DNA transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis.
Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.
Renigunta
Aparna
A
University of Giessen Lung Center (UGLC), Medical Clinic II, Biochemistry, Friedrichstrasse 24, D-35392 Giessen, Germany.
Krasteva
Gabriela
G
König
Peter
P
Rose
Frank
F
Klepetko
Walter
W
Grimminger
Friedrich
F
Seeger
Werner
W
Hänze
Jörg
J
D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D015696:Gene Products, tat / Q000737:chemistry / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D008168:Lung / Q000166:cytology* / Q000502:physiology; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes
D015696:Gene Products, tat; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C511561:Tat-RGD fusion protein; C047981:arginyl-glycyl-aspartic acid; D004247:DNA