastic (NT) liver tissue o f 4 pts with HCC, compared with normal and metastatic liver tissue (from colonic cancers), for the presence o f normal and variant ER transcript. RNA, extracted from liver, was reverse transcribed and amplified by PCR.using primers localized in exon 4 and 6. The normal transcript gives rise to an amplified segment o f 437 bp while the eDNA o f the variant E R transcript is about 142 bp shorter, Functional and quan'titative charaote~-ization o f ER were investigated by competitive binding o f 3H-moxestrol o f ER purified from liver ~/tosol. All 4 patients with HCC had normal E R transcripts in the N T tissue while 3 out o f 4 had abnormal ER transcript in the T tissue; o f these 3 patients, t w o had a mixture o f wild-type and abnormal E R transcript while o n e h a d only the variant form. The deleted ER transcript was not detected in control liver tissue nor in metatastic liver tissue. The demonstration o f a variant ER wanscript (therefore o f an E R mutated in the h0rmone-binding domain) in the tumour but not in the non-neoplastic liver tissue o f patients with HCC may constitute the molecular basis for the unresponsiveness o f HCC patients to antiestrogen therapy~ . (supported by grant 60%, 1993)
P2 C9/225 ] METABOLIC BASIS OF BILE ACID-INDUCED CHANGES DNA SYNTHESIS DURING LIVER REGENERATION
Bolx r J
Bruixf
A
Castells,
M
So16 t F Rivera r J
Rod6s. Liver Unit and Pathology and Hormonal Depts. Hospital Cl~nic. University of Barcelona. TGF-betal has not only fibrogenic activity, but may also exert an antiproliferative effect, which can modulate the growth and dissemination of HCC. This study was aimed to assess TGF betal mRNA expression in human small HCC (mean size 28mm) and in the surrounding non-tumoral cirrhotic liver using samples from 27 HCC patients submitted to surgery. mRNA expression was investigated by Northern Blot and its intensity was determined by densitometry. Results were compared against the mRNA expression £n normal healthy liver, mRNA for TGF-betal was increased in 14 of the 27 HCC samples, reaching a >2 fold increase in 3 of them. This was not related to the tumor data (size, capsule, differentiation degree) or to the etiology and TGF-betal mRNA expression of the underlying liver. Thereby, 17 cirrhotic samples showed increased expression, but only 1 of them reached a >2 fold value when compared vs normal liver. To correlate the increased mRNA expression with the presence of TGF-betal protein and to identify the actively producing cells, we performed immuno-histochemical stainings using antiTGF-betal monoclonal antibody. However, while cirrhotic samples showed non-specific staining in the fibrous tissue, HCC samples did not stain for the protein. These results indicate t h a t small HCC express mRNA for TGF-betal but at very low levels and that this feature is not related to a definite characteristic of the tumor or of the underlying cirrhotic liver. The negative results of the in~nunohistochemical stainings suggest that the translation of TGF-betal mRNA may be inhibited at a posttranscriptional level.
P2 C9/227 1 IN
Vi[lanueva, GR, Barbero ER, Monte MJ, Ssrrano MA, Marin JJG. Department of Physiology and Pharmacology. University of Salamanca. Spain. Previous studies on both mice "in vivo" and isolated regenerating rat livers (RRLs), have shown that the active DNA synthesis occurring during liver regeneration that follows partial (2/3) hepatectomy (PH) is inhibited by bile acids. Further investigation on the role of the metabolic pathways in taurocholate (TC)-induced changes in the replicative process has been performed. Using radiolabeled substrates, enzyme activity determinations were carried out in soluble liver extracts. Activities were calculated from the rate of reaction product appearance or substrate disappearance, as measured by scintillation counting of collected samples obtained from H.P.L.C. or separated from the reaction mixture by DEAr-cellulose disks. The presence of TC (0, 0.5, 5 or 10 mM) had no significant effect on the key enzymes for thymidine catabolism (dihydropyrimidine dehydrogenase) or for deoxynucleotide production by the "de novo" pathway (ribonucleotids reductase, RNR). By contrast, thymine production from thymidine (carried out by three different enzyme activities) was significantly (-45%) inhibited by TC (10 mM). Moreover, thymidine kinase (TK) activity, which is responsible for the salvage nucleotids pathway, was greatly increased (>20-fold) in RRLs 24 h after partial hepatectomy as compared with control rats. This activity was also significantly reduced (-20%) in the presence of TC (10 raM). To investigate the importance of TK inhibition on TC-sensitive DNA synthesis, experiments were performed on perfused RRLs. In order to achieve accumulation of ribonucleotide precursors for the "de novo" pathway, liver donors received the RNR inhibitor hydroxyurea (HU;. bolus: 1.69 ~mol/g b.w. plus infusion: 1.25 p.mol/h/g b.w.) from 14 h to 24 h after PH. After interruption of HU administration, DNA synthesis was rapidly increased, peaked (=5-fold) at 4 h, and decreased subsequently. DNA synthesis by isolated RRLs obtained from rats untreated with HU was significantly reduced (-40%) by portal TC infusion (25 nmoVmin/g liver).This effect was abolished by the RNR inhibition/release manoeuvre. Glycocholate and cholic acid were also found to have no effect under these conditions. In summary, these results indicate that the inhibition of enzyme activities involved in thymidine metabolism to TMP or to thymine may be affected by TC. They also suggest that bile acid-induced DNA synthesis inhibition is probably due, in part, to an impairment in the flux of DNA precursors through the nucleotide salvage pathway.
