Expression of human alpha-actinin in human hepatocellular carcinoma.

Little is known regarding gene expression during hepatocyte transformation. We have isolated an alpha-actinin complementary DNA from a human hepatocellular carcinoma library. This partial 2.4-kilobase complementary DNA has high homology with human placental and chicken nonmuscle alpha-actinins; our isolate contains the entire 3' noncoding region and it is within these sequences where the major differences between the vertebrate alpha-actinin complementary DNAs arise. Northern analysis revealed a 3.5-kilobase transcript in nonmuscle and a smaller 3.0-kilobase species in muscle tissue. Levels of alpha-actinin expression were low in normal liver and we investigated its expression during both hepatocyte proliferation and transformation. We found no increase during rat hepatocyte regeneration up to 24 h following two-thirds hepatectomy. However, high levels of alpha-actinin transcripts were observed in human hepatocellular carcinoma compared to noninvolved adjacent liver. We conclude that the alpha-actinin gene is highly expressed when hepatocytes have assumed the malignant phenotype.

Detection of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver tissue by the polymerase chain reaction.

DNA isolated from formalin-fixed, paraffin-embedded liver tissues from nine patients with hepatocellular carcinoma and six control patients was screened for hepatitis B virus (HBV) DNA with surface (S) and core (C) gene-specific primers by a modification of the polymerase chain reaction--southern blot technique (PCR-SB). PCR-SB results were correlated with histologic, immunohistochemical, and serologic findings. All cases with an established HBV etiology were positive by PCR-SB, as were three cases with negative immunohistochemistry and serology. Often there was selective amplification with one primer set and, in two cases, smaller than expected HBV amplification products suggesting internal deletions. The presence of a potent PCR inhibitor in nucleic acid preparations from tissue blocks that can be removed by Sephadex G-50 chromatography was confirmed. PCR-SB will be a powerful method for the diagnosis and follow-up of patients with HBV infection and may provide new insights into viral hepatocarcinogenesis.

Hepatic neoplasms in aflatoxin B1-treated, congenital duck hepatitis B virus-infected, and virus-free pekin ducks.

To assess the effects of the combination of persistent hepadnavirus infection and chemical carcinogen exposure, aflatoxin B1 (AFB) was administered p.o. for 60 days to congenitally duck hepatitis B virus (DHBV)-infected and virus-free Pekin ducks, starting at 3 days of age, during a 28-month study. Hepatic neoplasia occurred only in AFB-dosed ducks. Hepatocellular carcinomas or biliary carcinomas occurred in 4 of 8 DHBV-infected and 3 of 4 DHBV-free ducks, and hepatocellular adenomas developed in 2 DHBV-infected AFB-dosed ducks that survived 20 months or longer. Altered foci of hepatocytes similar to those observed in chemical carcinogen-dosed rodents, characterized by enlarged eosinophilic hepatocytes or vacuolated cytoplasm, occurred in AFB-dosed ducks. Cells in foci or hepatic neoplasms did not contain histochemically detectable gamma-glutamyltranspeptidase but were distinguished from uninvolved parenchyma by altered glycogen content. Immunohistochemical staining indicated that DHBV core antigen persisted in liver, spleen, pancreas, and, to a lesser extent, kidney of most congenitally infected ducks up to 28 months of age. Hepatic neoplasms contained only patches of hepatocytes were detectable viral antigen. Southern blot analysis of restriction endonuclease-digested neoplastic and normal liver DNA revealed high molecular weight forms of DHBV DNA consistent with integration of viral DNA into the genome of hepatic neoplasms from 3 of 4 DHBV-infected ducks but not nontumorous liver. These findings indicate that AFB is a potent hepatic carcinogen in ducks and that persistent congenital DHBV infection did not contribute significantly to the emergence of hepatic neoplasia in ducks under these conditions.

Unique structure of glycopeptide from alpha-fetoprotein produced in human hepatoma cell line, as determined by 1H-nuclear magnetic resonance spectroscopy.

