Human Α-fetoprotein Research Articles

Lectin Affinity Electrophoretic Demonstration of Tissue Specificity and Malignant Alteration of Human α-Fetoprotein Isoforms Produced in Transgenic Mice

Transgenic mouse which produces human α-fetoprotein (AFP) ubiquitously was used to study carbohydrate structures of AFP produced by various tissues as well as that in the serum. A series of tissues from the transgenic mouse were culturedin vitroand the AFP produced was analyzed by affinity electrophoreses with 4 kinds of lectins. Variable electrophoretic profiles of them suggested that the fine structures of the carbohydrate of human AFPs expressed in the mouse tissues were different. The characterization of human AFP produced by mouse hepatoma which developed in offspring between the AFP transgenic mouse and hepatoma-developing transgenic mouse indicated that the hepatoma AFP was distinct from the liver AFP and that the malignancy-specific phenotypic alteration was common to human and mouse.

Hepatocyte transplant combined with nonparenchymal cells in the mesenteric fat pad in mouse

Hepatoblastoma is the most frequent malignant pediatric liver tumor. Approximately 25% of hepatoblastoma patients cannot be cured with current treatment protocols. Additional treatment options must, therefore, be developed. Subcutaneous animal models for hepatoblastoma exist, but a more physiologic intrahepatic model is lacking.The α-fetoprotein-expressing hepatoblastoma-cell lines HepT1, HuH6 and the childhood hepatocellular carcinoma-cell line HepG2 were injected subcutaneously and intrasplenically into NMRI nu/nu mice. Tumor growth was monitored by measuring tumor size for subcutaneous and serum human α-fetoprotein levels for intra-abdominal tumors. Tumors were characterized microscopically.Subcutaneous tumor growth occurred in 70% (7/10) of mice injected with HuH6 and 50% (5/10) of mice injected with HepG2. HepT1 did not form tumors. Accumulation of serum α-fetoprotein reflected tumor growth. Intrasplenic growth was seen in 50% (14/27, HuH6) and 10% (3/10, HepG2) of the mice, with only HuH6 forming intrahepatic tumors in 25% (7/27) of the mice. Growth pattern and α-fetoprotein production were similar at the subcutaneous and intra-abdominal location. Intrahepatic grafting occurred by metastatic spread from the spleen, produced well-defined nodules, and was accompanied by a weakened expression of the hepatocyte marker carbamoylphosphate synthetase, and the canalicular markers CD10 and cytokeratin7. The expression of cytokeratin18 and -19, active caspase3, and β-catenin was increased. There were no lung metastases.We established an intrahepatic mouse model for human hepatoblastoma, in which tumor growth could be monitored by serum α-fetoprotein levels. Engrafting in the liver occurred by metastatic spread from the spleen and was accompanied by some loss of differentiation features.

Under buffer SFM observation of immunospecies adsorbed on a cyano grafted silicon substrate

Scanning force microscopy (SFM) in contact mode and in liquid medium has been employed to study immunospecies layers adsorbed on a silicon wafer. The silicon wafer has been grafted with a cyanosilane monolayer in order to create a surface with strong adhesive properties which prevent proteins being swept by the scan of the SFM tip. The force curves reveal that the adhesive force has been increased by a factor six without roughness modification (< 1 nm). After the incubation of the surface in a monoclonal antibody (mouse anti-human α-fetoprotein IgG) solution, SFM surface images suggest an homogeneous layer composed by ellipsoidal objects (40–60 nm in diameter, 6–13 nm in height). The substrate was moreover incubated in an antigenic solution (human α-fetoprotein): SFM images reveal that proteins have been added onto the antibody layer.

Suppression of Experimental Antigen-Induced Arthritis in Transgenic Mice Producing Human α-Fetoprotein

Experimental arthritis was induced in the knee joint of transgenic mice expressing human α-fetoprotein by immunization with methylated bovine serum albumin in Freund′s complete adjuvant. In the control experiment with normal C57BL/6 mice, definite arthritis was observed in 55.6% (5/9) of the mice, but in only 21.1% (4/19) of the transgenic mice. This result suggested that human α-fetoprotein functioned as an immunosuppressant to ameliorate the development of the immune disease.

