Phenotype and differentiation patterns of the oval cell lines OC/CDE 6 and OC/CDE 22 derived from the livers of carcinogen-treated rats.

An electron microscopic, immunocytochemical, and enzyme cytochemical analysis of the previously established oval cell lines OC/CDE 6 and OC/CDE 22 was performed to characterize the phenotype and differentiation patterns of long-term cultures of oval cells. It was found that alpha-fetoprotein, albumin, and cytokeratin 19 are present in all cultured cells. This indicates that oval cells constitute a population of immature cells expressing features of the antigenic phenotype of both the hepatocyte and bile ductular cell lineages. An electron microscopic examination revealed a gradual alteration in the ultrastructure of oval cells toward hepatocyte-like cells. The majority of the oval cells were positive for glucose-6-phosphatase activity. A particularly striking observation was that oval cells were heterogeneous in terms of peroxisome content. Only about 50% of the oval cells had peroxisomes in the cytoplasm, these cells probably being part of the hepatocyte lineage. The other cultured cells did not reveal catalase activity and probably represented cells committed to the bile ductular cell lineage. An addition of clofibrate to the culture medium resulted in a marked peroxisome proliferation in all oval cells, indicating that oval cells might be able to change their differentiation pathway depending on environmental influence toward the hepatocyte lineage. It is most intriguing that in oval cells with abundant cytoplasm peroxisome proliferation was accompanied by proliferation of the smooth endoplasmic reticulum (this is a morphological marker of mature hepatocytes). Taken together, our findings suggest that within the oval cell lines OC/CDE 6 and OC/CDE 22 cells undergoing a morphological and functional differentiation along the hepatocyte and bile ductular cell lineages are present.

Synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary aflatoxin B1 in woodchucks.

Interactive hepadnaviral and chemical hepatocarcinogenesis was studied in woodchucks inoculated as newborns with woodchuck hepatitis virus (WHV), which is closely related to the human hepatitis B virus. When the woodchucks reached 12 months of age, aflatoxin B1 (AFB1) was administered in the diet at dose levels of 40 micrograms/kg body weight/day for 4 months and subsequently 20 micrograms/kg body weight/day (5 days/week) for lifetime. WHV DNA was demonstrated by Southern blot hybridization in the serum and by PCR in the serum and/or liver tissue. The histo- and cytomorphology of the liver were investigated by light and electron microscopy. WHV carriers with and without AFB1 treatment developed a high incidence of preneoplastic foci of altered hepatocytes, hepatocellular adenomas, and hepatocellular carcinomas that appeared 6-26 months after the beginning of the combination experiment. Administration of AFB1 to WHV carriers resulted in a significantly earlier appearance of hepatocellular neoplasms and a higher incidence of hepatocellular carcinomas compared to WHV carriers not treated with AFB1. Neither hepatocellular adenomas nor carcinomas (but preneoplastic foci of altered hepatocytes) were detected in woodchucks receiving AFB1 alone, and no preneoplastic or neoplastic lesions were found in untreated controls. These results provide conclusive evidence of a synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary AFB1. Except for the frequent presence of ground glass cells containing surface antigen filaments in the infected woodchucks, the phenotype of preneoplastic foci of altered hepatocytes was similar in WHV carriers with and without exposure to AFB1 and in animals treated with AFB1 alone. Clear cell foci excessively storing glycogen and/or fat, amphophilic cell foci crowded with mitochondria and peroxisomes, and mixed cell foci composed of various cell types including basophilic cells rich in ribosomes predominated. The cellular phenotype in neoplastic lesions varied from clear, amphophilic, and mixed cell populations in highly differentiated adenomas and carcinomas to basophilic cell populations prevailing in poorly differentiated carcinomas. The striking similarities in altered cellular phenotypes of preneoplastic hepatic foci emerging after both hepadnaviral infection and exposure to AFB1 suggest closely related underlying molecular mechanisms that may be mainly responsible for the synergistic hepatocarcinogenic effect of these oncogenic agents.

