Cellular immunolocalization of alpha-fetoprotein in rat liver

Increased synthesis of alpha-fetoprotein (AFP) was induced in rat liver by the administration of 3'-methyl-4-dimethyl-aminoazobenzene. The indirect immunoperoxidase technique was used to detect AFP. Cellular localization of AFP was studied using a number of different fixation procedures. Serial sections stained with immunoglobulin served to determine the extent of diffusion of serum proteins into liver cells during fixation. Background staining was minimized when Lillie's neutral buffered formalin plus acetic acid was used as the fixative. After 3'-methyl-4-dimethylaminoazobenzene ingestion, bile duct cell proliferation occurred. The serum AFP was positive in all rats after 17 days on the diet. In rats with AFP-positive sera the immunohistochemical reaction in mature hepatocytes was positive while bile duct cells and small hepatocytes were negative for AFP.     

Immunohistochemical study of alpha-fetoprotein in the postnatal ontogeny of mice with a varying sensitivity to liver carcinogens]

The ontogenesis of alpha-fetoprotein (AFP) was studied in the C57BL, CC57BR, C3H/He, A/He and DD mice during the first 4 weeks of life. The AFP hepatocytes were determined by indirect immunofluorescent staining of liver cells isolated with sodium tetraphenyl borate; the content of AFP in blood was determined by the method of rado immunodiffusion. The methods utilized allowed to obtain the quantitative characteristics of the dynamics of AFP-positive cells in the liver and the content of AFP in the blood. In the newborn mice over 90% hepatocytes contain AFP, the intensity of luminescence being heterogenous. The number of bright-liminescent cells equals to 50% during the first day of life rapidly decreases and none of them are found by 9--11 days. The number of average- and weak-luminescent hepatocytes does not decrease during the first 10 days, but then gradually decreases and none of them are found by 23 days in the CC57BR and by 27 days in the DD and A/He mice. A comparison of the dynamics of AFP-positive cells in the liver and the content of AFP in the blood has shown that the bright-luminescent hepatocytes are the main producent of this protein in the early postnatal ontogenesis.     

25 years of the study of alpha-fetoprotein

alpha-Fetoprotein (AFP) is an embryonic serum protein. Only traces of AFP are present in blood of adult man or animal, but its level substantially increases during liver regeneration and in cases of primary liver carcinoma or embryonic carcinomas. Different aspects of AFP studies (localization of its synthesis, structure and functions, genetic control of its synthesis and its use for cancer diagnostics) are reviewed. The main consideration is given to the cellular mechanisms of regulation of AFP synthesis. Hypothesis of "structural repression of AFP" is substantiated. According to it formation of liver trabeculae leads to suppression of AFP synthesis in hepatocytes, whereas escape of hepatocyte from trabecula results in activation of AFP synthesis. A new experimental model is described: AFP synthesis is activated in primary hepatocyte culture and is suppressed during three-dimensional growth of hepatocytes in collagen gel or mixed culture with nonparenchymal liver cells.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Liver structure as a possible factor in the regulation of alpha-fetoprotein synthesis]

AFP-containing hepatocytes were shown to lose the membrane antigen, localized in the region of bile capillaries, due to the mouse liver regeneration induced by CCl4 and paracetamol poisoning. Both in the liver regeneration and early postnatal ontogenesis the cessation of the AFP synthesis in hepatocytes coincides with the appearance of the bile capillary antigen on the cell surface. It is suggested that the AFP synthesis cessation is the result of the arrangement of cell contacts characteristic of the definitive liver balk.     

"Contact inhibition" of alpha-fetoprotein synthesis and junctional communication in adult mouse hepatocyte culture

Synthesis of oncofetal serum protein alpha-fetoprotein (AFP) may be reexpressed in adult differentiated mouse hepatocytes both in regenerating liver and in primary monolayer culture of intact adult liver. We have found that appearance of AFP in these cultures was strongly correlated with the loss of junctional communication between hepatocytes as tested by the dye transfer method. When in hepatocyte culture the gradient of cell density was formed, and the cells in the center of the dense monolayer retained an epithelial morphology and junctional communication and were AFP-negative during 5 days of culture. At the periphery of the monolayer hepatocytes lost junctional communication by the third day of cultivation. They acquired fibroblast-like morphology, formed multilayered sheets, and started to produce AFP. These findings suggest that reexpression of AFP synthesis may be regulated through a process related to "contact inhibition" and junctional communication might play an important role in the phenomenon.     

Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.     

alpha 1-Fetoprotein production during the hepatocyte growth cycle of developing rat liver.

The relation of AFP production to DNA synthesis was investigated in newborn rat liver and in primary cultures of fetal rat hepatocytes, by combining immunoperoxidase AFP localization and autoradiography after ³H-thymidine labelling. The vast majority of AFP-positive hepatocytes did not incorporate ³H-thymidine after ≤4-h isotope pulses, suggesting that in the developing liver, essentially no production of AFP occurs in S, G₂ or M phases of the hepatocyte cell life cycle. Serial or continuous thymidine labelling experiments further indicated that post-mitotic hepatocytes constitute a sizable fraction of AFP-producing cells.     

