Application of high-performance liquid chromatography for the study of the microheterogeneity changes of mouse alpha-fetoprotein in fetal development

Changes in the microheterogeneity of mouse alpha-fetoprotein (MAFP) during fetal development were investigated by high-performance liquid chromatography (HPLC). A total of six distinct isoforms (Iso-1, Iso-2, Iso-3, Iso-4, Iso-5, Iso-6) of the heterogeneous MAFP were resolved from mouse amniotic fluid (MAF). Analysis of MAF collected at various times revealed that these isoforms were accumulated at different stages of the fetal development.     

alpha-Fetoprotein values in monkey (Macaca mulatta) pregnancy.

The pregnant rhesus monkey's (Macaca mulatta) potential as a model for understanding the dynamics of alpha-fetoprotein (AFP) metabolism in human pregnancy was evaluated. AFP levels in maternal and fetal serum and amniotic fluid were determined by radioimmunoassay. Significant correlations were found between decreasing maternal serum, fetal serum and amniotic fluid AFP concentrations and increasing gestational age. However, these data are not consistent with the AFP changes reported in human pregnancy. It appears that this animal has limited applicability as a model in this aspect of human pregnancy.     

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

Amniotic fluid cell morphology in early antenatal prediction of abortion and low birth weight.

The morphology of rapidly adherent (RA) amniotic fluid cells was examined in 201 pregnant women referred for amniocentesis because of two sequential high serum alpha-fetoprotein (AFP) concentrations. Out of 43 amniotic fluid samples containing increased amounts of AFP, 42 had neural or peritoneal cells predominating among the RA cells, the outcome being an infant with a neural-tube defect or exomphalos. In the other case with a raised amniotic fluid AFP concentration but only anterior placental cells the infant was normal. In 25 amniotic fluid samples containing normal amounts of AFP distinctive new patterns of RA cells were observed, termed fetal distress cells. These pregnancies resulted in five spontaneous abortions and 20 infants with birth weights under 2500 g. Fetal distress cells were not detected in any of the remaining 133 samples. One pregnancy was terminated because of a chromosomal abnormality, and there were seven twin pairs not recognised on ultrasonography before amniocentesis. The remaining 125 pregnancies went to term, resulting in infants with birth weights exceeding 2500 g. The results suggest that RA-cell morphology will prove to be of value in the early antenatal prediction of spontaneous abortion and low birth weight.     

Alpha-fetoprotein expression in intra- and extraembryonic fluids of developing chick embryo

The ontogeny of alpha-fetoprotein (AFP) has been studied in the chicken (from 7 days of incubation until 2 days after hatching) using (1) the two-dimensional immunoelectrophoresis technique, (2) the polyacrylamide gel electrophoresis, and (3) the high resolution two-dimensional polyacrylamide gel electrophoresis. The molecular weight of AFP was estimated at 71,000. AFP was seen as a heterogeneous population composed of four isoforms which slightly differ by their isoelectric points. Up to the 18th day of development, qualitative changes in AFP heterogeneity do not occur. Only traces of the two alkaline isoforms were observed in plasma of 2 days post-hatching chickens. AFP has been identified in allantoic and cerebrospinal fluids but is not present in amniotic fluid. At 7 days of embryonic age, all the plasma AFP species are present in cerebrospinal fluid.     

Characterization of the Growth Factor Activity of Amniotic Fluid on Cells from Hematopoietic and Lymphoid Organs of Different Life Stages

We investigated the proliferation-promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation-promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells from 2-week-old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1-year-old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti-mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti-mouse SCF antibody revealed a band at 30–32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type-dependently antagonized by TNF-α and TGF-β. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.     

Human alpha-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 alpha-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-gamma. 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.     

Separation of the estrogen-activated growth-regulatory forms of alpha-fetoprotein in mouse amniotic fluid.

