Elevation of serum alpha-fetoprotein and proliferation of oval cells in the livers of LEC rats

Alpha-fetoprotein (AFP) in the sera of 35 LEC (Long-Evans with a cinnamon-like coat color) rats between 7 and 25 weeks of age was evaluated by enzyme-linked immunosorbent assay (ELISA). Elevation of serum AFP and proliferation of oval cells in the liver were observed in most LEC rats, which suffered from acute hepatitis. On the other hand, the serum AFP level was within the normal range before the onset of hepatitis. Immunohistochemical staining for AFP revealed that some of the proliferating oval cells produced AFP. Morphometric analysis of AFP-positive cells and ELISA for serum AFP demonstrated that there was a statistically significant correlation between the number of AFP-positive cells in the liver and the concentration of AFP in the serum. Histological examination revealed the transition and differentiation of the oval cells to small hepatocytes. These results suggested that the phenomena which occurred in LEC rats suffering from acute hepatitis were similar to those that occurred during the early stage of azo dye hepatocarcinogenesis, although the extent of the oval cell proliferation and the elevation of serum AFP in LEC rats were not as great as those in rats treated with azo dye. This is the first report on a rat strain with proliferation of AFP-producing oval cells during its natural history.     

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.     

Elevation of Serum Alpha-fetoprotein and Proliferation of Oval Cells in the Livers of LEC Rats

Alpha-fetoprotein (AFP) in the sera of 35 LEC (Long-Evans with a cinnamon-like coat color) rats between 7 and 25 weeks of age was evaluated by enzyme-linked immunosorbent assay (ELISA). Elevation of serum AFP and proliferation of oval cells in the liver were observed in most LEC rats, which suffered from acute hepatitis. On the other hand, the serum AFP level was within the normal range before the onset of hepatitis. Immunohistochemical staining for AFP revealed that some of the proliferating oval cells produced AFP, Morphotnetric analysis of AFP-positive cells and ELISA for serum AFP demonstrated that there was a statistically significant correlation between the number of AFP-positive cells in the liver and the concentration of AFP in the serum. Histological examination revealed the transition and differentiation of the oval cells to small hepatocytes. These results suggested that the phenomena which occurred in LEC rats suffering from acute hepatitis were similar to those that occurred during the early stage of azo dye hepatocarcinogenesis, although the extent of the oval cell proliferation and the elevation of serum AFP in LEC rats were not as great as those in rats treated with azo dye. This is the first report on a rat strain with proliferation of AFP-producing oval cells during its natural history.     

Change in the ploidy state of rat liver cells during chemical hepatocarcinogenesis and its relationship to the increased expression of alpha-fetoprotein

The DNA content and ploidy state of cells isolated from the livers of rats exposed to the carcinogen 3'-methyl-4-dimethylaminoazobenzene for 10 and 20 weeks, as determined by flow cytometry, were correlated with the development of oval cells in the livers of treated animals and with serum levels of the oncoprotein alpha-fetoprotein (AFP). The study revealed that there was initially a steady rise in the AFP levels found in the carcinogen treated rats. Associated with this increase was a change in the ploidy pattern of the liver from an approximately equal mixture of tetraploid and diploid cells to a predominantly diploid state. Histologically, there was an increase in the number of oval cells during carcinogen treatment, and when stained immunohistochemically for AFP, these cells were positive. We conclude that the changing state of the diploid and tetraploid cell populations is due to the proliferation of oval cells and that these cells are responsible for the initial increase of serum AFP. The maintenance of the diploid population of cells at later periods of the study is a reflection of the persistence of a new cell type, possibly derived from oval cells. The effect of 3'-methyl-4-dimethylaminoazobenzene was not reversed if the animals were withdrawn from the diet at 10 weeks. In addition, in the cases of hepatocellular carcinoma that were found, a population of cells was detected by flow cytometry that contained altered DNA.     

Early increase of serum alpha-fetoprotein in spontaneous hepatocarcinogenesis in mice.