FIBROLAMELLAR CARCINOMA OF THE LIVER (FLC): COMPOSITION OF THE EXTRACELLULAR MATRIX (ECM) AND EXPRESSION OF CELL ADHESION MOLECULES (CAM). J-Y Scoazec. J-F Fleiou. A D'Errico. I Kozwaki. M Florentine. A-F Brinauier. W GH~ieni. A-M MancinL G Feldmarm. INSERM U327, Paris, France; H6pitaI Beanjun, Clichy, France; Policlinico S. Orsola, Bologna, Italy. Pibrolamellar carcinoma of the liver is a distinct clinico-pathological entity. It arises in young patients, without underlying chronic liver disease, and presents a distinctive histological picture. The prognosis of FI..C is better than that of the other types of primary liver carcinoma, since most tumors can be resected at diagnosis. The histogenesis of FLC remains debated. FLC has been considered as a highly differentiated variant of hepatocellular carcinoma or as a tumor of neuroendocrine derivation. To ftuher address this question, we analyzed: (a) the composition of the highly distinctive sU'oma of FLC, and (b) the expression of CAM by neoplastic cells. In 7 cases of FLC, presenting with a typical and homogeneous histological picture all over the tumor, we studied by immunohistochemJstry and Western blot (WB) the expression of: (a) the main ECM components, Co) the following cell-cell adhesion molecules: E-cadherin and GC4-reactive cadherin, beth detected on normal hepatocytes, N-cadherin and NCAM, present on neuroendocrine cells; (c) the integrin chains of the [~1 and ~J4 subfamilies. The ECM of FLC was characterized by the presence of high amounts of tenascin and the absence of detectable basement membranes around clusters of neoplastic cells. Neoplastic cells constantly expressed very low levels of Ecadherin. They expressed detectable levels of the GC4-reactive cadherin in only 3 cases. They were constantly negative for N-cadherin and NCAM. Neoplastic cells expressed the all31 integrin, characteristic of normal hepatocytes and absent from most other epithelial cells. As previously reported for other types of hepatocellular carcinomas, no expression of [~4 integrin was detected in any case. The v~rBpatterns of tenascin and cedherh= detected in FI..C were different from those observed in the normal liver tissue and varied from case to case, revealing an unexpected level of phenotypic heterogeneity in FLC. In conclusion, the pattern of cell adhesion molecules expressed by neoplastic cells in FLC is consistent with a hepatocyta derivation and lends no support for a neuroendocrine differentiation. Unexpectedly, FLC is characterized by some features usually associated with tumors of high grade of malignancy ( p r i c e of high levels o,f tanascin, absence of basement membrane, altered cadherin expression). This is in apparent contrast to the usually good prognosis of FLC, which is likely to result from the favorable clinical context in which this distinct variant of hepatocallular carcinoma occurs.