P-188 Cell-specific and non-cell-specific enhancer activity in a far upstream region of the human α-fetoprotein gene

P-188 Cell-specific and non-cell-specific enhancer activity in a far upstream region of the human α-fetoprotein gene

Homogeneous Immunoassay Using Photothermal Beam Deflection Spectroscopy

We developed a novel homogeneous immunoassay using photothermal beam deflection (PBD) spectroscopy. Antibody-coated glass beads having a particle size of 50 μm and antibody-coated colloidal gold ultrafine particles 20 nm in size were used, and an immune complex containing colloidal gold particles on antibody-coated glass beads was formed by immunological reaction in solution. An excitation beam was affected from below the sample, and a PBD signal was thus generated was measured. The PBD signal was not affected by nonreached antibody-floated colloidal gold particles which were suspended in the solution up to the colloidal gold concentration of 0.008%. This result indicates that homogeneous immunoassay is possible. Human α-fetoprotein was assayed to examine the sensitivity of this method, and a detection limit of 0.25 ng/mL was obtained. This was more sensitive by 1 order of magnitide that the latex agglutination method, the conventional homogeneous rapid assay. Some improvements of reagents and techniques can be expected to enhance the sensitivity further

ATBF1, a multiple-homeodomain zinc finger protein, selectively down-regulates AT-rich elements of the human alpha-fetoprotein gene.

ATBF1 is a 306-kDa protein containing four homeodomains, 17 zinc finger motifs, and several segments potentially involved in transcriptional regulation (T. Morinaga, H. Yasuda, T. Hashimoto, K. Higashio, and T. Tamaoki, Mol. Cell. Biol. 11:6041-6049, 1991). At least one of the homeodomains of ATBF1 binds to an AT-rich element in the human alpha-fetoprotein (AFP) enhancer (enhancer AT motif). In the present work, we analyzed the transcriptional regulatory activity of ATBF1 with respect to the enhancer AT motif and similar AT-rich elements in the human AFP promoter and the human albumin promoter and enhancer. Gel retardation assays showed that ATBF1 binds to the AFP enhancer AT motif efficiently; however, it binds weakly or not at all to other AT-rich elements in the AFP and albumin regulatory regions studied. Alterations of the enhancer AT motif by site-specific mutagenesis resulted in the loss of binding of ATBF1. Cotransfection experiments with an ATBF1 expression plasmid and the chloramphenicol acetyltransferase (CAT) gene fused to AFP promoter or enhancer fragments showed that ATBF1 suppressed the activity of AFP enhancer and promoter regions containing AT-rich elements. This suppression was reduced when the mutated AT motifs with low affinity to ATBF1 were linked to the CAT gene. The ATBF1 suppression of AFP promoter and enhancer activities appeared to be due, at least in part, to competition between ATBF1 and HNF1 for the same binding site. In contrast to the AFP promoter and enhancer, the albumin promoter and enhancer were not affected by ATBF1, although they contain homologous AT-rich elements. These results show that ATBF1 is able to distinguish AFP and albumin AT-rich elements, leading to selective suppression of the AFP promoter and enhancer activities.

Sequence analysis of the proximal promoter region of the human α-fetoprotein gene in hepatocellular carcinoma

We have examined whether or not mutations exist in the proximal promoter region of the human α-fetoprotein (AFP) gene in the hepatocellular carcinoma (HCC) tissue. Genomic DNA was extracted from four patients: one HCC tissue, one HCC and its corresponding non-cancerous (cirrhosis) tissues, one liver cirrhosis (LC) tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. Serum concentrations of AFP in the patients ranged from less than 5 to 10 138 ng/ml. Nucleotide sequence was determined by direct sequencing using a single-stranded DNA template that was produced first through the polymerase chain reaction (PCR) amplification and then asymmetric PCR. In one HCC tissue taken from the patient with a high concentration of serum AFP, nucleotides different from published ones were detected at −120 and −113. These changes, however, probably reflect a DNA polymorphism, because peripheral blood leukocytes of the same patient had the same changes. Including this patient, no mutations in the region from −160 to −10 were detected in the HCC specimens we have examined. These results suggest that the extremely proximal promoter region of the AFP gene where glucocorticoid-responsive element and HNF-1 binding sites exist is not responsible for the re-expression of AFP in HCC.