Determination of alpha-fetoprotein is used in diagnosis of tumors and neural tube defects. A good reliable source of alpha-fetoprotein would be an obvious advantage to the preparation of diagnostic reagents and their standardization. We have recently developed a method for the production of alpha-fetoprotein from a human hepatoma cell line. This method, which is suitable for scaling up, allowed us to produce 40 g of alpha-fetoprotein from culture supernatant liquid through a simple purification procedure. We have previously shown this protein to be identical to alpha-fetoprotein produced from other sources. However, because the presence of different glycoforms has been reported in alpha-fetoprotein preparations, both from human sources and from other species, it was important to establish the type and extent of glycosylation of alpha-fetoprotein prepared by our method. By using 1H-NMR spectroscopy we were able to establish that our product contains a single N-linked biantennary, fully sialylated complex-type oligosaccharide, typical of human hepatomas.

Studies on the polyisoprenoid composition in hepatocellular carcinomas and its correlation with their differentiation.

The levels of cholesterol, ubiquinone and dolichol and the polyprenol composition of dolichol in human hepatocellular carcinomas (hepatomas) with different degrees of differentiation were analyzed and compared with healthy liver tissue. Dolichols were also analyzed in liver metastases. The total level of cholesterol was increased, while the levels of dolichol and ubiquinone were decreased in all hepatomas, but no correlation between these levels and the degree of differentiation of the hepatomas could be observed. The level of dolichol decreased more in the hepatomas than in the liver metastases. The dolichol fraction from hepatomas with a low degree of differentiation contained higher relative amounts of short polyisoprenols (D17) and slightly lower relative amounts of D21 compared with healthy liver tissue, metastatic liver tumors or hepatomas with a high degree of differentiation. The significance of the lipid values found in the different groups is discussed.

Frequency of hepatic HBV-DNA in patients with cirrhosis and hepatocellular carcinoma: relation to serum HBV markers.

As part of a larger study designed to investigate the interaction of factors such as cirrhosis and hepatitis B virus infection as aetiological agents in the development of hepatocellular carcinoma, we investigated the status of hepatic HBV-DNA sequences in 156 cirrhotic patients. Forty-one were HBsAg seropositive and 18 (44%) of these had HBV-DNA sequences detectable in their livers. There are also 26 subjects who showed markers of a previous HBV infection (anti-HBs/anti-HBc), only one (4%) of whom had demonstrable hepatic HBV-DNA sequences. No sequences were found in any of the remaining 89 patients who were seronegative for all markers. Thus, liver HBV-DNA was only detected in the presence of a serum marker, usually HBsAg.

Immunohistochemical demonstration of liver fatty acid-binding protein in human hepatocellular malignancies.

Twenty-three hepatoblastomas of childhood, sixty-two adult hepatocellular carcinomas, and two hepatic sarcomas were examined immunohistochemically with the use of a polyclonal antibody against rat liver fatty acid-binding protein (L-FABP), which cross-reacts to human L-FABP. All the hepatoblastomas and half of the hepatic cell carcinomas contained L-FABP immunoreactive tumour cells, whereas two hepatic sarcomas were negative. The overall frequency of immunostained tumour cells was 43.5 per cent in hepatoblastomas and 18.6 per cent in hepatocellular carcinomas, respectively. Histologically well-differentiated areas contained more numerous immunopositive cells than undifferentiated or immature ones. These results indicate that L-FABP immunoreactivity is a new candidate for a tumour cell marker in hepatic cell malignancies, although its biological role has not been elucidated.

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells.

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.

The capsule surrounding primary liver tumors: wherefrom its prognostic significance?

Primary liver neoplasms in cirrhotic and non-cirrhotic patients were studied by electron microscopy and immunohistochemical methods for extracellular matrix (ECM) antigens. A capsule of variable thickness was present in many expanding hepatocellular carcinomas, while it was absent in those of small size, and either fragmented or absent in the infiltrating ones. In the capsules of early onset, fibronectin was the most frequent stromal glycoprotein. In the completely formed capsular structures, fibronectin, type-V collagen and laminin were the most common macromolecules seen. No differences were evident in the pattern of ECM macromolecules in the capsules surrounding hepatocellular carcinomas compared with those found in benign lesions, such as hepatocellular adenomas or focal nodular hyperplasia. The possibility is discussed that the capsule could be a result of the following sequence: tissue with expansive (not infiltrative) growth-mechanical compression-ischemic necrosis of surrounding tissues-repair process with ECM deposition.

