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Published OnlineFirst August 16, 2010; DOI: 10.1158/1541-7786.MCR-10-0090

Molecular
Canceresearch
Cycle, Cell Death, and Senescence

ction of Nonapoptotic Cell Death by Activated Ras

R

uires Inverse Regulation of Rac1 and Arf6

anti Bhanot, Ashley M. Young, Jean H. Overmeyer, and William A. Maltese

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huosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-ndent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can bed in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases,nd Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signalingay underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V)s cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state.ng Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activa--Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling

hrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicatinge decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V)ts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents these in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1). Together, the results suggest that perturbations of endosome morphology associated with Ras-inducedosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. Theseems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could
latter
suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.Mol Cancer Res; 8(10); 1358–74. ©2010 AACR.

tion othat tnisticaabovedrinkiendocdisrupspace.from tlogicanecrocartila

duction

reduced sensitivity of many types of cancer cells tootic cell death has stimulated interest in identifyingative nonapoptotic cell death pathways that mightd to kill tumor cells in a therapeutic context. Severalent forms of nonapoptotic cell death have beenbed based on specific cellular or molecular criteria.include autophagy-associated cell death (1-3), para-(4, 5), oncosis (6-8), necroptosis (9, 10), entosisand programmed necrosis (12, 13).ecade ago, Chi et al. (14) reported a unique form of

ell death that could be induced in glioblas-ic carcinoma cells by constitutive stimula-

similain gasVacAinducilaritieshareyet kntexts,that dprovecer ceWe

blastodepen

n: Department of Biochemistry and Cancer Biology,o College of Medicine, Toledo, Ohio

tary data for this article are available at Molecularnline (http://mcr.aacrjournals.org/).

thor: William A. Maltese, Department of Biochemistryy, Block Health Sciences Building, Mail Stop 1010,o College of Medicine, 3000 Arlington Avenue, Toledo,: 419-383-4161; Fax: 419-383-6228. E-mail: william.du

7786.MCR-10-0090

ssociation for Cancer Research.

; 8(10) October 2010

on February 24, 2021. © 20mcr.aacrjournals.org from

f Ras signaling pathways. We recently determinedhis form of cell death is morphologically and mecha-lly distinct from the various types of cell death noted(15). It entails a stimulation of macropinocytosis (cellng), combined with defects in clathrin-independentytic vesicle trafficking, ultimately resulting in cellulartion as large vacuoles expand to fill the cytoplasmicWe have termed this form of cell death “methuosis”he Greek methuo (to drink to intoxication). Morpho-lly, methuosis resembles type IIIA nonlysosomaltic cell death (16), originally observed in vacuolatedge during mineralization (17). It also bears somerity to the extensive endosomal vacuolization notedtric epithelial cells exposed to the Helicobacter pyloricytotoxin (18, 19) and in regions of liver necrosised by furosemide (20). Despite these superficial sim-s, it remains unclear whether these forms of cell deatha common molecular mechanism. Although it is notown if methuosis occurs in normal physiologic con-it is possible that understanding the molecular signalsrive this nonconventional form of cell death mayuseful for devising new approaches to eliminate can-lls that are refractory to apoptosis.have found that stimulation of vacuolization in glio-
ma cells by activated forms of H- and K-Ras does notd on conventional Ras effector pathways such as the
10 American Association for Cancer Research.

http://mcr.aacrjournals.org/

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EK-ERK kinase cascade or the phosphatidylinosi-′-kinase (PI3K)–Akt pathway (21). In consideringative mechanisms, we noted that previous studiesplicated the Rac1 GTPase as a positive regulator

cropinocytosis (22), phagocytosis (23), and cellularlization that occurs in response to the VacA cytotoxinBecause downstream targets of Ras include guaninetide exchange factors (GEF) that can stimulate acti-of Rac1 (24), we hypothesized that Ras might workgh Rac to trigger methuosis. Consistent with thisility, we observed that expression of constitutivelyRac1 (15), but not the related GTPases, Cdc42 or(21), could mimic the effects of Ras in glioblastomaBased on these findings, we initiated the presentto test the hypothesis that Rac1 is an essential down-mediator of Ras-induced methuosis. The resultsthe pathway for Ras-induced methuosis by estab-

g that the underlying perturbations in macropino-endosome trafficking that lead to cell vacuolizatione both activation of Rac1 and a coordinate Rac1-dent decrease in the pool of active Arf6, a GTPaseated in endocytic trafficking. The latter is mediatedgh an interaction between the active form of Rac1n Arf GAP, GIT1.

rials and Methods

ines for conditional expression of Ras andconstructs51 human glioblastoma cells were purchased fromCT Tumor Repository (National Cancer Institute).human osteosarcoma cells were obtained fromcan Type Culture Collection. HEK293T cells wereed from Systems Biosciences. Unless stated other-cell lines were grown in DMEM supplemented withfetal bovine serum (FBS), at 37°C with 5% CO2.eration of the U251-C18 cell line, which expresses-Ras(G12V) under the control of a tetracycline

nducible promotor, was described previously (15).251-C18 cells were maintained in DMEM sup-nted with 10% tetracycline-free FBS (Clontech),g/mL of G418 and 200 μg/mL of hygromycin.le U251 cell line that expresses myc-Rac1(G12V)the control of a tetracycline-inducible promoter,designated as U251-Rac1(G12V)tet, was generatedcribed previously (15).generate new stable cell lines that conditionally ex-activated Ras or dominant negative Rac1 or both,cells were infected with virus-enriched mediumning the regulatory vector pRetroX-Tet-On (Clon-which encodes the reverse tetracycline-regulatedctivator. Cells were selected in medium containingg/mL of G418, and clonal cell lines were tested innt transfection assays to determine which ones gaveghtest doxycycline-regulated gene expression. Theseesignated as U251 Tet-On. cDNAs encoding myc-
s(G12V) and FLAG-Rac1(T17N) were subclonedhe NotI/MluI sites of the expression vector, pRetroX-
ACTTnumb

acrjournals.org

on February 24, 2021. © 20mcr.aacrjournals.org wnloaded from

-Pur (Clontech). Retrovirus was produced by trans-g these constructs together with pRetroX-VSVG-nto 293 GP2 packaging cells (Clontech) usingectAMINE Plus reagent. Cells were maintained inM + 10% heat-inactivated FBS, using Biosafety Levelautions. Virus-enriched medium was passed through am filter and used to infect the U251Tet-On cells.were maintained in medium containing 1 μg/mL ofycin and 200 μg/mL of G418. For the expressiongenes of interest, 1 μg/mL of doxycycline was addedmedium.

ofection and retroviral expression of proteinsc-tagged H-Ras(G12V) and H-Ras(G12V, C186S)s were generated and cloned into the pCMV5sion vector as described previously (21). pCMV5-ac1(G12V) was provided by A.L. Wilson-DelfosseWestern Reserve University, Cleveland, OH).cells were nucleofected with an Amaxa Nucleofectorem, using Solution Tand Program T30, according toanufacturer's protocol (Lonza). For each reaction,of plasmid DNA was introduced into 3 × 106 cellsad been harvested in RPMI 1640 supplemented withheat-inactivated FBS. Following nucleofection, cellsplated and analyzed 18 to 20 hours later.constitutive expression of myc-Rac1(G12V) or myc-s(G12V) in various cell lines, the cDNAs encodingtter constructs were cloned into the pQCXI-H retro-ector (Clontech) using theNotI and BamHI sites. Ret-s was produced in 293GP2 packaging cells (Clontech)sfected with pQCXI-H-Rac1(G12V) and pRetroX--Env (Clontech), using LipofectAMINE Plus reagent.hours after transfection, the virus-enriched mediumollected, filtered, and used to infect the cells. Afterurs, the virus was removed and the cells were main-in DMEM with 10% FBS, 1 μg/mL puromycin,

00 μg/mL of hygromycin until they were examined.

hairpin RNA–mediated gene silencingsilencing expression of Tiam1, oligonucleotidesponding to nucleotides 5263 to 5283 [short hairpin(shRNA) no. 1, 5′-CCGTAGAGAATGTGTGTA-3′] in the 3′ untranslated region of huTiam1 (acces-umber U16296) or 3157 to 3177 of the coding(shRNA no. 2, 5′-CGCACCTACGTGAAGGATT-) were used. Each oligonucleotide was followed by ase loop region (CTTCCTGTCAGA) and the reverselement of the 21-bp gene-specific sequence. Thein sequences were flanked by BamHI and EcoRItion sites. The oligonucleotides were phosphorylatednnealed to their respective reverse complementarynces, then ligated into the pSIH1-H1-Puro lentivec-ystem Biosciences).NA constructs to suppress expression of Eps8 orwere made as described above except with differentucleotides: for Eps8, bases 1543 to 1563 (5′-CCA-
CTAATCGCCATATA-3′) of huEps8 (accessioner NM_004447). For GIT-1, bases 816 to 834
Mol Cancer Res; 8(10) October 2010 1359



