Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells.
The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.
Department of Hyperbaric Medicine, No 401 Hospital of PLA, Qingdao, China.
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Q
Sun
J
J
Xiong
J
J
Lu
S
S
Xu
Y
Y
Li
X P
XP
Zhong
G K
GK
Gao
H Q
HQ
Liu
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002846:Chromatography, Affinity; D006224:Cricetinae; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D015696:Gene Products, tat; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit