Document Detail


Comparative study on transduction and toxicity of protein transduction domains.
MedLine Citation:
PMID:  18223668     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND AND PURPOSE: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs. EXPERIMENTAL APPROACH: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines. KEY RESULTS: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat. CONCLUSIONS AND IMPLICATIONS: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.
Authors:
T Sugita; T Yoshikawa; Y Mukai; N Yamanada; S Imai; K Nagano; Y Yoshida; H Shibata; Y Yoshioka; S Nakagawa; H Kamada; S-I Tsunoda; Y Tsutsumi
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Publication Detail:
Type:  Comparative Study; Journal Article     Date:  2008-01-28
Journal Detail:
Title:  British journal of pharmacology     Volume:  153     ISSN:  0007-1188     ISO Abbreviation:  Br. J. Pharmacol.     Publication Date:  2008 Mar 
Date Detail:
Created Date:  2008-03-17     Completed Date:  2008-05-15     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7502536     Medline TA:  Br J Pharmacol     Country:  England    
Other Details:
Languages:  eng     Pagination:  1143-52     Citation Subset:  IM    
Affiliation:
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NIBIO), Ibaraki, Osaka, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amiloride / administration & dosage,  pharmacology
Antennapedia Homeodomain Protein / adverse effects,  metabolism*
Cell Line, Tumor
Cytochalasin D / administration & dosage,  pharmacology
Flow Cytometry
Fluoresceins / metabolism
Gene Products, rev / adverse effects,  metabolism*
Gene Products, tat / adverse effects,  metabolism*
Humans
Pinocytosis / physiology
Protein Transport
Viral Structural Proteins / adverse effects,  metabolism*
beta-Cyclodextrins / administration & dosage,  pharmacology
Chemical
Reg. No./Substance:
0/Antennapedia Homeodomain Protein; 0/Fluoresceins; 0/Gene Products, rev; 0/Gene Products, tat; 0/Viral Structural Proteins; 0/beta-Cyclodextrins; 0/herpes simplex virus type 1 protein VP22; 0/methyl-beta-cyclodextrin; 22144-77-0/Cytochalasin D; 2609-46-3/Amiloride
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