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Stromal cell lines from bone marrow and other blood-forming organs including fetal liver have been found to support hematopoiesis. In this paper, we demonstrate that rat liver biliary epithelial cells (RLEC), most likely originating from primitive bile ductules, are able to support long-term hematopoietic cell growth as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) production. RLEC have previously been shown to express a cell surface molecule named liver-regulating protein (LRP), which is involved in the long-term maintenance of hepatocyte functions in a coculture system. In addition, LRP-like molecules have been found in spleen, thymus, lymph nodes, and peripheral blood cells. In the present study, we found that hematopoietic cells and several stromal cell types from bone marrow were LRP-positive, and immunoprecipitation revealed polypeptides similar to those found in RLEC. We then investigated the biological role of LRP on hematopoiesis using short-term RLEC and bone marrow stromal cell culture systems. Addition of specific anti-LRP antibody to both systems reduced hematopoietic cell proliferation and committed progenitor production, whereas it did not directly affect the clonal proliferation and maturation of these progenitors in methylcellulose assays. Moreover, using diffusible chamber cultures that suppress direct contacts with hematopoietic cells, we observed low cell growth and no effect of monoclonal antibody (mAb) L8 treatment. All these results strongly argue for a cell proximity signal mediated by RLEC and bone marrow stromal cells and for the involvement of LRP-like molecules in this signal in liver and bone marrow hematopoietic function.

A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver-specific mRNAs and DNA-binding activity of ubiquitous and liver-enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3-methylcholanthrene-inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA-binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF-1 and HNF-4-like DNA-binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1-like DNA-binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer-binding protein and HNF-3-like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis-B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis-B virus replication and virion production, in agreement with the notion that HNF-1 activity is necessary for viral replication. We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis-B virus replication.

Rat liver epithelial cells (RLECs) isolated by trypsinization of the livers of normal 10 day old rats are largely used in co-culture with primary hepatocytes. The aim of the present study was to investigate the expression of biotransformation enzyme-encoding genes in three preparations of RLEC lines. Although no expression of cytochrome P450 1A1/2 (CYP1A1/2), CYP2B1/2, CYP2C6 or CYP3A mRNAs could be detected, we found that all of the different preparations of RLECs expressed a high level of CYP2E1 mRNA. We demonstrated the presence of the CYP2E1 apoprotein in microsomes of RLECs by immunoblot analyses, together with chlorzoxazone 6-hydroxylation, an activity known to be mainly catalyzed by CYP2E1. In addition, acetone treatment of these cells resulted in an increase in both CYP2E1 apoprotein and chlorzoxazone 6-hydroxylation activity levels. Finally, we showed the susceptibility of RLECs to N-methyl formamide- and diethylnitrosamine-induced toxicity, suggesting metabolic activation by CYP2E1. Thus, RLECs may cooperate with hepatocytes to CYP2E1-mediated metabolism in the co-culture model. In addition, transfection experiments with a CYP2E1 promoter construct, in which the proximal 539 bp containing the binding site for HNF1alpha were inserted upstream of the chloramphenicol acetyl transferase gene, demonstrated a strong induction upon co-transfection with an HNF1alpha expression plasmid. Thus, RLECs provide a useful tool for studying metabolism and cytotoxicity of CYP2E1 substrates in the absence of other expressed CYPs, and for analyzing CYP2E1 promoter function.

Id proteins are known as negative regulators of differentiation in various cell types. In this report, we show that the Id-1 gene was down-regulated during the development of rat liver. No Id-1 transcripts were detected in terminal differentiated hepatocytes. We have studied Id-1 expression in proliferating hepatocytes using an in vivo model of liver regeneration after partial hepatectomy and an in vitro growth factor-stimulated hepatocyte culture system. Strong activation of Id-1 was observed in mid-late G1 of the hepatocyte cell cycle at a time corresponding to a mitogen restriction point. These observations suggest that Id-1 is involved in the control of proliferation and differentiation in liver cells.