The Fatty Acid Binding Site of Human α-Fetoprotein

α-Fetoprotein (AFP) binds a series of endogenous fatty acids. To identify the fatty acid binding site, this protein, purified from umbilical cord blood by immunoaffinity chromatography, was covalently labeled using 12-(9-anthroyloxy)stearic acid conjugated with Woodward′s reagent K. After digestion with lysyl endopeptidase, the fatty acid-labeled peptide was isolated. Amino acid sequence analysis showed that this peptide consisted of the amino acid residue 210-227. The fact that no appreciable PTH-amino acid was detected at 14th cycle on the degradation suggested that Lys223 was modified with the fatty acid. The finding that lysyl endopeptidase did not cleave off at the Lys223 supported this suggestion. These results indicate that the carboxyl group of the bound fatty acid is located close to Lys223 in human AFP.

Purified human α-fetoprotein inhibits follicle-stimulating hormone-stimulated estradiol production by porcine granulosa cells in culture

We have investigated the effects of purified α-fetoprotein (AFP) on follicle-stimulating hormone (FSH)-stimulated estradiol production by porcine granulosa cells in monolayer culture. Granulosa cells isolated from small follicles of prepubertal pigs were cultured for 2 days in 5% fetal bovine serum for attachment and incubated for 3 days in medium containing androstenedione and various treatments. The media were then collected and assayed for estradiol by radioimmunoassay. Human AFP significantly ( P < 0.05) and dose-dependently inhibited FSH-stimulated estradiol production with 313 ng/ml AFP returning FSH-stimulated estradiol to basal levels; human serum albumin was without effect. AFP purified from either term cord blood or midtrimester amniotic fluid dose-dependently inhibited estradiol production stimulated by the combination of FSH and insulin-like growth factor-I. Furthermore, 125 ng/ml AFP inhibited estradiol production stimulated by cholera toxin, forskolin and cAMP. In contrast, extracellular accumulation of cAMP and progesterone production was not inhibited by AFP. These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit FSH-stimulated estradiol production by porcine granulosa cells in culture. Since AFP is known to augment growth factor-mediated cell growth, these data suggest that AFP inhibits differentiated functions (such as aromatase) while enhancing the proliferation of porcine granulosa cells.

High Level Expression of Human α-Fetoprotein in Transgenic Mice

Transgenic mice were produced by the microinjection of the cDNA of human alphafetoprotein (AFP) under control of the enhancer/promoter of the human β-actin gene. Among the 4 mouse lines where the transgene were stable transmitted to the progeny, 3 produced human AFP. The expression was not developmental stage-specific nor tissue-specific as predicted from the properties of enhancer/promoter. The serum human AFP levels of adult mice were 30∼600-fold higher than those of mouse AFP. These mice could be useful for the studies of possible biological functions of AFP during development as well as in malignancies.

