Studies of carcino-fetal proteins. II. Biochemical comparison of -fetoprotein from human fetuses and patients with hepatocellular cancer

α-fetoprotein from human fetuses and patients with a hepatocelhlar cancer was isolated and characterized. In accordance with earlier reports, the two proteins had similar electrophoretic mobilities and gave a reaction of immunological identity. They had very similar amino acid compositions. Each was composed of a single polypeptide chain with a molecular weight of 70,000. Protein from both sources had about 4 % carbohydrate with 2 moles of sialic acid per mole of protein. Tryptic digests of the isolated fetal and cancer α-fetoproteins were compared by peptic mapping. About 30 peptides were seen and the patterns given by the two proteins were indistinguishable. Thus both fetal and cancerous liver cells produce α-fetoproteins which are structurally indistinguishable and probably identical.     

Studies of carcino-fetal proteins. 3. Development of a radioimmunoassay for -fetoprotein. Demonstration of -fetoprotein in serum of healthy human adults

A radioimmunoassay (RIA) for human α-fetoprotein (α-FP) was developed. The sensitivity threshold of the method is 250 picograms per ml, which means that it is 20,000-40,000 times more sensitive than immunodiffusion, and covers completely the thousandfold concentration gap between the α-FP levels in normal human serum and the level detectable by immunodiffusion. Evidence for the presence of α-FP in serum of healthy human individuals is as follows: (I) Normal serum in 1:5 dilution inhibited the binding of labelled α-FP by antibody. (2) The activity inhibiting in RIA could be removed from serum by prior treatment with anti-α-FP immunoadsorbent. (3) At low pH α-FP could be eluted from immunoadsorbent that had been previously treated with serum. (4) After fractionation of normal human serum by electrofocusing the activity inhibiting in RIA was present in a fraction whose isoelectric point was that previously shown for fetal α-FP. The α-FP present in cancer is currently believed to be a product of an activated fetal gene which is suppressed at the adult stage. Our results show that this suppression is not complete in all cells. Measurement of α-FP at the radioimmunoassay level is considered to be a contribution to the early diagnosis of cancer.     

Studies on the physical, chemical and serological properties of haemagglutinins of dermatophytes

Trichophyton rubrum, T. violaceum, T. schoenleinii, Epidermophyton floccosum and Microsporum canis were shown to be able to agglutinate the RBC's of different animals and man. The reaction was proved to be specific by using the haemagglutination inhibition test (HI). The application of HI on the homologous, as well as the heterologous, serum revealed the cross-antigenic relationships between the 5 dermatophytes. The physical and chemical natures of the haemagglutinin were also investigated.

Zusammenfassung

Es wurde nachgewiesen, daß Trichophyton rubrum, T. violaceum, T. schönleinii, Epidermophyton floccosum und Microsporum canis imstande waren, Erythrozyten von ver-schiedenen Tieren und Mensch zu agglutinieren. Mit Hilfe des Hämagglutinationshem-mungstests wurde bewiesen, daß die Reaktion spezifisch war. Die 5 Dermatophyten zeigten auch Kreuzreaktionen. Die physikalischen und chemischen Eigenschaften der Hämagglutinine wurden auch untersucht.     

Isolation and properties of human alpha-fetoprotein from HepG2 cell cultures.

A relatively rapid 3-step fractionation method has been developed for the isolation of human alpha-fetoprotein from culture fluids of HepG2 cells applicable to large volumes. The protein exists as a complex with lipids or lipoproteins but an ethanol precipitation step is effective in separating it. Yields of 50-60% can be obtained from culture fluid containing 30-40 microg/ml. A minor fraction that appears to be a proteolytic product of the AFP is present in the final product.     

Purification and chemical characterization of alpha-fetoprotein from rat and mouse

Alpha-fetoprotein of the rat was immunochemically purified and characterized. Mouse α-fetoprotein, which cross-reacts with antibodies to rat α-fetoprotein, was also purified. Rat α-fetoprotein was heterogeneous in disc electrophoresis and isoelectric focusing and had two different isoelectric points at pH 4.76 and 5.05. Mouse α-fetoprotein was homogeneous when analyzed similarly. The molecular weight of both rat and mouse α-fetoprotein was 70,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The two specimens revealed a close similarity also in some other physicochemical, chemical and immunological properties.     

Differential expression of alpha-fetoprotein and gamma-glutamyltranspeptidase in chemical and spontaneous hepatocarcinogenesis.

The expression of two markers of fetal liver, alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase (GGT), was studied in chemical and spontaneous hepatocarcinogenesis in mice. Serum AFP concentration increased within 3 weeks 3 weeks from the commencement of feeding of o-aminoazotuluene. This early elevation subsided about 3 months after the beginning of the administration of the carcinogen. A new, sustained elevation of the serum AFP level followed at 5 to 6 months accompanied by the appearance of liver tumors. In immunofluorescence, some small oval cells and scattered adult-type hepatocytes contained AFP during the early stage of chemical carcinogenesis. During the later phase, AFP was detected in a few of the nodular areas, in solitary hepatocytes, and in groups of carcinoma cells. GGT activity in the liver increased within 1 week after the carcinogen regimen was started, preceding the early increase of AFP production. At the final stage, the chemically induced hepatomas contained about 80 times more GGT than did normal liver. In histochemical staining, proliferating oval cells and small areas of hepatocytes stained for GGT during the early weeks, and later most nodules, small areas of nonnodular parenchyma, and carcinomas contained GGT. During spontaneous carcinogenesis in male C3HeB/FeJ mice, premalignant lesions, accompanied by a slight increase of serum AFP, precede the appearance of liver tumors. No cells staining for AFP were detected during this early stage. Once overt liver cancers had developed, AFP was readily detectable in the tumors and was localized to some but not all carcinoma cells. The corresponding serum AFP levels were highly elevated. In contrast to the high levels of GGT found during chemical carcinogenesis, no elevation of GGT was found in livers at various stages of spontaneous carcinogenesis, including cancers in eight individual mice. These results indicate that the production of AFP and GGT is not turned on as a single "genetic package," and that these two markers differ in their behaviour in liver carcinogenesis.     

Production of alpha-fetoprotein, normal serum proteins, and human chorionic gonadotropin in stomach cancer: histologic and immunohistochemical analyses of 35 cases

By immunoperoxidase histochemical staining of formalin-fixed paraffin-embedded sections, the production of alpha-fetoprotein(AFP), albumin(ALB), transferrin(TF), alpha-1-antitrypsin(AAT), and human chorionic gonadotropin(HCG) was examined in 35 operatively resected stomach cancers with elevated serum AFP levels (higher than 20 ng/ml as determined by radioimmunoassay). Cells positive for AFP were found in 19 cases (54%). In 29 cases (83%), some tumor cells contained normal serum proteins (ALB, TF, or AAT). All 19 tumors with AFP-positive cells also stained positively for two or three kinds of normal serum proteins. In some cases, AFP and normal serum proteins were localized in the same cells. There were two cases in which metastatic tumors produced AFP, whereas the primary sites did not. In nine cases (26%), HCG was present in tumor cells and HCG- and AFP-positive cells were coexistent in six tumors. Histologic examination of AFP-producing stomach tumors revealed medullary or papillotubular arrangements with marked nuclear atypia and eosinophilic granular or clear cytoplasms containing no glycogen or mucin. Some tumors with medullary patterns resembled liver cell carcinomas. Concordant phenotypic expression of AFP and normal serum protein production appears to be a general feature of AFP-producing tumors such as liver cell carcinoma, yolk sac tumor, and stomach cancer.     

Studies of the intrinsic antiuterotropic activity of murine alpha-fetoprotein

We previously reported that uterine growth in immature mice given estradiol (E2) is strongly curtailed by co-administration of trace amounts of alpha-fetoprotein (AFP). We used a semiquantitative bioassay for this antiuterotropic activity to evaluate AFP purification and storage methods. AFP was isolated from Nya:NYLAR mouse amniotic fluid (MAF) by polyacrylamide gel electrophoresis plus Blue Sepharose affinity chromatography (PAGE/BS), and by affinity chromatographies employing immobilized E2 and rabbit anti-AFP. E2 (0.5 micrograms) and purified AFP (0.1-50 micrograms) were incubated in 0.10 ml PBS for 1 hr at 22 degrees C and given intraperitoneally to female pups, and their uterine weights determined 23 hr later. The antiuterotrophic action increased with AFP dose up to 1 microgram (AFP/E2 molar ratio = 0.008) and declined at higher doses. Greatest inhibition by 1.0 microgram AFP occurred in 15-18 day old NYLAR pups weighing 5-8 g, but the magnitude (approximately 3 mg) was insufficient for generating a dose/response standard curve. We therefore express the activity of a preparation as the % inhibition that 1.0 microgram of its immunoreactive AFP content imposes on the uterine growth that 0.5 micrograms E2 would elicit. NYLAR AFP was more effective in NYLAR pups than in Swiss or Colony 22 pups suggesting strain specificity. High activity (54-88% inhibition) was found in all 9 batches of PAGE/BS AFP prepared over a 2 year period. The anti-AFP affinity product had similar activity, while the immobilized E2 product showed less. AFP in MAF was inactive but treatment with Norit or dextran/Norit generated partial activity. Acid/Norit treatment to remove fatty acids produced fully active AFP but in low yield. Purified AFP in storage retained activity for 1-2 weeks at 4 degrees C, up to 6 weeks at -20 degrees C, and up to 4 months in dithiothreitol-supplemented buffer. The activity is thus inhibited by other MAF constituents and decays by a temperature sensitive process that is slowed by an antioxidant.     

Estrogen receptor and alpha-fetoprotein in human breast cancer: brief communication.

Concentrations of estrogen receptor (ER) and alpha-fetoprotein were determined by dextran-coated charcoal assay and analyzed with Scatchard plots and radioimmunoassay, respectively, in cytosols of 72 human breast cancers. The values for ER ranged from 0 to 340 fmoles/mg cytosol protein. The concentrations of alpha-fetoprotein, which were low in all tumor cytosols examined, ranged from less than 0.1 to 1.1 ng/mg cytosol protein. No positive relationship was found between ER and alpha-fetoprotein concentrations. These results show that ER, but not alpha-fetoprotein, usually accounts for most of the high estrogen-binding capacity in cytosols of human breast cancers.     

