Endogenous and Exogenous Alpha-Fetoproteins as Differential Markers of Cultured Neonatal Mouse Schwann Cells and Fibroblasts

Trojan, Jerzy; Boutry, J. M.; Hauw, J. J.; Lafarge‐Frayssinet, C.; Deugnier, Marie Ange; Hajeri-Germond, M.; Abramsky, Oded; Uriel, J

doi:10.1159/000111675

Cited by 4 publications

(6 citation statements)

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“…Their expression was maintained in cells characterized by active proliferation and differentiation, but it was diminished in neonates, and was practically lost in the adult tissues, including the liver. In the case of AFP, several studies have been performed in the past to analyze the uptake of this protein by different organs and cells [10][11][12][13][14][15][16][17]. However, direct studies of the expression of AFP-binding proteins in normal tissues have not been performed, mainly due to the fact that specific antibodies against these proteins are not available at the moment.…”

Section: Discussionmentioning

confidence: 99%

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Specific Uptake of Alpha-Fetoprotein and Albumin by Rat Morris 7777 Hepatoma Cells

et al. 1999

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Alpha-fetoprotein (AFP) is a major globulin of embryonic plasma and a physiological carrier of unesterified fatty acids. In the present work, we have characterized the interaction of AFP and albumin, a major serum protein of adult mammals which presents numerous biochemical analogies with AFP, with the plasma membrane of the rat Morris 7777 hepatoma cells. Time course analysis of the uptake of AFP and albumin by these cells showed a saturable profile at 4°C and 37°C. Saturable binding of ¹²⁵I-AFP or ¹²⁵I-albumin were observed when the concentration of these proteins increased (ranging from 0.3 to 4.5 µM). The Hill and Scatchard analysis revealed the existence of binding sites in the surface of hepatoma cells, with a k′d = 2.2 × 10^–6 M (2.9 × 10⁶ sites/cell) in the case of AFP and a k′d = 4.5 × 10^–6 M (3.9 × 10⁶ sites/cell) in the case of albumin. ¹²⁵I-AFP and ¹²⁵I-albumin bound to the cells were completely displaced in the presence of a 200-fold excess of unlabeled AFP or albumin, respectively, suggesting that these interactions were specific. We have observed crossed competition between AFP and albumin for their respective binding sites; no such crossed competition was observed when an excess of unlabeled transferrin was added. Pulse-chase experiments showed that about 50% of the AFP and 75% of the albumin taken up by the cells were released undegraded into the medium after 1 h. Cytochemical studies performed with covalent conjugates of AFP, albumin and transferrin with horseradish peroxidase have shown that AFP and albumin entered the cells via a vesicular system. This intracellular pathway is different from that of transferrin, a plasma protein whose internalization mediated by specific receptors via coated pits has been reported in other cells. The results presented here suggest that AFP and albumin interact with sites in the membrane of hepatoma cells, probably physically related, and then they are transported inside the cells by a mechanism different from that described for transferrin.

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Section: Discussionmentioning

confidence: 99%

“…AFP and albumin have been localized in identical structures of the developing nervous system and other tissues of the rat, mouse, human and baboon [11][12][13]. Previous studies have shown that the ability to internalize AFP, which is characteristic of developing cells [14][15][16], may reappear in neoplastic cells [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Specific Uptake of Alpha-Fetoprotein and Albumin by Rat Morris 7777 Hepatoma Cells

et al. 1999

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“…Expression of ATBF1 in brain and other nonhepatic tissues is interesting for several reasons. First, it has been shown that the AFP gene is expressed in brain and other nonhepatic tissues at particular stages of pre-and postnatal life (1,8,35,38,41). This suggests that nonhepatic AFP gene expression is developmentally regulated, possibly through a mechanism different from that operating in liver tissue (16).…”

Section: Discussionmentioning

confidence: 99%

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

Yasuda¹,

Mizuno²,

Tamaoki³

et al. 1994

Mol. Cell. Biol.

