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.     

Antigenic relationship between oval cells and a subpopulation of hepatic foci, nodules, and carcinomas induced by the "resistant hepatocyte" model system

Although proliferation of small ductular-like cells, designated oval cells, is often observed during the early stages of chemically induced hepatocarcinogenesis, their role during the carcinogenic process remains controversial. To investigate the possibility that oval cells may give rise to preneoplastic lesions that ultimately progress to hepatocellular carcinomas, we have carried out phenotypic analysis with a panel of monoclonal antibodies to determine if there is an antigenic relationship between oval cells and hepatic foci, nodules, and tumors induced by the resistant hepatocyte model system. In this model, rats are given a single dose (200 mg/kg) of diethylnitrosamine, followed by a brief exposure to 2-acetylaminofluorene and a partial hepatectomy. We found that approximately 10% of the early focal lesions observed 28 days after diethylnitrosamine expressed either one or both of the oval cell antigens designated OC.2 and OV-6. By 28 weeks after diethylnitrosamine, 16 of 16 hepatic nodules heterogeneously expressed OV-6 whereas 5-10% of the persistent nodules contained scattered small hepatocyte-like cells that expressed OC.2. Examination of resistant hepatocyte-induced primary hepatocellular carcinomas with an expanded panel of monoclonal antibodies demonstrated that most cells comprising 29 of 29 tumors expressed OV-6 and that 15-20% of the OV-6-positive tumors contained subpopulations of cells also expressing 3 additional oval cell antigens, OC.2, OC.3, and OV-1. All of the tumors examined expressed normal levels of the hepatocyte antigens, H.1 and HBD.1, and had dramatically reduced levels of H.2, H.4, and cell CAM 105 but showed elevated levels of the transferrin receptor, gamma-glutamyltranspeptidase, and the normal hepatocyte antigen, H.5. In conclusion, our findings demonstrate an antigenic relationship between oval cells and a subpopulation of hepatic foci, nodules, and tumors in the resistant hepatocyte model, suggesting that at least some primary tumors may be derived from oval cells in this model system.     

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.     

Proliferation of diploid hepatocytes and nonparenchymal (oval) cells during rat-liver regeneration in the presence of 2-acetylaminofluorene

Treatment of rats with the carcinogen 2-acetylaminofluorene (2-AAF) during liver regeneration (Solt-Farber protocol) induced a selective outgrowth of diploid, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes (3-4 times increase) as well as of nonparenchymal (oval) liver cells. After cessation of treatment the oval cells rapidly disappeared, while the population of diploid, GGT-positive hepatocytes declined more slowly over the subsequent ten weeks. In animals pretreated with the initiating carcinogen diethylnitrosamine (DEN) a large fraction of the diploid, GGT-positive hepatocytes persisted. The results differ from those obtained with our standard, sequential treatment protocol (2-AAF given after completed regeneration), where there is no hyperproliferation of oval cells and where GGT-positive hepatocytes are found only in DEN-pretreated animals (Saeter et al, Carcinogenesis 9: 581-587, 1988). Different experimental models of liver carcinogenesis may thus present different patterns of liver cell proliferation, which should be taken into account when general hypotheses on the cellular origin of liver cancer are proposed.     

Gamma-glutamyltransferase in putative premalignant liver cell populations during hepatocarcinogenesis.

The activity of gamma-glutamyltransferase, as measured quantitatively and by histochemical staining, was studied in different cell populations during the induction of liver cancer with 2-acetylaminofluorene (2-AAF) or diethylnitrosamine and compared with findings in fetal and in intact and regenerating adult liver. The enzyme activity is 20-fold higher in 12-week nodules than in control livers and 30-fold higher in 20-week nodules than in controls. A similar 30-fold increase in activity relative to control is present in hepatomas, induced by either 2-AAF or diethylnitrosamine, and in fetal hepatocytes. The enzyme shows increases in activity in foci of very early putative preneoplastic hepatocytes induced by a single dose of diethylnitrosamine and selected by low doses of 2-AAF plus partial hepatectomy. By 7 days, the foci show a 4-fold increase in enzyme activity, and by 3 weeks they are 40-fold higher than in the control liver. Histochemically, the foci are strongly positive for gamma-glutamyltransferase, especially in the bile canaliculi. By 21 days, the ductular (oval) cells induced by 2-AAF have disappeared. When stained for the enzyme activity, the foci stand out clearly against the negative background of the liver, allowing easy quantitation. It appears that gamma-glutamyltransferase is a useful marker for preneoplastic hepatocytes.     