Isolation and sequence of the human farnesyl pyrophosphate synthetase cDNA. Coordinate regulation of the mRNAs for farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and 3-hydroxy-3-methylglutaryl coenzyme A synthase by phorbol ester.

We report the isolation and nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA, an enzyme in the cholesterogenic pathway. Partial cDNAs for the human farnesyl pyrophosphate synthetase were isolated by screening human hepatoma (HepG2) and placental cDNA libraries with the rat liver cDNA for farnesyl pyrophosphate synthetase as a probe. Anchored polymerase chain reaction was used to isolate the 5'-end of the cDNA. The nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA has high identity (86%) to the rat liver cDNA. Treatment of the human monocytic leukemia cell line THP-1 with phorbol esters led to 2--7-fold increases in mRNA concentrations for the three cholesterogenic enzymes, farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and HMG-CoA synthase within 5 h. Immunoprecipitation of radiolabeled cells demonstrated that there was a corresponding increase in the rate of synthesis of all three proteins. The addition of cycloheximide to cells also led to increases in the mRNA concentrations of the three enzymes. Treatment of cells with phorbol esters and cycloheximide resulted in superinduction of all three mRNAs; HMG-CoA synthase mRNA levels increased 35-fold, farnesyl pyrophosphate synthetase 17-fold, and HMG-CoA reductase 16-fold 5 h after treatment. The mRNA levels returned to pretreatment levels by 20 h. Cells were also preincubated in the presence of a lipoprotein-deficient fraction of serum plus mevinolin to induce the levels of the three mRNAs. Addition of phorbol esters and cycloheximide to these derepressed cells led to further increases in the mRNA levels for all three enzymes. These results are consistent with the hypothesis that THP-1 cells contain a short-lived negative transcription factor which regulates transcription of the FPP synthetase, HMG-CoA reductase, and HMG-CoA synthase genes. Phorbol esters also regulate these same genes, presumably by modifying a common negative transcription factor and/or by inducing a positive transcription factor(s).

Cytologic, ultrastructural and immunologic features of intracytoplasmic hyaline bodies in fine needle aspirates of hepatocellular carcinoma.

Intracytoplasmic hyaline bodies in malignant cells from an aspirate of a liver mass are suggestive of hepatocellular carcinoma. Such inclusions were studied by light and electron microscopy and by immunocytochemistry in fine needle aspirates from five cases of hepatocellular carcinoma. Seen by light microscopy, the inclusions were round or ovoid and were surrounded by a prominent halo. By both light and electron microscopic immunocytochemistry, the hyaline bodies showed negative staining for alpha-fetoprotein, alpha-1-antitrypsin and cytokeratin. Ultrastructurally, they were not membrane bound and were composed of filamentous, finely granular material, resembling the early stages of Mallory bodies.

Altered glycosaminoglycan composition in reactive and neoplastic human liver.

We have investigated the glycosaminoglycan composition of normal human liver, focal nodular hyperplasia, hepatic adenoma, and hepatocellular carcinoma. Uronic acid increased about 4 fold in the benign and reactive lesions, and greater than 7 fold in the carcinoma. Whereas in focal nodular hyperplasia and adenoma dermatan sulfate was the predominant glycosaminoglycan, in hepatocellular carcinoma chondroitin sulfate was the predominant species; it increased 24 fold over normal liver and 3-5 fold over all the other tissues. HPLC analysis of chondroitinase ABC or AC digests showed a 58 fold increase in Delta-Di-OS disaccharides in hepatocellular carcinoma, indicating significant undersulfation of chondroitin sulfate. Surprisingly, the normal-appearing liver surrounding the carcinoma showed glycosaminoglycan changes similar to adenoma and nodular hyperplasia. These results thus indicate that specific glycosaminoglycan changes occur in hepatocellular carcinoma, and suggest for the first time that proteoglycan metabolism is also altered in the non-cirrhotic, hepatic parenchyma adjacent to liver carcinoma.

Accumulation of gangliosides with N-acetylneuraminosyl(alpha 2-6)lactosamine structure in primary human hepatoma.

