Chloride Transport in Glioma Growth

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519Physiology and Pathology of Chloride Transporters and Channels in the Nervous System 2009, E e ier I

Ch ri e Tra p rt i G i a Gr tha Ce I a i

Harald Sontheimer

C H A P T E R

I. I tr ucti 519

II. Gli ma a t eir Li eage 520

III. Gli ma Migrati a I va i 521

IV. Cl Tra p rt a Cell V lume Regulatii Gli ma Cell 521

V. C a ge i Cell V lume f I va i g CellRe uire Cl Efflux via ClC C a el 523

VI. Mec a i m f C l r t xi ActiGli ma I va i 524

VII. Cli ical U e f C l r t xi 5

VIII. Cell V lume C a ge A ciate itCell Pr liferati 526

IX. C clu i 52

Ack le geme t 528

Refere ce 529

o u t l i n e

I. InTRodUCTIon

Brain tumors fall into two principal categories, pri-mary and secondary. Primary tumors are often calledgliomas and originate in the brain. Secondary or meta-static brain tumors are peripheral cancers that invadethe brain. Together they account for well over 100,000new cancer cases diagnosed each year in the USA,of which approximately 40,000 are primary tumors(according to data from the Central Brain TumorRegistry of the United States, CBTRUS). In additionto their dissimilar origin, primary and secondary

brain tumors differ in many aspects of their etiologyand biology. For example, metastatic cancers are eas-ily distinguishable from normal brain tissue as theyrepresent the new growth of cancerous tissue with the

properties of the organ it originated from. Hence, theypresent as liver or lung cells growing within brain andtumors typically grow as confined solid masses. Thisis not the case for primary brain tumors which oftenlack clear boundaries between normal and malignant

brain tissue. A representative example is illustratedin Fig. 26.1A, which shows a cerebral glioma withcharacteristic diffuse margins. An important differ-ence between these two cancer types relates to howthe tumors spread and form metastasis. Metastatic

brain tumors disseminate hematogenously throughoutthe body and enter the brain through the vasculature.By comparison, primary brain tumors rarely metasta-size into the periphery but instead spread within the

brain often reaching distant sites such as the contralat-eral brain hemispheres or the spinal cord, as illustrated

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26. CHloRIdE TRAnsPoRT In GlIomA GRowTH And CEll InvAsIon520

in the example shown in Fig. 26.1B. These cancersspread by active cell migration without extravasatinginto the vasculature. This is reminiscent of neuronaland glial cell migration during brain development orstem cells and microglial cells in the adult brain sug-gesting that some of the underlying mechanisms ofmigration may be shared between these cells.

II. GLIoMAs And ThEIR LInEAGE

Although primary brain tumors can originate froma number of growth competent cells in the brain orspinal cord, the majority of them appear to derivefrom glial cells or their precursors. Reflecting this pre-sumed relationship these tumors are collectively calledgliomas. These include a diverse group of cancers that

may not always be of defined lineage. Among the dis-tinguishing features are immuno-positivity for cer-tain glial associated antigens ( