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Title: Downregulation of transferrin receptor surface expression by intracellular antibody

Abstract

To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

Authors:
 [1];  [2];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [3]
  1. Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China)
  2. (China)
  3. Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China). E-mail: guanxin_shen@yahoo.com.cn
Publication Date:
OSTI Identifier:
20979837
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 354; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2007.01.052; PII: S0006-291X(07)00064-2; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; APOPTOSIS; CELL CYCLE; ENDOPLASMIC RETICULUM; IRON; MICROSCOPES; MICROSCOPY; NEOPLASMS; PLASMIDS; POLYMERASE CHAIN REACTION; RECEPTORS; TRANSFERRIN; TUMOR CELLS

Citation Formats

Peng Jilin, Department of Immunology, Yunyang Medical College, Shiyan 442000, Wu Sha, Zhao Xiaoping, Wang Min, Li Wenhan, Shen Xin, Liu Jing, Lei Ping, Zhu Huifen, and Shen Guanxin. Downregulation of transferrin receptor surface expression by intracellular antibody. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.01.052.
Peng Jilin, Department of Immunology, Yunyang Medical College, Shiyan 442000, Wu Sha, Zhao Xiaoping, Wang Min, Li Wenhan, Shen Xin, Liu Jing, Lei Ping, Zhu Huifen, & Shen Guanxin. Downregulation of transferrin receptor surface expression by intracellular antibody. United States. doi:10.1016/j.bbrc.2007.01.052.
Peng Jilin, Department of Immunology, Yunyang Medical College, Shiyan 442000, Wu Sha, Zhao Xiaoping, Wang Min, Li Wenhan, Shen Xin, Liu Jing, Lei Ping, Zhu Huifen, and Shen Guanxin. Fri . "Downregulation of transferrin receptor surface expression by intracellular antibody". United States. doi:10.1016/j.bbrc.2007.01.052.
@article{osti_20979837,
title = {Downregulation of transferrin receptor surface expression by intracellular antibody},
author = {Peng Jilin and Department of Immunology, Yunyang Medical College, Shiyan 442000 and Wu Sha and Zhao Xiaoping and Wang Min and Li Wenhan and Shen Xin and Liu Jing and Lei Ping and Zhu Huifen and Shen Guanxin},
abstractNote = {To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.},
doi = {10.1016/j.bbrc.2007.01.052},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 354,
place = {United States},
year = {Fri Mar 23 00:00:00 EDT 2007},
month = {Fri Mar 23 00:00:00 EDT 2007}
}
  • 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
  • The effect of the synthetic diacylglcerol, sn-1,2-dioctanoylglycerol (diC/sub 8/), on the expression of the surface transferrin receptor reveals that exogenous diC/sub 8/ can act as an intracellular activator of protein kinase C and stimulate both down-regulation and increased receptor phosphorylation in a manner similar to that induced by the active tumor promotor, 4..beta..-phorbol 12,13-dibutyrate. Unlike the spontaneously irreversible effect noted when 4..beta..-phorbol 12,13-dibutyrate is added, this same effect mediated by diC/sub 8/ is brief, lasting only minutes, and is spontaneously reversible. The rate of reversibility is dependent on the concentration of diC/sub 8/ added, and it is associated with rapidmore » formation of a newly detected intracellular phospholipid that corresponds to sn-1,2-dioctanoyl phosphatidic acid. These data, in conjunction with findings that demonstrate that exogenous diacylglycerols (including diC/sub 8/) when added to cells do not stimulate cellular phospholipase A/sub 2/ or C, argue that protein kinase C is activated only briefly in this system since exogenous diC/sub 8/ is subject to rapid intracellular metabolism to phosphatidic acid.« less
  • 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
  • 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 wasmore » 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.« less
  • Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate {approximately} 4 hr after injection, suggesting a time-dependent sequesteringmore » of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.« less