Differential mechanisms of increased alpha 1-fetoprotein production in rats following carbon tetrachloride injury and partial hepatectomy.

Possible differences in the mechanisms of increased alpha1-fetoprotein (AFP) production following carbon tetrachloride (CCl4) intoxication and partial hepatectomy were studied with 5-week-old rats at the time of sacrifice. The maximum level of serum AFP reached in 4 days after a single dose of CCl4 was much higher than that after partial hepatectomy, although the incorporation of [3H]thymidine into liver DNA increased nearly to the same extent by either of these treatments. In the remnant after partial hepatectomy, the DNA synthesis that was further accelerated by treatment with a lower dose of thioacetamide was not associated with any further increases of serum AFP levels. However, CCl4 given to partially hepatectomized rats had an additive effect on increased AFP levels. The increases of serum AFP concentrations in CCl4-injured rats had an additive effect on increased AFP levels. The increases of serum AFP concentrations in CCl4-injured rats were depressed by Mitomycin C given in vivo, whereas the increases in partially hepatectomized rats were not. Treatment with 8-azaguanine inhibited both increase of serum AFP levels, although the inhibition was much less or was insignificant in partially hepatectomized rats. These results suggest that existence of different underlying mechanisms of the increased AFP production for the two experimental conditions.

Hormonal control of alpha-fetoprotein secretion in human hepatoma cell lines proliferating in chemically defined medium.

The regulation of alpha-fetoprotein (AFP) secretion and growth rate by various hormones in established human hepatoma (HuH-7, PLC/PRF/5, huH-1, huH-4, and KIM-1/c-4) and hepatoblastoma (HUH-6 Clone 5) cell lines was studied. These 6 cell lines replicated continuously in a chemically defined medium and secreted 84 ng (HuH-7) to 23 pg (huH-4) AFP per 24 h per 1 X 10(4) cells into the culture medium. The addition of insulin increased the growth rate of all examined cell lines and partially inhibited the AFP secretion in those cell lines except KIM-1/c-4, while the addition of dexamethasone inhibited the growth and stimulated the AFP secretion in all of the cell lines. The addition of 3,3',5-triiodothyronine inhibited the growth of all cell lines; however, different effects on the AFP secretion were observed depending on the cell lines used. Obviously, the AFP secretion was unrelated to the change in growth rate. When dexamethasone and N6-O2-dibutyryl cyclic AMP were added together, the AFP secretion was further stimulated. On the other hand, when dexamethasone and insulin were added simultaneously, the dexamethasone-mediated stimulation of AFP secretions was diminished. The data indicated that the regulatory mechanisms of AFP secretion by the hormones in the established human hepatoma and hepatoblastoma cell lines cannot be deduced according to the results of one cell line.

Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.

A human hepatoma cell line, HuH-7, which was established from a hepatocellular carcinoma, was found to replicate continuously in a chemically defined medium when the medium was supplemented with Na2SeO3. The cells grew better in this medium than in serum-containing medium without any adaptation period. Other established human hepatoma and hepatoblastoma cell lines, HuH-6 cl-5, PLC/PRF/5, huH-1, and huH-4, also grew in the defined medium. Although HLEC-1 cells failed to proliferate continuously with Na2SeO3 alone, they grew if a cell-free conditioned medium from HuH-7 cells was added to the medium. These cell lines, except the HLEC-1 cell line, produced the following human plasma proteins among those examined: albumin, prealbumin, alpha 1-antitrypsin, ceruloplasmin, fibrinogen, fibronectin, haptoglobin, hemopexin, beta-lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, Complement Components 3 and 4, and alpha 1-fetoprotein. Beside plasma proteins, the media from HuH-7, HuH-6 cl-5, PLC/PRF/5, and huH-1 contained anti-carcinoembryonic antigen-reactive proteins, and those from PLC/PRF/5, huH-1, and huH-4 medium contained hepatitis B surface antigen. These proteins were detected during periods of serial cultivation over 9 months under the above culture conditions. The hepatoma cell lines grown in the fully defined synthetic medium may provide a new approach for investigating the growth and metabolism of human hepatoma cells in vitro.

Profiles of carbohydrate-metabolizing enzymes in human hepatocellular carcinomas and preneoplastic livers.

