Hormonal regulation during secretion of alpha-fetoprotein in hepatoma cells grown in synthetic medium

The variant cell line of H4-II-E-C3 cells derived from the Reuber H-35 hepatoma cells has been established using protein- and lipid-free synthetic medium. This H4-II-E-C3-V line can synthesize and secrete considerable amounts of alpha-fetoprotein (AFP) and albumin. The addition of 5 X 10(-7) M dexamethasone to the medium stimulated the excretion of AFP without increasing total AFP synthesis, whereas 8.7 X 10(-8) M insulin inhibited the excretion of AFP without a significant inhibition of intracellular AFP synthesis. However, neither dexamethasone nor insulin altered either the cellular or secreted levels of albumin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. AFP appeared in the medium after 10 min, and 50% of the protein was secreted after 110 min. The rate of secretion of AFP was much slower than that of albumin, 50% of which was secreted after 25 min. Dexamethasone, 5 X 10(-7) M, caused a marked enhancement in the rate of AFP secretion, with 50% released after 75 min. Insulin, 8.7 X 10(-8) M, by contrast, caused a marked delay in AFP secretion with only 20% released after 180 min and then a plateau was approached. Since the intracellular AFP was excreted 55% after 180 min the remaining 25% of newly made AFP was suggested to be degraded during secretion. The kinetics of movement of AFP during secretion and endoglycosidase H treatment of intracellular and secreted AFP suggested that insulin impeded the transport of AFP from the rough endoplasmic reticulum to the Golgi apparatus.     

Expression of human alpha-fetoprotein in yeast.

Human alpha-fetoprotein (AFP) was expressed in Saccharomyces cerevisiae, with a plasmid containing the cDNA sequence for human AFP fused with the rat AFP signal peptide. The recombinant AFP was purified from the yeast lysate by DEAE-cellulose and immunoaffinity chromatography. The amino acid composition and the molecular weight of the recombinant AFP were similar to those of hepatoma AFP. N-terminal amino acids sequence analysis indicated that the signal peptide had been processed. The recombinant and hepatoma AFP reacted identically in Ouchterlony immunodiffusion and radioimmunoassay tests. These observations indicated that the yeast recombinant protein had the properties of native AFP.     

Pattern analysis of serum alpha-fetoprotein in the early diagnosis of hepatocellular carcinoma in liver cirrhosis

In a surveillance program for hepatocellular carcinoma (HCC), serum alpha-fetoprotein (AFP) was determined every 4 months in 164 patients with liver cirrhosis. Ultrasonography (US) was performed yearly or as dictated by abnormal AFP levels. During a follow-up of 32.5 +/- 20.8 months HCC was identified by US in 16 patients. In 9 of them the AFP levels rose steadily over 4 months, increasing 7, 8 and 12 months in 3 cases before the lesion became detectable by US. In 4 patients tumors developed despite persistently normal AFP levels. Nine more patients showed abnormal fluctuations of AFP but HCC was not detected. AFP sensitivity was higher at a low cut-off point (40 ng/ml) while specificity of the test appeared higher at the 200 ng/ml cut-off point. An AFP value rising steeply over a few months appeared more reliable than a fixed preset threshold in indicating carcinomatous transformation. Screening for AFP can be expected to uncover about 3/4 of HCC developing in cirrhotics with few false-positive reactions. The test may have a unique role in identifying a subset of liver tumors whose early expression is AFP production.     

Effect of alpha-fetoprotein on the growth of human hepatoma cells in vitro]

The stimulatory activity of human alpha-fetoprotein (AFP) on the growth of mouse hepatoma-22 cells had been reported in our previous paper. The present work aimed at further investigation of the effect of AFP on human hepatoma cell growth by MTT colorimetric assay. The results showed that AFP could stimulate the growth of SMMC-7721 human hepatoma cells in vitro. The present results also showed that the stimulatory effect of AFP on the growth of SMMC-7721 cells was decreased by the anti-serum of AFP. The anti-AFP antibody alone could suppress the growth of SMMC-7721 cells. On the other hand, AFP and anti-AFP antibody had no effect on the growth of HL-60 human leukemia cells, indicating that the tumor cell growth stimulating effect of AFP was not simply due to non-specific addition of exogenous protein and this effect of AFP showed strict tumor cell specificity. In addition, MCF-7 human breast cancer cell growth was also promoted by AFP and inhibited by anti-AFP antibody. Because AFP cell-surface receptors have been detected in MCF-7 breast cancer cells, and AFP could also be produced and secreted by MCF-7 cells, the possibility may be considered: AFP may bind with its receptors on tumor cell membrane for the purposes of growth stimulation.     

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.     

Alpha-fetoprotein enhances the proliferation of human hepatoma cells in vitro

Although the biological functions of alpha-fetoprotein ( AFP ) have been extensively studied, little is known about its effect on tumor cell growth. Our previous work has found that human AFP significantly stimulates the growth of mouse hepatoma cells in vitro. The purpose of the present study is to observe the effect of AFP on the proliferation of human hepatoma cells in vitro. Using a MTT- microculture tetrazolium assay, we found that the proliferation of human hepatoma cells was enhanced by in vitro treatment of AFP. However, the same concentrations of AFP had no effect on HL - 60 human leukemia cell proliferation, indicating that the human hepatoma cell proliferation - promoting role of AFP was not simply due to non-specific addition of exogenous protein and the proliferation enhancement of AFP showed certain tumor cell specificity. On the other hand, the growth stimulation of AFP could be diminished by rabbit anti - human AFP antibody. The anti- AFP antibody alone suppressed the growth of BEL - 7404 human hepatoma cells, not affecting HL - 60 cell proliferation. BEL - 7404 cell proliferation was not inhibited by normal rabbit immunoglobulins to demonstrate the specificity of anti-AFP effect. Taken together, it is concluded that AFP enhances the proliferation of human hepatoma cells in vitro, and this effect is seemingly mediated by an AFP/receptor autocrine pathway.     