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CACACCCATTGACTATGC-3′) and 1070 to 1090AGGTGGATCGAAGAGAAAAT-3′) of huGIT1sion number NM_014030; ref. 25). Control shRNAucts used oligonucleotides targeting Chiridius poppei(5′-GGAGGAGCTGCACAGCAA-3′) or Aequoreaa GFP (5′-GCAAGCTGACCCTGAAGTTC-3′),r of which match sequences in the human genome.NA lentivirus was produced by simultaneous trans-of 293TN cells with shRNA-pSIH1-H1-Puro vec-PackH1-Rev, pPackH1-Gag, and pPackVSV-Gm Biosciences), using LipofectAMINE Plus reagentogen). Virus collected between 24 and 48 hours af-nsfection was passed through a 0.45-μm filter ando infect U251 C-18 cells that were ∼50% confluent.were exposed to the virus for 2 to 3 days. One dayemoval of the virus, doxycycline was added to them (1 μg/mL) to induce myc-H-Ras(G12V) expres-and cells were analyzed 2 to 3 days later by phasest microscopy or Western blotting.

orphology and viabilitydetermine the percentage of cells with cytoplasmicles, phase contrast images of live cells were capturedn Olympus IX70 microscope equipped with a digitala and SPOT imaging software (Diagnostics Ins-nts, Inc.). For each culture, multiple photographsaken of different fields of cells and at least 100 cellsscored for vacuolization. Cells containing three orphase-lucent vacuoles with diameters ≥0.5 μmh >10 smaller vacuoles (0.1-0.5 μm) were counteditive.l viability was determined using the 3-(4,5-hylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideas described previously (26). PARP cleavage in thece or absence of zVAD-fmk (Bachem Biosciences)etermined by Western blot analysis.ere replicate assays were done, statistical significancenges in cell morphology or viability was assessed bynt's t test.

rn blot analysis9E10 monoclonal antibody against the myc epitopeiochem/EMD Biosciences) and the antibody to(BD Biosciences) were purchased from the indicateds. Antibody against α-tubulin was obtained from. The antibody against PARP was obtained fromiosciences. Methods for protein determination,AGE, and Western blot analysis have been describedChemiluminescence signals were quantified using anInnotech FluorChem HD2 imaging system.

nofluorescencels were prepared for immunofluorescence as de-d previously (28). Myc-tagged proteins were detectedrabbit polyclonal antibody (EMD Biosciences) con-d to FITC. FLAG-tagged proteins were detected
a mouse monoclonal antibody, M2, from Sigma,ed by goat anti-mouse IgG conjugated with Alexa
RacRac1

ancer Res; 8(10) October 2010


568 (Invitrogen). Cells were examined with a Nikone TE2000-U fluorescence microscope equipped withtal camera and NIS-Elements AR software (Nikonments, Inc.), and the percentage of vacuolated cellsetermined by counting at least 70 cells in photomi-aphs from randomly selected fields. Criteria foring cells as vacuolated were the same as those notedfor phase contrast micrographs.

e of fluid phase tracersocytic compartments were labeled with the fluid phaseLucifer yellow, as described previously (15). For label-lls with dextran-Alexa Fluor 568 (AF568), U251 C-18ere grown in 60 mm dishes for 3 days in phenol red–edium in the presence or absence of 1.0 μg/mL ofycline and 20 μmol/L of EHT 1864, with medium re-ent each day. To quantify dextran uptake, cells wereated with 0.5 mg/mL of dextran-AF568 (Invitrogen)minutes, washed with HBSS, and harvested by trypsi-n. Cells were suspended in a final volume of 500 μL,00 μL of cell suspension was transferred to a black-, black-bottomed microplate and analyzed on a Spec-x M5 plate reader (Molecular Devices) with 568 nmtion and 603 nm emission filters. Background fluores-was subtracted using values obtained from cells incu-in the absence of dextran-AF568.

ated ras expression with the ProteoTuner systemLenti-X ProteoTuner System was purchased from

ech. Myc-tagged H-Ras cDNA constructs (G12V or, C186S) were subcloned into the EcoRI-BamHI re-on sites of the lentiviral expression vector, pLVX-er Green, placing the Ras constructs in frame withquence encoding the 12 kDa destabilization domainand upstream from an IRES, which allows constitu-xpression of ZsGreen1. Lentivirus was produced in93T cells (American Type Culture Collection) usingctAMINE Plus reagents (Invitrogen), and pPack-H1iral Packaging System (System Biosciences). Forty-hours after transfection, the medium containing thewas collected and filtered through a 0.45-μm mem-. U251 glioblastoma cells were infected with the fil-virus to express DD-myc-H-Ras(G12V). Four daysnfection, Shield1 was added to the medium at concen-s of 30 or 100 nmol/L to stabilize the DD tag andvarying levels of Ras expression. Twenty-four hours af-addition of Shield1, overall infection efficiency in alles was determined to be >85% by counting the per-e of green fluorescent cells. The amount of DD-myc-s expressed was determined by immunoblotting withyc monoclonal antibody or anti-Ras antibody M-90Cruz Biotechnology). Cell morphology was assessedase contrast microscopy. The relative amount of acti-Rac1 was assayed in cell lysates as described below.

and Arf6 activation assays
1 activation assays were done using the EZ-DetectActivation Kit (Thermo Scientific Pierce). The assay
Molecular Cancer Research



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GST-fusion protein containing the p21-bindingin of p21-activated protein kinase 1 (PAK1) to selec-bind active Rac1 in whole cell lysates. The activeis then collected on glutathione agarose beads andified by immunoblot analysis using anti-Rac1 mono-antibody (Thermo Scientific Pierce). The value forRac1 collected on the beads was normalized to thentration of protein in the cell lysate. On a separatenoblot, the total Rac1 in a sample of the cell lysateetermined and normalized to the protein concentra-The normalized results were then expressed as thef active Rac1 to total Rac1 in the sample. The sameach was used to quantify active Arf6, except that theused GST-GGA3 (Golgi-associated gamma adaptinntaining Arf binding protein 3) to pull down activeand a monoclonal antibody against Arf6 for subse-immunoblot analysis (Thermo Scientific Pierce).e replicate assays were done, statistical significancenges in activation of Rac1 or Arf6 was assessed bynt's t test.analysis of Rac1 or Arf6 in stable cell lines, activationwere done on cells harvested on the 3rd day after theon of doxycycline to induce expression of H-Ras). Control cells were maintained without doxycy-Where indicated, EHT 1864 (provided by ExonHitpeutics, Paris, France) was added to the culturem at a concentration of 25 μmol/L to block Rac1tion. Both doxycycline and EHT 1864 were replaceday after treatment until the assays were done. For celles transiently nucleofected with H-Ras constructs,c1 or Arf6 activation assays were done 18 to 24 hoursucleofection.

noprecipitationimmunoprecipitation of myc-Rac(G12V), cells wered with HBSS and homogenized in immunoprecipi-buffer: 10 mmol/L of Tris-HCl (pH 7.4), 1 mmol/LCl2, 150 mmol/L of NaCl, 0.75% v/v NP40, withlete Mini Protease Inhibitors (Roche). Lysates wered by centrifugation at 10,000 × g for 30 minutes, and 1/10th of the supernatant solution was re-for checking the expression of myc-Rac1(G12V)

ndogenous GIT1. The remainder of the solutioncubated with rabbit anti-myc agarose beads (Sigma)hours at 4°C. The beads were washed three timesmmunoprecipitation buffer and the bound proteinsubjected to SDS-PAGE and immunoblot analysis forac1(G12V) or GIT1, using monoclonal antibodiesabove.

lts

itutively active Rac1 mimics the effect of actives in stimulating nonapoptotic cell deathr previous studies showed that overexpression ofitutively active Rac1(G12V) in glioblastoma cells
a striking accumulation of vacuoles that incorporateid-phase marker, Lucifer yellow, similar to what we
in neuRac3

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ed with H-Ras(G12V) (15). This is not a generalof all Ras-related GTPases, as active Cdc42(G12V)hoA(G14V) did not cause vacuolization (21). Theulation of vacuoles induced by Rac1(G12V) repre-the first observable step in a pathway that ultimatelyto cell detachment from the substratum and disrup-f the cell membrane. However, in these earlier stud-e did not establish if the loss of cell viability causedc1(G12V) was caspase-independent, as was the case-Ras(G12V) (15). To examine this possibility, we in-ced myc-Rac1(G12V) into the U251 glioblastomane using a retroviral vector. Within 48 hours, thession of Rac1(G12V) caused extreme cytoplasmiclization (Fig. 1A), and within 5 to 7 days, thereloss of cells from the substratum and a decrease inumber of viable cells compared with cells infectedthe empty viral vector (Fig. 1D). Although caspasetion could be detected by examining PARP cleavagedetached cells (Fig. 1C), addition of the generale inhibitor, zVAD-fmk, at a concentration that com-y blocked PARP cleavage did not protect the cellsRac1(G12V)-induced vacuolization and cell death1A, C, and D). In this respect, the effects of Rac1) were essentially identical to the cell death pheno-

induced by activated Ras, which we have termedosis (15). Identical results were obtained when theexperiment was done with a stable U251 cell lineich myc-Rac1(G12V) was conditionally expressedponse to doxycycline (Supplementary Fig. S1).