Activities of key carbohydrate-metabolizing enzymes in biopsied human tissues of hepatocellular carcinoma and related conditions were determined by established methods. Among the enzymes analyzed, fetal-type liver enzymes (low-Km hexokinase, glucose 6-phosphate dehydrogenase, and pyruvate kinase-M2) showed increased activities, and adult-type liver enzymes [glucose 6-phosphatase, fructose 1,6-bisphosphatase, high-Km hexokinase (or glucokinase), and pyruvate kinase-L] showed decreased activities, resulting in undifferentiated enzyme patterns not only in fetal livers and hepatocellular carcinomas but also in livers of acute and chronic hepatitis and liver cirrhosis with or without tumors. Hepatocellular carcinomas showed a general tendency of having greater enzyme deviations than hepatitic and cirrhotic livers. The extent of the enzyme deviation in hepatocellular carcinomas varied considerably from one enzyme to another for each tumor tissue as compared with that in the benign liver diseases. Thus, the phenotypic heterogeneity was important for discriminating between the neoplastic and inflammatory changes in differentiation markers. The enzyme patterns of tumors and their corresponding host cirrhotic livers were unrelated, suggesting that the cirrhotic liver has a significance as preneoplastic state only in terms of having a high incidence of evolving hepatocellular carcinoma.

Sugar chains of human cord serum alpha-fetoprotein: characteristics of N-linked sugar chains of glycoproteins produced in human liver and hepatocellular carcinomas.

Human serum alpha-fetoprotein (AFP) is elevated in not only hepatocellular carcinoma (HCC) but also benign liver diseases. AFP produced in HCC and benign liver diseases was separated into several isoforms corresponding to different sugar chain structures by several types of lectin affinity electrophoresis, and the HCC-specific AFP isoform was discriminated from those of benign liver diseases. Because a small amount of HCC-specific AFP isoform was detected in cord serum AFP, the whole sugar chain structures of human cord serum AFP were determined, as follows: Neu5Ac alpha 2-->Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2 -->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4R1 and R2, Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2-->6Gal beta 1 -->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4R1 and R2, and Neu5Ac alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2 -->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4R1 and R2 in the ratio of 81.6:8.9:9.5. R1 and R2 denote GlcNAc beta 1-->4GlcNAcOT (subscript OT represents an NaB3H4-reduced oligosaccharide) and GlcNAc beta 1-->4(Fuc alpha 1-->6)GlcNAcOT, respectively, and the ratio between R1 and R2 in the respective fractions was approximately 19:1. In contrast, the sugar chain structure of HCC highly specific AFP isoform was found to comprise a monosialyl-biantennary sugar chain with additional fucosylation of the proximal N-acetylglucosamine. Fucosylation of AFP produced in fetal liver increased in inverse proportion to the gestation period, in weeks, indicating that fucosylation of AFP in HCC may be related to the dedifferentiation of human hepatocytes through malignant transformation.

A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepatocellular carcinoma.

Lectin-affinity electrophoretic separation of serum alpha-fetoprotein (AFP) was carried out using AFP Differentiation Kits, which used Lens culinaris agglutinin-A (Kit L) and erythroagglutinating phytohemagglutinin (Kit P). Separated AFP bands were detected with a sensitive antibody-affinity blotting technique and determined quantitatively by densitometry, and the results were expressed as percentages of the intensity of total AFP bands. Sera from 424 patients with acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, and extrahepatic tumors were assayed for proportion of AFP present as Lens culinaris agglutinin-A-reactive AFP (AFP-L3) and erythroagglutinating phytohemagglutinin-reactive AFPs (AFP-P4+P5). From the maximum Youden indices determined, cutoff levels were set at 15% for both AFP-L3 and AFP-P4+P5 to discriminate between patients with chronic hepatitis and liver cirrhosis and patients with hepatocellular carcinoma. AFP-L3 and AFP-P4+P5 showed sensitivities of 55.3 and 61.0% at specificities of 93.9% and 82.3%, respectively. Thirty-eight % of tumors that measured less than 20 mm in diameter were positive for AFP-L3 and AFP-P4+P5. AFP-L3 exceeded the cutoff level of 15% 4.0 +/- 4.9 months before detection of hepatocellular carcinomas by imaging techniques with a sensitivity of 48% and a specificity of 81%. Thus, these tests are useful for the early detection of hepatocellular carcinomas in patients with hepatitis or liver cirrhosis.

A tetrazolium method for staining peroxidase labels in blotting assays.

