A precursor--product relationship exists between oval cells and hepatocytes in rat liver

Oval cell proliferation was induced in twelve male Fischer ratsby administration of 2-acetylaminofluorene (2-AAF) for 2 weeksand by performing partial hepatectomy one week after the beginningof 2-AAF administration. Albumin expression in liver was studiedby using in situ hybridization of ³H-labeled rat albumin riboprobe.Radiolabeled thy-midine was administered to a group of animalsat day 6 after partial hepatectomy. The animals were killedat 0, 3, 7, 9, 11 and 13 days after partial hepatectomy. Bothoval cells and basophilk hepatocytes showed a prominent expressionof albumin, whereas albumin expression hi acidophilic preexistinghepatocytes was decreased. Oval cell nuclei were exclusivelylabeled one day after administration of [³H]thymidine. At day9, 11 and 13 basophilic hepatocytes became labeled and the areaoccupied by these cells increased. This is the first demonstrationof the transfer of radiolabeled thymidine from oval ceDs tonewly formed hepatocytes in vivo. Thus the precursor—productrelationship between oval cells and basophilic hepatocytes hasbeen established.     

Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers

The expansion and differentiation of oval cells in the acetylaminofluorene(AAF)/partial hepatectomy (PH) model was studied utilizing pulse-chaselabeling with both tritiated thymidine ([³H]TdR) and bromodeoxyuridine(BUdR). Animals in which a significant decrease in serum albuminand increase in alanine aminotransferase and bilirubin wereobserved demonstrated the most prominent differentiation ofoval cells into hepatocytes. Administration of [³H]TdR or BUdR,either individually or together, to the animals on day 6 afterpartial hepatectomy resulted in labeling of the majority ofthe oval cells by days 7 and 9 after PH. A striking differencein the distribution of [³H]TdR- and BUdR-labeled cells in thedouble labeling experiments was observed on day 11, at whichtime the number of [³H]TdR-labeled cells increased 6-fold andthat of double labeled cells decreased 2-fold. Furthermore,on day 11 the basophilic foci were weakly positive for BUdRand negative at later time points in animals receiving BUdRalone or together with [³H]TdR. In contrast, the cells in basophilicfoci as well as transitional cells were positive for [³H]TdR.Cells heavily labeled with both [³H]TdR and BUdR were presentat all time points, indicating an inhibition of the proliferativeactivity. Pulse labeling of rat liver epithelial cells withBUdR in vitro demonstrated that immunodetection of BUdR waslost after three or more cell divisions. We conclude that theBUdR tagging method is particularly sensitive to label dilutionduring cell cycling and may not be suitable for establishmentof a precursor-product relationship between cell lineages whenthe progenitor population proliferates more than three times.     

Induction of foci of altered hepatocytes by a single injection of azaserine to rats.

The induction of foci of altered, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes by azaserine was investigated. After injection of a single dose of azaserine, many foci developed in male Wistar rats fed a choline-devoid (CD) diet containing acetylaminofluorene (AAF), but only a few in rats fed a choline-supplemented (CS) diet containing AAF. Similar results were obtained in rats fed a plain CD diet or a plain CS diet and injected with a single dose of azaserine after a partial hepatectomy. These findings indicate that azaserine is an effective initiator of liver carcinogenesis in rats, and that a CD diet acts as a strong promoter of the evolution initiated liver cells to foci of altered, GGT-positive hepatocytes.     

In situ hybridization studies on expression of albumin and alpha-fetoprotein during the early stage of neoplastic transformation in rat liver

The expressions of albumin and alpha-fetoprotein (AFP) genes were studied in early preneoplastic liver lesions produced by the Solt-Farber protocol using "in situ" hybridization with single stranded RNA probes. In normal rat liver, albumin was expressed at a lower level in the centrilobular than in the periportal areas of the liver acinus, whereas the bile duct epithelium did not show any expression. Five weeks after initiation with diethylnitrosamine, islands of hepatocytes were present which showed heterogeneous expression of albumin and were surrounded by cells comprised of albumin negative hepatocytes and oval cells. gamma-Glutamyltranspeptidase positive foci of enzyme altered cells were located in albumin positive areas. Albumin expression gradually decreased in permanent nodules but increased in the hepatocytes outside the nodules during the first five months after initiation with diethylnitrosamine. Remodeling nodules, which were partly gamma-glutamyltranspeptidase and albumin positive, were also present. However, no consistent correlation was found between gamma-glutamyltranspeptidase positive and albumin negative areas during the first 5 months after initiation. Occasionally, cells showing an elevated expression of albumin were found in permanent nodules. These cells were located in the vicinity of oval type cells, which also showed a weak expression of albumin. AFP was expressed at high level in oval cells 5 weeks after the initiation. However, oval cells observed at later time points, either around the neoplastic nodules or inside the nodules showed only low expression of AFP. Hepatocytes in the enzyme-altered foci and in neoplastic nodules were always negative for AFP. The presence of strongly albumin positive cells inside the neoplastic nodules in close proximity to oval type cells suggests that these cells may be derived from primitive "stem-cell"-like oval cells.     

