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The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls

Dries Vercauteren UGent, Roosmarijn Vandenbroucke UGent, Arwyn T Jones, Joanna Rejman UGent, Jo Demeester UGent, Stefaan De Smedt UGent, Niek Sanders UGent and Kevin Braeckmans UGent (2010) MOLECULAR THERAPY. 18(3). p.561-569
abstract
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 (M beta CD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent M beta CD 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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
TRANSFECTION EFFICIENCY, RNA INTERFERENCE, DEPENDENT INTERNALIZATION, CELL-LINE, INTRACELLULAR TRAFFICKING, CLATHRIN-INDEPENDENT ENDOCYTOSIS, CAVEOLAE-MEDIATED ENDOCYTOSIS, MAMMALIAN-CELLS, HELA-CELLS, DELIVERY
journal title
MOLECULAR THERAPY
Mol. Ther.
volume
18
issue
3
pages
561 - 569
Web of Science type
Article
Web of Science id
000275454500015
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
7.149 (2010)
JCR rank
10/158 (2010)
JCR quartile
1 (2010)
ISSN
1525-0016
DOI
10.1038/mt.2009.281
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1063955
handle
http://hdl.handle.net/1854/LU-1063955
date created
2010-10-26 10:57:02
date last changed
2012-06-26 14:32:10
@article{1063955,
  abstract     = {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 (M beta CD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent M beta CD 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.},
  author       = {Vercauteren, Dries and Vandenbroucke, Roosmarijn and Jones, Arwyn T and Rejman, Joanna and Demeester, Jo and De Smedt, Stefaan and Sanders, Niek and Braeckmans, Kevin},
  issn         = {1525-0016},
  journal      = {MOLECULAR THERAPY},
  keyword      = {TRANSFECTION EFFICIENCY,RNA INTERFERENCE,DEPENDENT INTERNALIZATION,CELL-LINE,INTRACELLULAR TRAFFICKING,CLATHRIN-INDEPENDENT ENDOCYTOSIS,CAVEOLAE-MEDIATED ENDOCYTOSIS,MAMMALIAN-CELLS,HELA-CELLS,DELIVERY},
  language     = {eng},
  number       = {3},
  pages        = {561--569},
  title        = {The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls},
  url          = {http://dx.doi.org/10.1038/mt.2009.281},
  volume       = {18},
  year         = {2010},
}

Chicago
Vercauteren, Dries, Roosmarijn Vandenbroucke, Arwyn T Jones, Joanna Rejman, Jo Demeester, Stefaan De Smedt, Niek Sanders, and Kevin Braeckmans. 2010. “The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls.” Molecular Therapy 18 (3): 561–569.
APA
Vercauteren, Dries, Vandenbroucke, R., Jones, A. T., Rejman, J., Demeester, J., De Smedt, S., Sanders, N., et al. (2010). The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. MOLECULAR THERAPY, 18(3), 561–569.
Vancouver
1.
Vercauteren D, Vandenbroucke R, Jones AT, Rejman J, Demeester J, De Smedt S, et al. The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. MOLECULAR THERAPY. 2010;18(3):561–9.
MLA
Vercauteren, Dries, Roosmarijn Vandenbroucke, Arwyn T Jones, et al. “The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls.” MOLECULAR THERAPY 18.3 (2010): 561–569. Print.