We have previously reported that murine fetal alpha-fetoprotein (AFP) incubated for 1.0 h at room temperature in the presence of high concentrations of estradiol (E2) generates a growth-regulatory product designated AFP/E2. Subsequently we developed a bioassay in the immature mouse uterus to measure both the growth-inhibitory and growth-enhancing properties of AFP. In the present study, we have employed this bioassay to monitor each of the amniotic fluid-derived AFP isolates fractionated by various chromatographic and electrophoretic techniques. The objective of this investigation was to partition and isolate the various molecular forms of AFP contained in amniotic fluid and determine whether the growth-regulatory activities resided with one or more of the fractions. AFP was fractionated by three different chromatographic/electrophoretic methods: E2 affinity chromatography, preparative polyacrylamide-gel electrophoresis (PAGE), and high-performance liquid chromatography (HPLC); and one immunoaffinity method: gel-entrapped antibody filtration (GAF). Whereas E2 affinity chromatography separated the biological activity of AFP into inhibitory and possibly enhancing activities, PAGE purification yielded three fractions: an inhibitor, an enhancer, and a fraction without growth-regulatory activity. Immunoaffinity separation yielded an AFP product with only inhibitory activities. In comparison, fractionation by HPLC produced seven AFP fractions in which only three displayed growth-regulatory activities: two inhibitory and one enhancing. After subsequent HPLC rechromatography of these fractions, none displayed any biological activity. Thus, murine AFP derived from amniotic fluid is composed of potential heterofunctional forms that, depending on their relative abundance in the preparation, constitute a mixture capable of either (a) growth inhibition, (b) no effect, or (c) growth enhancement.     

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.     

Alpha fetoprotein is more than a hepatocellular cancer biomarker: from spontaneous immune response in cancer patients to the development of an AFP-based cancer vaccine

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with a poor prognosis and limited therapeutic options. Due to its overexpression in the majority of HCCs, alpha-fetoprotein (AFP) represents one of the most useful markers for hepatocarcinomas and for monitoring patients' response to therapy. Although it was earlier reported that AFP has immunosuppressive properties, it has been recently demonstrated that AFP induces spontaneous T and B cells responses in HCC patients. The characterization of AFP-immunogenic epitopes gives the opportunity to design AFP-based cancer vaccines for human HCC. The activity of AFP-based vaccines has been investigated in HCC mouse models in order to develop novel strategies to treat patients with HCC. This review will discuss the rationale for using the AFP-based vaccination strategy and recent results corroborating the usefulness of AFP vaccines as a potential tool for cancer therapy.     

Rat alpha-fetoprotein: isolation, radioimmunoassay and fetal-maternal distribution during pregnancy.

A method is described for the isolation of mg quantities of two forms of rat alpha-fetoprotein (AFP) from amniotic fluid by preparative disc-gel column electrophoresis using a continuous elution system. AFP isolated by this method is suitable for use as an antigen, can be labelled for use in a radioimmunoassay and serves as a reference standard. The characteristics of a new antiserum to AFP are also described. The protocol for a radioimmunoassay is outlined which permits the measurement of AFP in several fetal-maternal physiological compartments throughout gestation. Levels of AFP in fetal liver and fetal plasma suggest that secretion of AFP from liver occurs soon after synthesis with minimal hepatic storage. The pattern for AFP in maternal serum parallels that observed in amniotic fluid and fluctuations in maternal serum levels of AFP appear to be buffered by AFP accumulation in amniotic fluid. Fetal clearance of AFP under normal conditions may be relatively constant from Days 11-20 of gestation since an amniotic fluid: maternal serum AFP ratio of 30:1 is maintained during this period.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Alpha-fetoprotein and human lymphocyte subpopulations.

Peripheral blood lymphocytes were incubated with varying concentrations of alpha-fetoprotein and enumerated for total and "active' T lymphocytes, B lymphocytes, and their proliferative responses to phytohemagglutinin. No significant effect was observed on total T or B lymphocyte proportions. However, there was a dose-related increase in proportions of the so called "active" T lymphocytes. The response of human lymphocytes to phytohemagglutinin was markedly depressed. The alteration in the proportion of active T cells and the inhibition of T lymphocyte response to phyto hemagglutinin by alpha-fetoprotein occurred at higher concentrations than are present in amniotic fluid, serum of pregnant women, or serum of adults, but well within the range reached in fetal serum. The immunoregulatory role of alpha-fetoprotein is discussed.     

Prenatal diagnosis of congenital anomalies in an intrauterine growth retarded fetus

Summary Chromosome analysis of amniotic fluid cells and amniotic fluid alpha-fetoprotein determinations were used to investigate a fetus with severe intrauterine growth retardation in the third trimester. The karyotype was 47,XY,18+ and increased alpha-fetoprotein levels indicated the presence of congenital malformations. We suggest that when severe fetal growth retardation is detected early in the antepartum course, amniotic fluid alpha-fetoprotein and amniotic fluid cell chromosome studies be done to determine if congenital anomalies may be an etiological factor.     