tion of methuosis by activated H-Ras depends ontion of endogenous Racpreceding observations, coupled with reports thatn activate Rac1, suggested that Rac lies downstreamRas in the signaling pathway for methuosis. To beginthis possibility, we asked if overexpression of consti-

ly active H-Ras(G12V) could stimulate the activationdogenous Rac in U251 glioblastoma cells. Cellsnucleofected with an expression vector encodingmyc-H-Ras(G12V) or the farnesylation-incompetent-Ras(G12V, C186S), which is relatively ineffectiveucing vacuolization (ref. 21 and Fig. 2A and B).ool of activated endogenous Rac GTPase was thenred by an established assay that takes advantage oflective interaction of Rac-GTP with the p21 bindingin of the Rac effector, PAK1 (Fig. 2C and D). Thes of this experiment established that in cells overex-ng H-Ras(G12V), vacuolization was accompaniedobust activation of endogenous Rac. Neither vacuoli-nor activation of endogenous Rac occurred in cellssing H-Ras(G12V) that contained a COOH-termi-steine substitution that prevents farnesylation andrane association of the protein. Activation of Rac2ot examined in glioblastoma cells because Rac2 is ex-d mainly in cells of hematopoietic lineage (29). Rac3ngly expressed in neuronal tracts of the brain (30) and
ronal cell lines (31), but we were unable to detectexpression in glioblastoma cells, using an antibody



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ic for Rac3 (ref. 32; data not shown). Therefore, for allr studies, we inferred that Rac1 was the predominantof Rac being measured in glioblastoma cells.merous studies have documented the ability of ac-d Ras to function as an oncogene, promoting celleration and cell survival through activation of theEK-ERK and PI3K-Akt signaling pathways (33,ur earlier results indicated that Ras-induced cyto-ic vacuolization does not require the activation ofr PI3K signaling pathways (21). This suggests thatduction of methuosis by H-Ras(G12V) might occurhen the level of activated Ras reaches a thresholdent to stimulate Rac1 activation. To examine thisility, we used the ProteoTuner system to express-Ras(G12V) as a fusion protein with an NH2-al destabilizing domain that promotes rapid protea-degradation. By adding increasing amounts of the

nyl tetrazolium bromide (MTT) assays over a period of 7 d to compare theirults shown in the bar graph are from three independent experiments (meagnificant at P ≤ 0.04.

molecule, Shield1, which binds to the DD and im-egradation, we were able to obtain graded increases

of enextens



expression level of myc-H-Ras(G12V) in paralleles (Fig. 3A). Under these conditions, the extent ofr vacuolization (Fig. 3C and D) correlated with thent of Rac1 activation (Fig. 3B).verify a direct link between Ras-induced activationc1 and the extensive cytoplasmic vacuolization thates methuosis, we used the compound EHT 1864,has been established as a selective inhibitor of Racses (35). The compound acts by displacing boundtide, thereby preventing interaction of Rac with itsstream effectors (35). The inhibitor studies werewith a stable glioblastoma cell line, U251-C18in which conditional expression of myc-H-Ras) is obtained in response to the addition of doxy-e (Fig. 4A). In the absence of the Rac inhibitor,ycline-induced expression of H-Ras(G12V) waspanied by a significant increase in the activation

y with and without zVAD (the inhibitor was replenished every day).). The decreases in viable cells at day 5 and day 7 (±zVAD)

E 1. Expression of activated Rac1 induces caspase-independent death in U251 glioblastoma cells. A, cells were infected with retrovirusg myc-Rac1(G12V) or virus without a cDNA insert (empty vector, EV). On the day after infection, 50 μmol/L of z-VAD-fmk was added to halfultures, and the others were maintained without the caspase inhibitor. On day 6 after infection (A) live cells were examined by phase contrastopy, (B) Western blot analysis was done to verify expression of myc-Rac1(G12V), and (C) attached and detached cells were harvested for analysisP cleavage. There were no detached cells in the EV controls. D, cells expressing Rac1(G12V) were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,

viabilit

dogenous Rac1 (Fig. 4B), and this coincided withive vacuolization (Fig. 4C and D). When the Rac




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itor, EHT 1864, was added together with doxy-e, there was no diminution of myc-H-Ras(G12V)sion (Fig. 4A), but the Ras-dependent increase inRac1 was blocked (Fig. 4B). Most notably, inhibit-e activation of Rac1 strongly suppressed Ras-inducedlization (Fig. 4C and D) and significantly reducedr uptake of the fluid phase marker, Dextran-AF568E).obtain an independent line of evidence that Rac1ions downstream from H-Ras for initiation ofosis, we attempted to stably suppress the expressionogenous Rac1 with shRNA. However, we found thatf Rac1 adversely affected cell attachment and pro-ion. As an alternative, we took advantage of thenown ability of the dominant-negative Rac1) to interfere with the function of endogenous

(22, 36). For these studies, we used the pRetroX--Puro vector, in conjunction with the RetroX-n system to generate U251 cell lines capable oftional expression of myc-H-Ras(G12V) alone,-Rac1(T17N) alone, or myc-H-Ras(G12V) +-Rac1(T17N). As shown in Fig. 5A, within 48 hoursing doxycycline to the medium, phase contrast mi-py revealed that the cells expressing H-Ras(G12V)xtensively vacuolated and had begun to detach fromsh, whereas the cells expressing Rac1(T17N) showedimal level of vacuolization, similar to the cells incu-
without doxycycline. Most notably, coexpressingminant-negative Rac1 construct
(wher

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s markedly suppressed vacuolization compared withxpressing H-Ras(G12V) alone. To account for varia-in transfection efficiency, immunofluorescencescopy was done to identify only those cells that weresing myc-H-Ras(G12V), FLAG-Rac1(T17N), or theination of both constructs (Fig. 5B). The percentageuolated cells was then calculated using only thed cells. The results confirmed the qualitative impres-of cellular vacuolization based on phase contrastscopy of the live cells. Taken together with the pre-studies with the Rac1 inhibitor, these observations

rt the idea that activation of a Rac1-mediated path-s necessary for the early perturbations of endocyticking that lead to cellular vacuolization associatedethuosis.

lation of Rac1 activation by H-Ras(G12V) doesepend on conventional exchange factors inastoma cellsnext turned our attention to the question of hows(G12V) might promote the activation of Rac1 inlioblastoma cells. A number of Rac GEFs can beted by PI(3,4,5)P3, the product of PI3K (37). Thus,ossibility is that Ras(G12V) stimulates Rac1 activa-ndirectly through positive modulation of PI3K. Wee that this is unlikely because our previous studiesshown that overexpression of PTEN in U251 cells
e PTEN is mutated) suppresses PI3K signaling but
together with active does not prevent Ras-induced vacuolization (21). A more

E 2. Expression of activatedcauses an increase in theof active Rac1 in U251toma cells. A, cells wereected with expressionencoding the indicatednstructs. After 24 h, liveere examined by phaset microscopy anded for Western blot analysisk the expression ofgged Ras proteins. B, cellsored for vacuolizationribed in Materials ands. C, pull-down assaysvated Rac1 were done onfrom two pooled 10-cmof cells. Bottom blots,ndogenous Rac1 thatto the GST-PAK1. Toptal Rac1 in an aliquot of thete. D, the immunoblotfor active and total Rac1antified and the units forac1 were normalized toc as described in Materials




likely(G12VAmonout adomaof RacTo

inducanti-TThe s(Fig.exprescondiger thsis, deSos

H-RaknownGrb2,tyrosiGEFscaffoif suc

Ras-inwe usreducto indDespithe mto tha(Fig. 6Tak

mostEps8/tion in

Ras-inamouAs w

by ovvationendocseriesbetwe

URuolils oblasprehiels oteoTexpmyshod to-myas.eld14.5r thctivl Raate

rphotrass that were vacuolated was determined.

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possibility is that at high expression levels, H-Ras) can directly stimulate the activity of a Rac GEF.g the members of the Rac GEF family, Tiam1 standss a candidate because it contains a Ras-bindingin and has been reported to promote the activation1 in a Ras-dependent manner (24).explore the possible contribution of Tiam1 to Ras-ed methuosis, we used a lentiviral vector to introduceiam1 shRNA into the U251-C18 stable cell line.hRNA suppressed Tiam1 to undetectable levels6A), but did not prevent doxycycline-induciblesion of myc-H-Ras(G12V) (Fig. 6B). Under thesetions, expression of H-Ras(G12V) continued to trig-e vacuolar phenotype that is characteristic of methuo-spite the suppression of Tiam1 (Fig. 6B).1 is another GEF that has the potential to respond tos(G12V) by activating Rac1. Although Sos1 is bestfor its ability to form a complex with the adapter,to facilitate activation of Ras in response to receptorne kinase signals (38), it can also behave as a Racwhen assembled into a tricomplex with Eps8 and a
lding protein, E3b1 (Abi-1) (39, 40). To determineh a complex might be responsible for mediating
activeclathr



duced Rac1 activation and cellular vacuolization,ed shRNA directed against Eps8 to dramaticallye the level of the protein in U251-C18 cells priorucing the expression of H-Ras(G12V) (Fig. 6A).te the near complete suppression of Eps8 expression,orphologic response of knockdown cells was similart observed in control cells with normal levels of Eps8B).en together, the preceding results indicate that the twoobvious Ras-responsive Rac GEFs, Tiam1 and Sos1/E3b1, are not essential for the initiation of vacuoliza-glioblastoma cells expressing H-Ras(G12V).

duced activation of Rac1 causes a decrease in thent of active Arf6e considered possible molecular mechanisms where-

erexpression of H-Ras(G12V) and consequent acti-of endogenous Rac1 might cause dysfunction ofytic compartments, our attention was drawn to aof studies that have defined functional connectionsen Rac1 and another small GTPase, Arf6. In its

FIGvaclevegliotheof SleveProthethenotuseDDH-RShiandafteof atotain Mmoconcell

state, Arf6 promotes membrane recycling fromin-independent endosomes (CIE) to the plasma



E 3. Rac1 activation and cellularzation correlate with increasingf H-Ras(G12V) expression in U251toma cells. A, cells were grown insence of increasing concentrationsld1 to obtain graded expressionf DD-myc-H-Ras(G12V) using theuner system. The blot showsressed Ras protein detected withc antibody. In a separate blot (datawn) an antibody against H-Ras wasdetermine the ratio of expressed

c-H-Ras(G12V) to endogenousThe results were 1.4 with no, 3.0 with 30 nmol/L of Shield1,with 100 nmol/L of Shield1. B, 1 d

e addition of Shield1, the amounte endogenous Rac1, normalized toc1, was determined as describedrials and Methods. C and D, celllogy was assessed by phaset microscopy and the percentage of


membcompfactor(arfap

Mostfrom

FIGURin the pverifiedindepenP < 0.01864 (+active RRac actof three+EHT 1dextrandeterm


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rane (41, 42). Arf6 and Rac1 colocalize in theseartments, which also contain nucleotide exchange

-AF568 was measured in the +Dox cells expressing myc-H-Ras(G12V), with or witinations on three cultures. The decrease in dextran uptake in the cells treated wit

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interestingly, Arf6 is required for trafficking of Rac1endosomes to the plasma membrane, so that when

s (ARNO and DOCK180/Elmo) and effectorstin-2/POR1 and PI4P 5-kinase) for both proteins.

active Rac1(Q61L) is expressed together with inhibitoryArf6(T27N), both proteins accumulate in vacuole-like

E 4. Blocking Rac1 activation in glioblastoma cells prevents Ras-induced vacuolization. A, the stable cell line, U251-C18, was incubated for 3 dresence or absence of doxycycline (1 μg/mL), with or without the Rac1 inhibitor, EHT 1864 (25 μmol/L). Inducible expression of myc-H-Ras(G12V) wasby Western blot analysis. B, Rac activation assays were done on cells subjected to each condition. The results are the mean (±SD) of threedent determinations done on separate cultures. The increase in active Rac1 in the cells expressing H-Ras(G12V) (+Dox alone) was significant at01 (*) compared with the cells not expressing Ras (−Dox). The decrease in active Rac1 in the cultures expressing H-Ras(G12V) in the presence of EHTDox, +EHT 1864) was significant at P < 0.001 (**) compared with the corresponding control (+Dox, −EHT 1864). The decrease in the basal level ofac1 in the −Dox cultures treated with EHT 1864 was significant at P < 0.05 (***). C and D, at the same time that cultures were harvested for theivation assays, parallel cultures were examined by phase contrast microscopy to determine the percentage of cells that were vacuolated. The resultsseparate experiments (mean ± SD) are shown. The suppression of vacuolization by the Rac inhibitor in the cultures expressing H-Ras(G12V) (+Dox,864) was significant at P < 0.0001 (*) compared with the cells expressing H-Ras(G12V) without the Rac inhibitor (+Dox, −EHT 1864). E, uptake of

hout EHT 1864. The results are the mean ± SD of separateh the Rac inhibitor was significant at P < 0.04 (*).




structArf6 ainducof Arderivewe hyby H-in theof RacTo

to det(G12Vpaniedwas aGST-boundamouexpresthe incondiin actspecifthe Rpressiof doeffectnot exthe R(Fig.the cesuggesoccursincreaTo

Rac1of H-glioblrus-mside-bmentathe osglioblcausedand acidingincorp

Rac1We

Rac1towarGTPaeffectoexpresonly aArf6.and mArf6 (

FIGURof vacuconditioconstruas descof the cB, imm(G12V)

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ures in HeLa cells (36). A recent study has shown thatlso plays a role in the trafficking of macropinosomesed by Ras(G12V) in HeLa cells, and that perturbationf6 function leads to the accumulation of CIE-d vacuoles that contain Ras (43). For these reasons,pothesized that cytoplasmic vacuolization inducedRas(G12V) in glioblastoma cells might entail changesactivation state of Arf6, in addition to the activation1.test this hypothesis, we used the U251-C18 cell lineermine if doxycycline-mediated induction of H-Ras) expression and cellular vacuolization were accom-by changes in the amount of active Arf6. The latter

ssessed in pull-down assays using a fusion protein,GGA3, which interacts specifically with the GTP-form of Arf6. The results showed that the relative

nt of active Arf6 decreased by ∼50% in the cellssing H-Ras(G12V) (Fig. 7A and B), contrasting withcrease in Rac1 activation observed under identicaltions (Fig. 4A and B). To determine if the decreaseive Arf6 in the cells expressing H-Ras(G12V) wasically related to the activation of Rac1, we addedac inhibitor, EHT 1864, at the same time that ex-on of H-Ras(G12V) was induced by the additionxycycline. By itself, EHT 1864 had no significanton the baseline level of active Arf6 in cells that werepressing active H-Ras (−Dox; Fig. 7B). However,ac inhibitor prevented the decrease in active Arf67B) and the accumulation of vacuoles (Fig. 7C) inlls expressing H-Ras(G12V). These results stronglyt that the decline in the amount of active Arf6 thatin cells expressing H-Ras(G12V) is caused by the

se in the amount of active Rac1.rule out the possibility that the reciprocal changes inand Arf6 which occur in response to the expressionRas(G12V) might be a unique feature of the U251astoma cell line, we compared the effects of retrovi-ediated H-Ras(G12V) expression in U251 cellsy-side with U20S human osteosarcoma cells (Supple-ry Fig. S2). The results showed that the response ofteosarcoma cell line was even more dramatic than theastoma cell line. That is, Ras(G12V) expressiona 10-fold increase in the amount of active Rac160% decrease in the amount of active Arf6, coin-with the accumulation of cytoplasmic vacuoles thatorated the fluid-phase tracer, Lucifer yellow.

affects Arf6 via the Arf GAP, GIT1next addressed the question of how the activation ofmight result in a shift in the nucleotide state of Arf6d the inactive GDP-bound form. Active Racses operate through a large number of downstreamrs to influence actin dynamics, cell migration, genesion, cell survival, and endocytosis (44). However,few Rac1 effectors offer potential connections withArfaptin-2/POR1 is a mediator of actin dynamics
embrane ruffling that can bind to both Rac1 and45, 46). However, arfaptin-2 binds preferentially to
The pescoringconstru



E 5. Expression of dominant-negative Rac1 blocks the inductionolization by active H-Ras. U251 cell lines capable ofnal expression of myc-H-Ras(G12V), FLAG-Rac1(T17N), or bothcts, were generated using the pRetroX-Tight-Pur Tet-On systemribed in Materials and Methods. A, phase contrast microscopyells after 2 d with or without doxycycline in the medium.unofluorescence microscopy shows expression of myc-H-Rasand/or FLAG-Rac1(T17N) only in the presence of doxycycline.rcentage of vacuolated cells in each culture was determined by
only the cells with visible expression of the myc and/or FLAGcts. The results are based on counting a minimum of 70 cells.



the ineffectto linArf6Rac1.(alsoit canknownthe PAturn apaxilliendosa receespecicase oGIT1presumGIT1withmedia

ToRac1(myc-Rence osis (15inducmyc-GIT1no deimmuspeciftionalcells e(G12(G12Vindicawith GTo

FIGURor Eps8preventof gliobmediateor Eps8as descMethodcell lysashRNAof Tiamcompacontrolα-tubulcompaproteinB, whepresencexpress(bottomdecreascells lacompaVacuoliphase cand quMateria


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active GDP form of Rac1 (47) and has no knownon Arf6 GTP hydrolysis. A more likely candidatek Rac1 to the observed decrease in GTP-boundwould be an Arf6 GAP that could interact withAmong the large family of Arf GAPs (48), GIT1known as Cat-1 or p95-APP1) stands out becauseassociate with both Rac1 and Rac3. Rac1-GDP isto associate with GIT1 through its interaction withK-interacting Rho exchange factor, βPIX. This inffects the association of the GIT1/PIX complex withn, with consequences for cell-matrix adhesion andomal trafficking (49-51). However, evidence fromnt study indicates that the GTP forms of Rac1 andally Rac3 could also interact with GIT1 (52). In thef Rac3, interaction of the GTP-bound form withwas direct, and results in a reduction of Arf6 activity,ably through stimulation of the GAP function of(52). We hypothesized that if Rac1-GTP interaction
GIT1 has the same effect, GITtor of the Rac1-dependent decli
ls and Methods.