Oval cells--hepatocytes relationships in Dipin-induced hepatocarcinogenesis in mice.

During Dipin-induced hepatocarcinogenesis in mice there is powerful and prolonged proliferation of oval cells which are arranged in duct-like structures. Ultrastructure and differentiation pathways of oval cells depend on their location in the liver lobule. The major part of oval cells is represented by duct lining cells morphologically similar to biliary epithelial cells. They form the system of branching anastomozing ducts and expand into the parenchyma from portal to central veins. Later these ducts disintegrate. In the periportal areas, three stages of oval cell differentiation can be distinguished: (1) low differentiated cell similar to cells of terminal biliary ductules in their size and ultrastructure, (2) transitional cells and (3) young hepatocytes. Cells with ultrastructural characteristics of sequential stages of hepatocyte differentiation are located within the ducts surrounded by the basal lamina. Our data suggest that oval cells are the committed cell precursors capable of differentiating into hepatocytes or biliary epithelial cells in the periportal microenvironment.

[The stem-cell reserve of the liver]. / Stvolovoi rezerv pecheni.

This paper considers the problem of epithelial stem cells of the liver and possibilities of its experimental solution. Authors' own data obtained with the model of induced hepatocarcinogenesis in mice are discussed; the experiments were performed using electron microscopy, autoradiography and immunochemistry. In accordance with these data, Gering cells are stem cells of the liver, and oval cells correspond to committed precursors capable to differentiation in either hepatocellular or cholangiolar direction under the conditions of periportal microenvironment. We have also compared hepatocyte differentiation in preneoplastic mouse liver and regenerating pancreas of adult rats (Rao et al. Amer. J. Pathol. 1989. V. 134. P. 1069-1086). We also discuss stem cell compartment organization in organs having glandular structure and the possibility of existence in the adult of non-committed multipotent cells capable of producing various types of differentiation in tissues having common origin during embryogenesis.

[The formation of oval-cell ducts during hepatic carcinogenesis in mice. Its relationship to the pre-existing canals of Hering]. / Obrazovanie protokov oval'nykh kletok v protsesse gepatokantserogeneza u myshei. Sviaz' s predsushchestvuiushchimi kanalami Geringa.

It has been shown that a population of the oval cells is formed in mouse liver during the dipin-induced carcinogenesis (Radaeva, Factor, 1990b). This paper deals with the origin of the oval cells and their proliferation potential depending on localization in the liver lobule. Series of semithin liver sections were studied under the light microscope and detected labeled cells analyzed under electron microscope on serial ultrathin sections. We found that proliferation of cells of terminal bile ductules (Hering [correction of Gering] canals) takes place at the early stages of liver carcinogenesis. These cells and first labeled oval cells had similar size and morphology and jointly formed the ducts. Oval cell population was heterogeneous in terms of proliferative potential. Proportion of proliferating cells (38-45%) in the oval cells of Hering [correction of Gering] canals and small ducts surrounding portal tracts remained similar throughout the period of formation of the oval cell population. In the oval cells infiltrating the parenchyma, the proportion of proliferating cells appeared to depend on the intensity of the oval cell response: it attained the maximum (62%) on intermediate stage and decreased to the minimum (22%) at the peak of the reaction. These data suggest that Hering [correction of Gering] canals probably give origin to the ducts formed by oval cells.

Changes in catalase and glucose-6-phosphatase distribution patterns within oval cell compartment as possible differentiation markers during viral hepatocarcinogenesis in woodchucks.