Gangliosides of hepatomas have been analyzed by using a monoclonal antibody directed to N-acetylneuraminosyl(alpha 2-6)lactoneotetraosylceramide (sialyl(alpha 2-6)paragloboside), which was prepared by injecting the monosialoganglioside fraction of human meconium into BALB/c mice. The monoclonal antibody, named MSG-15, was found to bind sialyl(alpha 2-6)paragloboside, but it failed to react with other gangliosides, including N-acetylneuraminosyl(alpha 2-3)lactoneotetraosylceramide (sialyl (alpha 2-3)paragloboside) and "Ii"-type gangliosides. MSG-15 was found to recognize NeuAc alpha 2-6Gal beta structure of the ganglioside. Gangliosides obtained from human hepatomas were analyzed by immunostaining on high-performance thin-layer chromatography plates using the monoclonal antibody MSG-15. All primary hepatoma samples used in this study (nine samples) were found to contain sialyl(alpha 2-6)paragloboside, which accounted for 13-31% of the monosialoganglioside fractions in the hepatomas. Furthermore, MSG-15 recognized several monosialogangliosides in addition to sialyl(alpha 2-6)paragloboside. These gangliosides apparently also contain a terminal NeuAc alpha 2-6Gal beta structure. Other ganglioside fractions obtained from hepatoma and meconium were immunostained on thin layer chromatography plates with MSG-15. Additionally, another monoclonal antibody (H-11), which recognizes terminal lactosamine structure, was used to immunostain these fractions after sialidase treatment. Bands stained with both monoclonal antibodies showed similar mobilities to each other in the di- and trisialoganglioside fractions as well as monosialoganglioside fraction. In control liver, GM3 ganglioside accounted for 92% of monosialoganglioside fraction, and sialyl(alpha 2-6)paragloboside accounted for less than 1% of the fraction. Immunohistochemical study by using MSG-15 in tissue sections from hepatocellular carcinoma and normal liver tissues demonstrated that only hepatocellular carcinoma cells gave a positive reaction. These results suggest that the biosynthetic pathway of gangliosides containing NeuAc alpha 2-6Gal beta 1-4GlcNAc beta structure is activated in hepatoma cells.

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for alpha-fetoprotein from hepatocellular carcinoma.

Alpha-fetoprotein in sera from patients with hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak), which passed through the column without adsorption, was found in both healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma. The second and third peaks were reactive with L. culinaris agglutinin and found only in patients with hepatocellular carcinoma. For alpha-fetoprotein in the second and third peaks, a novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) was developed. Alpha-fetoprotein in test serum was reacted with dinitrophenyl affinity-purified anti-alpha-fetoprotein IgG, and the complex formed was trapped onto affinity-purified (antidinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. The polystyrene balls were washed to eliminate substance(s) other than alpha-fetoprotein in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine. The eluted complex containing alpha-fetoprotein in the second and third peaks was trapped onto L. culinaris agglutinin-coated polystyrene balls and reacted with affinity-purified anti-alpha-fetoprotein Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene balls was assayed by fluorimetry. The maximal volume of serum that could be used without interference was 20 microliters, which was 100-fold larger than that in the previous enzyme immunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Hamster liver chromatin immunospecific non-histone proteins.

Three antisera were prepared against non-histone protein classes named NHCP1, NHCP2 and dehistonized chromatin (with different affinity to DNA) from hamster liver. Two main antigenic bands of MW 17,000 and 36,000 were specific in the NHCP1 fraction and one antigen of MW 56,000 was specific for the NHCP2 fraction from nuclease-sensitive and especially nuclease-resistant chromatin. Other NHCP2 liver antigens of MW 22,000, 27,000, 30,000, 36,000, 37,000, 40,000, 45,000, 46,000, 51,000, 98,000 and 100,000 were present only in nuclease-resistant chromatin of hamster liver. Immunologically specific hamster liver non-histone proteins within the NHCP1 and NHCP2 fractions seem to be restricted to nuclease-resistant chromatin fraction of this tissue. The above mentioned liver specific antigens are absent or present only at trace amounts in analogous Kirkman-Robbins hepatoma fractions.

[Fibrolamellar liver carcinoma]. / Das fibrolamelläre Leberkarzinom.