Hepatocellular Carcinoma
P2C9/226]
I P2 c91224 J V A R I A N T LIVER ESTROGEN RECEPTOR (ER) IN PATIENTS WITH H E P A T O C E L L U L A R CARCINOMA (HCC) M A Y CAUSE UNRESPONSIVENESS TO ANT[ESTROGENS
TRANSFORMING GROWTH FACTOR BETA1 (TGF-BETAI) IN HUMAN HEPATOCELLULAR CARCINOMA (HCC). COMPARISON WITH CIRRHOTIC AND NORMAL LIVER. L
L.Carneilini, A.Cn'ottola~ P.Buttafoco,A.DuFani I.Ferretti, F.Manen~. EVilla. Chair o f Gastroenterology, University o f Modena, Italy. Increased levels o f liver ERs have been bflen demonstrated in HCC; therefore anti-estrogens have been used as adjuvants in advanced HCC. However, this palliation therapy has been unsatisfactory.As it has been shown in mammary cancers unresponsive to tamoxifen, ERs may have abnormal hormone-binding domains as a result o f an alternatively spliced transcript; we therefore investigated the neoplastic (T) and non-neoplastic (NT) liver tissue o f 4 pts with HCC, compared with normal and metastatic liver tissue (from colonic cancers), for the presence o f normal and variant ER transcript. RNA, extracted from liver, was reverse transcribed and amplified by PCR.using primers localized in exon 4 and 6. The normal transcript gives rise to an amplified segment o f 437 bp while the eDNA o f the variant E R transcript is about 142 bp shorter, Functional and quan'titative charaote~-ization o f ER were investigated by competitive binding o f 3H-moxestrol o f ER purified from liver ~/tosol. All 4 patients with HCC had normal E R transcripts in the N T tissue while 3 out o f 4 had abnormal ER transcript in the T tissue; o f these 3 patients, t w o had a mixture o f wild-type and abnormal E R transcript while o n e h a d only the variant form. The deleted ER transcript was not detected in control liver tissue nor in metatastic liver tissue. The demonstration o f a variant ER wanscript (therefore o f an E R mutated in the h0rmone-binding domain) in the tumour but not in the non-neoplastic liver tissue o f patients with HCC may constitute the molecular basis for the unresponsiveness o f HCC patients to antiestrogen therapy~ . (supported by grant 60%, 1993)
P2 C9/225 ] METABOLIC BASIS OF BILE ACID-INDUCED CHANGES DNA SYNTHESIS DURING LIVER REGENERATION
Bolx r J
Bruixf
A
Castells,
M
So16 t F Rivera r J
Rod6s. Liver Unit and Pathology and Hormonal Depts. Hospital Cl~nic. University of Barcelona. TGF-betal has not only fibrogenic activity, but may also exert an antiproliferative effect, which can modulate the growth and dissemination of HCC. This study was aimed to assess TGF betal mRNA expression in human small HCC (mean size 28mm) and in the surrounding non-tumoral cirrhotic liver using samples from 27 HCC patients submitted to surgery. mRNA expression was investigated by Northern Blot and its intensity was determined by densitometry. Results were compared against the mRNA expression £n normal healthy liver, mRNA for TGF-betal was increased in 14 of the 27 HCC samples, reaching a >2 fold increase in 3 of them. This was not related to the tumor data (size, capsule, differentiation degree) or to the etiology and TGF-betal mRNA expression of the underlying liver. Thereby, 17 cirrhotic samples showed increased expression, but only 1 of them reached a >2 fold value when compared vs normal liver. To correlate the increased mRNA expression with the presence of TGF-betal protein and to identify the actively producing cells, we performed immuno-histochemical stainings using antiTGF-betal monoclonal antibody. However, while cirrhotic samples showed non-specific staining in the fibrous tissue, HCC samples did not stain for the protein. These results indicate t h a t small HCC express mRNA for TGF-betal but at very low levels and that this feature is not related to a definite characteristic of the tumor or of the underlying cirrhotic liver. The negative results of the in~nunohistochemical stainings suggest that the translation of TGF-betal mRNA may be inhibited at a posttranscriptional level.
