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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}
}