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Resume La therapie cellulaire est une strategie therapeutique d'avenir pour le traitement des insuffisances hepatocellulaires et pour la correction de certains deficits enzymatiques congenitaux. Des etudes recentes montrent qu'il est possible de restaurer certaines ou la totalite des fonctions hepatiques chez l'animal apres injection d'hepatocytes dans la rate ou la veine porte. Un foie bioartificiel extracorporel hybride pourrait egalement suppleer transitoirement les fonctions hepatiques defaillantes dans les cas des insuffisances hepatocellulaires, maintenir les patients en survie et permettre une regeneration spontanee du foie natif, ou agir transitoirement en vue d'une transplantation hepatique orthotopique dans de bonnes conditions. Certaines difficultes d'utilisation de systemes de culture d'hepatocytes a plat, dans des microfibres ou sur des microporteurs nous ont conduit a developper un systeme d'immobilisation des hepatocytes dans des billes d'alginate. Ce systeme a ete utilise avec succes pour corriger le deficit de conjugaison de la bilirubine chez des rats Gunn. L'application d'un tel systeme chez l'homme requiert la preparation d'hepatocytes en grande quantite. Nous avons isole des hepatocytes de jeune porc par perfusion enzymatique du foie total, qui ont ete immobilises dans des billes d'alginate puis inocules dans un bioreacteur. Les hepatocytes porcins exprimaient des fonctions hepatospecifiques a un haut niveau, telles que celles impliquees dans la detoxication et la biotransformation, etaient immunoproteges et pouvaient etre cryocongeles. Ce systeme est prometteur pour la mise au point d'un systeme de suppleance hepatique transitoire chez l'homme.

Several novel differentiated cell lines have been derived from a human hepatocarcinoma named HBG. Analysis of their functional properties evidenced a gradual differentiation process as they became confluent and a remarkable stability of the whole quiescent population for at least 6 weeks. However, when replated at low density after several weeks of quiescence, the differentiated cells were able to rapidly reverse to active proliferation, accompanied by transient dedifferentiation. Demonstration that the differentiated hepatic cells were growth-arrested in G1 phase was provided by the increased number of cells with 2C DNA content and decreased expression of S-phase markers. Characteristic features of oncogenes and cell cycle genes were defined during the differentiation process: (a) a biphasic expression of c-myc, with the latter wave covering the quiescence period; (b) opposite kinetics of c-Ki-ras and of N-ras expression with a pattern of changes paralleling that of c-myc; and (c) a decrease of cyclin D1 protein expression and of the cyclin D1-associated kinase activity. The mechanisms by which quiescent differentiated cells might reinitiate active proliferation were analyzed by studying several genes involved in cell growth and death regulation. We found: (a) a point mutation and loss of the specific activity of the tumor suppressor gene p53 without alteration of the apoptotic response to transforming growth factor beta1; (b) a gradual decrease of retinoblastoma protein, which was constantly present, mainly in a hyperphosphorylated form; and (c) an increase of cyclin-dependent kinase inhibitor p27 expression in confluent differentiating cells, as expected, whereas, surprisingly, a disappearance of the p21 protein was observed in parallel. These data may reflect specific mechanisms of cell cycle regulation in liver parenchymal cells through which these cells can proceed to control their reversible differentiation program.

Liver regulating protein (LRP) is an integral plasma membrane protein that plays a critical role in maintaining the differentiated phenotype of adult rat hepatocytes by mediating cell-cell interactions with rat liver epithelial cells. Using a specific monoclonal antibody (MAb L8) capable of inhibiting the interactions between these two cell types, the cellular distribution of LRP was analyzed in the liver. Various cell types, including hepatocytes and several sinusoidal cells, were found to be positive, whereas vascular endothelial cells and bile duct cells were consistently negative. This observation led us to question whether cells of nonhepatic origin would also express LRP. We show that MAb L8 immunoreactive material was detected in only three groups of tissues and corresponded to molecules similar to LRP but with different molecular weights. LRP-like molecules were demonstrated on acinar cells of the exocrine pancreas and on all hemopoietic cells regardless of their localization in the organism. LRP-like molecules were also expressed by germ cells and surrounding feeder cells in the testis and ovary in a stage-dependent manner. These results demonstrate the existence of a family of LRP proteins and strongly suggest a critical role for these molecules in regulating cell-cell communication in specific tissues.

Laminin deposition is increased in fetal liver and in a variety of liver diseases, including the development of carcinoma. We investigated the role of the hepatocyte in the synthesis of both laminin and its 32- to 67-kd receptor in normal adult, fetal, and diethylnitrosamine-treated rat livers, and in adult rat hepatocyte primary cultures. Laminin was localized by immunoelectron microscopy in the endoplasmic reticulum of hepatocytes in fetal-derived and in 18-month-old diethylnitrosamine-treated rat livers. Steady-state mRNA levels for the three chains of laminin (A, B1, and B2) and the laminin receptor (LBP-32) were examined. Northern-blot analyses showed that hepatocytes at all stages lacked the A-chain mRNA. B1-chain mRNA was undetectable in normal adult hepatocytes, while significant levels of B1-chain mRNA were found in fetal hepatocytes and in adult hepatocyte primary culture. In hepatocytes from diethylnitrosamine-treated rats, B1-chain mRNAs were abundant and were present mainly in nodular formations rather than in the nontumorous areas. B2-chain mRNAs were barely detectable in either normal adult or fetal hepatocytes. In diethylnitrosamine-treated rats, the steady-state B2-chain mRNA level was higher in nodules than in nontumorous areas. In primary culture, B2-chain mRNAs were present as early as 4 hours after adult hepatocyte seeding, and dramatically increased during the following 2 days. Only low levels of laminin-receptor (LBP-32) mRNAs were present in normal adult hepatocytes, whereas the levels were high in the fetal and in the tumor-containing livers. In diethylnitrosamine-treated rats, LBP-32 mRNAs were more abundant in nodular formations rather than in nontumorous areas. In hepatocyte primary culture, the expression of the LBP-32 mRNA dramatically increased during the first 24 hours. These results show that in hepatocytes, expression of laminin chains and its receptor LBP-32 are not coordinated and depends on the maturation of the cells. In addition, they suggest that the expression of B1 and B2 chains in adult hepatocytes is related to changes of the normal phenotype and/or the pericellular environment.

A rapid and transient induction of the c-fos transcript followed 4 hr later by long-term increase in the c-myc transcript was observed after disruption of the liver tissue with collagenase or EDTA perfusion and after in vitro detachment of the cell-sheet of liver cells in culture. This increase of c-fos and c-myc transcripts could result from both an increased gene transcription and a stabilization of the corresponding mRNAs, as suggested by the effects of cycloheximide and actinomycin D. This increase was dependent on the extent of cell dissociation and isolation and was not the effect of dissociating agents. These findings strongly argue in favor of the role of cell-cell interactions in the induction of these two oncogenes. They are consistent with observations made on injured cells, or cells entering the G1 phase or undergoing differentiation since all these situations involve morphological changes and cell-cell contact modifications. In addition, evidence is given for a constant expression of the c-myc gene in normal hepatocytes maintained in non-proliferative primary culture. This unexpected c-myc maintenance raises the question of the role of this oncogene in hepatocyte differentiation.

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The liver epoch like other tissue epochs occurs after that different events have induced heterogeneity in embryonic cells which results in distinct evolutionary processes. These events and those of organogenesis like "induction" are deeply dependent on cell-cell communications. Cell-cell interactions involve either soluble factors (hormones, growth factors), extracellular matrix or plasma membrane proteins responsible for cell-cell recognition and/or adhesion. All these plasma membrane signals are transduced to the nucleus and modulate the expression of groups of genes. To be functionally stable along the adult stage the liver has to maintain an ordered activity of cell renewal. This balance between proliferation and differentiation is, at least in part, controlled by cell-cell communications. Therefore, it is not surprising that intercellular communications are altered during hepatocarcinogenesis. They involve changes in the distribution of junctions, in the amounts of extracellular matrix components and/or growth factors which all result in modifying the differentiation/proliferation balance. Cell culture models have been used for these different studies; new in vitro systems should be set up in the near future by taking advantage of the targeted hepatocarcinogenesis in transgenic mouse.