Hepatic lineages isolated from developing rat liver show different ways of maturation

Immunocytochemical analysis revealed that different hepatic cell types exist during liver development: (i). cells co-expressing the stem-cell marker Thy1 and the hepatic lineage marker CK-18 and (ii). cells only expressing CK-18 (hepatoblasts). In this study we separated the different hepatic cells and analyzed gene-expression and phenotype. Fetal rat livers were digested by collagenase solution. OX43- and OX44-positive hematopoietic cells were depleted and Thy1-positive cells were enriched using Magnetic cell sorting. The different cell compartments were analyzed by RT-PCR and immunocytochemistry for Thy1, CK-18, AFP, and albumin. Hepatoblasts expressed albumin at all times and AFP in the early stages. Thy1-enriched cells expressed CK-18 at all times, albumin in the early, and AFP in the late stages. Thy1-positive cells from fetal livers express liver specific genes. The data suggest that Thy1-positive hepatic cells develop towards hepatic stem cells, and hepatoblasts develop towards mature hepatocytes of the adult liver.     

Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury

Restoration of centrolobular injury induced by carbon tetrachloride (CCl4), when hepatocyte proliferation is inhibited by treatment with N-2-acetylaminofluorene (AAF), is accomplished by proliferation of ductular progenitor cells, that arise intraportally and extend into the liver lobule. This pattern contrasts to the restitutive proliferation of hepatocytes when AAF is not administered, and the proliferation of non-ductular periportal oval cells follows periportal necrosis induced by allyl alcohol. The expanding ducts stain for alphafetoprotein (AFP), OV-6, pan-cytokeratin (CKPan), and laminin. The neoductular proliferation is accompanied by fibronectin-positive Kupffer cells and desmin-positive stellate (Ito) cells, which may play critical roles not only in controlling proliferation and differentiation of ductular progenitor cells, but also in reestablishing hepatic cord structure. When AAF is discontinued 7 days after injury, clusters of small hepatocytes appear next to the neoductules. Some of these small hepatocytes, as well as some larger hepatocytes adjacent to the ducts, stain for AFP and for carbamoylphosphate synthetase I (CPS-I), suggesting that the ductular progenitor cells may differentiate into hepatocytes when AAF is withdrawn. The restitutive process is facilitated by clearing of the central necrotic zone by infiltrating macrophages and co-migration of mature hepatocytes, with Kupffer cells and stellate cells, into the necrotic zone.     

Localisation and synthesis of α-fetoprotein in the rabbit

Summary The cellular location and sites of synthesis of -fetoprotein (AFP) in the foetal, neonatal and maternal rabbit, were studied by the fluorescent antibody technique and by culturing tissuesin vitro with labelled amino acids. AFP was found to be localised intracellularly within liver hepatocytes and yolk sac endoderm of the foetus, and within the maternal uterine epithelium. Analysis of extracts of the cultured tissues for incorporation of radioactivity into serum proteins separated by polyacrylamide gel electrophoresis or analysed by autoradiography of immuno-precipitation lines, confirmed that the foetal liver and yolk sac splanchnopleur were the principal sites of primary synthesis of AFP. Localisation of AFP in the uterine epithelium and other foetal organs was consistent with a secondary derivation from the uterine fluid or from the blood circulation. These findings are discussed in relationship to findings in man and other mammals.Supported by an award from the Medical Research Council to whom grateful acknowledgement is made.     

In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes.     

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

Synthesis of alpha-fetoprotein (AFP) and cell proliferation in regenerating livers of NMRI mice after partial hepatectomy. An immunohistochemical and autoradiographic study with 3H-thymidine

To get more information concerning the relation between cell proliferation and the synthesis of alpha-fetoprotein (AFP), liver regeneration and AFP production was followed simultaneously in short intervals in NMRI mice after two-thirds hepatectomy using autoradiographic and immunohistochemical methods. The results indicate that AFP synthesis is not linked closely to cell proliferation in the sense that each proliferating hepatocyte synthesizes AFP during a definite phase of the cell cycle. However, AFP production, as measured by the index of anti-AFP positive hepatocytes, occurs in a very small population of parenchymal cells when the bulk of the hepatocytes has just passed the S-phase and is considered to be in the G2-, M or early G1-phase of the cell cycle when the cells are still in an undifferentiate state.     

Occurrence of alpha-fetoprotein-containing hepatocytes in human embryos and fetuses: an immunohistochemical study using the light and ultrastructural immunoperoxidase methods

The occurrence of alpha-fetoprotein (AFP)-containing hepatocytes in 12 human embryos and fetuses ranging from 30-32 days of estimated ovulation age (Streeter's horizon XIV) to 16-17 weeks of estimated menstrual age was investigated using the direct (horseradish peroxidase (HRP)-labeled anti-human AFP horse IgG(Fab')2) and/or indirect immunoperoxidase methods. Under light microscopy, the AFP-containing hepatocytes were successfully demonstrated in paraffin-embedded liver tissues fixed with 4% paraformaldehyde(PFA)-picric acid solution containing 0.5% glutaraldehyde (GA). As a result of the studies of the human fetal livers at different developmental stages, only a small number of AFP-containing hepatocytes were initially identified immunohistochemically at the stage of Streeter's horizon XIX. Ultrastructural immunohistochemistry (the block-staining method using anti-human AFP horse IgG(Fab')2) revealed that the immunoreactive products against AFP were demonstrated on the ribosomal granules of the rough endoplasmic reticulum (rER), outer nuclear envelopes, free ribosomes, and Golgi's apparatus, and also in the cisternal lumina of the ER. No reactive products were noted in the nuclei or mitochondria. Our observations confirmed the presence of AFP-producing ability of the hepatocytes and the ultrastructural localization of the sites of protein synthesis in the early stage of development of human livers. Furthermore, we describe the extreme usefulness of the block-staining method for demonstrating the subcellular localization of AFP using HRP-labeled reduced anti-AFP antisera on liver tissues fixed with 4% PFA-picric acid solution containing 0.5% GA.