Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.     

The lectin binding sites on the membranes of the nuclear envelope, mitochondria and the cell surface of human lymphoblastoid cells

The membranes of the cell surface, the endoplasmic reticulum, outer and inner mitochondrial leaflet and nuclear envelope were isolated from three human lymphoblastoid cell lines. Membrane components were separated by dodecyl sulfate polyacrylamide gel electrophoresis and the gels incubated with the radioiodinated lectins from lentil, castor bean, scarlet runner bean, gorse seed and Roman snail. After gel slicing and counting, the molecular weights of the lectin binding sites were determined. About 20 glycoproteins were identified as constituents of the plasma membrane, a similar glycoprotein distribution was observed in the endoplasmic reticulum. The outer mitochondrial membrane contained some impurities from the plasma membrane, the inner mitochondrial membrane lacked specific lectin receptors. Two prominent glycoproteins with molecular weights of 70 000 and 60 000 were identified with the castor bean lectin in the nuclear envelope.     

Transferrin binding by human lymphoblastoid cell lines and other transformed cells

Viable cells of 18 human cell lines, including 15 transformed cell lines of malignant and lymphoblastoid origin, were examined by an indirect immunofluorescence method for their ability to bind purified transferrin and transferrin in normal human serum. The specificity of the reaction was investigated by study of the binding reactions of several other serum proteins, including albumin, α-1-antitrypsin, and α-2-macroglobulin. Membrane binding of human transferrin was demonstrated in less than 5% of normal peripheral blood mononuclear cells or cultured diploid fibroblasts, but in more than 80% of the cells from 13 of the transformed lines, and the data obtained indicated that this binding reaction reflected the presence of specific receptors for transferrin.     

High affinity binding sites on plasma membrane obtained from the lymphoblastoid cultured 1301 cell line for highly radioactive serum thymic factor

The interaction of the synthetic serum thymic factor (FTS, facteur thymique sérique) with a plasma membrane preparation of human T lymphocytes from the lymphoblastoid T cell line 1301 was studied using ³H-labelled FTS (specific activity 120 Ci/mmol). The binding is temperature dependent and function of the concentration of both ³H-labelled FTS and membrane proteins. At 37°C, using 1 nM of ³H-labelled FTS a steady state is observed within 80 min. The binding is reversible, specific and saturable. Scatchard analysis reveals the existence of at least two binding sites with respective K_d of the order of 0.516±0.2 nM and 110±27.8 nM with concentrations of 0.186±0.045 pmol and 2.026±0.367 pmol per mg of membrane protein.     

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces...     

Transferrin receptors on human B and T lymphoblastoid cell lines.

Experiments demonstrating the existence of receptors for iron-saturated transferrin on both B and T lymphoblastoid cell lines of human origin are described. Binding of 125I-labeled transferrin is rapid, saturable and reversible. It can be specifically inhibited by unlabeled transferrin but not by other proteins. The number of receptors on T cell lines determined by Scatchard analysis is almost double the number on B cell lines but the binding affinities are equal. The putative transferrin receptor can be removed from the cell by the proteolytic enzymes papain and trypsin, and is re-expressed during overnight incubation at 37 degrees C. Resynthesis is inhibited by puromycin. The receptor can be solubilized by deoxycholate, and retains transferrin binding capacity when non-covalently attached to an amphipathic matrix consisting of deoxycholate-coupled poly(L-lysyl) Agarose.     

The relationship between tumor promoter binding and epstein-barr virus induction in human lymphoblastoid cell lines

The tumor promoter TPA (12-0-tetradecanoyl-phorbol-13-acetate) efficiently induces the synthesis of Epstein-Barr virus (EBV) antigens in EBV-genome-harboring human lymphoblastoid cells. We attempted to obtain information on the binding of TPA to cells and on the relationship between TPA-binding and EBV induction by the use of tritiated TPA (³H-TPA). Our data show: (1) In the absence of cells TPA can bind to serum completely within 60 minutes. (2) Cells can compete for a proportion of serum-bound TPA. (3) Binding of TPA to cells reaches equilibrium within 60 minutes and is higher at 37° than 0°C. In the absence of serum, the rate of binding is about twice as high as in the presence of serum. (4) Dissociation of TPA from cells also seems to be rapid. When the cells are incubated with cold TPA after prior treatment with ³H-TPA, followed by washing, a much higher rate of release of labeled TPA is observed than in cultures to which fresh medium is added exclusively. Dissociation is higher in the presence of serum than in the absence of it. If radiolabeled cells are analyzed after serial washing, the rate of cell-associated/noncell-associated radioactivity indicates that the proportion of molecules required for EBV induction is dissociated rapidly.     