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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 a-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.The regulatory regions of all genes so far studied contain multiple cis-acting elements which interact with trans-acting factors (22,32). Liver-specific gene expression is also achieved through a combinatorial action of regulatory DNA elements and interacting transcription factors (20,21,30). One characteristic feature of a number of liver-specific promoters is the presence of DNA sequences having a well-organized array of 10 to 11 adenine (A) and thymine (T) residues sandwiched by a guanine (G) and a cytosine (C) (AT-rich element) (4-7, 11, 15, 23, 26, 40). These elements have been shown to be the binding sites of a liver-enriched transcription factor, HNF1 (also called LF-B1 and APF) (3-6, 12, 31). Mutations of the AT-rich element cause drastic reduction of liver-specific transcriptional activity both in vitro and in vivo (21, 28, 33, 42), indicating that the AT-rich element and HNF1 play a crucial role in liver-specific promoter activation.In the promoters of a-fetoprotein (AFP) genes of mice, rats, and humans, two HNF1-binding sites are conserved. In the enhancers, on the other hand, the AT-rich element is present in the human AFP gene but not in the mouse and rat genes (13). The human AFP enhancer AT motif is also unique in that HNF1 binds to it 10-fold...

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“…At that time it was not known whether these proteins were synthesized, or were internalized from extracellular sources (Tilghman et al, 1979;Uriel et al, 1981). We have recently demonstrated that cells which specifically internalized exogenous AFP, i.e., neurons of embryonic chick retinas (Hajeri-Germond et al, 1991), rat and mouse Schwann cells (Trojan et al, 1992), and cultured rat PBM cells (Esteban et al, 1993), were precisely those expressing endogenous alphafetoprotein.…”

Section: Discussionmentioning

confidence: 99%

“…Anti-AFP and anti-SA antibodies (and the corresponding FITC labelled conjugates) were used, in indirect immunocytochemical labelling, as described by Trojan et al [1992].…”

Section: Immunocytochemistrymentioning

confidence: 99%

Expression of serum albumin and of alphafetoprotein in murine normal and neoplastic primitive embryonic structures

Trojan¹,

Naval

Johnson

et al. 1995

Molecular Reproduction Devel

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Alphafetoprotein (AFP), a major serum protein synthesized during the embryo-fetal and postnatal period (in the yolk sac, then in the liver), is also an oncoprotein. The intracellular presence of AFP and of serum albumin (SA) in normal and neoplastic neural crest and neural tube derivatives was previously demonstrated. In this work we have studied the comparative expression of AFP and SA in primitive neuroectoblastic structures of mouse embryos (6 and 7 days "post coitum") and mouse teratocarcinomas (derived from the PCC4 cell line). Using immunofluorescence technique, antibodies to SA gave a positive reaction in embryos of 7 days, while AFP was not detected during this period. By mRNA in situ hybridization, SA mRNA gave a strong signal in both 6 and 7 day embryos, whereas AFP mRNA gave a weak signal only in 7-day embryos. The distribution of SA and AFP and their mRNAs was investigated in primitive neuroectoblastic structures of the teratocarcinomas by in situ hybridization and immunostaining. Only SA protein was detectable by immunostaining. SA mRNA gave a strong signal in differentiating structures as well as in undifferentiated cell clusters. AFP mRNA was observed only in differentiating structure. Dot-blot hybridization indicated that the level of SA transcripts was at least 6-fold higher than that of AFP transcripts in the teratocarcinomas investigated. In teratocarcinoma-bearing mice injected intraperitoneally with 125I-radiolabeled SA and AFP, significant accumulations of both SA and AFP were demonstrated in the tumors, SA being about 3-fold higher than that of AFP after normalization to quantity of uptake in liver. External in vivo photoscanning confirmed this relationship of accumulated radiolabeled proteins. The last observation could be useful in vivo for diagnosis of teratocarcinoma. We conclude that the expression of SA relative to AFP and the external cellular uptake of SA relative to AFP are similar in normal embryonic developing tissues and in the corresponding morphologically neoplastic tissues of the teratocarcinomas. The same SA:AFP relationship constitutes an oncofetal marker of primitive neuroectoblastic structures.

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Endogenous and Exogenous Alpha-Fetoproteins as Differential Markers of Cultured Neonatal Mouse Schwann Cells and Fibroblasts

Cited by 4 publications

References 12 publications

Specific Uptake of Alpha-Fetoprotein and Albumin by Rat Morris 7777 Hepatoma Cells

Specific Uptake of Alpha-Fetoprotein and Albumin by Rat Morris 7777 Hepatoma Cells

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

Expression of serum albumin and of alphafetoprotein in murine normal and neoplastic primitive embryonic structures

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