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explore this possibility, we used the stable U251-G12V)tet cell line in which constitutively activeac1(G12V) is conditionally expressed in the pres-f doxycycline, resulting in the induction of methuo-). As shown in Fig. 8A, when myc-Rac1(G12V) wased in these cells, immunoprecipitation of the activeRac1(G12V) protein revealed that endogenouswas associated with it. If doxycycline was omitted,tectable myc-Rac1(G12V) was expressed, and thenoprecipitate did not contain GIT1, ruling out non-ic pull down of GIT1 with the myc antibody. Addi-ly, when the same experiment was done with U251xpressing myc-H-Ras(G12V) instead of myc-Rac1V), no GIT1 was detected in the myc-H-Ras) immunoprecipitate (Fig. 8B). These observationste that the active form of Rac1 is able to associateIT1 in glioblastoma cells.assess the functional importance of GIT1 for the

1 might be a keyne in active Arf6.

induction of methuosis, we generated two stable U251 celllines in which expression of GIT1 was strongly suppressed

E 6. Suppression of Tiam1expression does notRas-induced vacuolizationlastoma cells. A, shRNA-d knockdown of Tiam1expression was doneribed in Materials ands. Western blots of wholetes show that the specifics reduced the expression1 or Eps8 by >90%red with the respectives. The loading controls,in and LDH, confirm thatrable amounts of cellwere applied to the lanes.n cells were grown in thee of doxycycline to induceion of myc-H-Ras(G12V)blots), there was noe in vacuolization of thecking Tiam1 or Eps8,red with the control.zation was assessed byontrast microscopyantified as described in




by shmRNline eobservphenoby mydetermrequirthe in(G12Vby retlishedroundblastologiescontrothesetion incells icontrKD cof actithe Gof cytexpressuppoArf6-Gat leasof GITin thefactorthe m

Discu

Thepoor,chemois tharenderof cellof theapoptto sloreportundermethuof conblastoand oThe

displafluid-f(15).(lameare inunderconst

FIGURdecreaof gliobpreseninhibito(G12V)were dnormalresultsseparatH-Ras(the celinhibitopull-dowere hexamincells th(mean

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RNAs targeting different sequences in the GIT1A (Fig. 9A). As a control, we used a stable U251 cellxpressing shRNA against GFP. We have previouslyed that myc-Rac1(G12V) could induce a methuosistype that is essentially identical to that inducedc-H-Ras(G12V) (ref. 15 and Fig. 1). Therefore, toine if the interaction of active Rac1 with GIT1 ised for the decline in the pool of active Arf6 andduction of vacuolization, we introduced myc-Rac1) into the control and GIT1 knockdown cell linesroviral transduction (Fig. 9). In contrast with pub-studies in which suppression of GIT1 caused celling and detachment of neuronal cells (31), the glio-ma cell lines lacking GIT1 exhibited cell morpho-and growth rates that were comparable to thel cells. When myc-Rac1(G12V) was expressed incell lines, the control cell line showed a ∼50% reduc-the amount of active Arf6, compared with the same

nfected with empty retroviral vector (Fig. 9B). Inast, expression of Rac1(G12V) in the two GIT1ell lines caused a negligible reduction in the amountve Arf6 (Fig. 9B). The preservation of active Arf6 inIT1 KD cells was associated with a reduced amountoplasmic vacuolization and cell death in response tosion of Rac1(G21V) (Fig. 9C-E). These findingsrt the idea that the reduction in the amount ofTP caused by activation of Rac1 could be mediated,t in part, through stimulation of the GAP function1. They also reinforce the notion that the reductionpool of active Arf6 is an important contributingto the endosomal trafficking defects that underlieethuosis phenotype.

ssion

prognosis for patients with glioblastoma remainsdespite efforts to improve surgical, radiological, andtherapeutic treatment strategies. A major problemt glioblastoma cells harbor genetic mutations thatthem resistant to apoptosis (53, 54). The discoverydeath mechanisms that do not depend on activationclassical mitochondrial and death receptor–mediatedotic pathways could present new opportunitiesw the progression of these aggressive tumors. In this, we have begun to define the signaling pathwayslying a nonconventional form of cell death termedosis, which can be triggered by ectopic expressionstitutively activated Ras in a broad spectrum of glio-ma cell (15), as well as gastric carcinoma cells (14)steosarcoma cells lines (Supplementary Fig. S2).hallmark cytopathologic feature of methuosis is the

cement of much of the cellular cytoplasmic space byilled vacuoles derived from macropinosomes or CIEMacropinosomes are formed when membrane rufflesllipodia) enclose pockets of extracellular fluid andternalized (55, 56). We previously showed that cells
going methuosis in response to the expression ofitutively active H-Ras(G12V) exhibit increased
additio(+Dox,express



E 7. Expression of activated H-Ras causes a Rac1-dependentse in the amount of active Arf6, which is essential for vacuolizationlastoma cells. A, U251-C18 cells were incubated for 3 d in thece or absence of doxycycline (1 μg/mL), with or without the Rac1r, EHT 1864 (25 μmol/L). Inducible expression of myc-H-Raswas verified by Western blot analysis. B, Arf6 activation assaysone on cells subjected to each condition, with active Arf6ized to total Arf6 as described in Materials and Methods. Theare the mean (± SD) of three independent determinations done one cultures. The decrease in active Arf6 in the cells expressingG12V) (+Dox alone) was significant at P < 0.01 (*) compared withls not expressing Ras (−Dox) or the cells incubated with the Racr, EHT 1864. Representative immunoblots of the raw Arf6 GTPwns are shown below the bar graph. C, at the time that culturesarvested for the Arf6 activation assays, parallel cultures wereed by phase contrast microscopy to determine the percentage ofat were vacuolated. The results of three separate experiments± SD) are shown. The suppression of vacuolization caused byn of the Rac inhibitor to cultures expressing H-Ras(G12V)
+EHT 1864) was significant atP < 0.0001 (*) comparedwith the cellsing H-Ras(G12V) without the Rac inhibitor (+Dox, −EHT 1864).



macroever, ubackcompcells usomesThus,necroswith cThe

notypdepen(Figs.ulatorand cevidenicle trcytosiwith sreceptof Raendocing mthat invatednousby Ralargethe in

Ouinvolvfickinnocyttendwith lclinetion inFig. Sof Arfbecaudoesshowof GIT(Fig. 8clingfindinof RaccominmacroArf6-Gmay psivelylysosocellsbetwelysosolabeleH-RacompinducLysoTlysosoacquiFig. Sendosfickintion oendosIn supdirectVerynameformtermindict tRab7-late ention omisesbasisAt

contrimight seem contradictory to the observations of Porat-Shliom et al. (43), who found that active Arf6(Q67L)

FIGURaddingstable Ucollecteof whowere suagainstreceiveof GIT1inducedassocia


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pinocytotic uptake of fluid phase tracers (15). How-nlike normal macropinosomes, which either recycleto the plasma membrane or merge with lysosomalartments (57, 58), labeled vesicles internalized intondergoing methuosis do not seem to fuse with lyso-or release their contents at the cell surface (15).large vacuoles accumulate and eventually cause ais-like rupture of the cell that cannot be preventedaspase inhibitors.present studies establish that the methuosis phe-e induced by overexpression of activated H-Rasds on downstream activation of the Rac1 GTPase2–5). Rac1 is widely recognized as an important reg-of actin dynamics, integrin-mediated cell adhesion,ell migration (44, 59), but there is also abundantce implicating Rac1 in the control of endocytic ves-afficking (60, 61). Active Rac1 seems to affect endo-s at multiple levels. On one hand, Rac1 interactsynaptojanin-2 and downregulates clathrin-mediatedor endocytosis (62, 63). However, the active formc1 has the opposite effect on clathrin-independentytosis, stimulating membrane ruffling and promot-acropinocytosis (22). Our results strongly suggestglioblastoma cells in which overexpression of acti-

H-Ras drives the continuous activation of endoge-Rac1, the chronic stimulation of macropinocytosisc1 is an important factor in the biogenesis of the

to express myc-H-Ras(G12V) to establish that GIT1 does notte with activated H-Ras.