P2 C9/227 1 IN
Vi[lanueva, GR, Barbero ER, Monte MJ, Ssrrano MA, Marin JJG. Department of Physiology and Pharmacology. University of Salamanca. Spain. Previous studies on both mice "in vivo" and isolated regenerating rat livers (RRLs), have shown that the active DNA synthesis occurring during liver regeneration that follows partial (2/3) hepatectomy (PH) is inhibited by bile acids. Further investigation on the role of the metabolic pathways in taurocholate (TC)-induced changes in the replicative process has been performed. Using radiolabeled substrates, enzyme activity determinations were carried out in soluble liver extracts. Activities were calculated from the rate of reaction product appearance or substrate disappearance, as measured by scintillation counting of collected samples obtained from H.P.L.C. or separated from the reaction mixture by DEAr-cellulose disks. The presence of TC (0, 0.5, 5 or 10 mM) had no significant effect on the key enzymes for thymidine catabolism (dihydropyrimidine dehydrogenase) or for deoxynucleotide production by the "de novo" pathway (ribonucleotids reductase, RNR). By contrast, thymine production from thymidine (carried out by three different enzyme activities) was significantly (-45%) inhibited by TC (10 mM). Moreover, thymidine kinase (TK) activity, which is responsible for the salvage nucleotids pathway, was greatly increased (>20-fold) in RRLs 24 h after partial hepatectomy as compared with control rats. This activity was also significantly reduced (-20%) in the presence of TC (10 raM). To investigate the importance of TK inhibition on TC-sensitive DNA synthesis, experiments were performed on perfused RRLs. In order to achieve accumulation of ribonucleotide precursors for the "de novo" pathway, liver donors received the RNR inhibitor hydroxyurea (HU;. bolus: 1.69 ~mol/g b.w. plus infusion: 1.25 p.mol/h/g b.w.) from 14 h to 24 h after PH. After interruption of HU administration, DNA synthesis was rapidly increased, peaked (=5-fold) at 4 h, and decreased subsequently. DNA synthesis by isolated RRLs obtained from rats untreated with HU was significantly reduced (-40%) by portal TC infusion (25 nmoVmin/g liver).This effect was abolished by the RNR inhibition/release manoeuvre. Glycocholate and cholic acid were also found to have no effect under these conditions. In summary, these results indicate that the inhibition of enzyme activities involved in thymidine metabolism to TMP or to thymine may be affected by TC. They also suggest that bile acid-induced DNA synthesis inhibition is probably due, in part, to an impairment in the flux of DNA precursors through the nucleotide salvage pathway.
FIBROLAMELLAR CARCINOMA OF THE LIVER (FLC): COMPOSITION OF THE EXTRACELLULAR MATRIX (ECM) AND EXPRESSION OF CELL ADHESION MOLECULES (CAM). J-Y Scoazec. J-F Fleiou. A D'Errico. I Kozwaki. M Florentine. A-F Brinauier. W GH~ieni. A-M MancinL G Feldmarm. INSERM U327, Paris, France; H6pitaI Beanjun, Clichy, France; Policlinico S. Orsola, Bologna, Italy. Pibrolamellar carcinoma of the liver is a distinct clinico-pathological entity. It arises in young patients, without underlying chronic liver disease, and presents a distinctive histological picture. The prognosis of FI..C is better than that of the other types of primary liver carcinoma, since most tumors can be resected at diagnosis. The histogenesis of FLC remains debated. FLC has been considered as a highly differentiated variant of hepatocellular carcinoma or as a tumor of neuroendocrine derivation. To ftuher address this question, we analyzed: (a) the composition of the highly distinctive sU'oma of FLC, and (b) the expression of CAM by neoplastic cells. In 7 cases of FLC, presenting with a typical and homogeneous histological picture all over the tumor, we studied by immunohistochemJstry and Western blot (WB) the expression of: (a) the main ECM components, Co) the following cell-cell adhesion molecules: E-cadherin and GC4-reactive cadherin, beth detected on normal hepatocytes, N-cadherin and NCAM, present on neuroendocrine cells; (c) the integrin chains of the [~1 and ~J4 subfamilies. The ECM of FLC was characterized by the presence of high amounts of tenascin and the absence of detectable basement membranes around clusters of neoplastic cells. Neoplastic cells constantly expressed very low levels of Ecadherin. They expressed detectable levels of the GC4-reactive cadherin in only 3 cases. They were constantly negative for N-cadherin and NCAM. Neoplastic cells expressed the all31 integrin, characteristic of normal hepatocytes and absent from most other epithelial cells. As previously reported for other types of hepatocellular carcinomas, no expression of [~4 integrin was detected in any case. The v~rBpatterns of tenascin and cedherh= detected in FI..C were different from those observed in the normal liver tissue and varied from case to case, revealing an unexpected level of phenotypic heterogeneity in FLC. In conclusion, the pattern of cell adhesion molecules expressed by neoplastic cells in FLC is consistent with a hepatocyta derivation and lends no support for a neuroendocrine differentiation. Unexpectedly, FLC is characterized by some features usually associated with tumors of high grade of malignancy ( p r i c e of high levels o,f tanascin, absence of basement membrane, altered cadherin expression). This is in apparent contrast to the usually good prognosis of FLC, which is likely to result from the favorable clinical context in which this distinct variant of hepatocallular carcinoma occurs.