Serum alpha-fetoprotein and variant alkaline phosphatase in human hepatocellular carcinoma

Alpha-fetoprotein and variant alkaline phosphatase were studied in a group of patients with hepatoma. The incidence of positive alpha-fetoprotein was 67.6% in confirmed cases. Variant alkaline phosphatase was present only in patients with positive fetoprotein test, and it appeared in only 28.6% of the cases.     

Preparation of conjugates of adriamycin with antibodies to alpha-fetoprotein and their properties

For its selective accumulation into tumor cells, the antitumor drug adriamycin (ADM) was attached to specific antibodies reactive against alpha-fetoprotein, a carcinoembryonic protein, and the antibody and antitumor activities of the resulting conjugate were investigated. Using the glutaraldehyde or carbodiimide binding methods, preparations containing 4-5 moles of ADM per mole of antibody were obtained. They retained 60-75% of the original antibody activity. Upon incubation of AH-66 ascites hepatoma cells with the conjugates, an increased number of dead cells and inhibited uptake of 3H-thymidine by the cells were observed. The conjugates were more effective against tumor cells than the free drug, and it was further presumed that the cytotoxic action of ADM on normal cells could be reduced by binding it to a macromolecular protein.     

Developmental changes in carbohydrate moiety of human alpha-fetoprotein.

Human AFP purified from fetal serum and amniotic fluid was separated into three different variants by chromatography on concanavalin A insolubilized on Sepharose (Con A--Sepharose). The three variants were indistinguishable in immunodiffusion and radioimmunoassay. Sera from patients with yolk-sac tumor and amniotic fluid from early pregnancy were found to contain a high proportion (15-45%) of AFP which does not bind to Con A, while AFP in fetal and newborn sera, and in amniotic fluid from late pregnancy, contained less (2-6%) of this variant. The use of a large excess of Con A--Sepharose and the fact that the non-bound AFP consistently eluted as non-bound in rechromatography showed that this AFP is non-reactive with Con A. Fractionation of radiolabelled AFP from cord serum in a mixture with amniotic fluid verified the difference in the amount of the Con-A nonreactive variant in AFP from these two sources. These results suggest that AFP synthesized by the yolk-sac tissue and by the liver are glycosylated differently. The variant may prove to be diagnostically useful.     

Specific uptake of alpha-fetoprotein by malignant human lymphoid cells

We have studied the ability of human B- and T-lymphoblastoid cell lines, as well as of peripheral lymphoid cells from leukemia patients, to take up alpha-fetoprotein (AFP) and other serum proteins. Two technical approaches have been employed, both using fluorescent protein derivatives (FITC-proteins): microscopic examination of labelled cells using epifluorescent illumination and quantitation of endocytosed proteins by fluorescence-activated cell sorting (FACS). Compared to human resting T-lymphocytes, all T- and B-cell lines tested exhibited positive staining for fluoresceinated AFP and transferrin (Tf) and a significant increase, up to 100-fold, of the number of AFP and Tf molecules endocytosed per cell. Labelling was prevented or strongly diminished by a 100-fold excess of unlabelled protein. Preliminary results with peripheral lymphoid cells harvested from leukemia patients showed the presence of AFP- and Tf-positive cells in the blood of all patients examined. Intensity of labelling was related to the type of leukemia cells and/or the degree of cell maturation. Most cell lines exhibited positive staining for alpha 2-macroglobulin (alpha 2M) and also, to a lesser extent, for serum Vitamin D3 binding protein (DBP). In contrast, no labelling was observed with FITC-serum albumin (FITC-Alb) or FITC-ovalbumin (FITC-OVA). Comparative uptake of several FITC-proteins by a single cell population revealed significant quantitative and qualitative differences.     

Functional properties of proteins coded by three human alpha-interferon genes and a pseudogene

We have characterized the functional properties of four highly purified recombinant human class I alpha-interferon subtypes whose biological activities have not been described previously. We selected biological and biochemical activities that may discriminate between different functions of these molecules. We found that the alpha subtypes could be discriminated only by antiviral-host range specificity and natural killer cell activation. Differences in their antiproliferative activity were cell line dependent. Competitive binding, antiproliferative activity in agar, enhancement of expression of HLA-ABC, elevation of 2'-5'-oligoadenylate synthetase levels and enhancement of phosphorylation of the Mr 69,000 protein kinase did not allow discrimination among the alpha I subtypes on the tested cell lines.     

The prevention of chemical hepatocarcinogenesis in rats by combining active and passive immunization with alpha-fetoprotein

The rats were immunized actively with mouse alpha-fetoprotein and passively with a horse antiserum against rat alpha-fetoprotein. The immunized rats fed 3'-methyl-4-dimethylaminoazobenzene containing diet. The development of hepatocellular carcinoma in livers were completely prevented.