The concentration of serum alpha-fetoprotein (AFP) was followed in C3H mice having a high incidence of spontaneous liver-cell cancer. No general elevation of serum AFP level with age was seen in mice without tumor. With a single exception, mice bearing hepatocellular carcinomas had increased serum AFP levels. In some mice this increase followed a biphasic course. Mice killed within 1 month of the time when an elevation of serum AFP was first observed had small tumors or no detectable tumor. Premalignant lesions were present in the livers of 11 out of 16 mice that had elevated AFP but no cancer, while only one out of 14 mice with normal AFP had such alterations. Our results strongly suggest that spontaneous hepatocarcinogenesis proceeds through almost the same premalignant lesions as chemically induced carcinogenesis, and that an increase in AFP production occurs early during this process, often preceding macroscopic lesions. Autologous antibodies to AFP were produced in a group of C3H mice by immunization with rat AFP. These anti-AFP antibodies reduced the amount of serum AFP but had no effect on the incidence of spontaneous hepatomas.     

Alpha-fetoprotein and gamma-glutamyltranspeptidase in mice. Effect of Raf gene.

The regulation of the high physiological level of serum alpha-fetoprotein (AFP) in BALB/c/J mice was studied. Serum AFP concentration in 7- to 12-week-old BALB/c/J mice varied from 460 ng/ml up to 2,790 ng/ml and in other strains tested from 10 ng/ml to 400 ng/ml. In contrast to the difference in serum AFP level, activity of the fetal enzyme gamma-glutamyltranspeptidase (GGT) in the liver of BALB/c/J mice was similar to that found in other mice. In all newborn mice, the initially high GGT activity decreased almost 100-fold during the first 10 days. The serum AFP concentration stayed relatively stable during this period. AFP decreased to the adult level between the 10th and 30th days. Partial hepatectomy and administration of carbon tetrachloride increased the serum AFP level in all mice. The elevation of AFP was 10 times higher in BALB/c/J mice than in mice with low basal AFP level. A slightly elevated GGT activity in the liver was observed in only 10 out of 40 mice after carbon tetrachloride treatment, but none of the mice after partial hepatectomy showed an elevation. These results suggest that the gene ("Raf" gene) controlling the reduced decrease of serum AFP level in young BALB/c/J mice enhances the "turning on" of production of AFP in the regenerating liver. This gene does not, however, control the expression of the other carcinofetal liver marker, GGT.     

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.     

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.     

Localization of alpha1-fetoprotein and DNA-synthesis in liver cell populations during experimental hepatocarcinogenesis in rats.

Six-, 12- and 20-week-old rats were fed N-nitrosomorpholine (NNM) at low concentrations (6 mg/kg/day) or high concentrations (20 mg/kg/day) for 6 or 12 weeks. Irrespective of the age of the rats, both NNM schedules resulted in development of hepatomas and during the early stages of hepatoma induction, liver histotoxic patterns depended only on the dose of carcinogen employed. Necrosis of hepatocytes and proliferation of small, oval-shaped cells occurred when high doses of NNM were applied. Parallel to the proliferation of oval-shaped cells, resurgence of alpha₁-fetoprotein (AFP) in rat sera was observed and production of this protein was confined to the oval-shaped cells as shown by immunoperoxidase staining. During proliferation of bile duct epithelium, induced by galactosamine injections, those cells could also stain for AFP, and proliferation of oval-shaped cells concomitant with intracellular AFP staining resulted from restitution of heavily damaged liver. In pulse-chase labelling experiments with ³H-thymidine, oval-shaped cells were seen in continuous development towards hepatocytes, and distinct areas of hyperplastic appearance occurred. Normal hepatocytes and hyperplastic areas did not stain for AFP. Upon low-dose feeding of NNM, no cellular AFP and no serum AFP were detected unless hepatoma cells had developed. At the stage of malignant conversion, distinct AFP-staining nodules were localized which consisted of neoplastic hepatocytes. AFP-staining and non-AFP-staining nodules were seen concomitantly in the same animal. AFP was localized only in neoplastic hepatocytes. Pulse-labelling experiments with [³H]thymidine showed the proliferative character of hepatoma cells from which the AFP-staining population in particular was involved. No correlation was found between the presence of AFP and histological grading of hepatomas. The wide range of both serum AFP levels and their rising rates in individual rats indicated the highly heterogeneous character of the induced hepatomas with respect to AFP production.     

Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.      

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.     

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.     

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.     

{gamma}-Glutamyltranspeptidase-conferred resistance to hydroquinone induced GSH depletion and toxicity in isolated hepatocytes

Hepatocyte resistance against glutathione (GSH) depleting xenobioticswas studied in an in vitro model. Hepatocytes were isolatedfrom carcinogen treated rats that had received phenobarbitalfor three weeks. Isolated cells were incubated in GSH containingbuffer with hydroquinone, which depleted GSH. Cells were thenseeded on collagen coated plates and cultured overnight in completemedium. Attached cells were stained and the proportion of -glutamyltranspeptidase(GGT)-positive cells was counted. It was found that toxicityrelated to GSH depletion increased the proportion of GGTpositivecells from 10–15% up to 40–60%, indicating thatthe toxicity mainly affected GGT-negative cells. GSH added tothe buffer was essential for this effect. It is concluded thatGGT may protect GGT-positive hepatocytes from GSH depletionand toxicity early during liver carcinogenesis.     

Differential cytokeratin and alpha-fetoprotein expression in morphologically distinct epithelial cells emerging at the early stage of rat hepatocarcinogenesis

The various liver cell populations emerging during the transitory reappearance of alpha-fetoprotein (AFP) in serum of 3'-methyl-4-dimethylaminoazobenzene-ingesting rats were analyzed in situ and in vitro on isolated cell preparations in terms of their cytokeratin and AFP expression using single and double indirect immunofluorescence microscopy. A polyclonal guinea pig antibody raised against cow hoof prekeratin, which recognized a Mr 52,000 cytokeratin, was found to react with bile ductular epithelial cells and oval cells but not with hepatocytes. A monoclonal antibody against a Mr 55,000 cytokeratin reacted not only with bile ductular and oval cells but also with hepatocytes. In contrast, a polyclonal antibody against porcine eye lens vimentin reacted with sinusoidal cells and stroma cells. To assess further the heterogeneity of the emerging cell populations, liver cells were isolated after 4 weeks of treatment and fractionated according to cell size and ploidy level into 4 fractions (I to IV) by velocity sedimentation at 1 X g. A cell-type analysis using AFP and albumin as functional markers revealed the presence of AFP-producing cells in Fraction IV at a mean velocity equivalent to that of newborn diploid rat hepatocytes, whereas most of the albumin-producing cells were distributed in Fractions I to III at velocities similar to those of adult tetraploid rat hepatocytes. A similar analysis based on the differential expression of Mr 52,000 and Mr 55,000 cytokeratins and vimentin in bile ductular and other diploid epithelial cells, hepatocytes, and mesenchymal cells showed that large cells in Fractions I to III were tetraploid hepatocytes, whereas viable cells present in Fraction IV were diploid epithelial cells and mesenchymal cells in proportion of 62 and 38%, respectively. These cell populations could be resolved further by changing the sedimentation time. A subsequent examination of the Mr 55,000 cytokeratin-containing diploid epithelial cells in Fraction IV using double immunofluorescence microscopy resolved three cell populations with respect to Mr 52,000 cytokeratin and AFP expression, namely, two cell populations expressing either protein marker and a third one containing both markers. These results suggest a ductular origin of oval cells and a possible relation to immature hepatocytes.     