A sensitive staining method of horseradish peroxidase-labeled immunoglobulins on nitrocellulose membrane was established by employing a reaction chain leading to formazan formation with phenol as a substrate of peroxidase and NADH as a hydrogen donor to reduce nitro blue tetrazolium. Higher concentrations of NADH relative to phenol were necessary to increase the intensity of staining and to ensure a wide dose-response range of color production with respect to the applied enzyme activities. By an optimized tetrazolium method in combination with antibody-affinity blotting, as low as 4 ng/ml alpha-fetoprotein was detected and 3-4-fold greater color intensities in a working assay range as compared with those of existing methods were obtained. The present technique of peroxidase staining may prove to have a wide application for the enzyme immunoassay using blotting modalities.

Allomyrina dichotoma lectin-nonreactive alpha-fetoprotein in hepatocellular carcinoma and other tumors: comparison with Ricinus communis agglutinin-I.

Allomyrina dichotoma lectin (allo A) with a specificity to beta-D-galactose was used to fractionate human alpha-fetoprotein (AFP) by affinity electrophoresis. AFP from cord sera and serum of a patient with fulminant hepatitis showed single bands with a high affinity for allo A. Some patients with hepatocellular carcinoma and patients with gastric cancer and yolk sac tumor had two additional AFP bands, one weakly reactive and the other nonreactive with allo A. Patterns of AFP bands obtained with Ricinus communis agglutinin-I (RCA-I) and erythroagglutinating phytohemagglutinin from Phaseolus vulgaris were entirely different from those obtained with allo A. Of the two common bands reactive with RCA-I, the weakly reactive one was relatively intense in some malignant patients and the strongly reactive one was detected in patients with extrahepatic tumors. Thus, affinity electrophoresis with those lectins provides a potentially useful adjunct for the discrimination between benign and malignant conditions with increased serum levels of AFP.

Tissue of origin of elevated alpha-fetoprotein in ataxia-telangiectasia.

Although only a single gene exists for alpha-fetoprotein (AFP), differential glycosylation generates several different forms and these are associated with different tissues of origin, namely, liver, gastrointestinal tract, and yolk sac. This microheterogeneity of serum AFP was studied in seven patients with ataxia-telangiectasia (AT) in order to determine the tissue of origin of their elevated AFP levels. Concanavalin A (Con A), Lens culinaris agglutinin A (LCA-A), and erythroagglutinating phytohemagglutinin (E-PHA) affinity electrophoresis and antibody-affinity blotting were used to fractionate AFP. It was found that serum AFP in AT patients was composed mainly of Con-A band 2 (AFP-C2), LCA-A band 1 (AFP-L1), and E-PHA band 2 (AFP-P2). This profile of AFP species in AT patients is similar to those seen in neonates and patients with chronic hepatitis, but clearly different from AFP originating in hepatocellular carcinoma, gastric carcinoma, and yolk sac tumour cells. These data are compatible with a hepatic origin for the elevated AFP in AT patients. Since no evidence exists for ongoing liver damage in these patients, we suggest that the AFP gene in the AT liver may be under aberrant transcriptional control, perhaps secondary to a defect of DNA regulatory proteins which are necessary for hepatic maturation.

Two distinct cell lines derived from a human osteosarcoma.

Two cell lines were established from a human osteosarcoma transplanted into athymic nude mice after the second (O9N2) and fifth passages (HuO9). Both cell lines expressed 1,25(OH)2D3-responsive alkaline phosphatase activity and produced tumors in the dorsum of nude mice that were histologically similar to the original tumor. However, the morphological and growth characteristics of the two cell lines differed. O9N2 cells were large and polygonal, whereas HuO9 cells showed spindle shapes. HuO9 cells had a higher growth rate and saturation density than O9N2 cells. The c-myc oncogene was amplified 4- to 8-fold in HuO9 cells but not in O9N2 cells. Both cell lines had a homozygous internal deletion, lacking the 7.4-kb HindIII fragment in the Rb gene. The results suggest the importance of the c-myc oncogene in the growth and morphological control of human osteosarcoma cells and of the Rb gene in the pathogenesis of the tumor.

D-Glucose anomeric preference of hexokinases in higher animals.

The D-glucose anomeric preference of hexokinases isolated from rat liver, brain, and skeletal muscle, and bovine retina was studied using the glucose-6-phosphate dehydrogenase-NADP system. The ratios of maximum phosphorylation rates of beta-D-glucose to those of alpha-D-glucose were 1.33, 1.46, and 1.54 for hexokinase types I, II, and III from rat liver, 1.45 and 1.63 for type I from rat brain and bovine retina, 1.53 for type II from rat skeletal muscle, and 0.55 (when determined at 5 mM) for type IV (glucokinase) from rat liver, respectively.

Lectin affinity electrophoresis in a yolk sac tumour in the vagina with yolk sac tumour-type glycoform of alpha fetoprotein.