cytoplasmic vacuoles that eventually compromisetegrity of the cell. 1 Unpub

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r previous studies have indicated that methuosises dysfunction in other steps of the endosomal traf-g pathway beyond the initial stimulation of macropi-osis. During methuosis, fluid-filled endocytic vesiclesto accumulate instead of being recycled or mergedysosomes (15). Our current results show that a de-in the pool of active Arf6 is important for vacuoliza-duced by activation of Rac1 (Fig. 7; Supplementary2). It is likely that this is due to loss of function6-GTP, rather than gain of function of Arf6-GDPse overexpression of the GDP-locked Arf6(T27N)not induce vacuolization in U251 cells.1 We alsothat the decline in active Arf6 requires the presence1 (Fig. 9), an Arf6 GAP that can interact with Rac1). Because activation of Arf6 is important for recy-macropinosomes back to the cell surface (58), thesegs suggest a model wherein chronic activation1 plays a dual role, stimulating the formation of in-g macropinosomes while simultaneously impedingpinosome recycling by decreasing the pool ofTP. The resulting overload of fluid-filled vesiclesromote their abnormal fusion to generate progres-larger vacuoles which, in the absence of effectivemal clearance, fill the cytoplasm and disrupt the(Fig. 10). The postulated block in traffickingen clathrin-independent late endosomes andmes is supported by the observation that vacuolesd with fluorescent dextran in glioma cells expressings(G12V) do not merge with LysoTracker-positiveartments (15). Furthermore, although the Ras-ed vacuoles are not sufficiently acidic to sequesterracker or acridine orange, and they do not containmal enzymes like cathepsin B, some of them dore LAMP1 (15) and Rab 7 (Supplementary3). These features are consistent with arrest at a lateomal stage. The molecular basis for the latter traf-g defect remains to be defined, but Rac1 modula-f Rab7, which functions in trafficking between lateomes and lysosomes, seems to be a likely possibility.port of this concept, Sun et al. (64) have reported ainteraction between Rac1 and Rab7 in osteoclasts.recently, Frasa et al. (65) described a novel proteind Armus, which associates with the GTP-boundof Rac1 and inactivates Rab7 through its COOH-al GAP domain. Based on this observation, we pre-hat chronic activation of Rac1 might interfere withmediated trafficking events at the interface betweendosomes and lysosomes (Fig. 10). Future investiga-f the role of signaling between Rac1 and Rab7 pro-to provide additional insights into the molecularfor endosomal vacuolization in methuosis.first glance, our finding that inactivation of Arf6butes to vacuolization of cells expressing active Ras

E 8. Active Rac1 interacts with the Arf6 GAP, GIT1. A, 3 d afterdoxycycline to induce expression of myc-Rac1(G12V) in a251 cell line, cells were lysed and the myc-tagged protein wasd on agarose beads conjugated with anti-myc antibody. Samplesle-cell lysate and proteins eluted from the myc-agarose beadsbjected to Western blot analysis with monoclonal antibodiesGIT1 or myc. Immunoprecipitation of lysate from cells that did notdoxycycline was done to control for nonspecific precipitation. B, the same experiment was done with stable U251 cells

lished observation.




induc(G12Vinate ainducsing Hnascenintern

and Pwhichcontraendogcharac

FIGURStable(GIT1 awere sucell lineexpressfor threon thecontrasbar gracell lineusing Mexpresssignific

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es the formation of vacuoles in cells expressing H-Ras). However, the vacuoles induced by active Arf6 orig-t a different stage of the endocytic pathway than thoseed by inactivation of Arf6. Specifically, in cells coexpres--Ras(G12V) with Arf6(Q67L), vacuoles arise because

ed as a percentage of the same cells infected with empty vector. The increasedant at P < 0.007 relative to the shRNA control cell line.



I(3,4,5)P3, but have not yet acquired Rab5 or Rab7,mark early and late endosomes, respectively (43). Inst, vacuoles that form when H-Ras(G12V) activatesenous Rac1, with concordant inactivation of Arf6, haveteristics of a late endosomal compartment that has ac-

t macropinosomes are trapped at an early stage afteralization, resulting in vesicles that contain PI(4,5)P2

quired LAMP1 (15) and Rab7 (Supplementary Fig. S3). Thiswould be consistent with our model (Fig. 10) wherein CIEs

E 9. Suppression of GIT1 expression prevents the Rac1(G12V)-mediated decrease in active Arf6 and protects glioblastoma cells from methuosis.cell lines were generated from U251 cells infected with lentivirus encoding shRNA against GFP (control) and two different shRNAs against GIT1and b). A, the control and GIT1 knockdown cell lines were infected with retrovirus encoding myc-Rac1(G12V), and 3 d after infection, the cellsbjected to Western blot analysis to check expression of myc-Rac1(G12V) and GIT1. B, on the 4th d after infection, the control and GIT1 knockdowns were assayed for the amount of active Arf6 as described in Materials and Methods. The results show the amount of active Arf6 in the cellsing myc-Rac1(G12V), expressed as a percentage of the value obtained for the same cell line infected with empty retroviral vector (mean ± SDe experiments). The differences between each of the GIT1 knockdown cell lines (*) and the control cell line were significant at P < 0.004. C and D,4th d after infection with empty or myc-Rac1(G12V) retroviral vectors, the control and GIT1 knockdown cell lines were examined by phaset microscopy to determine the percentage of cells that were vacuolated. The results of three separate experiments (mean ± SD) are shown in theph. The suppression of Rac1-induced vacuolization in the GIT1 knockdown cell lines (*) was significant at P < 0.0001 compared with the control. E, on the 5th d after infection with empty or myc-Rac1(G12V) lentiviral vectors, the control and GIT1 knockdown cells were assayed for cell viabilityTT. Assays were done on quadruplicate cultures in a 96-well plate, and the results for the cells infected with myc-Rac1(G12V) retrovirus were

viability observed in each of the GIT1 knockdown cell lines was




fail toafter tThe

has beconnecytosirecentRac1nentsthe exprommitogways i

endocRac1part,nucleoRas tonor Eppossibexchaways mAno

mote

FIGURthresho(3) As tconseqendosomechanby Fras ce to fdisplac and d


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recycle and arrest at a stage before lysosomal fusion, buthe Rab5/Rab7 conversion (66).ability of activated Ras to stimulate macropinocytosisen recognized for a number of years (67, 68), but thection between dysfunctional Ras-induced macropino-s and cell death in specific types of cancer cells has onlyly been appreciated. The present study implicates theand Arf6 GTPases as important downstream compo-in this process. A major unanswered question is whypression of constitutively activated Ras mutants canote cell proliferation and survival through classical

ism for a block in lysosomal fusion could be Rac1 stimulation of the Raba et al. (65). (5) The accumulated CIE and late endosomal vesicles coalese most of the cytoplasm and lead to cell detachment from the substratum

en-activated protein kinase and PI3K signaling path-n some types of cells, but can also cause disruptions of

also trthat l

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ytic trafficking and cell death through alterations ofand Arf6 in others. The answer might lie, at least inin cell type–specific variations in the expression oftide exchange factors that can link the activation ofRac1. In this regard, our finding that neither Tiam1s8 was essential for Ras to induce methuosis raises theility that other poorly characterized nucleotide

nge factors (e.g., Tiam2/Stef ) or novel signaling path-ight play such a role in glioblastoma cells.ther unsolved puzzle is why Rac1 activation can pro-cell motility and invasiveness (59, 69, 70), yet can

, Armus, resulting in inactivation of Rab7, as recently describedorm progressively larger vacuolar structures, which ultimatelyisruption of cell membrane integrity.

E 10. A hypothetical model for Ras-induced methuosis suggested by the present findings. (1) Expression of constitutively activated H-Ras reaches ald sufficient to stimulate the activity of an unidentified Rac1 GEF. (2) The resulting increase in the pool of active Rac1 enhances macropinocytosis.he pool of active Rac1 increases beyond normal physiologic levels, Rac1-GTP associates with GIT1 and stimulates Arf6 GAP activity. Theuent decrease in the pool of active Arf6 impairs recycling of CIEs. (4) CIEs derived from macropinosomes acquire some characteristics of latemes (Rab7 and LAMP1), but fail to merge with lysosomes, due to trafficking defects at the late endosome/lysosome boundary. One possible

7 GAP

igger cytopathologic changes in vesicular traffickingead to cell death. Our results suggest that a key to




underthe baphysioGDPcell adRac1ulatinlike thThe mto placell adGDPwith RHowe(G12VwhereactivaRac1(tweenover tteractireduce(G12Vby knmay hby stiIt is

mightcells (clear ision oand ceof cellof H-(15).with gpossibcell linremaiWe haobtainsion inunderinducmethumightincludGAPstissue-of no

attenthumaof Arftissueing mOu

on ceectopibelievlargertionalsomalthe Hmannlargeendosvacuobecauand incase,myeliperiphmulatthrinobserArf6-dmechcytopUltimpathwcan tropporwell to