Cytochemical analysis at the ultrastructural level was performed to characterize expression of catalase and glucose-6-phosphatase (G6Pase) activity as possible differentiation markers in oval cells proliferating during hepatocarcinogenesis induced in woodchucks by chronic infection with the woodchuck hepatitis virus (WHV) and additional treatment with aflatoxin B1 (AFB1). Oval cells from WHV-carriers treated with AFB1 showed two types of catalase-positive organelles: 1) microperoxisomes appearing as small strongly osmiophilic bodies corresponding to those present in biliary cells from control woodchucks, 2) peroxisomes with a hepatic staining pattern resembling those of mature hepatocytes but lacking a nucleoid. While in oval cells penetrating into the parenchyma a catalase-positive reaction product was restricted to rare microperoxisomes, in close vicinity to the portal tract about 30% of the oval cells produced peroxisomes with a hepatic staining pattern, indicating the existence of two different populations within the oval cell compartment. Peroxisomes with a hepatic staining pattern formed clusters and exhibited pleomorphism with marked variation in shape and size, the size sometimes coming up to that of normal hepatocellular peroxisomes. Serial sections revealed the complex organization of these peroxisomes. They consisted of several interconnected segments forming a peroxisomal reticulum. These findings are consistent with the hypothesis that a subpopulation of oval cells represents committed precursor cells capable of differentiating into hepatocytes. Activity of G6Pase was not demonstrable in this subpopulation of oval cells and became positive only in transitional cells. Differential expression of catalase and G6Pase activity in a stepwise fashion within the oval cell compartment appear to mark differentiation of oval cells into hepatocytes. Thus, elevated expression of catalase may be a useful early marker for the distinction of different subpopulations of oval cells committed to hepatic cell lineages before definitely changing their phenotype, whereas expression of G6Pase activity seems to begin later, accompanying morphological changes towards the phenotype of mature hepatocytes.

Origin and fate of oval cells in dipin-induced hepatocarcinogenesis in the mouse.

We have studied the development and differentiation of oval cells in the Dipin model of hepatocarcinogenesis in the mouse and compared this process to generation of biliary epithelial cells by bile duct ligation using light and electron microscopy. The Dipin model of hepatocarcinogenesis consists of a single injection of an alkylating drug, Dipin (1,4-bis[N,N'-di(ethylene)-phosphamide]-piperazine), followed by partial hepatectomy. The Dipin treatment resulted in irreversible damage and gradual death of hepatocytes by necrosis and apoptosis. Earlier work provided evidence that regeneration of parenchyma occurred via oval cell proliferation and subsequent differentiation into hepatocytes that replaced the degenerating hepatocytes. Both autoradiographic and morphological data indicated that oval cells were derived from ductular cells of Hering canals. The first oval cells labeled with [3H]thymidine were similar in size and ultrastructure to ductular cells of Hering canals with whom intracellular connections existed. The proliferation of ductular cells of Hering canals gave rise to a new system of oval cell ducts that spread into the liver acinus. In the periportal areas, the transition of oval cells into hepatocytes was observed inside the ducts. Both growth patterns and ultrastructure of oval cells were different from the biliary epithelial cells in bile duct-ligated liver. Also, oval cells retained the property to interact with adjacent hepatocytes through desmosomes and intermediate junctions. Oval cell population was heterogeneous in terms of proliferating potential. A proportion of proliferating cells (38 to 45%) in the Hering canals and small oval cell ducts located in the periportal areas was similar throughout the period of oval cell development. The extent of proliferation of oval cells decreased from 62% at the stage of active migration into the acinus to 22% at maximum formation of oval cell ducts. These data suggest that in the mouse liver cells of the terminal biliary ductules harbor the hepatic stem cell compartment from which oval cells, capable of differentiating into hepatocytes, may be derived.

NEU overexpression in the furan rat model of cholangiocarcinogenesis compared with biliary ductal cell hyperplasia.