AIMS: To investigate a potential diagnostic use of alpha fetoprotein (alpha FP) isoform analysis by lectin affinity electrophoresis to distinguish between endodermal sinus tumours arising in the vagina in infants from those at other sites. METHODS: alpha FP in the serum of a patient with a vaginal endodermal sinus tumour was analysed for its isoforms by lectin affinity electrophoresis. The isoforms were compared with that of cord serum, sera of hepatoid adenocarcinoma of the uterus, and endodermal sinus tumour of the ovary. RESULTS: The isoforms of alpha FP obtained by lectin affinity electrophoresis in the serum of the patient with vaginal endodermal sinus tumour differed from the isoforms of alpha FP in the cord serum of normal neonates, and sera of patients with hepatoid adenocarcinoma of the uterus or endodermal sinus tumour of the ovary. CONCLUSIONS: Endodermal sinus tumour arising in the vagina could be distinguished from that in the ovary by the lectin affinity electrophoresis, and a potential diagnostic use of alpha FP isoform analysis by the lectin affinity electrophoresis for the detection of the endodermal sinus tumour in infants was demonstrated.

Microheterogeneity of rat alpha-fetoprotein.

A purified and homogeneous preparation of rat AFP, as judged by both electrophoresis on Cellogel and immunoelectrophoresis, was separated into two components, AFPa and AFPb, by disc electrophoresis on 7% polyacrylamide gel. These two components had a definite difference in electrostatic net charge and gave only a single band on SDS-electrophoresis. Immunological reactivity or electrophoretic separation or mobility of the two components could be altered by treatment with either sulfhydryl inhibitors or reducing agents but not by treatment with protein denaturants. Electrophoresis of neuraminidase-treated AFP on 5% polyacrylamide gel yielded clearly separable, slower moving four to six and finally two components depending on the time of incubation with neuraminidase. The time-dependent conversion of faster into slower migrating components of both AFPa and AFPb upon neuraminidase treatment was confirmed by reelectrophoresis of separated and similarly treated AFPa and AFPb. Two bands of sialized or desialized AFP were also observed on isoelectric focusing. Both AFPa and AFPb treated with and without neuraminidase gave single fused precipitin lines against the antiserum in Ouchterlony double-diffusion analysis. On the basis of the changes in electrophoretic mobilities of the intermediates following neuraminidase treatment, AFPa and AFPb were estimated to have at least 2.5 and 4.5 molecules of sialic acid per molecule, respectively.

Distinct molecular species of human alpha-fetoprotein due to differential affinities to lectins.

Resolution of human alpha-fetoprotein (AFP) into four distinct molecular species was demonstrated by a combination of two affinity chromatographies with crossed-immuno-affino-electrophoresis (CIAE) using concanavalin A (Con A) and Lens culinaris hemagglutinin (LcH)-A and LcH-B as affinity media. Of the four AFPs, AFP1 had no affinity for Con A, LcH-A, or LcH-B; AFP2 showed a high affinity for Con A, a low affinity for LcH-A, and an intermediate affinity for LcH-B (or a low affinity, depending on the lot of LcH-B preparations used); AFP3 revealed strong affinities for all of the three lectins; and AFP4, a trace component of hepatoma AFP in the present study, showed no interaction with Con A, but a definite interaction with LcH-A or LcH-B. These results were based on the determination of dissociation constants (Kd) of AFP-lectin complex by CIAE on isolated preparations of the three major hepatoma AFPs. These AFPs had identical electrophoretic mobilities of 0.86-0.87 (relative to human albumin) in the absence of lectins. The calculated mobilities of AFP2 and AFP3 were both reduced to 0.50-0.58 by saturation with lectins, but these two AFPs were clearly separated by 1 mg/ml LcH-A or LcH-B because of their large differences in Kd.

Age and sex-dependent alterations of serum amylase and isoamylase levels in normal human adults.

Total amylase and salivary- and pancreatic-type isoamylase levels were assayed in sera from 606 apparently healthy adults of different sex and age groups. There were significant differences in both total amylase and isoamylase levels, depending on age and sex, one of the characteristics being that levels of these three enzymes were significantly higher in the elderly group in both men and women than in other age groups. Another feature was that all of these enzyme levels were significantly greater in women in the third and fourth decade than in men. Age and sex differences should be taken into consideration in the evaluation of mild hyperamylasemia.

Hexokinase isozyme pattern in CCl(4)-injured rat liver.

