title: Transduction of peptides and proteins into live cells by cell penetrating peptides.
abstract: Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.
authors.affiliation: Jena Bioscience GmbH, Loebstedter Str. 80, 07749 Jena, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Mussbach
authors.forename: Martin
authors.initials: M
authors.lastname: Franke
authors.forename: Ansgar
authors.initials: A
authors.lastname: Zoch
authors.forename: Buerk
authors.initials: B
authors.lastname: Schaefer
authors.forename: Siegmund
authors.initials: S
authors.lastname: Reissmann
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D014161:Transduction, Genetic
chemical_list: D057846:Cell-Penetrating Peptides; D010455:Peptides; D011506:Proteins; D034741:RNA, Small Interfering