Proteins in the amniotic and allantoic fluids of fetal pigs

Seven proteins were identified in the amniotic and allantoic fluids of fetal pigs (Sus scrofa domesticus) using crossed immunoelectrophoresis: albumin, fetuin, transferrin, alpha-fetoprotein (AFP), alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin. The total protein concentrations were determined by the method of Bradford and individual protein concentrations by radial immunodiffusion or rocket immunoelectrophoresis. Transferrin and fetuin were the major proteins in amniotic fluid during the second trimester of gestation and together with AFP and albumin accounted for the majority of the total protein in amniotic, but not allantoic fluid.     

Alpha-fetoprotein can regulate growth in the uterus of the immature and adult ovariectomized mouse

This is a report of development of (1) a 3-day immature mouse bioassay for alpha-fetoprotein (AFP) to increase the working range in uterine wet weights over-coming seasonality, and (2) a bioassay for AFP performed with ovariectomized adult mice to increase sensitivity. Mouse AFP was isolated from amniotic fluid and purified by polyacrylamide gel electrophoresis followed by Blue Sepharose chromatography. The uterine growth evoked by the injection of 1.0 microgram AFP together with excess molar oestradiol (0.5 microgram) over a 23-h period was compared in immature ovariectomized adult Nylar mice. The 3-day assay with immature mice was rendered usable in any season, with sensitivity comparable to the 1-day assay. Increased sensitivity, however, was demonstrated by utilization of AFP in a 1-day assay with the adult ovariectomized mouse.     

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 detection of neural tube closure defects by exfoliative cytology of amniotic fluid

Cytospin preparations of amniotic fluid samples from 200 pregnancies, taken between 16 and 20 weeks' gestation, were examined without knowledge of the fluid alpha-fetoprotein (AFP) level. The specimens were taken because of the possibility of neural tube closure defect. All but eight cases showed predominantly squamous cells, amnion cells, macrophages and blood cells. AFP levels in these fluids were within the normal range in 178 cases, unequivocal in 11, undetectable in 2 and raised in 1; none of the babies in these cases had a neural tube closure defect. In eight cases a large population of small cells with dark nuclei and a population of large, foamy macrophages were present in addition to the other cell types; all of these babies had a neural tube closure defect (five anencephaly and three anencephaly with spina bifida). This technique may provide a useful additional method of diagnosis of neural tube closure defects.     

Alpha-fetoprotein and albumin in experimentally-induced exencephaly in the rat.

Alpha-fetoprotein and albumin were quantified in the sera and amniotic fluids from control, Vitamin A-treated non-exencephalic and Vitamin A-treated exencephalic rat fetuses. Exencephaly was associated with amniotic fluid alpha-fetoprotein concentrations which were significantly elevated over those of Vitamin A-treated non-exencephalic and of untreated fetuses. Amniotic fluid albumin concentrations also were higher in the exencephalic fetuses than in the non-exencephalic fetuses. Serum alpha-fetoprotein and albumin concentrations were lower in the exencephalic than in the non-exencephalic fetuses. The results are cosistent with simple diffusion across a defective barrier as the cause of elevated amniotic fluid alpha-fetoprotein concentrations in the presence of open neural tube defects. This experimental model of neural tube defects result in changes in amniotic fluid alpha-fetoprotein similar to those changes found in human amniotic fluid alpha-fetoprotein concentrations in the presence of neural tube defects.     

The control of serum protein synthesis in hepatoma-fibroblast hybrids.

Hybrids between mouse hepatoma cells (which secrete several serum proteins) and mouse or rat fibroblasts (which do not secrete these proteins) produce transferrin and the third component of complement (C3) like the parental hepatoma cells, while they do not secrete either albumin or alpha-fetoprotein (AFP). This lack of albumin and AFP secretion is probably due to a lack of synthesis, rather than to a simple defect in secretion. The cessation of albumin and AFP production is not dependent upon the parental fibroblast nor upon the selection conditions; it is best explained by a shut-off synthesis and could thus reflect the existence of a regulatory mechanism. This would imply a difference between the control of albumin and AFP synthesis and that of transferrin and C3 synthesis. On the other hand, in agreement with Peterson and Weiss (1972), hybrids between rat hepatoma cells and mouse fibroblasts continue to product rat albumin. This suggests that the mouse hepatoma cells differ from the rat hepatoma cells in the way they control albumin production.