In vitro fusion of endocytic vesicles: Effects of reagents that alter endosomal pH

Pless, Dorothy D.; Wellner, Robert B.

doi:10.1002/(sici)1097-4644(199607)62:1<27::aid-jcb4>3.0.co;2-3

Cited by 33 publications

(18 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lysosomotropic agents, such as chloroquine, have traditionally been used as reagents for distinguishing the route of entry of a virus. Such agents are nonspecific weak bases that diffuse across membranes in a concentration-dependent manner and thereby neutralize the pH of endocytic vesicles (16,43). Viruses unaffected by lysosomotropic agents are said to enter by fusing in a pH-independent manner with the plasma membrane, and viruses whose infectivity was decreased or blocked by these agents are said to enter the host cell by fusing with endocytic vesicles in a pHdependent manner (25,38).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Endosomal/Lysosomal Degradation Increases the Infectivity of Human Immunodeficiency Virus

Fredericksen

Wei

Yao

et al. 2002

J Virol

192

165

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Inhibition of Endosomal/Lysosomal Degradation Increases the Infectivity of Human Immunodeficiency Virus

Fredericksen

Wei

Yao

et al. 2002

J Virol

192

165

View full text Add to dashboard Cite

“…As a weak base, chloroquine increases endosomal pH by sequestering protons (Pless and Wellner 1996). To examine whether alkalization of endosomes by chloroquine is required for induction of cytokines in human astroglial cells, we evaluated the effect of other lysosomotropic agents which can increase endosomal pH via different mechanisms.…”

Section: Effect Of Other Lysosomotropic Agents On Cytokine Mrna Exprementioning

confidence: 99%

Chloroquine induces activation of nuclear factor‐κB and subsequent expression of pro‐inflammatory cytokines by human astroglial cells

Park

Kwon

Choi

et al. 2003

Journal of Neurochemistry

View full text Add to dashboard Cite

Chloroquine, an antimalarial lysosomotropic base, is known for its anti-inflammatory effects and therefore used for treatment of autoimmune diseases. Given its anti-inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. In this study, we examined whether chloroquine has an anti-inflammatory effect in the CNS by determining the in vitro effects of chloroquine on LPS-induced expression of cytokines by glial cells. We observed that (i) chloroquine augmented LPS-induced expression of proinflammatory cytokines such as lymphotoxin (LT)-b, tumor necrosis factor (TNF)-a, interleukin (IL)-1a, IL-1b and IL-6 in human astroglial cells, while the same treatment suppressed LPS-induced expression of cytokines in monocytic and microglial cells; (ii) chloroquine alone induced expression of pro-inflammatory cytokines in a dose-and time-dependent manner in astroglial cells; (iii) other lysosomotropic agents such as ammonium chloride and bafilomycin A 1 had minimal effects on cytokine expression; and (iv) chloroquine induced the activation of nuclear factor-kappa B in astroglial cells, which is a required component of chloroquine induction of cytokines. These results suggest that chloroquine may evoke either anti-or pro-inflammatory responses in the CNS depending on the cellular context.

show abstract

“…Cellular fluorescence was reduced by 50%-70% on incubation at 4°C, showing the energy dependence of dendriplex uptake. Six endocytosis inhibitors were used to study the mecha nism of dendriplex uptake: methylβcyclodextrin (MβCD), a cholesteroldepleting cyclic oligomer of glycopiranoside used as a general endocytosis inhibitor; 45,46 chlorpromazine, a cationic amphipathic molecule that inhibits formation of clathrincoated pits; 47 genistein, a specific tyrosine kinase inhibitor that causes local disruption of the actin cytoskeleton and avoids recruit ing of dynamin II, indispensable for both caveolinmediated endocytosis and Rhomediated endocytosis; 48,49 chloro quine, a weak base that increases endosomal and lyso somal pH, causing lysosomal disruption 50 and reduction of clathrindependent endocytosis; 51 cytochalasine D, which disrupts actin filaments and inhibits actin polymerization and membrane ruffling, affecting both phagocytosis and pinocytosis; 52,53 and nocodazole, which disrupts microtubules and inhibits translocation of endosomes and lysosomes, 54 blocking clathrindependent endocytosis but not affecting SV40 uptake to caveosomes, and preventing its sorting from caveosomes to endoplasmic reticulum. 55 Cell viability in the presence of the above inhibitors was determined first in order to establish optimal concentrations for their use.…”

mentioning

confidence: 99%

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Perez¹,

Cosaka²,

Romero³

et al. 2011

IJN

View full text Add to dashboard Cite

Background: Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intra cellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silenc ing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methylβcyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolindependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methylβcyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrindependent and caveolindependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrindependent and caveolindependent endocytosis vs only caveolindependent endocytosis in T98G cells.

show abstract

In vitro fusion of endocytic vesicles: Effects of reagents that alter endosomal pH

Cited by 33 publications

References 50 publications

Inhibition of Endosomal/Lysosomal Degradation Increases the Infectivity of Human Immunodeficiency Virus

Inhibition of Endosomal/Lysosomal Degradation Increases the Infectivity of Human Immunodeficiency Virus

Chloroquine induces activation of nuclear factor‐κB and subsequent expression of pro‐inflammatory cytokines by human astroglial cells

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Contact Info

Product

Resources

About