title: Comparative study on transduction and toxicity of protein transduction domains.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.affiliation: Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NIBIO), Ibaraki, Osaka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Sugita
authors.forename: T
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Y
authors.initials: Y
authors.lastname: Mukai
authors.forename: N
authors.initials: N
authors.lastname: Yamanada
authors.forename: S
authors.initials: S
authors.lastname: Imai
authors.forename: K
authors.initials: K
authors.lastname: Nagano
authors.forename: Y
authors.initials: Y
authors.lastname: Yoshida
authors.forename: H
authors.initials: H
authors.lastname: Shibata
authors.forename: Y
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: S
authors.initials: S
authors.lastname: Nakagawa
authors.forename: H
authors.initials: H
authors.lastname: Kamada
authors.forename: S-I
authors.initials: SI
authors.lastname: Tsunoda
authors.forename: Y
authors.initials: Y
authors.lastname: Tsutsumi
mesh_terms: D000584:Amiloride / Q000008:administration & dosage / Q000494:pharmacology; D051943:Antennapedia Homeodomain Protein / Q000009:adverse effects / Q000378:metabolism*; D045744:Cell Line, Tumor; D015638:Cytochalasin D / Q000008:administration & dosage / Q000494:pharmacology; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D015694:Gene Products, rev / Q000009:adverse effects / Q000378:metabolism*; D015696:Gene Products, tat / Q000009:adverse effects / Q000378:metabolism*; D006801:Humans; D010873:Pinocytosis / Q000502:physiology; D021381:Protein Transport; D015678:Viral Structural Proteins / Q000009:adverse effects / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: D051943:Antennapedia Homeodomain Protein; D005452:Fluoresceins; D015694:Gene Products, rev; D015696:Gene Products, tat; D015678:Viral Structural Proteins; D047392:beta-Cyclodextrins; C073413:herpes simplex virus type 1 protein VP22; C108732:methyl-beta-cyclodextrin; D015638:Cytochalasin D; D000584:Amiloride