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.

Lectins and immunohistochemistry of colorectal cancer, its recurrences and metastases.

In 31 patients resected specimens from primary colorectal cancers, corresponding liver metastases and local recurrences were investigated for the staining pattern of lectins (PNL, UEA, WGA, HPA, SBA, RCA) and tissue antigens (CEA, SP, ACT) by immunohistochemistry. Comparison of staining patterns showed a loss of marker expression from normal colonic mucosa to colorectal primary carcinomas, and a tendency to marker loss from the primary tumour to liver metastases. However, even a neo-expression of markers not present in the primary tumour could be observed. For clinical use, serum markers observed in patient follow-up may be valuable even where the findings are negative at the time of primary tumour surgery. In contrast to the heterogenous marker map of primary tumours and metastases, comparison of primary and locally recurrent tumour revealed a staining pattern that was almost always identical. This supports the hypothesis that locoregional recurrences develop from remnant cells of the primary tumour left behind at surgery. There is no support for the thesis that locoregional recurrences arise from mucosal changes at the anastomosis or from suture material.

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.

Chromogranin A, neuron-specific enolase and synaptophysin as neuroendocrine cell markers in the diagnosis of tumours of the gastro-entero-pancreatic system.

Neuroendocrine (NE) tumours of the gastro-entero-pancreatic tract were analysed immunohistochemically for the expression of chromogranin A, neuron-specific enolase and synaptophysin. In all cases at least one marker was present and in 17 out of 19 investigated neoplasms, at least one of the three markers could be demonstrated in more than 75% of the NE tumour cells. Monoclonal antibody chromogranin A stained a much higher proportion of NE cells in tumours with hormonal activity than in hormonally inactive ones. Immunostaining of the primary tumour as compared to its respective metastases was almost identical. Thus, chromogranin A, neuron-specific enolase and synaptophysin identify NE tumours and their metastases regardless of their localization and their state of hormonal activity. As 'panendocrine' markers of NE tumours they are of special diagnostic value in NE tumours that do not produce hormones and peptides.

Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen.

BACKGROUND AND METHODS: The role of hepatitis B virus (HBV) in the course of patients with primary liver cancer who are negative for hepatitis B surface antigen has been debated. We used the polymerase chain reaction to evaluate 28 such patients for the presence of DNA and RNA sequences of the virus; 22 of these patients had associated cirrhosis. The patients were from areas with different prevalences of HBV infection (South Africa, Italy, France, and Japan). RESULTS: Antibodies to the surface and core antigens of HBV were detected in 10 of the 23 patients tested. HBV DNA sequences were detected in 17 of the 28 patients, including 8 of the 10 with HBV antibodies and 6 of 13 without HBV serologic markers. HBV RNA molecules were found in four of five tumors tested. CONCLUSIONS: Our investigation indicates that transcriptionally active HBV genomes are present in various geographic areas among patients with liver cancer who are negative for hepatitis B surface antigen. This observation is consistent with an etiologic role for the virus in the development of these tumors.

Mutational analysis of the H-ras oncogene in spontaneous C57BL/6 x C3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens.

The frequency and mutational profile of H-ras gene activation were determined in spontaneous liver tumors of male C57BL/6 x C3H/He mice and in tumors induced with the genotoxic hepatocarcinogen benzidine.2 HCl or the nongenotoxic hepatocarcinogens phenobarbital, chloroform, and ciprofibrate. DNA sequence analysis of the H-ras gene from representative tumors revealed that 32 of 50 (64%) spontaneous tumors and 13 of 22 (59%) benzidine.2 HCl-induced tumors contained a point mutation in codon 61. Tumors induced with the nongenotoxic agents had a much lower frequency of codon 61 mutations, i.e., phenobarbital, 1 of 15 (7%); chloroform, 5 of 24 (21%), and ciprofibrate, 8 of 39 (21%). No mutations were observed at codons 12, 13, and 117 in tumors from any of the groups. Only three base pair substitutions within codon 61 were found. The one most frequently detected in all of the groups was a C.G to A.T transversion at the first nucleotide position, occurring at a 59%, 85%, 100%, 80%, and 88% frequency in the spontaneous tumors and in the tumors induced with benzidine 2.Hcl, phenobarbital, chloroform, and ciprofibrate, respectively. In these same groups an A.T to G.C transition or an A.T to T.A transversion at the second nucleotide position occurred at a frequency of 34%, 8%, 0%, 0%, and 12%, and 6%, 8%, 0%, 20%, and 0%, respectively. The number of tumors carrying an activated H-ras gene in the nongenotoxic treatment groups is within the range that would be expected if those animals had not received any treatment. This indicates that the activation of the H-ras gene in those tumors is probably the result of a spontaneous event. The data suggest that these toxicologically and pharmacologically diverse nongenotoxic hepatocarcinogens increase the frequency of liver tumors but do not induce mutations in the H-ras gene. Instead these agents appear to interact with a population of cells that do not contain an activated H-ras gene. This suggests that the mechanisms of tumor development by these nongenotoxic carcinogens differ at least partially from the mechanisms responsible for the development of spontaneous tumors or those induced by a typical genotoxic agent.

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.

A novel DNA lesion in neoplastic livers of feral fish: 2,6-diamino-4-hydroxy-5-formamidopyrimidine.

DNA from neoplastic hepatic tissues of feral fish environmentally exposed to carcinogens was shown to contain the guanine-derived lesion 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). This modification of DNA appears to arise from the attack of the hydroxyl radical at C-8 of guanine. Concentrations of the lesion ranged from 0.97 to 5.11 nmol/mg DNA. FapyGua was not detected in nonneoplastic tissue and has not been previously reported to occur in the DNA of animal tissues. The present findings support the view that reactive oxygen species damage DNA in living systems and thus may play an important role in the formation of neoplastic tissues.

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.

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.

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.

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.

A clinicopathologic study of primary hepatic carcinoid tumors.

Six cases of primary hepatic carcinoid tumors were studied with combined immunocytochemical and electron microscopic techniques. Positive tumor immunostaining with PHE5, LK2H10, neuron-specific enolase (NSE), serotonin, gastrin, and insulin antibodies was observed. At the ultrastructural level, cytoplasmic dense granules were seen in all the cases tested. This finding supports a putative origin of these carcinoids found in the liver from a pluripotential stem cell. The clinical course and follow-up of these cases suggests that this unusual hepatic neoplasm has a more favorable prognosis than other forms of hepatic cancer.

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.

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).

Study on the relation of Se, Mn, Fe, Sr, Pb, Zn, Cu, and Ca to liver cancer mortality from analysis of scalp hair.

This project made use of Chongming Island, a high prevalence area for liver cancer, with an uneven geographical distribution, to study the relation between trace elements and high liver cancer incidence. A comparative study of Se, Mn, Fe, Sr, Pb, Zn, Cu and Ca contents of scalp hair of normal persons living in areas with different incidences of liver cancer, and a case-control investigation matched with sex and age were made. The selenium level is relatively low compared with other locations in China, which might indicate Se deficiency on the island. Iron and Mn show an obvious difference, indicating that the availability of these elements was less in the high cancer incidence part of the island than in the low incidence part. The hair iron content of patients with liver cancer is clearly lower than that of normal controls. Selenium, Mn and Fe should be taken into consideration in liver cancer prevention research.