Immunohistochemical studies have suggested that the tyrosine kinase growth factor receptor p185neu is overexpressed in a high percentage of human cholangiocarcinomas. To establish the specificity and temporal relationship between the expression of this receptor in cholangiocarcinogenesis, we investigated c-neu expression in precancerous cholangiofibrotic tissue and subsequently derived primary and transplantable cholangiocarcinomas originated in the livers of furan-treated rats. Proliferated bile ductules formed in rat models of bile ductular hyperplasia and the cell types of normal adult rat liver were also analyzed for c-neu expression. c-neu expression was not detected in normal adult rat liver by either Western blotting, immunohistochemistry, or in situ hybridization. In comparison, all of the cholangiocarcinomas analyzed, which were characterized by intestinal-type mucin-producing neoplastic glands, exhibited a prominent band with a molecular weight 185 kd, corresponding to p185neu. Only the neoplastic glandular epithelia of the cholangiocarcinomas showed a strong immunoreactivity for p185neu, which was predominantly localized to their cell surface but also observed cytoplasmically. In situ hybridization further revealed the cytoplasm of the tumor glandular epithelial cells to be strongly positive for c-neu mRNA transcripts. Of particular interest was our finding that c-neu is expressed early in furan cholangiocarcinogenesis, being more pronounced in the metaplastic intestinal glands of cholangiofibrotic tissue than in hyperplastic biliary epithelial cells in either the same tissue or in hyperplastic bile ductule tissue. Our results demonstrate that c-neu overexpression is a prominent feature of intestinal-type cholangiocarcinomas as well as of metaplastic intestinal glands that precede their development and is detected at lower levels in hyperplastic biliary epithelia. The overexpression of c-neu in the metaplastic and malignant neoplastic glands also correlated with their increased proliferating cell nuclear antigen (PCNA) labeling indices relative to those of hyperplastic biliary ducts and ductules and also appeared to correlate with their intestinal glandular pattern of differentiation.

Overexpression of C-NEU and C-MET during rat liver cholangiocarcinogenesis: A link between biliary intestinal metaplasia and mucin-producing cholangiocarcinoma.

Based on limited but compelling immunohistochemical data demonstrating individual overexpression of the tyrosine kinase growth factor receptors, c-erbB-2 and c-met, in significant percentages of human cholangiocarcinoma (ChC), we investigated if combined overexpression of both c-neu, the rat homologue of c-erbB-2, and c-met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), might represent a characteristic, early event associated with furan-induced cholangiocarcinogenesis in rat liver. Specifically, through the use of immunohistochemistry, in situ hybridization (ISH), and Western and Northern blotting, we found that both c-neu and c-met are prominently overexpressed in intestinal metaplastic lesions in early putative precancerous cholangiofibrotic tissue formed in the livers of rats after 6 weeks of furan treatment when compared with normal and hyperplastic intrahepatic biliary epithelia. We further demonstrated that c-neu and c-met are concordantly overexpressed in neoplastic glandular epithelia in later-developed primary "intestinal-type" of ChC formed in the livers of furan-treated rats, as well as in subsequently derived transplantable mucin-producing tumors. Overexpression of c-neu and c-met correlated with increased proliferating cell nuclear antigen (PCNA)-labeling indices, which were determined to be three to four times higher in intestinal metaplastic glands in precancerous cholangiofibrotic tissue and in neoplastic glands in the primary "intestinal type" of ChC than in hyperplastic bile ductular structures within either cholangiofibrotic or bile duct-ligated (BDL) livers. The c-neu and c-met receptor proteins overexpressed in different in vivo passages of a transplantable ChC each contained immunoreactive phosphotyrosines, indicating an activated state. However, we did not detect evidence of either gene amplification of c-neu or c-met or of a common transmembrane-activating mutation in c-neu expressed in transplantable ChC. Our findings indicate that altered expression of c-neu and c-met occurs relatively early in the process of furan-induced cholangiocarcinogenesis in rat liver and may play a potentially important role in its pathogenesis. They further indicate a common alteration in tyrosine kinase growth factor receptor expression linking early putative precancerous intestinal metaplastic lesions in liver to later-developed mucin-producing biliary cancer.

Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated "intestinal-type" biliary cancer chemically induced in rat liver.

