The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.
Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.
Vercauteren
Dries
D
Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University, Ghent, Belgium.
Vandenbroucke
Roosmarijn E
RE
Jones
Arwyn T
AT
Rejman
Joanna
J
Demeester
Joseph
J
De Smedt
Stefaan C
SC
Sanders
Niek N
NN
Braeckmans
Kevin
K
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000737:chemistry; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007790:Lactosylceramides / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D014168:Transferrin / Q000737:chemistry; D047392:beta-Cyclodextrins / Q000737:chemistry
D015703:Antigens, CD; D002966:Clathrin; D005456:Fluorescent Dyes; D007790:Lactosylceramides; D014168:Transferrin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C009744:CDw17 antigen