モノクローナル抗体結合リポソームを用いての癌ターゲティング療法の開発

Monoclonal antibodies against human α-fetoprotein (AFP) or carcinoembryonic antigen (CEA) were conjugated to liposomes containing adriamycin (ADM), and their therapeutic effects were experimentally studied in vivo on AFP or CEA positive human solid tumors maintained in BALB/c nu/nu mice. The anti-tumor effect of antibody-conjugated liposomes containing ADM was immunologically specific and was greater than that of unconjugated liposomes containing ADM or that of ADM alone. Furthermore, tissue distribution studies revealed the selective delivery of ADM to tumors occurred with immunoliposomes. However, it was also revealed the liposomes are preferentially accumulated to the reticuloendothelial cells. To improve the therapeutic effects, we further prepared the following three types of liposomes and their therapeutic activities were assessed. (1) Fab′ fragment was introduced instead of whole molecule of antibody. These liposomes are expected to keep the antigen-binding activity of antibody intact. Actually, the therapeutic effect of liposomes with Fab′ fragment was greater than that of liposomes with whole molecule. (2) GMI gangliosides were incorporated into liposomes and with these liposomes the accumulation of ADM to reticuloendothelial cells was reduced. (3) Temperature-sensitive liposomes were prepared with dipalmitoylphosphatidylcholine. Combination of temperature sensitivity and monoclonal antibodies for targeting therapy is now in progress.

In vivo transient rise in plasma free fatty acids alters the functional properties of α-fetoprotein

Previous in vitro studies have shown that unsaturated fatty acids (UFA) induce conformational changes in rodent and human α-fetoprotein (AFP). To determine whether such changes in the binding and immunological properties of rat AFP also occur in vivo, plasma free fatty acid (FFA) concentrations were increased in young male rats (15, 21 and 28 days old) by acute i.v. injection of heparin (200 IU/kg). Plasma estrogens (estrone and estradiol) did not change after injection of heparin. There was a large increase in plasma FFA 10–20 min post-heparin injection, with a return to normal 60 min later. This transient rise in FFA plasma was associated with a 50% drop ( P < 0.001) in the binding of estradiol to rat AFP of 15-, 21- and 28-day-old rats by reducing the number of binding sites ( P < 0.001), leaving the affinity constant ( K a ) unchanged. FFA extracts from post-heparin plasma induced similar changes in estradiol binding to purified rat AFP. The rise in plasma FFA induced a loss of AFP immunoreactivity, in 21- ( P < 0.001) and 28-day-old rats ( P < 0.001), but not in 15-day-old rats. This age-dependent response correlated with the FFA/AFP molar ratio (38 in 15-day-old rats, 388 in 21-day-old rats, and 5600 in 28-day-old rats). These results indicate that an in vivo rise in FFA induces rapid and reversible conformational changes in AFP which may modulate the endocrine and immune function of this oncofetal protein.

A Human α-Fetoprotein Enhancer-Binding Protein, ATBF1, Contains Four Homeodomains and Seventeen Zinc Fingers

We have isolated a full-length cDNA encoding a protein (ATBF1) that binds to an AT-rich motif in the human alpha-fetoprotein gene enhancer. The amino acid sequence deduced from the cDNA revealed that this is the largest DNA-binding protein (306 kDa) known to date, containing four homeodomains, 17 zinc finger motifs, and a number of segments potentially involved in transcriptional regulation. Although the exact function of this protein has not been determined, these structural features suggest that ATBF1 plays a transcriptional regulatory role.

Phytoestrogens: new ligands for rat and human α-fetoprotein

The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat α-fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA > equol > enterolactone > enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants ( K d) for phytoestrogens binding to AFP were: K d NDGA = 5 ± 1.2 · 10 −7 M, K d equol = 6.7 ± 0.8 · 10 −6 M, K d enterolactone = 1.7 ± 0.4 · 10 −5 M and K d enterodiol = 2.2 ± 0.6 · 10 −5 M. The K d for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of estrone binding sites remained unchanged. This, plus the results of double-reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased K d), but high concentrations induced conformational changes in rat AFP, as both K d and the number of binding sites ( n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development.

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human α-fetoprotein

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human α-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.