Recently, we observed that Met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), is overexpressed in epithelial cells of both early-appearing intestinal metaplastic glands in precancerous hepatic cholangiofibrotic tissue and neoplastic glands in later developed intestinal-type of cholangiocarcinoma originated from the furan rat model of cholangiocarcinogenesis when compared with normal and hyperplastic intrahepatic biliary epithelia. We now show that HGF/SF is also aberrantly expressed in a manner closely paralleling that of its receptor in the neoplastic epithelial cells of furan-induced rat cholangiocarcinomas and in a majority of metaplastic epithelial cells within earlier formed precancerous hepatic cholangiofibrotic tissue. Using in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), we further showed specific expression of HGF/SF messenger RNA (mRNA) in a novel rat cholangiocarcinoma epithelial cell line overexpressing Met. This cholangiocarcinoma cell line, termed C611B, was established from tumorigenic cells isolated from a furan-induced transplantable tumor. Moreover, we detected by in situ hybridization strong expression of HGF/SF mRNA transcripts in the cancerous epithelial glands of cholangiocarcinoma developed in recipient rats after in vivo cell transplantation of C611B cells. In contrast, mRNA transcripts and protein immunoreactivity for this cytokine were not detected in hepatocytes and biliary epithelial cells in adult normal rat liver nor in rat hyperplastic intrahepatic biliary epithelium. Our results clearly show that HGF/SF becomes aberrantly expressed in cholangiocarcinoma epithelium and in putative precancerous intestinal metaplastic epithelium induced in the liver of furan-treated rats.

Hepadnaviral hepatocarcinogenesis: in situ visualization of viral antigens, cytoplasmic compartmentation, enzymic patterns, and cellular proliferation in preneoplastic hepatocellular lineages in woodchucks.

BACKGROUND/AIMS: Hepadnaviral hepatocarcinogenesis induced in woodchucks with and without dietary aflatoxin B1 has been established as an appropriate animal model for studying the pathogenesis of human hepatocellular carcinoma in high-risk areas. Our aim in this study was the elucidation of phenotypic cellular changes in early stages of this process. METHODS: Woodchucks were inoculated as newborns with woodchuck hepatitis virus (WHV), and partly also exposed to aflatoxin B1. Sequential hepatocellular changes in the expression of viral antigens, ultrastructural organization, cellular proliferation and apoptosis were studied in situ by electron microscopy, enzyme and immunohistochemistry. RESULTS: A characteristic finding in WHV-infected animals (with and without aflatoxin B1) was proliferative areas of minimal structural deviation, which predominated periportally, comprised glycogen-rich, amphophilic, and ground-glass hepatocytes, and expressed the woodchuck hepatitis core and surface antigens. Two main types of proliferative foci emerged from minimal deviation areas, glycogenotic clear cell foci and amphophilic cell foci (being poor in glycogen but rich in mitochondria), giving rise to the glycogenotic-basophilic and the amphophilic preneoplastic hepatocellular lineages. A gradual loss in the expression of viral antigens appeared in both lineages, particularly early in the glycogenotic-basophilic cell lineage. Whereas glycogenosis was associated with an enzymic pattern suggesting an early activation of the insulin-signaling pathway, amphophilic cells showed changes in enzyme activities mimicking a response of the hepatocytes to thyroid hormone, which may also result from early changes in signal transduction. CONCLUSION: Preneoplastic hepatocellular lineages in hepadnaviral and chemical hepatocarcinognesis show striking phenotypic similarities, indicating concordant and possibly synergistic early changes in signaling.

A new method for flat-embedding large native cryostat sections for targeting small preneoplastic lesions in comparative ultrastructural and ultracytochemical investigations.

Ultrastructural studies of rare and small cellular lesions in pathologically altered tissue are difficult to perform by applying conventional electron microscopic preparation. The search for lesions, often consisting of only a few cells in randomly obtained small specimen blocks, is time consuming and often without success. The methodological requirements for comparative enzyme cytochemical and morphological studies, i.e., preservation of both enzyme activity and ultrastructure, are divergent. By processing large native cryostat sections for electron microscopy, small preneoplastic focal lesions were successfully targeted in liver and kidney. Glucose-6-phosphatase, alkaline phosphatase, acid phosphatase, catalase, and cytochrome c oxidase activities were distinctly localized to endoplasmic reticulum, canalicular membrane, lysosomes, peroxisomes, and mitochondria, respectively, in the morphologically altered cells. Fixation of serial cryostat sections and enzyme reactions were both carried out through a semipermeable membrane except those for cytochrome c oxidase, which was demonstrated after fixation through the membrane by floating the section in incubation medium containing cytochrome c. Thereafter, the sections were flat embedded and polymerized between epoxy resin disks and aluminum dishes fitting exactly together. The objects of interest were identified in the light microscope, cut out, and reembedded in reversed gelatine capsules. By using this technique an ultrastructural preservation was achieved similar to that seen after immersion fixation. The enzyme activities were clearly localized without diffusion of the reaction product or unspecific deposits. The procedure permits precise targeting and complex studies of rare and small lesions, and opens new perspectives for the use of cryo-preserved tissue.