In vivo differentiation of rat liver oval cells into hepatocytes

The Solt-Farber protocol, in the absence of an initiating agent, was used to examine the precursor-product relationship between oval cells and hepatocytes in rat liver. The animals were administered 2-acetylaminofluorene (AAF) by gavage for 2 wk combined with partial hepatectomy 1 wk after administering AAF Two dose levels of AAF were used: 9- and 21-mg total dose for animals in Groups I and II, respectively. [3H]Thymidine was administered i.p. to one-half of the animals at Day 6 post-partial hepatectomy. Animals were sacrificed 7, 9, 11, and 13 days after surgery. Only oval cells became labeled on Day 7 in both groups. On Day 9 both labeled oval cells and labeled basophilic hepatocytes were present in Group I, whereas in Group II only oval cells remained labeled. On Days 11 and 13 both oval cells and basophilic hepatocytes were labeled in both groups. The total amount of radioactivity in Group II livers remained the same on Day 9 when only labeled oval cells were present and on Days 11 and 13 when both labeled oval cells and labeled basophilic hepatocytes were present. The calculated half-life for basophilic hepatocytes was about 50 h. The differentiation of oval cells into basophilic hepatocytes was delayed in Group II as compared to Group I, and the higher dose of AAF also induced the formation of both intestinal metaplasia and bile duct formation. In situ hybridization with an alpha-fetoprotein probe showed a strong expression in groups of typical oval cells and in cells arranged in duct-like structures. In addition a transient expression of AFP was also observed in the areas of basophilic hepatocytes 9 to 11 days after partial hepatectomy. Administration of AAF decreased the level of albumin mRNA in preexisting hepatocytes and caused a significant decrease of serum albumin. In contrast, oval cells showed a strong albumin expression, and basophilic hepatocytes formed islands of albumin-expressing cells. Oval cells and the foci of early basophilic hepatocytes lacked glucose-6-phosphatase activity. At Day 13 significant numbers of basophilic hepatocytes were positive for glucose-6-phosphatase. Oval cells were strongly gamma-glutamyltranspeptidase positive, whereas the foci of basophilic hepatocytes were negative for gamma-glutamyltranspeptidase. Only occasionally were transiently gamma-glutamyltranspeptidase-positive hepatocytes observed in basophilic foci. In summary our data indicate that oval cells can differentiate to hepatocytes and may have an important physiological function as a source of major serum proteins when hepatocytes are unable to synthesize these proteins.     

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.     

Diagnostic utility of the HepPar1 antibody to differentiate hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration samples

BACKGROUND: The cytopathologic distinction between hepatocellular carcinoma (HCC) and metastatic carcinoma (MC) in the liver can be problematic, especially in patients with poorly differentiated HCC, in whom a trabecular pattern, bile production, and Mallory bodies may not be apparent on small fine-needle aspiration (FNA) samples. HepPar1 (OCH1E5) is a monoclonal antibody specifically developed to react with hepatocytes. It rarely reacts with bile duct and nonparenchymal liver cells. METHODS: FNA samples (cell blocks) from 75 liver tumors were selected. These included 50 moderate to poorly differentiated HCC cases, 5 cholangiocarcinoma (CC) cases, and 20 MC cases (4 from the breast, 4 from the stomach, 4 from the pancreas, and 8 from the colon). Immunohistochemical staining for HepPar1 was performed to differentiate HCC from MC. RESULTS: The HepPar1 antibody was positive in 50 of 50 HCC cases (100%). The positivity was cytoplasmic, diffuse, and granular. All 5 cases of CC were found to be negative (0%). Although focal positivity within tumor cells was noted in one case, cytologically these were entrapped normal hepatocytes between the tumor cells. In addition, 3 of 20 MC cases (15%) also were positive for HepPar1. All three cases originated from gastric primary tumors and exhibited diffuse, granular cytoplasmic staining. CONCLUSIONS: The results of the current study demonstrate that HepPar1 is an effective marker with which to differentiate between HCC and CC and/or MC. HepPar1 was found to demonstrate 100% positivity in HCC cases, compared with 0% and 15% positivity, respectively, in CC and MC cases. In addition, HepPar1 is extremely helpful in limited tissue samples from FNA. Although 15% of the MC cases in the current study were found to be positive, with the help of clinical correlation and other immunohistochemical stains a definite diagnosis could be rendered. Potential pitfalls include residual benign hepatocyte staining within a non-HCC malignancy, as was observed in one of the CC cases in the current study.     