A reliable protocol for direct detection of lectin binding sites on the plasma membrane of a single living sperm cell in maize

. A novel protocol for direct detection and localization of lectin binding sites on the surface of single sperm cells is presented. Fluorescence-conjugated lectins and single cell manipulation techniques were employed in this protocol. Advantages of the protocol include that sperm cell membranes are well protected and artifacts avoided by minimizing movement of the medium. Damage of the sperm membrane was reduced using a droplet-staining/washing method. Any morphological change may be readily recorded.     

Differences in the incorporation of bromodeoxyuridine by human lymphoblastoid cell lines.

Long term human lymphoblastoid lines differ in their ability to grow in medium containing bromodeoxyuridine (BrdU) and to incorporate analog into their DNA. Eight Burkitt's lymphoma cell lines divided at least twice in BrdU-containing medium and made DNA in which over 90% of the thymidine residues were substituted with analog in both strands. Three infectious mononucleosis-derived lines and 24 lines transformed in vitro were inhibited by BrdU after one cell division and made only hybrid DNA in which one strand was substituted with analog. One out of eight normal individuals from whom long term lines were prepared gave cell lines which divided at least twice in BrdU and gave DNA in which both strands were substituted with analog. It would appear that intrinsic cellular factors regulate the response to BrdU and that Burkitt's tumor lines are characterized by their ability to make stable doubly substituted DNA containing a high proportion of halogenated analog.     

Ca binding to the human red cell membrane: Characterization of membrane preparations and binding sites

Summary Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10^–6 to 10^–3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×10⁵ and 6×10³ M^–1, and right side out vesicles exhibit three independent binding sites with association constants of 2×10⁵, 1.4×10⁴ and 3×10²M^–1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×10⁵, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×10⁵ per equivalent cell.     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.     

Identification of functional binding sites for progesterone in rat Leydig cell plasma membrane.

Steroid hormones influence cell functions by binding to intracellular receptors and then acting within the nucleus. There is now evidence that steroids affect cell functions also via interaction with plasma membrane receptors in a number of different cell types. In this regard, progesterone appears to be one of the most active steroids. In this paper, we evaluate the effects of progesterone on rat Leydig cell functions, determining variations of ion homeostasis and testosterone production. This steroid was able to effect a depolarization of the plasma membrane that was due to an influx of sodium (Na+) from the external medium since it was absent when extracellular Na+ was iso-osmotically substituted with choline chloride or sucrose. The determination of intracellular sodium concentration ([Na+]i) with the Na+ -sensitive fluorescent dye sodium-benzofuran-isophtalate (SBFI) confirmed these observations. Progesterone did not modify Leydig cell intracellular calcium concentration ([Ca2+]i) at any dose tested. Furthermore, using a cell impermeant progesterone conjugate, we demonstrated that progesterone was able to stimulate Leydig cell steroidogenesis in a dose-dependent manner. The exclusion of calcium (Ca2+) from the extracellular medium did not modify the depolarizing action of progesterone and its steroidogenetic effect while in Na+ -free medium (sucrose supplemented) progesterone-stimulated effects were completely blunted. Finally, using fluorescence microscopy with a fluorescein isothiocyanate-coupled cell impermeant progesterone conjugate, we identified plasma membrane binding sites for progesterone in rat Leydig cells. These results suggest that rat Leydig cells possess progesterone receptors located on the plasma membrane, which when occupied achieves a plasma membrane depolarization, dependent on an influx of Na+ from the external medium, and the subsequent activation of steroidogenesis.     

Interferon production potentials of various human lymphoblastoid cell lines

A number of human lymphoblastoid cells were examined concerning their ability to produce spontaneously liberated and virus-induced interferon (IFN). It was found that, in addition to B cells, various T and nonT-nonB lymphoblastoid cells responded well to Sendai virus infection to form IFN, the characterization of which has been recently reported (20). One B lymphoblastoid cell line from an infectious mononucleosis (IM) patient produced a large amount of IFN-alpha and might become an alternative source of IFN production. Among 68 cell lines examined, 35 cell lines liberated 10 U/ml or more of IFN spontaneously in culture fluid. The presence of Epstein-Barr virus (EBV) genome or its activation appears to have no correlation with the spontaneous liberation of IFN. Spontaneously produced IFN from three cell lines was characterized as IFN-alpha. Comparatively higher amounts of IFN were produced in cells from IM patients than those from Burkitt's lymphoma cases or healthy adults. Spontaneously produced IFN was detected more easily in cells transformed by EBV alone than in those transformed by EBV and a tumor promoter, TPA.     