Oval cell lines OC/CDE 6 and OC/CDE 22 give rise to cholangio-cellular and undifferentiated carcinomas after transformation.

BACKGROUND: There is compelling evidence for a parenchymal origin of the predominant cell lineage leading from preneoplastic clear and acidophilic glycogen storage foci through mixed and basophilic cell populations to hepatocellular carcinomas in the rat. However, a controversial question remains to be answered: Do the basophilic cell foci invariably originate from parenchymal cells or do oval cells also have the potential to give rise to this type of focus and progress to hepatocellular neoplasms? Oval cells are nonparenchymal epithelial cells with scant cytoplasm and ovoid nuclei that first appear in the periportal areas of the liver lobules and thereafter invade the whole parenchyma when animals are exposed to high doses of a wide range of chemical carcinogens. EXPERIMENTAL DESIGN: Two oval cell lines, OC/CDE 6 and OC/CDE 22, which had been established from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 or 22 weeks, were transformed either by leaving the cells in confluence for a long time period (OC/CDE 6) or by treating the cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. The transformed cells were injected subcutaneously in newborn rats and the tumors developing in these animals were analyzed histopathologically, ultrastructurally, and immunohistochemically. RESULTS: The two transformed oval cell lines gave rise to carcinomas, in which cholangiocellular, adenoid and solid tumor formations were observed. Subpopulations of these tumors expressed cytokeratins 7, 8, 18, and 19, but were albumin- and alpha-fetoprotein-negative. Areas within the carcinomas derived from transformed OC/CDE 22 cells representing undifferentiated liver tumor formations were also identified. Cells within these areas had lower nucleus/cytoplasm ratios than cells in the solid growing tumor formations, stained positive for cytokeratins 8 and 18 and were cytokeratin 7- and 19-, albumin- and alpha-fetoprotein-negative. Ultrastructurally, these cells did not resemble those of differentiated hepatocellular carcinomas. CONCLUSIONS: It has been shown that oval cells are precursor cells of carcinomas containing cholangiocellular, adenoid and solid formations which may be largely undifferentiated. However, the transformed OC/CDE 6 or OC/CDE 22 cells do not serve as precursor cells of differentiated hepatocellular carcinomas.

Establishment and characterization of a nontumorigenic cell line derived from a human hepatocellular adenoma expressing hepatocyte-specific markers.

In the present study the establishment and characterization of a nontumorigenic liver epithelial cell line (HACL-1) derived from a human hepatocellular adenoma is described. The HACL-1 cells have a finite life span (i.e., they proliferate for a period of 2 months and then senesce), show cell-cell contact inhibition, do not grow in soft agar, are not tumorigenic when injected in nude mice, and possess a normal diploid karyotype. The cultured cells resemble hepatocytes, but exhibit some features of dedifferentiation. At the ultrastructural level the cells are endowed with round or oval nuclei, abundant cytoplasmic organelles, and varying amounts of glycogen. The rough endoplasmic reticulum is disorganized, while peroxisomes and matrix granules within mitochondria are lacking. HACL-1 cells are cytokeratin 18-positive as well as (transiently) albumin- and alpha-fetoprotein-positive, but do not express cytokeratin 19. Furthermore, no mutations were observed in exons 5-8 of the tumor suppressor gene p53. Taken together these results show that HACL-1 cells are nontumorigenic proliferating liver epithelial cells, which might prove to be of great value in future studies on diverse aspects of human liver cell biology and carcinogenesis.