Four fibrolamellar liver carcinomas were surgically removed and were postoperatively examined. Three patients are alive roughly three years from surgery, and there are no signs of imminent recurrence, while the fourth case was diagnosed only two months back. The carcinomas had developed in non-cirrhotic livers which also produced negative responses to serological tests for hepatitis B. In flow cytometry, DNA indices were indicative of diploidy in two cases and aneuploidy in the other two. The highest DNA index value was recorded from the smallest tumour which could be assigned to the category of "minute HCC". No correlation was found to exist either between age, sex, and DNA index. Positive CEA reaction was immunohistochemically recorded from few tumour cells, whereas negative AFP responses were exhibited by all four tumours. Appearance of AAT in tumour cells was detected in three cases. High degree of differentiation, similarity between tumour and liver cells, and oncocytoid nature of cells were revealed by optical light and electron microscopy. This high degree of differentiation was additionally confirmed by two factors: glucose-6-phosphatase activity was preserved in all four tumours, adenosinetriphosphatase activity was histochemically detectable from certain points of the tumour cell membrane. Gamma-glutamyl-transpeptidase activity, too, was very strongly pronounced in all tumour cells, which, however, cannot be interpreted as a sign of differentiation. Membrane-bordered "dense-core" granules were visible in few tumour cells in two cases. Intensive granular serotonin reactions were immunohistochemically recorded from the majority of tumour cells in the same cases. Our histochemical and ultrastructural parameters have produced clear-cut evidence to the hepatocyte nature of FLC cells. Yet, the presence of secretory granules and positive serotonin reaction might possibly support the assumption that the FLC originates from those pluripotent cells of the liver which may develop in two directions, depending on the individual case, to become either hepatocytes or neurosecretory cells.

Absence of growth-hormone receptor in hepatocellular carcinoma and cirrhotic liver.

Hepatocellular carcinoma is a hormone-sensitive tumor. It has been reported that thyroxine and prolactin significantly stimulated hepatoma growth, whereas growth hormone failed to do so. To learn whether the growth hormone receptor is present in human hepatocellular carcinoma, we used radioreceptor assays in samples of human hepatocellular carcinoma. The liver tissues adjacent to hepatocellular carcinoma (mostly cirrhotic) and control liver tissues (taken during various surgical procedures) were also studied. The study results showed that the affinity constant and capacity of high-affinity growth hormone receptor in normal liver tissues were 6.6 +/- 2.0 x 10(10) mol/L-1 (mean +/- SE, n = 7) and 20.7 +/- 11.5 fmol/mg protein, respectively. The affinity constant and capacity of low-affinity growth hormone receptor in normal liver tissues were 8.9 +/- 3.3 x 10(9) mol/L-1 and 64.7 +/- 32.1 fmol/mg protein, respectively. The absence of growth hormone receptor in human hepatocellular carcinoma and cirrhotic liver samples may explain the absence of growth hormone in the stimulation of hepatoma growth and the decrease of somatomedin levels in cirrhosis.

Isolation and characterization of a hepatoma-associated abnormal (des-gamma-carboxy)prothrombin.

Hepatoma-associated abnormal (des-gamma-carboxy)prothrombin (HAPT) is a newly described tumor marker for hepatocellular carcinoma. HAPT has been measured in the blood of patients with hepatoma by immunoassay but has not been isolated or characterized. This paper describes the quantitative isolation and structural characterization of HAPT. Purified HAPT has the same molecular weight, amino-terminal sequence, and amino acid analysis (exclusive of gamma-carboxyglutamic acid) as native prothrombin and abnormal prothrombin isolated from the blood of patients taking sodium warfarin. HAPT is heterogeneous in gamma-carboxyglutamic acid (Gla) content with an average of 5 Gla residues/molecule compared to 10 Gla residues for native prothrombin and 2 Gla residues for abnormal prothrombin. HAPT is glycosylated in a manner equivalent to that for native prothrombin when evaluated by a concanavalin A-binding assay. These studies find structural identity between HAPT and abnormal prothrombin. Therefore the findings support the hypothesis that HAPT results from an acquired defect in the posttranslational vitamin K-dependent carboxylation of the prothrombin precursor and not an intrinsic defect in the prothrombin precursor molecule.