Low selection of preneoplastic hepatocytes after treatment with the 2-acetylaminofluorene diet-partial hepatectomy regimen in the liver of hepatocarcinogenesis-resistant DRH strain rats

In hepatocarcinogenesis-resistant DRH rats, preneoplastic hepatocytic lesions are smaller than those of usual rats during carcinogenesis. When preneoplastic hepatocytes from DRH and Donryu (original strain of DRH) were reciprocally transplanted into the livers of DRH and Donryu treated with 2-acetylaminofluorene (2-AAF) diet/two-thirds hepatectomy (PH), the Donryu cells formed small colonies within the DRH liver, whereas the DRH cells formed large colonies within the Donryu liver. The DRH liver showed less degree of oval cell proliferation after treatment with 2-AAF and PH, and DRH hepatocytes were more resistant to the growth-inhibitory effect of 2-AAF after PH. Furthermore, DRH hepatocytes were generally resistant to cytotoxicity of hepatotoxins. The tissue environment of the DRH liver, therefore, is less effective for selective growth of preneoplastic hepatocytes during the carcinogen treatment, which is probably a major cause of the hepatocarcinogenesis-resistance in DRH rats.     

Is there a liver stem cell?

The role of a putative liver stem cell in liver regeneration and carcinogenesis is reviewed. There is increasing evidence that there is a liver stem cell that has the capacity to differentiate into parenchymal hepatocytes or into bile ductular cells. These stem cells may be activated to proliferate after severe liver injury or exposure to hepatocarcinogens. They are not activated by moderate liver injury, which is repaired by proliferation of mature hepatocytes. Exposure to most chemical hepatocarcinogens results in proliferation of a small morphologically indistinct cell population termed "oval cells." These cells have been shown to have the capacity to differentiate into hepatocytes or into ductular cells. The origin of these cells appears to be from transition duct cells, but there is also evidence of an even less mature periportal liver stem cell. Study of the development of these cells during carcinogenesis indicates that liver cancer arises from oval cells by aberrant differentiation of stem cells.     

Transforming activity of DNA from rat liver tumors induced by the carcinogen methyl(acetoxymethyl)nitrosamine

Altered c-Ha-ras genes have been frequently detected in the DNA of spontaneous or chemically induced mouse liver tumors. To determine if ras gene mutation is a frequent event during liver carcinogenesis in rats, we examined the transforming activity of DNA from liver tumors that developed in rats injected with methyl(acetoxymethyl)nitrosamine (DMN-OAc) after a partial hepatectomy. Three weeks after the injection of DMN-OAc, rats were fed a diet containing phenobarbital. This carcinogen acts only on replicating liver cells. Six of eight tumor DNAs induced the transformation of NIH 3T3 cells. The transforming activity was stable upon a second round of transfection, and the transformants were tumorigenic in nude mice. Southern blot analysis of transformant DNAs showed that the transforming activity was not due to the acquisition of a ras (Ha, Ki, or N), neu, myc, A-raf, v-raf, erbA, or erbB gene of rat origin. Several transformants' restriction enzyme sensitivity was analyzed, and their activity indicated that similar transforming sequences were present in at least two tumors and that one tumor contained two different transforming sequences. These results suggest that during hepatocarcinogenesis induced in rats by DMN-OAc, alterations in the ras gene family occur infrequently or not at all and that several different genes (which are not homologous to common oncogenes) become activated and are capable of transforming NIH 3T3 cells.     

Carcinohistogenesis and expression of alpha fetoprotein in experimental hepatocarcinoma

In1973,WhenAnthony,etal.inUganda[']werestudyinghumanhepatocirrhosisandhepatocarcinomatheyfirstreportedthatatypicalhyperplasia(AH)ofhepatocyteswasprecancerosisofhepatocellularcarcinoma(HCC).Roncalli,etal.[2]dividedAHintolargehepatocyticandsmallhepatocyticAH,andtheyconsideredthelatterwascloselyrelatedtoformingHCC.In1988,Xu,etal.[3]pointedoutthatHCCoccursfromproliferativenodulesofbasophilicandacidophilichepatocytes.Indeed,carcinohistogenesis,inparticularthechangeofalpha-fetoprotein(AFP)i…