Effect of phenobarbital on the gamma-glutamyltranspeptidase activity and the remodeling of nodules induced by the initiation-selection model

The effect of subsequent administration of phenobarbital on the gamma-glutamyltranspeptidase (GGT) activity and on the remodeling of nodules induced by the Solt-Farber procedure was examined in rats. GGT-nodules were initiated by diethylnitrosamine (DENA) followed by selection with 2-acetylaminofluorene (2AAF) in the diet and a partial hepatectomy (PH). Phenobarbital (500 ppm in the drinking water), administered to rats that were previously treated according to the Solt-Farber procedure, (1) increased the persistence of the GGT-nodules, (2) increased the percentage of the liver occupied by GGT positive cells, (3) increased the area of GGT activity per nodule and (4) increased the incidence of eosinophilic lesions. Subsequent treatment with phenobarbital did not alter the incidence of either GGT-nodules or hepatocellular carcinoma. Thus, phenobarbital increased the GGT activity of nodules induced by the Solt-Farber procedure and slowed both the loss of GGT activity by these nodules and their concurrent remodeling, but had no effect on the occurrence of cancer.     

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.     

Sparse Distribution of Hepatocyte Growth Factor-producing Cells inside Hepatocellular Foci in Rats Treated with Hepatocarcinogens

The distribution of hepatocyte growth factor (HGF)–synthesizing cells in rat liver during development of glutathione S –transfcrasc P form (GST–P)–positive nodules after diethylnitrosamine initiation followed hy promotion with 2–acetylaminofluorene plus partial hepatectomy (PH) was investigated using in situ hybridization, HGF–produclng cells were iioii–parenchymal in nature, and were suspected to be mainly of Kupffer type. They were mostly located outside GST–P–positive lesions, in the surrounding parenchyma. In the oval cell proliferation phase 1 week after PH, they increased and they were mainly localized around the portal triads. It is concluded that HGF is directly involved in an endogenous paracrine growth pathway controlling proliferation in oval cells and in normal, hut not GST–P–positive, hepatocytes.     

Presence of alpha-fetoprotein-positive cells in hepatocellular foci and microcarcinomas induced by single injections of diethylnitrosamine in infant mice

Single injections of diethylnitrosamine (5 and 50 micrograms/g body weight) in male C57BL/6J X C3HeB/FeJ F1 mice when they were 15 days old resulted in the induction of RNA-rich hepatocellular foci and nodules that contained alpha-fetoprotein (AFP)-positive hepatocytes after 20 and 28 weeks. The focal lesions were composed of 1- to 2-cell-thick plates of hepatocytes or closely packed clusters of cells, but they did not show the histological patterns that are diagnostic of trabecular hepatocellular carcinoma. AFP-positive hepatocytes were found in almost one-fourth (14 of 60) of the foci and nodules in a serially sectioned block of liver from a mouse given one injection of 50 micrograms/g body weight diethylnitrosamine and killed at 28 weeks. In general, the presence or absence of AFP-positive cells correlated with the size of the foci and nodules. All six nodules with diameters greater than 1.5 mm contained AFP-positive cells, while all 12 foci smaller than 0.24 mm in diameter were negative for AFP. However, among the 42 foci that were intermediate in size, there were 8 AFP-positive foci, the sizes of which appeared rather randomly distributed among the negative foci. Reactive changes in hepatocytes could be ruled out as a cause of the induction of AFP because the foci first appeared many weeks after the administration of diethylnitrosamine in these mice. Since bile ductules or oval cells, which occasionally appeared in these foci, were lacking entirely in AFP and since ductules are absent from the early-appearing and smallest foci, we believe that in this model the AFP-positive foci arise only from hepatocytes. The presence of AFP in the focal lesions and in tumor thrombi that extended from them into hepatic vein branches supports the hypothesis that some foci undergo progression to invasive microcarcinomas and that these in turn are precursors of late-appearing (after 1 year) metastasizing trabecular hepatocellular carcinomas.     