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standing the paradoxical effects of Rac1 might lie inlance between active Arf6 and Rac1. Under normallogic conditions, Arf6 cycles between the inactivestate and the active GTP state. Arf6-GTP promoteshesion and migration by facilitating translocation ofto the leading edge of the cell membrane and stim-g activation of Rac1 via nucleotide exchange factorse DOCK180/Elmo complex (71) or Kalirin (72).ultifunctional scaffold protein, GIT1, also seems

y an important role in Rac1 trafficking to points ofhesion (49, 73). GIT1 can associate with both theand GTP forms of Rac1, but the indirect interactionac1-GDP through βPIX seems to predominate (52).ver, based on the coimmunoprecipitation of Rac1) with GIT1 (Fig. 8), we speculate that in cellsoverexpression of H-Ras(G12V) drives constitutivetion of endogenous Rac1, or in cells where activeG12V) is highly expressed, a direct interaction be-the GTP form of Rac1 and GIT1 might be favoredhe “normal” interaction with Rac1-GDP. Direct in-on between Rac3-GTP and GIT1 has been shown toArf6 activity (52). Our observation that the Rac1)-induced decrease in active Arf6 was prevented

ockdown of GIT1 (Fig. 9) suggests that Rac1-GTPave a similar ability to reduce the pool of active Arf6mulating the GAP activity of GIT1.important to note that although our model (Fig. 10)apply to glioblastoma cells and other types of cancerosteosarcoma and gastric carcinoma), it remains un-f all cell types have the potential to respond to expres-f activated Ras or Rac1 by undergoing vacuolizationll death. We previously reported that in some typess (HEK293 and human skin fibroblasts), expressionRas(G12V) did not elicit the methuosis phenotypeHowever, the results obtained in the present studyraded H-Ras(G12V) expression (Fig. 3) raised theility that the lack of response previously seen in somees could have been due to the level of Ras expressionning below the threshold needed for Rac1 activation.ve since revisited this issue using a retroviral vector tonearly identical levels of H-Ras(G12V) overexpres-U251, U20S, and HEK293T cells, and found thatthese conditions, the HEK 293 cells could in fact beed to undergo morphologic changes typical ofosis (Supplementary Fig. S4). Other factors thatinfluence the responses of particular cell types mighte the expression level of GIT1 relative to other Arf6or the expression level of Arf6 itself. The question of
specific variations in these proteins in different types
ls has not received muchRece

OnlineF

ers? Ann N Y Acad Sci 2000;926:1–12.ckshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int Jchem Cell Biol 2004;36:2405–19.

3. Gosu

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ion. However, the recent finding that high-graden gliomas and glioma cell lines have elevated levels6 compared with low-grade tumors or normal brain(74) suggests that significant variations in Arf6 signal-echanisms may occur in some types of cancer cells.r initial characterization of methuosis has focusedlls in which this form of death can be triggered byc expression of activated Ras or Rac1. However, wee that methuosis may represent just one example of agroup of cytopathologies characterized by dysfunc-trafficking and vacuolization of early or late endo-compartments. For instance, in cells exposed to. pylori VacA toxin (75) or the PI3K inhibitor, wort-in (76), cells are disrupted by the accumulation ofvacuoles that arise from the reorganization of lateomes and lysosomes. Interestingly, the induction ofles by VacA seems to depend on Rac1 signalingse it is potentiated by expression of activated Rac1hibited by dominant-negative Rac1 (18). In anothercultured cells overexpressing a peripheral nerven protein, Gas3/PMP22 (growth arrest–specific 3/eral myelin protein 22), have been found to accu-e vacuoles derived from an Arf6-dependent noncla-endosome recycling compartment (77). Thesevations suggest that perturbation of Rac1- andependent trafficking pathways might be a commonanism in various cytopathologies associated withlasmic vacuolization and necrosis-like cell death.ately, as more is learned about the relevant signalingays, the identification of drug-like compounds thatigger methuosis in tumor cells may provide newtunities for treatment of cancers that do not respondconventional agents that work by inducing apoptosis.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank Dr. Laurent Désiré and ExonHit Therapeutics for providing EHTd Dr. Amy Wilson-Delfosse for the Rac1(G12V) construct.

Support

grant R01 CA115495 (W.A. Maltese).costs of publication of this article were defrayed in part by the payment ofarges. This article must therefore be hereby marked advertisement ince with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 03/03/2010; revised 07/27/2010; accepted 08/05/2010; publishedirst 08/16/2010.
rmal and transformed cel
rencesrsch W, Ellinger A, Gerner C, Frohwein U, Schulte-Hermann R.grammed cell death (PCD). Apoptosis, autophagic PCD, or

zuacik D, Kimchi A. Autophagy as a cell death and tumorppressor mechanism. Oncogene 2004;23:2891–906.
erandio S, de Belle I, Bredesen DE. An alternative, nonapoptoticm of programmed cell death. Proc Natl Acad Sci U S A 2000;97:376–81.



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ng Y, Li X, Wang L, et al. An alternative form of paraptosis-likel death, triggered by TAJ/TROY and enhanced by PDCD5 overex-ssion. J Cell Sci 2004;117:1525–32.jno G, Joris I. Apoptosis, oncosis, and necrosis. An overview ofl death. Am J Pathol 1995;146:3–15.mp BF, Berezesky IK, Chang SH, Phelps PC. The pathways of cellath: oncosis, apoptosis, and necrosis. Toxicol Pathol 1997;25:82–8.arez Y, Gonzalez L, Cuadrado A, et al. Kahalalide F, a new marine-rived compound, induces oncosis in human prostate and breastcer cells. Mol Cancer Ther 2003;2:863–72.gterev A, Huang Z, Boyce M, et al. Chemical inhibitor of nonapop-ic cell death with therapeutic potential for ischemic brain injury.t Chem Biol 2005;1:112–9.n W, Li L, Qiu S, et al. Shikonin circumvents cancer drug resis-ce by induction of a necroptotic death. Mol Cancer Ther 2007;641–9.erholtzer M, Mailleux AA, Mouneimne G, et al. A nonapoptotic cellath process, entosis, that occurs by cell-in-cell invasion. Cell7;131:966–79.ntichaki P, Tavernarakis N. Death by necrosis. Uncontrollabletastrophe, or is there order behind the chaos? EMBO Rep 2002;04–9.inger AL, Thompson CB. Death by design: apoptosis, necrosisd autophagy. Curr Opin Cell Biol 2004;16:663–9.i S, Kitanaka C, Noguchi K, et al. Oncogenic Ras triggers cellcide through the activation of a caspase-independent cell deathgram in human cancer cells. Oncogene 1999;18:2281–90.ermeyer JH, Kaul A, Johnson EE, Maltese WA. Active ras triggersath in glioblastoma cells through hyperstimulation of macropino-osis. Mol Cancer Res 2008;6:965–77.rke PH. Developmental cell death: morphological diversity andltiple mechanisms. Anat Embryol 1990;181:195–213.hweichel J-U, Merker HJ. The morphology of various types of cellath in prenatal tissues. Teratology 1973;7:253–66.tchin NA, Cover TL, Akhtar N. Cell vacuolization induced by thecA cytotoxin of Helicobacter pylori is regulated by the Rac1Pase. J Biol Chem 2000;275:14009–12.zuki J, Ohnishi H, Wada A, et al. Involvement of syntaxin 7 in hu-n gastric epithelial cell vacuolization induced by the Helicobacterori-produced cytotoxin VacA. J Biol Chem 2003;278:25585–90.lker RM, McElligott TF. Furosemide induced hepatotoxicity.athol 1981;135:301–14.ul A, Overmeyer JH, Maltese WA. Activated Ras induces cytoplas-vacuolization and non-apoptotic death in glioblastoma cells via

vel effector pathways. Cell Signal 2007;19:1034–43.ley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The smallP-binding protein rac regulates growth factor-induced membranefling. Cell 1992;70:401–10.stellano F, Montcourrier P, Chavrier P. Membrane recruitment ofc1 triggers phagocytosis. J Cell Sci 2000;113:2955–61.bert JM, Lambert QT, Reuther GW, et al. Tiam1 mediates Ras

ivation of Rac by a PI(3)K-independent mechanism. Nat Cell Biol02;4:621–5.ra K, Nam JM, Kojima C, Mochizuki N, Sabe H. EphA2 engages1 to suppress Arf6 activity modulating epithelial cell-cell contacts.l Biol Cell 2009;20:1949–59.ul A, Maltese WA. Killing of cancer cells by the photoactivatabletein kinase C inhibitor, calphostin C, involves induction of endo-smic reticulum stress. Neoplasia 2009;11:823–34.ng X, Overmeyer JH, Maltese WA. Functional specificity of themmalian Beclin-Vps34 PI 3-kinase complex in macroautophagysus endocytosis and lysosomal enzyme trafficking. J Cell Sci06;119:259–70.hnson EE, Overmeyer JH, Gunning WT, Maltese WA. Geneencing reveals a specific function of hVps34 phosphatidylino-ol 3-kinase in late versus early endosomes. J Cell Sci 2006;119:19–32.d PD, Butler JS, Skalnik DG. Identification of a genomic fragment
t directs hematopoietic-specific expression of Rac2 and analysisthe DNA methylation profile of the gene locus. Gene 2004;341:–33.
CDce

