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Title: Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

Abstract

Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. The authors studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. These data indicate that TfR expression is regulated by twomore » independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.« less

Authors:
; ;
Publication Date:
Research Org.:
Univ. of Texas Health Science Center, San Antonio
OSTI Identifier:
6790275
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Cell. Physiol.; (United States); Journal Volume: 129:3
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; MONOCLONAL ANTIBODIES; RADIORECEPTOR ASSAY; PHORBOL ESTERS; BIOCHEMISTRY; TRANSFERRIN; DNA REPLICATION; GROWTH; INHIBITION; IODINE 125; LYMPHOCYTES; RECEPTORS; ANIMAL CELLS; ANTIBODIES; BETA DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CARCINOGENS; CHEMISTRY; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; ESTERS; GLOBULINS; GLOBULINS-BETA; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; LEUKOCYTES; MATERIALS; MEMBRANE PROTEINS; METALLOPROTEINS; NUCLEI; NUCLEIC ACID REPLICATION; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PROTEINS; RADIOISOTOPES; SOMATIC CELLS; TRACER TECHNIQUES 550601* -- Medicine-- Unsealed Radionuclides in Diagnostics

Citation Formats

Alcantara, O., Phillips, J.L., and Boldt, D.H. Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms. United States: N. p., 1986. Web. doi:10.1002/jcp.1041290310.
Alcantara, O., Phillips, J.L., & Boldt, D.H. Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms. United States. doi:10.1002/jcp.1041290310.
Alcantara, O., Phillips, J.L., and Boldt, D.H. 1986. "Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms". United States. doi:10.1002/jcp.1041290310.
@article{osti_6790275,
title = {Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms},
author = {Alcantara, O. and Phillips, J.L. and Boldt, D.H.},
abstractNote = {Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. The authors studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.},
doi = {10.1002/jcp.1041290310},
journal = {J. Cell. Physiol.; (United States)},
number = ,
volume = 129:3,
place = {United States},
year = 1986,
month =
}
  • We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular /sup 59/Fe incorporation is inappropriately low. Althoughmore » shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of /sup 59/Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation.« less
  • The authors have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, they have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle. They demonstrate here that this procedure is useful for a human leukemic cell line. They enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment,more » or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, they show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks transferrinfection, as does incubation of the cells at 18{degree}C.« less
  • It has been suggested that effects of anti-transferrin receptor antibodies o cell growth and receptor expression are the result of varying degrees of receptor crosslinking by bi- and multivalet binding agents. In order to study this question directly, the authors have cultured murine lymphoma cells in mon- and divalent fragments from IgG and IgM monoclonal anti-transferrin receptor antibodies and in intact antibodies. The studies presented here demonstrate that effects of antibody binding on transferrin receptor distribution, metabolism, and function depend, at least in part, on antibody valence, and therefore on the degree of crosslinking of receptors by antibody. They foundmore » that monovalent antibody fragments did not significantly alter cell growth, receptor surface expression, intracellular localization, or degradation. Divalent antibody caused a uniform down-regulation of cell-surface receptor expression, which was accompanied by increased degradation only when antibody Fc was present. Normal receptor cycling apparently continued, despite the reduction in surface expression. Culture in multivalent IgM antibody, however, resulted in accumulation of antibody-complexed receptor on the cell surface without internalization and caused profound inhibition of cell growth. Thus, they show two mechanisms by which different degrees of antibody crosslinking can influence transferrin receptor function: by receptor down-regulation and blocking internalization.« less
  • Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEEGEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. The authors show here that there is a strict correlation between the levels of immunoreactive polypeptide and extractable calcium- and phospholipid-dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was foundmore » to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13-dibutyrate protein kinase C was essentially undetectable by 40 hours; the half-life of this down-regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium- and phospholipid-dependent kinase activity precisely parallels the phorbol ester induced down-regulation of binding and responsiveness in Swiss 3T3 cells.« less
  • Human rIL-4 specifically induces the expression of the low affinity receptor for IgE (Fc epsilon R2/CD23) on normal B cells and on the Burkitt lymphoma cell line Jijoye. IL-4 does not induce the generation of the second messenger cAMP in Jijoye cells. PGE2 (at 10(-7) M) was found to inhibit by 50% the IL-4 mediated Fc epsilon R2/CD23 induction on Jijoye cells. The PGE2 half maximum inhibitory concentration (1 nM) was comparable to that inducing a half maximal increase of intracellular cAMP (4nM PGE2). 8-bromo-cAMP (10(-3) M), forskolin (10(-5) M), and cholera toxin (100 ng/ml), which increase intracellular cAMP levels,more » also inhibited by 40 to 80% the IL-4 induced Fc epsilon R2/CD23 expression on Jijoye cells. PGE2 8-bromo-cAMP, forskolin, and cholera toxin also inhibited the IL-4-induced Fc epsilon R2/CD23 expression on normal B lymphocytes. Taken together these data suggest that PGE2 inhibits the IL-4 induced Fc epsilon R2/CD23 through an increase of intracellular cAMP. In contrast, IFN-gamma, which strongly inhibits IL-4-mediated Fc epsilon R2/CD23 expression on Jijoye cells, did not increase intracellular cAMP levels and thus probably acts through another mechanism. IFN-gamma and PGE2 did not inhibit binding of IL-4 to its receptor. It could be excluded that IFN-gamma and PGE2 were acting via an alteration/desensitization of the IL-4R inasmuch as 24 h pre-incubation of Jijoye cells with these agents affected neither the affinity of 125I-IL-4 for its receptor (Kd = 0.8 to 1.5 x 10(-10) M) nor the maximal number of binding sites per Jijoye cells (Bmax = 390 to 550). Furthermore, IFN-gamma and PGE2 did not affect the internalization and degradation of 125I-IL-4. These data demonstrate that PGE2 and IFN-gamma inhibit the IL-4-mediated induction of Fc epsilon R2/CD23 on B lymphocytes via different mechanisms that do not alter the interaction of IL-4 with its receptor.« less