Microfluorometric determination of DNA adducts in immunofluorescent-stained liver tissue from rats fed 2-acetylaminofluorene

The intensities of immunofluorescence in nuclei stained by an antiserum specific for the DNA adduct N-deoxyguanosin(8-yl)aminofluorene (dG-8-AF), were quantified by microfluorometry in frozen liver sections from male Fischer rats fed 2-acetylaminofluorene (AAF). Results of previous studies demonstrated that dG-8-AF is the predominant adduct (80-100%) formed in livers of rats fed AAF continuously, and that nuclei of hepatocytes and bile duct epithelial cells in rats fed AAF exhibit an adduct-specific immunofluorescence. In the present investigation, nuclear staining for dG-8-AF was quantified by microfluorometry in liver sections from male Fischer rats fed 0.02% AAF continuously for 2, 4, 8, 12, 16, 20, and 28 days. Microfluorometric determinations of the intensities of nuclear immunofluorescence staining within periportal, midzonal, and centrilobular hepatocytes and bile duct epithelial cells revealed that levels of the dG-8-AF adduct increased in these cells during AAF feeding, reaching a plateau by 12 days. However, significant differences were detected in dG-8-AF levels within cells of each lobular area. Nuclei of periportal hepatocytes exhibited the most intense immunofluorescence, nuclei of centrilobular hepatocytes and bile duct epithelial cells emitted the least intense fluorescence, and nuclei of midzonal hepatocytes exhibited an intermediate fluorescence intensity. Quantitation of whole-liver levels of the dG-8-AF adduct by RIA, after extraction of DNA, also revealed that adduct accumulation reached a plateau by 12 days of AAF feeding. Thus, similar profiles of adduct accumulation were obtained by microfluorometric analysis of immunofluorescence staining within frozen liver sections, and by RIA analysis of DNA extracted from whole livers. The periportal concentration of DNA adducts in livers of rats continuously fed a carcinogenic dose of AAF may be an important early event in AAF-induced liver tumorigenesis.     

Expression of Lewis blood group antigens in cancerous and non-cancerous liver

The expression of Lewis blood group antigens, Lewisa, Lewisb, Lewisx and Lewisy, in 40 non-cancerous livers and in 20 hepatocellular carcinomas (HCC), 5 cholangiocarcinomas (CC), and 6 combined hepatocellular and cholangiocarcinomas (Comb) was studied by immunohistochemistry using monoclonal antibodies. Although normal hepatocytes did not express any of the four Lewis antigens, Lewisy expression was detected in some hepatocytes at the periphery of pseudolobules in cirrhotic liver. While Lewisx in bile duct epithelial cells was undetectable in normal livers, it was detected in some cases with non-cancerous liver diseases. Lewisx and Lewisy were detected in 30% of HCC and 100% of CC and Comb. Non-cancerous bile duct epithelial cells in almost all instances were stained strongly by anti-Lewisa and/or anti-Lewisb antibody. Proliferating bile ductules within Glisson's sheath and pseudolobules could be clearly detected by Lewisa and Lewisb staining. Lewisa and Lewisb were never detected in non-cancerous hepatocytes, and were only rarely detected in HCC, but were expressed in most cases of CC and Comb. These results suggested that Lewisa and Lewisb are useful markers for differentiation towards biliary epithelial cells in the liver, and that Lewisx and Lewisy expression might be associated with states of increased or altered cell proliferation.     

Immunofluorescent study on alpha-fetoprotein-producing cells in the early stage of 3'-methyl-4-dimethylaminoazobenzene carcinogenesis.

The study was carried out to identify alpha-fetoprotein (AFP)-producing cells in the hepatic tissue by immunofluorescent antibody techniques during the early stage of 3'-methyl-4-dimethylaminoazobenzene ingestion. After 1 to 3 weeks, cells fluorescent to AFP were undetectable in cholangiolar cells ("oval cells") and also in degenerated megalocytic hepatocytes. After 4 to 7 weeks AFP appeared in rat sera, and "transitional cells" and small hepatocytes proliferated markedly in the periportal areas of hepatic lobules. AFP was exclusively detected in the majority of the transitional cells and a small portion of the small hepatocytes. Some fluorescent cells appeared in small groups, and others were randomly distributed in the periportal areas. The typical oval cells and the megalocytic hepatocytes were not fluorescent. When AFP in sera became undetectable, the regenerated hepatocytes matured considerably and were not brightly fluorescent. In the hepatic tissue, where AFP-producing cells were observed by fluorescent antibody technique, hematopoietic cells were frequently observed but they were not fluorescent.     