Activities of hexokinase isozymes in carbon tetrachloride (CCl(4))-injured rat liver were determined quantitatively by DEAE-cellulose column chromatography and compared with those of regenerating liver, fetal liver and ascites hepatoma cells (AH 130). The CCl(4)-injured liver revealed an isozyme distribution with predominant Types I, II and III (3.2, 8.8 and 6.8 times higher than the control values, respectively) and with undetectable activity of Type IV hexokinase (glucokinase). Although the isozyme pattern generally resembled that of fetal liver or hepatoma cells, the relatively high activity of hexokinase Type III in CCl(4) treatment characterizes the pattern of hexokinase isozyme in acue liver damage.

Ectopic production of a salivary type amylase by adenocarcinoma cells: demonstration by a culture technique.

Characterization of an elevated amylase activity in ascitic fluid obtained from a patient with carcinomatous peritonitis is described; ninety-one percent of the increased amylase activity in the fluid was of salivary type and the remainder of pancreatic type, when studied by ion-exchange chromatography. Culture of ascitic cells successfully demonstrated morphologically characterized tumor cells surviving for at least 31 days. During that period, significant amylase activities were detected in the culture media, and the isozyme pattern was a single band whose electrophoretic mobility corresponded to salivary amylase. The data obtained indicate that the ascites amylase of salivary type was produced ectopically by the tumor cells.

Cathodic isozyme of serum creatine kinase in a case of stomach cancer complicated by disseminated intravascular coagulation.

A rare isozyme of serum creatine kinase (CK) migrating cathodic to CK-MM on electrophoresis was found in a 30-year-old male with stomach cancer complicated by disseminated intravascular coagulation leading to massive upper gastrointestinal bleeding and marked anemia. Serum CK activity rose to a maximum of 374 U/l without detectable CK-MB isoenzyme. The patient was also characterized by a marked increase in serum lactate dehydrogenase (all isozymes elevated) and by preferential leakage of mitochondrial aspartate aminotransferase and glutamate dehydrogenase, indicating the presence of extensive tissue damage involving mitochondria. Skeletal muscle mitochondria were considered the most likely source of the additional CK isozyme.

Microheterogeneity of Kasahara isozyme.

The microheterogeneity of Kasahara isozyme was investigated by affinity electrophoresis with Con A as the affinity ligand in combination with polyacrylamide gradient gel electrophoresis. On two-dimensional Con A-containing agarose gel electrophoresis, the Kasahara isozyme was separated into three molecular species. Kasahara isozyme electrophoresed as two distinct bands with enzyme activity on polyacrylamide gradient gel, but liver, intestinal or placental alkaline phosphatase showed only one distinct spot or band on both electrophoreses. One of the three molecular species of Kasahara isozyme separated by Con A-containing agarose gel electrophoresis was extracted from the gel and applied to the polyacrylamide gradient gel electrophoresis again, resulting in the same electrophoretic pattern as that of the original Kasahara isozyme. These findings indicated that the Kasahara isozyme consists of at least four molecular species. The same analysis was conducted with alkaline phosphatase of the HuH-6 cl-5 cell line, which has been reported to release an alkaline phosphatase closely resembling the Kasahara isozyme, and the results were compared with those obtained with the Kasahara isozyme.

Comparison of carbohydrate structures of serum alpha-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis.

Serum alpha-fetoprotein (AFP) is a glycoprotein of which the sugar chain is considered to show structural changes with malignancies. Microheterogeneity of the serum AFP carbohydrate structure was studied in samples from 35 patients with benign and malignant diseases. Sera were digested directly, extensively, and sequentially with sialidase. beta-galactosidase and beta-N-acetylhexosaminidase. Before and after digestion, sera were examined by means of lectin affinity electrophoresis using eight lectins. Relationships between AFP carbohydrate structures and liver diseases were elucidated by the lectin-reactive profiles and the effect of glycosidase digestion. More than 94% of the AFP carbohydrate structures found in patients with benign and malignant liver diseases were biantennary complex-type oligosaccharides. Changes in the AFP carbohydrate structures at the early stage of hepatocellular carcinoma revealed the addition of alpha 1-->6 fucose to the reducing terminal N-acetylglucosamine and monosialylated AFPs. In both advanced hepatocellular carcinoma and AFP producing extrahepatic malignancies, AFP carbohydrate structures were characterized as the further addition of beta 1-->4 N-acetylglucosamine and heterogeneity in the galactose and N-acetylglucosamine residues. Sequential glycosidase digestion and lectin affinity electrophoresis is useful for analysing the carbohydrate structures of serum glycoprotein.