Rat alpha-fetoprotein in experimental utero-placental ischaemia.

Retardation of growth and death of fetal rats were produced after uteroplacental ischaemia was induced by surgical ligation of the uterine arteries. Changes in maternal plasma levels of alpha-fetoprotein (AFP) were measured by radioimmunoassay. In rats in which the uterine blood supply was totally occluded, the resultant increase in maternal plasma AFP was due to resorption of fetal elements, because AFP levels in maternal rat plasma did not increase following hysterectomy in a control group. Maternal plasma AFP levels in rats with a partly occluded blood supply (and therefore some dead and some live fetuses) paralleled those of sham-operated rats, suggesting that increased placental transfer of AFP to maternal plasma may have offset the anticipated decline of AFP due to a decreased number of live fetuses.     

一个抗真菌蛋白在绿色木霉中的分泌表达

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。     

Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons.

The requirements for activation of the mouse alpha-fetoprotein (AFP) gene in transient heterokaryons were investigated. For this purpose, the 7-kilobases of DNA flanking the 5' end of the AFP gene were linked to a mouse major histocompatibility complex (MHC) class I structural gene. The fusion gene was stably integrated at different sites into mouse L-cells, which do not transcribe the AFP gene. Transient heterokaryon fusions demonstrated that the silent AFP-MHC gene and the endogenous AFP gene were activated by factors present in HepG2 cells, a liver-derived cell line, but not by those in HeLa cells. Activation was detected at the protein level in single heterokaryons by using monoclonal antibodies against the cell surface protein and at the mRNA level in populations of cells. The AFP promoter alone was sufficient for activation could be used for DNA transfer strategies to identify genes which can activate AFP promoter elements in trans.     

The expression of alpha-fetoprotein and albumin genes in rat liver during chemical carcinogenesis

The effect of the hepatocarcinogen 3′-methyl-4-dimethylaminoazobenzene on α-fetoprotein (AFP) and albumin gene expression in rat liver was studied. Serum concentrations of AFP and albumin were measured. Amounts of AFP mRNA and albumin mRNA in rat livers were determined by hybridization of total cytoplasmic RNAs to their cDNAs. Dramatic increases in serum AFP concentrations coincided with increases in AFP biosynthesis and amount of AFP mRNA in livers of carcinogen-treated rats. In contrast, no or little change in albumin mRNA concentration was found in livers of rats treated with 3′-methyl-4-dimethylaminoazobenzene. Concomitantly, there was little change in liver albumin biosynthesis or serum albumin concentrations during hepatocarcinogenesis.     

The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi

The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.     

Variation in spontaneous chromosomal damage as a function of biologic rhythms in women

Summary The alphafetoprotein (AFP) concentration in maternal serum was determined before and after chorionic villus sampling (CVS). A significant increase of 20% or more in the pre-CVS level was noted immediately after sampling in 59% of 837 pregnancies indicating some degree of feto-maternal haemorrhage. The increase in the AFP concentration in maternal serum was correlated with the weight of the tissue sample but not with the number of sampling attempts. A correlation of AFP increase and frequency of spontaneous abortions following CVS was suggested only in the group with an AFP increase of more than 100% or with a continuing rise in the first hour following CVS. CVS in early pregnancy obviously did not interfere with maternal serum AFP screening for neural tube defects in the second trimester. Although AFP measurement before and after CVS seems to have no immediate diagnostic application, in the research phase of CVS it may help to identify those procedures that are the least traumatic.     

Alpha-fetoprotein in Abortion

The clinical value of maternal serum alpha-fetoprotein (AFP) as a guide to the outcome of threatened abortion was assessed. After the thirteenth week of gestation, abortion occurred more frequently (10/12) in women with abnormal serum AFP levels than in those (2/12) whose AFP concentrations were within the normal range. Low levels were present in women with blighted ovum and high concentrations were associated with intrauterine fetal death. In legal first and second trimester abortions, the circulating maternal AFP levels in postabortion samples were often higher than before abortion, irrespective of whether abortion was performed instrumentally or induced with prostaglandins. Maternal serum AFP levels provide a new means for prediction of the outcome of threatened abortion.     

Generation of monoclonal antibodies to alpha-fetoprotein and application in solid-phase enzyme immunoassay

By using an improved hybridoma technique, monoclonal antibodies specific to alpha-fetoprotein (AFP) were generated. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were initially cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for subculture. Out of 800 colonies recovered after two cell fusion experiments, 16 were shown to exhibit affinity to AFP by radioimmunoassay. Six hybrid cell lines which showed high affinity and specificity were selected for further evaluation. From the results of a cross-matching procedure, two pairs of antibodies (AFP 3 and AFP 05; AFP 3 and AFP 013) reacting with discrete antigenic determinants were identified for preparing solid-phase sandwich enzyme immunoassay (EIA) kits. The association constants between AFP and these three antibodies (AFP 3, AFP 05, and AFP 013) were 2.0, 3.7, and 3.8 X 10(9) M-1, respectively. The immunoglobulin subclass of them was determined to be IgG1. The EIA procedure designed could be performed within 40 min in a one-stage incubation and 70 min in a two-stage incubation. The incubation time was shown to be equal to or shorter than that of any other known commercial kits and the sensitivity was less than 1 IU/ml. In order to avoid the high-dose hook effect which occurred in the one-stage incubation procedure, a two-stage incubation protocol was advised.     

Regulation of rat liver maturation in vitro by glucocorticoids.

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.