Distribution of alpha-fetoprotein- and albumin-containing cells in the livers of Fischer rats fed four cycles of N-2-fluorenylacetamide.

Immunofluorescent localization of alpha-fetoprotein (AFP)- and albumin-containing cells was determined in the livers of Fischer rats fed 0.05% N-2-acetylfluorenamide for four 2-weeks-on, 1-week-off cycles. After this exposure multiple changes in the liver include over 1000 neoplastic nodules/liver, as well as extensive production of so-called oval cells and focal zones of atypical hepatocellular hyperplasia. Approximately 1% of the oval cells contain AFP, and about half of the zones of atypical hyperplasia include cells that contain AFP, but none of the neoplastic nodules or normal hepatocytes have any AFP-containing cells. Since up to 60% of the hepatocellular carcinomas developing from this regimen will predictably produce AFP, it is tentatively concluded that hepatocellular carcinoma may arise not only from "premalignant" neoplastic nodules but also from oval cells or the atpyical differentiation of hepatocytes.     

Initiation and selection of resistant hepatocyte nodules in rats given the pyrrolizidine alkaloids lasiocarpine and senecionine

The biological mechanisms by which pyrrolizidine alkaloids contribute to initiation and nodule selection (promotion) steps in hepatic carcinogenesis were studied in male Fischer 344 rats. Lasiocarpine at single or double dosages (up to 80 mumol/kg) delayed hepatic regeneration for at least 8 weeks after partial hepatectomy (PH). This regimen of lasiocarpine and PH had a strong selective influence on the growth of gamma-glutamyltranspeptidase (gamma-GT)-positive hepatocyte nodules in rats previously initiated with diethylnitrosamine. However, both lasiocarpine (up to 80 mumol/kg) and senecionine (up to 160 mumol/kg) were inactive as initiators of gamma-GT-positive nodules in rats exposed to a similar selection regimen consisting of 2-acetylaminofluorene and PH. When lasiocarpine or senecionine was given 12 h after PH, very few nodules were initiated. Lasiocarpine pretreatments reduced the initiating activity of diethylnitrosamine and N-nitrosomethylurea in rats subsequently selected with 2-acetylaminofluorene and PH. Resistant nodules selected with lasiocarpine had the typical resistant nodule phenotype (positive for gamma-GT and epoxide hydrolase) and also lacked pyrrolizidine alkaloid-induced megalocytosis. Lasiocarpine treatment also resulted in small regenerative nodular proliferations of hepatocytes that were distinct from resistant nodules because they were negative for gamma-GT and epoxide hydrolase and unrelated to diethylnitrosamine pretreatments. These studies suggest that the hepatocarcinogenicity of pyrrolizidine alkaloids can be better explained by their strong selection (promotion) influence on initiated hepatocytes, rather than by their very weak initiating activity.     

Concomitant and isolated expression of TGF-alpha and EGF-R in human hepatoma cells supports the hypothesis of autocrine,paracrine, and endocrine growth of human hepatoma

Single and double immunohistochemical staining for transforming growth factor (TGF)-alpha and epidermal growth factor-receptor (EGF-R) was done in order to identify the localization of TGF-alpha and EGF-R in human hepatocellular carcinoma (HCC). Single immunohistochemical staining for TGF-alpha showed immunoreactivity in the cytoplasm of hepatoma cells in 22 of 30 cases of HCC. The localization of TGF-alpha was heterogeneous from HCC cells to HCC cells. In the surrounding regenerative nodules, the hepatocytes were mildly to moderately positive for TGF-alpha. The proliferating bile ductules and peripheral nerves were also immunopositive for TGF-alpha. Single immunohistochemical staining for EGF-R demonstrated a linear localization of EGF-R along the cell membrane of the HCC cells in 21 of the 30 cases of HCC. In the regenerative nodules, the hepatocytes also showed linear staining along the cell membrane. Double staining for TGF-alpha and EGF-R in 12 cases of HCC showed a concurrent localization of TGF-alpha and EGF-R in some hepatoma cells and isolated localization of the two substances of other HCC cells. These combinations either abruptly moved around or intermingled with each other. These immonohistochemical results thus support the theory of an autocrine, paracrine, and endocrine mechanism of TGF-alpha and EGF-R on the proliferation of human hepatocellular carcinoma. © 1995 Wiley-Liss, Inc.