Downregulation of transferrin receptor surface expression by intracellular antibody.
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 the first time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.
Peng
Ji-Lin
JL
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
Wu
Sha
S
Zhao
Xiao-Ping
XP
Wang
Min
M
Li
Wen-Han
WH
Shen
Xin
X
Liu
Jing
J
Lei
Ping
P
Zhu
Hui-Fen
HF
Shen
Guan-Xin
GX
D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000494:pharmacology; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000276:immunology*; D013347:Subcellular Fractions / Q000276:immunology
D007135:Immunoglobulin Variable Region; D011990:Receptors, Transferrin