54. Fu

acrjournals.org


rbetta S, Gualdoni S, Albertinazzi C, et al. Generation and charac-ization of Rac3 knockout mice. Mol Cell Biol 2005;25:5763–76.jdo-Milasinovic A, Ellenbroek SI, van Es S, van d V, Collard JG.c1 and Rac3 have opposing functions in cell adhesion and differ-tiation of neuronal cells. J Cell Sci 2007;120:555–66.lis A, Corbetta S, Cioce A, de C, I. Differential distribution of Rac1d Rac3 GTPases in the developing mouse brain: implications for ae of Rac3 in Purkinje cell differentiation. Eur J Neurosci 2003;18:17–24.mpbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. In-asing complexity of Ras signaling. Oncogene 1998;17:1395–413.hl K. Oncogenic Ras in tumour progression and metastasis. Biolem 2005;386:193–205.utes A, Onesto C, Picard V, Leblond B, Schweighoffer F, Der CJ.ecificity and mechanism of action of EHT 1864, a novel small mol-ule inhibitor of Rac family small GTPases. J Biol Chem 2007;282:666–78.dhakrishna H, Al Awar O, Khachikian Z, Donaldson JG. ARF6 re-irement for Rac ruffling suggests a role for membrane trafficking inrtical actin rearrangements. J Cell Sci 1999;112:855–66.lch HC, Coadwell WJ, Stephens LR, Hawkins PT. Phospho-sitide 3-kinase-dependent activation of Rac. FEBS Lett 2003;6:93–7.N, Batzer A, Daly R, et al. Guanine-nucleotide-releasing factoros1 binds to Grb2 and links receptor tyrosine kinases to Ras sig-lling. Nature 1993;363:85–8.ocenti M, Zippel R, Brambilla R, Sturani E. CDC25(Mm)/Ras-F1 regulates both Ras and Rac signaling pathways. FEBS Lett99;460:357–62.ocenti M, Tenca P, Frittoli E, et al. Mechanisms through whichs-1 coordinates the activation of Ras and Rac. J Cell Biol 2002;6:125–36.ters PJ, Hsu VW, Ooi CE, et al. Overexpression of wild-type andtant ARF1 and ARF6: distinct perturbations of nonoverlappingmbrane compartments. J Cell Biol 1995;128:1003–17.dhakrishna H, Donaldson JG. ADP-ribosylation factor 6 regulatesovel plasma membrane recycling pathway. J Cell Biol 1997;139:–61.rat-Shliom N, Kloog Y, Donaldson JG. A unique platform for H-s signaling involving clathrin-independent endocytosis. Mol Biolll 2008;19:765–75.rridge K, Wennerberg K. Rho and Rac take center stage. Cell04;116:167–79.n Aelst L, Joneson T, Bar-Sagi D. Identification of a novel Rac1-eracting protein involved in membrane ruffling. EMBO J 1996;15:78–86.Souza-Schorey C, Boshans RL, McDonough M, Stahl PD,n Aelst L. A role for POR1, a Rac1-interacting protein, in ARF6-diated cytoskeletal rearrangements. EMBO J 1997;16:5445–54.in OH, Exton JH. Differential binding of arfaptin 2/POR1 to ADP-osylation factors and Rac1. Biochem Biophys Res Commun 2001;5:1267–73.ue H, Randazzo PA. Arf GAPs and their interacting proteins.ffic 2007;8:1465–75.Cesare A, Paris S, Albertinazzi C, et al. p95-1 links membranensport to Rac-mediated reorganization of actin. Nat Cell Biol00;2:521–30.rner CE, Brown MC, Perrotta JA, et al. Paxillin LD4 motif bindsK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP pro-n: a role in cytoskeletal remodeling. J Cell Biol 1999;145:851–63.ris S, Longhi R, Santambrogio P, de C, I. Leucine-zipper-mediatedmo- and hetero-dimerization of GIT family p95-ARF GTPase-tivating protein, PIX-, paxillin-interacting proteins 1 and 2.chem J 2003;372:391–8.jdo-Milasinovic A, van der Kammen RA, Moneva Z, Collard JG.c3 inhibits adhesion and differentiation of neuronal cells by mod-ng GIT1 downstream signaling. J Cell Sci 2009;122:2127–36.ii N, Maier D, Merlo A, et al. Frequent co-alterations of TP53, p16/
KN2A, p14arf, PTEN tumor suppressor genes in human gliomall lines. Brain Pathol 1999;9:469–79.rnari FB, Fenton T, Bachoo RM, et al. Malignant astrocytic



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Bhanot et al.

Mol C1374

Do


oma: genetics, biology, and paths to treatment. Genes Dev 2007;:2683–710.anson JA, Watts C. Macropinocytosis. Trends Cell Biol 1995;5:4–8.naldson JG, Porat-Shliom N, Cohen LA. Clathrin-independentdocytosis: a unique platform for cell signaling and PM remodeling.ll Signal 2009;21:1–6.coosin EL, Swanson JA. Macropinosome maturation and fusionh tubular lysosomes in macrophages. J Cell Biol 1993;121:11–20.nt BD, Donaldson JG. Pathways and mechanisms of endocyticycling. Nat Rev Mol Cell Biol 2009;10:597–608.an AY, Coniglio SJ, Chuang YY, et al. Roles of the Rac1 andc3 GTPases in human tumor cell invasion. Oncogene 2005;24:21–9.ley AJ. Rho proteins: linking signaling with membrane trafficking.ffic 2001;2:303–10.mons M, Rusk N. Control of vesicular trafficking by Rho GTPases.rr Biol 2003;13:R409–18.maze C, Chuang TH, Terlecky LJ, Bokoch GM, Schmid SL.gulation of receptor-mediated endocytosis by Rho and Rac.ture 1996;382:177–9.lecz N, McCabe PC, Spaargaren C, Qiu R, Chuang Y, Symons M.naptojanin 2, a novel Rac1 effector that regulates clathrin-diated endocytosis. Curr Biol 2000;10:1383–6.n Y, Buki KG, Ettala O, Vaaraniemi JP, Vaananen HK. Possible roledirect Rac1-Rab7 interaction in ruffled border formation of osteo-sts. J Biol Chem 2005;280:32356–61.sa MA, Maximiano FC, Smolarczyk K, et al. Armus is a Rac1ector that inactivates Rab7 and regulates E-cadherin degradation.rr Biol 2010;20:198–208.k J, Ghigo E, Kalaidzidis Y, Zerial M. Rab conversion as a mech-
sm of progression from early to late endosomes. Cell 2005;122:–49.r-Sagi D, Feramisco JR. Induction of membrane ruffling and fluid-
AltrecGa



ase pinocytosis in quiescent fibroblasts by ras proteins. Science86;233:1061–8.lsh AB, Bar-Sagi D. Differential activation of the Rac pathway by-Ras and K-Ras. J Biol Chem 2001;276:15609–15.ely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV. Cdc42 andc1 induce integrin-mediated cell motility and invasiveness through3)K. Nature 1997;390:632–6.wards DC, Sanders LC, Bokoch GM, Gill GN. Activation of-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin

toskeletal dynamics. Nat Cell Biol 1999;1:253–9.nty LC, Ravichandran KS, Casanova JE. The DOCK180/Elmomplex couples ARNO-mediated Arf6 activation to the downstreamtivation of Rac1. Curr Biol 2005;15:1749–54.o TH, Eipper BA, Donaldson JG. Arf6 recruits the Rac GEF Kalirinthe plasma membrane facilitating Rac activation. BMC Cell Biol07;8:29.mont RT, Perry SJ, Schmalzigaug R, Roseman JT, Xing Y, ClaingThe GIT/PIX complex: an oligomeric assembly of GIT family ARFPase-activating proteins and PIX family Rac1/Cdc42 guaninecleotide exchange factors. Cell Signal 2004;16:1001–11., Wang J, Ng SS, et al. Adenosine diphosphate-ribosylation fac-6 is required for epidermal growth factor-induced glioblastoma

ll proliferation. Cancer 2009;115:4959–72.linari M, Galli C, Norais N, et al. Vacuoles induced by Helicobacterlori toxin contain both late endosomal and lysosomal markers.iol Chem 1997;272:25339–44.aves BJ, Bright NA, Mullock BM, Luzio JP. The effect of wort-nnin on the localisation of lysosomal type I integral membranecoproteins suggests a role for phosphoinositide 3-kinase activityregulating membrane traffic late in the endocytic pathway. J Celli 1996;109:749–62.ies R, Nobbio L, Edomi P, Schenone A, Schneider C, Brancolini C.erations in the Arf6-regulated plasma membrane endosomal
ycling pathway in cells overexpressing the tetraspan proteins3/PMP22. J Cell Sci 2003;116:987–99.



2010;8:1358-1374. Published OnlineFirst August 16, 2010.Mol Cancer Res Haymanti Bhanot, Ashley M. Young, Jean H. Overmeyer, et al. Requires Inverse Regulation of Rac1 and Arf6Induction of Nonapoptotic Cell Death by Activated Ras

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