Pregnancy Hormone Levels Signal Trisomy 21, Improved Screening, Lower Costs Possible

ADVANCES in biochemistry are helping scientists identify pregnant women who are at high risk for carrying a fetus with Down's syndrome. Scientists now recognize three substances in the mother's serum—human chorionic gonadotropin, unconjugated estriol, and α-fetoprotein (AFP)—the concentrations of which, when coupled with the woman's age-associated risk, allow a more accurate calculation of the likelihood that she is carrying an affected fetus. The prenatal blood test, administered during the second trimester and followed by amniocentesis and chromosome analysis on those mothers who are at risk, detects about 67% of all affected fetuses in the general pregnant population—a twofold to threefold improvement from the standard screening method that employs only AFP serum levels and age-associated risk. Identifying Younger Patients The new method helps to identify younger patients who are at high risk and should have amniocentesis performed but may not be offered the procedure under standard screening methods. It can also

Human α-fetoprotein (AFP) causes a selective down regulation of monocyte MHC class II molecules without altering other induced or noninduced monocyte markers or functions in monocytoid cell lines

Human α-fetoprotein (AFP) purified from human amniotic fluid was investigated for its effect on human monocytoid cell lines, including U 937 cells with established subclones. The impact of AFP on the expression of surface markers (MHC class I and II, CD4, CD18, CD45, Fc receptors for IgG) was analyzed using known inducers of monocyte-macrophage differentiation such as phorbol esters and IFN-γ. Furthermore we investigated the effect of AFP on the induction of macrophage antibody-dependent cell-mediated cytolytic activity (ADCC). AFP did selectively induce a rapid down regulation of surface MHC class II expression. No evidence of alterations was found in the endogenous or differentiation-induced expression of other markers on the surface on monocytes, nor did AFP affect the functional maturation of surface Fc receptors or the ability to express ADCC.

Chromatofocusing profile of purified human α-fetoprotein and albumin differs from those of crude samples: Effect of protein concentration on the elution of the sample

Chromatofocusing was utilized to characterize charge microheterogeneity of purified human α-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with p Is 4,57 (52%), 4,27 (43%), and <4,00 (5%), respectively. In contrast, 10 μg of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With ≥5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with ≥5 mg unlabeled HSA. When carrier proteins of varying p I values were chromatofocused along with purified AFP, it was observed that only those proteins withp Is in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.

Structure of the gorilla α-fetoprotein gene and the divergence of primates

The sequence of the gorilla α-fetoprotein gene, including 869 base pairs of the 5′ flanking region and 4892 base pairs of the 3′ flanking region (24,607 in total), was determined from two overlapping lambda phage clones. The sequence extends 18,846 base pairs from the Cap site to the polyadenylation site, and it reveals that the gene is composed of 15 exons, which are symmetrically placed within three domains of α-fetoprotein. The deduced polypeptide chain is composed of a 19-amino-acid leader peptide, followed by 590 amino acids of the mature protein. The RNA polymerase II binding site, TATAAAA, and the promoter element, CCAAC, are positioned at −21 and −65 from the Cap site, respectively. The polyadenylation signal, AATAAA, is located in the last exon, which is untranslated. The sequence for the gorilla α-fetoprotein gene was compared with that of the previously published human α-fetoprotein gene ( P. E. M. Gibbs, R. Zielinski, C. Boyd, and A. Dugaiczyk, 1987, Biochemistry 26: 1332–1343). Four types of repetitive sequence elements were found in identical positions in both species. However, one Alu and one Xba DNA repeat within introns 4 and 7, respectively, of the human gene are absent from orthologous positions in the gorilla. The Alu and the Xba DNA repeats probably emerged in the human genome after the human/gorilla divergence and became established novelties in the human lineage. There are 363 21,523 mutational changes between human and gorilla, amounting to 1.69% DNA divergence between the two primate species. The value of 1.69% is lower than the 2.27% obtained from melting temperatures of hybrids between human and gorilla genomic DNA (C. G. Sibley and J. E. Ahlquist, 1984, J. Mol. Evol. 26: 99–121). At the protein level, Homo sapiens differs from Gorilla gorilla only at 4 of 609 amino acid positions (0.665) in the α-fetoprotein sequence. This difference signifies a lower rate of molecular divergence for the α-fetoprotein gene in primates, as compared to rodents.