Spontaneously transformed rat pancreatic epithelial oval cells give rise to ductal type adenocarcinomas

Oval epithelial cells that proliferate in the pancreas of the rats maintained on a copper-deficient diet are considered as stem cells with a potential to differentiate into hepatocytes. We isolated these oval cells from a copper-deficient rat and maintained these as a cell line in our laboratory. During 13th passage, oval cells showed increased growth and cellular pleomorphism and were analyzed for spontaneous transformation by anchorage-independent growth in soft agar and tumorigenicity in nude mice. Oval cells formed large colonies in soft agar and developed tumors in nude mice after subcutaneous transplantation. The tumors, by light and electron microscopy, showed features of well differentiated ductal-type adenocarcinomas. The phenotypic properties of these tumors included expression of neutral mucins, keratin filaments, carcinoembryonic antigen and glutathione S-transferase-pi and absence of gamma-glutamyltranspeptidase. The results of this study demonstrate that spontaneously transformed oval cells can form typical ductal-type adenocarcinomas. These observations are of particular interest, since bonafide ductal adenocarcinomas have not been described in the rat pancreas before.     

The resistant hepatocyte model of carcinogenesis in the rat: the apparent independent development of oval cell proliferation and early nodules

The early cellular changes in the Solt-Farber resistant hepatocytemodel of carcinogenesis have been studied to clarify the relationshipof oval cell proliferation to the development of early hepatocytenodules. Cellular proliferation, intermediate filament profilesand the expression of specific cytochrome P450 enzymes wereexamined. At 24 h after partial hepatectomy (PH) many of thebile ductular cells were in S phase, but over the next few daysDNA synthesis progressively decreased in the portal bile ductsand was more common in arborizing ductules (oval cells) radiatingfrom the portal areas. These cells strongly expressed cytokeratins8 and 19 and vimentin, and from 1 week after PH they frequentlyunderwent differentiation either into hepatocytes, expressingcytochrome P450 enzymes, or into intestinal-type cells. Fivedays after PH, numerous basophilic foci were discernible, andthese expanded rapidly. The ductular cells swirled around thefoci, but their antigenic profile clearly indicated that thesecells were not involved in the development of these early nodules.In normal hepatocytes, cytokeratin 8 immunoreactivity was distinctlymembranous in location, and could only be readily detected inperiportal hepatocytes. In the basophilic hepatocyte foci, overexpressionof cytokeratin 8 was consistently associated with cells organizinginto acini, with expression reminiscent of authentic bile ducts,possibly indicating a structure-function relationship. In conclusion,early foci and nodules in this model are derived from resistanthepatocytes and not ductular oval cells, the latter being afacultative multipotential stem cell compartment.     

Enzyme histochemical and immunohistochemical characterization of oval and parenchymal cells proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet.

Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor-product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes.     

Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers

Oval cells and biliary epithelial cells were isolated from livers of rats fed a choline-deficient diet containing 0.1% ethionine and from normal rat livers, respectively. Nonparenchymal cell suspensions prepared from these livers by collagenase perfusion followed by digestion of undissociated tissue with 0.1% collagenase, 0.1% Pronase, and 0.004% DNase I were separated into six fractions by centrifugal elutriation. Cells in each fraction were characterized histochemically for gamma-glutamyl transpeptidase, peroxidase, alkaline phosphatase, and glucose-6-phosphatase activities, and for albumin and alpha-fetoprotein by immunocytochemical methods. Cells from Fraction 5 of the elutriation procedure had various features predicted for oval cells and were selected for further studies. The cell yield in this fraction, from each preneoplastic liver, was 5.7 X 10(7) cells, 93 +/- 2% of which were gamma-glutamyl transpeptidase positive, 6 +/- 1% peroxidase positive, 61% albumin positive, and 29% alpha-fetoprotein positive. Cells in this fraction have a median diameter of 13.1 micron and are diploid and cycling. The majority of these cells has morphological features characteristic of biliary epithelial cells, although some cells display features intermediate between duct cells and hepatocytes. Nucleic acid hybridization using specific probes revealed that these cells contain albumin and alpha-fetoprotein messenger RNAs, while hepatocytes from normal and preneoplastic liver contain only albumin messenger RNA. Biliary cells obtained from normal livers do not contain albumin messenger RNA. The large-scale purification and characterization of cell populations from preneoplastic livers is an important step in elucidating the cellular derivation of liver tumors.     

Initiation-promotion-initiation. Induction of neoplastic foci within islands of precancerous liver cells in the rat

The hypothesis that during the promotion phase of carcinogenesis a second rare event leads to a promoter-independent tumour cell was tested in an initiation-promotion-initiation type of experiment. Precancerous (island) cells induced in rat liver by 10 mg/kg N-nitrosodiethylamine given 24 h after partial hepatectomy were promoted by a protocol consisting of 2-acetylaminofluorene/partial hepatectomy. Administration of 25-100 mg/kg N-ethyl-N-nitrosourea served as second initiater. Microscopic foci of neoplastic cells were observed within the precancerous islands 66 days later; no such foci were noted in the appropriate controls. Deficiency of adenosine triphosphatase and glucose-6-phosphatase marker enzymes in the foci was more pronounced than in the surrounding island cells; glycogen storage was decreased and cytoplasmic basophilia slightly increased; gamma-glutamyltranspeptidase staining was negative or decreased with respect to the surrounding island cells, which exhibited a partially positive reaction. We conclude that a secondary change produced by N-ethyl-N-nitrosourea in precancerous island cells leads to focus-forming cells which grow, in the absence of promoter, into foci of neoplastic phenotype. Similar rare, initiation-like events might be involved in the process of tumour promotion in general.     

Comparison of oval cells induced in rat liver by feeding N-2-fluorenylacetamide in a choline-devoid diet and bile duct cells induced by feeding 4,4'-diaminodiphenylmethane

Oval cells and duct-like structures produced in the livers of rats fed N-2-fluorenylacetamide in a choline-devoid diet differ from bile ducts produced after feeding 4,4'-diaminodiphenylmethane. Rapid elevations of serum alpha-fetoprotein (AFP) occur after feeding N-2-fluorenylacetamide in a choline-devoid diet; no elevations of AFP are seen during 4,4'-diaminodiphenylmethane feeding. The duct-like structures associated with oval cells frequently contain AFP and albumin and are faintly delineated by laminin, whereas normal bile ducts and ducts induced by 4,4'-diaminodiphenylmethane do not contain AFP or albumin and are delineated by an intensely staining layer of laminin. Zones of oval cell proliferation label intensely for fibronectin, whereas zones of bile duct proliferation label much less intensely. It is concluded that the "tubuloform degeneration" seen after carcinogen exposure does not necessarily represent differentiation to true bile duct structures and that oval cells may neither derive from nor differentiate into bile ducts. Oval cells have characteristics more like fetal hepatocytes than ductular cells and may represent a "stem cell"-like population with potential for loss of growth control and malignant transformation.     

卵圆细胞在实验性肝癌发生过程中的演变特征

背景与目的:卵圆细胞在肝癌发生过程中的作用至今还不十分明了.本研究拟通过动态的方法观察卵圆细胞在肝癌发生过程中的演变规律,揭示卵圆细胞与肝癌发生之间的关系.方法:构建实验性肝癌的大鼠诱癌模型,运用常规HE染色、免疫组织化学和爱新蓝特殊染色等方法,动态观察卵圆细胞在肝癌发生过程中的演变规律.结果:HE和免疫组化结果显示,在诱癌的第4周即可见散在的卵圆细胞在门管区附近出现.卵圆细胞OV-6免疫染色呈阳性.在诱癌的第8周和第14周,OV-6阳性细胞逐渐增多,并向肝小叶实质内深入,将肝组织分割成假小叶状.到诱癌的第17周和第24周,多个癌灶出现,同时OV-6阳性细胞的总体数量下降,癌灶内可观察到OV-6阳性细胞.爱新蓝特殊染色显示,诱发的肿瘤属混合性肝癌,其中胆管上皮细胞癌爱新蓝染色呈阳性,肝细胞癌染色呈阴性.结论:卵圆细胞在肝癌的发生过程中可能扮演重要的角色.     

Prolonged ornithine decarboxylase induction in regenerating carcinogen-treated liver

A cascade of events leading to hypertrophy has been proposed and implicated in growth regulation in a variety of normal and neoplastic cells and tissues. There is a tightly coupled temporal sequence: (a) cyclic adenosine 3':5'-monophosphate-dependent protein kinase (cAPK) activation; (b) ornithine decarboxylase (ODC) induction; and (c) the accumulation of the organic cation, spermidine, resulting in an increased spermidine/spermine ratio characteristic of both normal and neoplastic growth. The specific activation of type I cAPK has been implicated to ODC induction, and the amounts of type I and type II cAPK alter as a function of growth and transformation. Therefore, we wished to study the alterations in these biochemical parameters as well as that of a putative marker of preneoplastic hepatocytes, gamma-glutamyltranspeptidase, in a rapid multistep hepatocarcinogenesis system. We found a marked and prolonged increase in the cAPK ratio followed by a similar pattern of ODC induction after a single carcinogenic dose of diethylnitrosamine and again in response to partial hepatectomy. Liver foci were detectable within four days of partial hepatectomy in animals that received the entire carcinogen regimen, and the foci contained significant and increasing amounts of gamma-glutamyltranspeptidase activity. The increase in ODC activity was followed closely by an increased spermidine/spermine ratio. Total type I activity in the cytosol decreased most dramatically at the time of foci formation, suggestive of selective activation and turnover. These data suggest that the prolonged activation of cAPK and elevation of ODC may be necessary for hepatocarcinogenesis.     

Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats

An oval cell-enriched population was isolated using two isopyknic centrifugation steps in Percoll gradients from the livers of young adult male rats maintained for 6 to 12 weeks on a choline-deficient diet containing 0.05% DL-ethionine. This cell population equilibrated sharply at densities ranging between 1.07 and 1.08 g/ml, possessed a mean cell diameter in fixed-cell smears of 13.6 micron, and showed viabilities of greater than 95% as judged by trypan blue dye exclusion. Contamination of this population by hepatocytes and Kupffer cells was determined to be less than 1% and between 4 and 14%, respectively. gamma-Glutamyl transpeptidase activity was demonstrated both biochemically and histochemically to be the most constant marker for evaluating the oval cell-enriched population isolated at various times over the 6 to 12 weeks of the choline-deficient/DL-ethionine dietary regimen. In contrast, the percentages of nonhepatocytic cells showing labeling for DNA synthesis and for alpha-fetoprotein were both found to be the highest in the oval cell-enriched population isolated at 6 weeks and lowest in that obtained at 12 weeks of dietary treatment. Furthermore, at 10 to 11 weeks, 19.2% of the nonhepatocytic cells in this population were positive for albumin, while 2.1% were positive for glucose-6-phosphatase activity, indicating some cells to be intermediate in function between the oval cell and the hepatocyte. In comparison, hyperplastic bile ductular epithelial cells in tissue preparations isolated from the livers of rats previously subjected to 13 weeks of chronic feeding of the noncarcinogenic cholestatic agent, 1-naphthyl isothiocyanate, or at 8 to 13 weeks following bile duct ligation were found to be strongly positive for gamma-glutamyl transpeptidase activity, as well as to be positive for alkaline phosphatase activity, but to be essentially negative for glucose-6-phosphatase activity, glycogen content, and albumin production. However, an occasional bile ductular cell in these preparations was found to exhibit a strong cytoplasmic binding of [6,7-3H]estradiol, an indirect measure of alpha-fetoprotein production. Also, a low, but demonstrable amount of DNA synthesis was noted in the bile ductular cells present in these preparations. Furthermore, a viable cell population highly enriched in bile ductular epithelial cells was isolated by isopyknic centrifugation in Percoll following enzymatic dissociation of the hyperplastic tissue preparation from bile duct ligated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Profound postinitiation enhancement by short-term severe methionine, choline, vitamin B12, and folate deficiency of hepatocarcinogenesis in F344 rats given a single low-dose diethylnitrosamine injection