Instability of Mex- phenotype in human lymphoblastoid cell lines

Three lymphoblastoid cell lines (LCLs) had extremely low activities of O6-alkylguanine-DNA alkyltransferase (O6-AGT), and were classified as Mex-. They were highly sensitive to cell killing by 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a hydrochloride (ACNU), whereas NMO2, a Mex+ LCL with a high O6-AGT activity, was resistant to the agent. Small fractions of these Mex- LCLs survived the treatment with 10 micrograms/ml of ACNU for 24 h, and the surviving cells were found to be resistant to subsequent treatments with the agent. In addition, they contained O6-AGT activities comparable to that of NMO2 and were therefore regarded as Mex+. These results suggest that the Mex- phenotype in LCLs is unstable.     

Isolation and characterization of mitochondria from human B lymphoblastoid cell lines.

Mitochondria were isolated from detergent-treated Epstein-Barr virus-transformed human lymphocytes to examine their potential use in the study of the functional expression of genetic disorders of the respiratory chain. The increase of cytochrome c oxidase activity in the mitochondrial fraction indicated a 6-fold purification of intact mitochondria. Polarographic and spectrophotometric studies revealed that the isolated mitochondria were functionally well preserved. Furthermore, the isolated mitochondria supported an active in organello protein synthesis, which was dependent on the presence of a respiratory substrate generating ATP and was essentially abolished by chloramphenicol or by a specific respiratory chain inhibitor, such as antimycin. Thus, B lymphoblastoid cell lines constitute a valuable source of mitochondria to investigate mitochondrial functions in patients affected by respiratory chain disorders.     

Glycosylation of developing human glomeruli: lectin binding sites during cell induction and maturation

Expression of cellular glycoconjugates during differentiation of human fetal kidney was studied using fluorochrome-labeled lectins. Each lectin revealed a characteristic binding pattern during the phenotypic change of the nephrogenic mesenchyme and during distinct stages of nephron development. The uninduced mesenchymal cells were positive for Pisum sativum (PSA), Concanavalin A (ConA), Wistaria floribunda (WGA), and Ricinus communis (RCA-I) lectins. However, these lectins failed to react with the uninduced cells of the S-shaped bodies, whereas Maclura pomifera (MPA), Triticum vulgaris (WGA) and, after neuraminidase treatment, Arachis hypogaea (PNA) agglutinins bound intensely to the presumptive podocytes. During later stages of nephrogenesis, MPA positively on the podocytes weakened and could not be observed in adult kidney glomeruli. Binding sites for Helix pomatia (HPA) agglutinin in glomeruli were also expressed only transiently during nephrogenesis. During further development PSA, ConA, WFA, and RCA-I reacted with mesangial cells in addition to the glomerular basement membranes. The segment-specific lectin binding patterns of the tubuli emerged in parallel with the appearance of brush border and Tamm-Horsfall antigens of the proximal and distal tubuli. The results show that nephron site-specific saccharides appear in a developmentally regulated manner and in parallel with morphologic maturation of the nephron. Lectins therefore appear to be useful tools for study of induction and maturation of various nephron cell types.     

Asparagine-linked sugar chains of plasma membrane glycoproteins from healthy and common variable immunodeficiency B lymphoblastoid cell lines

Asparagine-linked sugar chains of plasma membrane glycoproteins, which are formed by glycosylation during B cell maturation, were examined with B lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus derived from healthy controls and patients with common variable immunodeficiency (CVI). Both two patients with CVI showed hypogammaglobulinemia and impaired B cell functions. LCLs from healthy controls and the patients showed CD19+ and HLA/DR+ in the cell surface and secreted IgM. In both healthy controls and the patients, the main oligosaccharide in asparagine-linked sugar chains of the membrane glycoproteins of LCLs was biantennary sugar chain with bisected GlcNAc (Gal₂-GlcNAc₂-Man₃-GlcNAc-GlcNAc-Fuc-GlcNAc_OT). Biantennary sugar chain with an-fucosyl residue linked at the proximal GIcNAc was seen but biantennary sugar chain without an-fucosyl residue at the proximal GlcNAc was little detected in each LCL. There was no difference in quality and quantity of asparagine-linked sugar chains between healthy controls and the patients. These results suggest that glycosylation during B cell maturation may not be impaired in patients with CVI.