The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Schwoebel, Eric; Ho, Thai H.; Moore, Mary Shannon

doi:10.1083/jcb.200111077

Cited by 121 publications

(126 citation statements)

References 46 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…Instead, a small decrease was detectable (67736% of transferrin and 80723% of CTB uptake, as compared to 100% uptake under control conditions). Moreover, when cells were maintained at 41C or were depleted for ATP (with sodium azide and deoxyglucose; Schwoebel et al, 2002), conditions that block endocytosis, the C 6 -SM-stimulated doxorubicin uptake remained unaffected. Thus, we conclude that the C 6 -SM effect is unrelated to endocytosis.…”

Section: Resultsmentioning

confidence: 99%

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

et al. 2004

View full text Add to dashboard Cite

Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC 50 toxicity values seven-to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABCtransporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol -water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

show abstract

Section: Resultsmentioning

confidence: 99%

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

et al. 2004

View full text Add to dashboard Cite

show abstract

“…For energy depletion studies, GFP-RCC1 tsBN2 cells were incubated in glucose-free DMEM (Invitrogen) containing penicillin (100 U/ml), streptomycin sulfate (100 g/ml), 10 mM HEPES, pH 7.3, and 10% fetal bovine serum [hereafter referred to as gluc (Ϫ) media] containing 10 mM sodium azide and 6 mM 2-deoxy-d-glucose (Schwoebel et al, 2002). Cells were incubated 20 -30 min in energy depletion buffer before fluorescence recovery after photobleaching (FRAP) analysis.…”

Section: Cell Treatmentsmentioning

confidence: 99%

“…The linker histone H1 has a much faster nuclear mobility than the core histones (albeit with a mobility still markedly slower than GFP-RCC1) (Lever et al, 2000;Misteli et al, 2000), but there is no evidence that RCC1 interacts with histone H1. In addition, the nuclear mobility of GFP-histone H1 is markedly decreased upon energy depletion of the cells due to an inhibition of histone H1 phosphorylation (Dou et al, 2002).To determine whether the observed movement of GFP-RCC1 on and off chromatin is affected by energy levels, cells were energy depleted by incubating them in glucose-free media containing sodium azide and 2-deoxyglucose for 20 -30 min before photobleaching (Schwoebel et al, 2002). Instead of slowing down GFP-RCC1 movement, energy depletion increased the rate of fluorescence recovery after photobleaching approximately twofold compared with untreated cells (t 1/2 value of 5.4 Ϯ 0.3 s; Figure 2C).…”

mentioning

confidence: 99%

The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport

Cushman

Stenoien

Moore

2004

MBoC

Self Cite

View full text Add to dashboard Cite

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or ␣-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo. INTRODUCTIONRegulator of chromosome condensation (RCC1) stimulates guanine nucleotide exchange by the small GTPase Ran (Bischoff and Ponstingl, 1991a). Inside the cell, RCC1 is essential for the efficient conversion of RanGDP to RanGTP. RanGTP in turn is essential for several key cellular processes, including nucleocytoplasmic transport, regulation of spindle formation and nuclear envelope reassembly at mitosis, and prevention of rereplication of DNA during S phase (Dasso, 2002;Yamaguchi and Newport, 2003).The local concentration of RanGTP is a positional cue used by nuclear import and export complexes to distinguish between the cytoplasm and nuclear interior, and this regulates the assembly and disassembly of these transport complexes in the correct compartment. RanGTP is kept high inside the nucleus by localization of RCC1 to chromatin in the nuclear interior, and low inside the cytoplasm by the RanGAP (GTPase activating protein) that is restricted to that compartment. The differential placement of these two Ran accessory factors forms a gradient of RanGTP across the nuclear pore complex essential for most nuclear transport during interphase . All nuclear carriers of the karyopherin-␤ (Kap-␤) (importin ␤) family bind RanGTP Gorlich and Kutay, 1999). Binding of RanGTP is required for Kap-␤ export carriers to simultaneously bind their cargo inside the nucleus. These export complexes disassemble after encounter with the RanGAP (and a cofactor RanBP1) in the cytoplasm and the subsequent conversion of complexed RanGTP to RanGDP. Conversely, import complexes consisting of the carrier with bound cargo assemble only in the absence of RanGTP (the cytoplasm) and disassemble upon encoun...

show abstract

“…It is widely held that the maintenance of the Ran protein gradient is dependent on energy levels in the cell (Schwoebel et al, 2002). RCC1 will catalyze exchange of either GDP or GTP on Ran with equal efficiency (Bischoff and Ponstingl, 1991).…”

Section: The Ran Protein Gradient Is Not Strictly Dependent On Guaninmentioning

confidence: 99%

“…In tsBN2 cells, RCC1 is temperature sensitive, and when grown at the nonpermissive temperature, these cells lose the Ran protein gradient, presumably due to a requirement for Ran to be in the GTP-bound form in order to concentrate in the nucleus under steady-state conditions (Ren et al, 1993). When cells are treated with sodium azide and deoxyglucose, Ran rapidly delocalizes, again presumably because of the loss of ATP and due to the nucleotide diphosphate kinase GTP levels (Schwoebel et al, 2002).…”

Section: The Ran Protein Gradient Is Not Strictly Dependent On Guaninmentioning

confidence: 99%

Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

Kelley

Paschal

2007

MBoC

View full text Add to dashboard Cite

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10 -20 min) and late (30 -60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response. INTRODUCTIONCells subjected to UV radiation or oxidative, mechanical, or aniso-osmotic stress undergo both short-and long-term adaptation. Hyperosmotic stress induces rapid dehydration that drives an increase in intracellular salt concentration and a decrease in cell volume, placing physical strain on both the cytoskeleton and the plasma membrane (Lang et al., 1998). In response to these changes, cells rapidly initiate a stress signaling cascade and attempt to restore iso-osmolarity through transport of inorganic ions and initiate an accompanying reorganization of the actin cytoskeleton (Haussinger, 1996;Di Ciano et al., 2002). Short-term recovery of cell volume is facilitated by increasing intracellular concentrations of inorganic ions; however, elevated levels of intracellular salts can be detrimental to protein structure and function (Yancey et al., 1982;Russo et al., 2003). Long-term adaptation to hyperosmotic stress is achieved through signal transduction pathways that communicate with the metabolic and transcriptional machinery, resulting in the production or accumulation of organic osmolytes that increase intracellular osmolarity without adversely affecting protein structure or function (O'Neill, 1999).One of the most thoroughly studied stress kinases is the mitogen-activated protein kinase (MAPK) p38. p38 and its yeast homologue Hog1p are activated in response to a wide variety of adverse environmental conditions. These include osmotic, UV, and mechanical stress. Cytokines, such as interleukins and tumor necrosis factor ␣ are also known to activate p38 (Ono and Han, 2000). Hog1p and p38 are both phosphorylated on a threonine, glycine, tyrosine (TGY) motif within the kinase activation loop (Thr 180 an...

show abstract

The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Cited by 121 publications

References 46 publications

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport

Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

Contact Info

Product

Resources

About