The potential promoting and/or complete carcinogenic activity of a methyl group-deficient (MD) diet lacking methionine, choline, vitamin B12, and folate on liver tumor induction in weanling male F344/NCr rats was examined. Each of 50 rats per group received one injection 20 mg diethylnitrosamine [(DENA) CAS: 55-18-5; N-nitrosodiethylamine]/kg body weight at 4 weeks of age, and then each was maintained on a methyl group-adequate (MA) diet for 52 weeks (groups 2 and 5) or on an MD diet for 15 weeks followed by the MA diet for 37 weeks (group 4). Controls received injections of saline and were maintained on the same two respective diet regimens (groups 1 and 3, respectively). Histologic results from sacrifices at 6, 10, 15, 22, 39, and 52 weeks revealed early development of foci of eosinophilic gamma-glutamyltransferase (GGT)-positive hepatocytes by week 6 in DENA-MD diet-treated rats, with subsequent development of a diffuse hyperplasia of hepatocytes, oval cell proliferation, cholangiofibrosis, nodular cirrhosis, and neoplastic nodule (NN) formation and, at 52 weeks, hepatocellular carcinomas (HCC) in 13 of 15 rats. Similar but significantly fewer lesions were observed at slightly later sacrifice times in the livers of saline-MD diet-treated rats, with development of NN in 5 of 12 rats and an HCC in 1 of 12 rats at 52 weeks. DENA-treated rats on MA diets developed relatively few GGT-positive foci, and none developed any neoplastic lesions. Except for basophilic foci, areas and foci of cellular alteration containing glycogen-rich hepatocytes frequently exhibited diminished uptake of injected iron and decreased glucose-6-phosphatase and ATPase contents focally or throughout. This study indicates that a relatively brief exposure of both untreated and DENA-treated weanling rats to a severely MD diet produces classical preneoplastic and neoplastic lesions in their livers.     

Initiation by bleomycin of hepatocellular foci in the rat.

The antitumor antibiotic, bleomycin, was tested for activity as an initiator of hepatocellular foci and neoplasms in rats. The compound was administered in a single dose via the portal vein 4 h after the proliferative stimulus of a two-thirds partial hepatectomy. Rats were subsequently fed diet containing phenobarbital for up to 41 weeks to promote the development of initiated hepatocytes. Bleomycin-treated livers displayed significantly increased frequencies of basophilic hepatocellular foci and hepatocellular foci which retain glycogen during fasting. Foci that express glutathione-S-transferase (placental form) were not initiated by bleomycin. Hepatocellular neoplasms were infrequently seen in bleomycin-treated livers (5% incidence). The results suggest that oxygen radical-mediated DNA damage may initiate, within populations of proliferating hepatocytes, new lineages of altered hepatocytes that form foci but have low probability of progressing to neoplasms during promotion with phenobarbital.     

Production of monoclonal antibodies to preneoplastic liver cell populations induced by chemical carcinogens in rats and to transplantable Morris hepatomas

Monoclonal antibodies (moabs) to neoplastic and preneoplastic liver cells in rats have been selected to follow cellular changes in the livers during chemical carcinogenesis. The moabs were induced by immunizations of BALB/c mice with four partially purified liver cell preparations: 1) oval cells induced in male Fischer rats fed 0.05% N-2-acetylaminofluorene in a choline deficient diet: 2) preneoplastic gamma-glutamyltranspeptidase positive hepatocytes induced by i.p. injection of diethylnitrosamine into male Fischer rats followed by 0.02% N-2-acetylaminofluorene and partial hepatectomy (Solt-Farber model): 3) sharply dissected neoplastic nodules induced in male Fischer rats by five 2-week cycles of 0.05% N-2-acetylaminofluorene diet: and 4) Morris hepatomas 7777 and 5123 passaged in male Buffalo rats. The hybridomas were screened by enzyme linked immunosorbent assay or by indirect immunofluorescence on composite cryostat sections of fetal and adult rat liver, liver containing neoplastic nodules, and Morris hepatoma 7777. Positive clones were limit diluted and partially characterized by indirect immunofluorescence on cryostat sections of other preneoplastic and neoplastic rat livers as well as normal rat tissues. Two moabs to oval cells, two moabs to hepatocytes, and one moab to hepatomas have been selected for further study.