Phenotypic and karyotypic changes induced in cultured rat hepatic epithelial cells that express the "oval" cell phenotype by exposure to N-methyl-N'-nitro-N-nitrosoguanidine.

A diploid population of cultured rat hepatic epithelial cells that expresses the "oval" cell phenotype was exposed briefly and repetitively to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the effect on more than 20 phenotypic properties was evaluated during the neoplastic transformation of the population. MNNG treatments of this hepatic epithelial cell population resulted in a progressively increasing phenotypic alteration and heterogeneity including changes in specific activities of several cellular enzymes and expression of isozymes, synthetic functions, and various in vitro growth properties. Changes in phenotypic expression were clustered episodically and were associated with major karyotypic changes. The development of increasing phenotypic heterogeneity preceding and accompanying tumorigenicity in cultured liver epithelial cells in vitro and the specific phenotypes that occur resemble superficially the pattern of phenotypic changes that occur in hepatocytes during chemical hepatocarcinogenesis in vivo. The results of this study provide the basis for future investigations to further elucidate the mechanistic and linkage relationship between specific pretumorigenic and paratumorigenic phenotypes and tumorigenicity

Temporal Analysis of Hepatocyte Differentiation by Small Hepatocyte-Like Progenitor Cells during Liver Regeneration in Retrorsine-Exposed Rats

Liver regeneration after two-thirds surgical partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). We have examined expression of the major liver-enriched transcription factors, cytochrome P450 (CYP) enzymes, and other markers of hepatocytic differentiation in SHPCs during the protracted period of liver regeneration after PH in retrorsine-exposed rats. Early-appearing SHPCs (at 3–7 days after PH) express mRNAs for all of the major liver-enriched transcription factors at varying levels compared to fully differentiated hepatocytes. In addition, SHPCs lack (or have significantly reduced) expression of mRNA for hepatocyte markers tyrosine aminotransferase and α-1 antitrypsin, but their expression levels of mRNA and/or protein for WT1 and α-fetoprotein (AFP) are increased. With the exception of AFP expression, SHPCs resembled fully differentiated hepatocytes by 14 days after PH. Expression of AFP was maintained by most SHPCs through 14 days after PH, gradually declined through 23 days after PH, and was essentially absent from SHPC progeny by 30 days after PH. Furthermore, early appearing SHPCs lack (or have reduced expression) of hepatic CYP proteins known to be induced in rat livers after retrorsine exposure. The resistance of SHPCs to the mitoinhibitory effects of retrorsine may be directly related to a lack of CYP enzymes required to metabolize retrorsine to its toxic derivatives. These results suggest that SHPCs represent a unique parenchymal (less differentiated) progenitor cell population of adult rodent liver that is phenotypically distinct from fully differentiated hepatocytes, biliary epithelial cells, and (ductular) oval cells

Liver Regeneration in Rats with Retrorsine-Induced Hepatocellular Injury Proceeds through a Novel Cellular Response

The adult rodent liver contains at least two recognized populations of cells with stem-like properties that contribute to liver repair/regeneration under different pathophysiological circumstances: (i) unipotential committed progenitor cells (differentiated hepatocytes and biliary epithelial cells) and (ii) multipotential nonparenchymal progenitor cells (oval cells). In retrorsine-induced hepatocellular injury the capacity of fully differentiated rat hepatocytes to replicate is severely impaired and massive proliferation of oval cells does not occur. Nevertheless, retrorsine-exposed rats can replace their entire liver mass after 2/3 surgical partial hepatectomy through the emergence and expansion of a population of small hepatocyte-like progenitor cells that expresses phenotypic characteristics of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes, but differ distinctly from each type of cell. The activation, proliferation, and complete regeneration of normal liver structure from small hepatocyte-like progenitor cells have not been recognized in other models of liver injury characterized by impaired hepatocyte replication. We suggest that the selective emergence and expansion of small hepatocyte-like progenitor cells observed in the retrorsine model reflect a novel mechanism of complete liver regeneration in the adult rat. Furthermore, we suggest that these cells may represent a novel progenitor cell population that (i) responds to liver deficit when the replication capacity of differentiated hepatocytes is impaired, (ii) expresses an extensive proliferative capacity, (iii) can give rise to large numbers of progeny hepatocytes, and (iv) can restore tissue mass

Plasticity of the neoplastic phenotype in vivo is regulated by epigenetic factors

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor

NFkappaB prevents apoptosis and liver dysfunction during liver regeneration

Although NF�B binding activity is induced during liver regeneration after partial hepatectomy, the physiological consequence of this induction is unknown. We have assessed the role of NF�B during liver regeneration by delivering to the liver a superrepressor of NF�B activity using an adenoviral vector expressing a mutated form of I�B�. This adenovirus (Ad5I�B) was almost exclusively expressed in the liver and inhibited NF�B DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. After partial hepatectomy, infection with Ad5I�B, but not a control adenovirus (Ad5LacZ), resulted in the induction of massive apoptosis and hepatocytes as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5I�B but not Ad5LacZ decreased the mitotic index after partial hepatectomy. These two phenomena, increased apoptosis and failure to progress through the cell cycle, were associated with liver dysfunction in animals infected with the Ad5I�B but not Ad5LacZ, as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NF�B during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression. (J. Clin