Ultrastructural studies of the intracellular translocation of endocytosed alpha‐foetoprotein (AFP) by cytochemistry and of the uptake of <sup>3</sup>H‐arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells

Geuskens, Maurice; Naval, Javier; Uriel, J

doi:10.1002/jcp.1041280307

Cited by 36 publications

(31 citation statements)

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“…ceptor binding but prevents endocytosis (34). Next, the cells were incubated at 37°C, which triggers endocytosis of receptorbound fetuin (35). As shown in Fig.…”

Section: Fig 2 Two-dimensional Sds-page and Western Blotting Of Promentioning

confidence: 99%

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

Wajih¹,

Borrás

Wang

et al. 2004

Journal of Biological Chemistry

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Matrix ␥-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent ␥-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-␥-carboxylated MGP and fully ␥-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptormediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

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“…ceptor binding but prevents endocytosis (34). Next, the cells were incubated at 37°C, which triggers endocytosis of receptorbound fetuin (35). As shown in Fig.…”

Section: Fig 2 Two-dimensional Sds-page and Western Blotting Of Promentioning

confidence: 99%

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

Wajih¹,

Borrás

Wang

et al. 2004

Journal of Biological Chemistry

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“…AFP may compete with albumin for binding in different cell types [15,[27][28][29]. Cytochemical and kinetic studies, carried out in several tumoral cells as well as in normal human T lymphocytes [18,27,30,31] have shown that the AFP was taken up by a receptor-mediated endocytosis mechanism similar to that reported for transferrin [19,28,31].…”

Section: Introductionmentioning

confidence: 99%

“…After three washes with cold PBS, cells were fixed for 15 min at 4°C, in 1.6% glutaraldehyde prepared in 0.1 M cacodylate buffer, pH 7.2. Afterwards, they were washed and incubated with diaminobenzidine (Sigma) substrate as previously described [19]. Postfixation in 2% OsO 4 in cacodylate buffer, for 40 min at room temperature, dehydration in ethanol and embedding in Epon were carried out in situ.…”

Section: Binding and Uptake Of Afp Albumin-and Transferrin-hrp Conjumentioning

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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“…Immature cells from most fetal structures internalize afp, and it has been suggested that the purpose of that internalization-and one of the functions of this protein-is to transport and deliver polyunsaturated fatty acids into fetal cells 6,7 . The afp uptake ceases when embryonic cells and tissue structures approach a high degree of differentiation, even if the blood concentration of afp is still high or increasing [8][9][10] .…”

Section: Introductionmentioning

confidence: 99%

Increased Alpha-Fetoprotein Receptor in the Serum of Patients with Early-Stage Breast Cancer

Moro¹,

Tcherkassova²,

Stieber³

2012

Current Oncology

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The alpha-fetoprotein (afp) receptor (recaf) is an oncofetal antigen found in most types of cancer. Using a competitive radioimmunoassay, we measured the concentration of serum recaf in three sets of samples. Set 1 was blind and consisted of 119 normal subjects, 43 breast cancer patients (stages i and ii), and 20 patients with benign breast conditions. In this set, the assay discriminated normal from cancer samples with a receiver operating characteristic for the area under the curve (ROCAUC) of 0.983; with 95% specificity and 93% sensitivity at a cut-off of 4.6 K (arbitrary) recaf units; and with 72% sensitivity and 100% specificity at a cut-off of 7.3 K units. At 7.3 K units, the specificity for benign breast conditions was 85%, and the sensitivity was 72% (ROCAUC was 0.773). Carcinoembryonic antigen and cancer antigen 15-3 respectively showed 39% and 41% sensitivity, with 95% specificity in comparisons of normal with cancer samples, and 34% and 44% sensitivity, with 85% specificity in comparisons of benign with cancer samples. Set 2 consisted of 353 normal, 30 benign, and 64 cancer samples (stages ii and iii). The recaf assay sensitivity in discriminating normal from cancer samples was 97%, with 97% specificity. Benign compared with cancer samples showed 87% sensitivity, with 97% specificity. Set 3 included only 40 normal and 40 cancer samples. The assay sensitivity was 89%, with 100% specificity. Sets 2 and 3 were not tested with carcinoembryonic antigen and cancer antigen 15-3. These results strongly suggest that the recaf assay could be used for detecting breast cancer in its early stages.

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Ultrastructural studies of the intracellular translocation of endocytosed alpha‐foetoprotein (AFP) by cytochemistry and of the uptake of ³H‐arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells

Cited by 36 publications

References 31 publications

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

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

Increased Alpha-Fetoprotein Receptor in the Serum of Patients with Early-Stage Breast Cancer

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Ultrastructural studies of the intracellular translocation of endocytosed alpha‐foetoprotein (AFP) by cytochemistry and of the uptake of 3H‐arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells

Cited by 36 publications

References 31 publications

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

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

Increased Alpha-Fetoprotein Receptor in the Serum of Patients with Early-Stage Breast Cancer

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Ultrastructural studies of the intracellular translocation of endocytosed alpha‐foetoprotein (AFP) by cytochemistry and of the uptake of ³H‐arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells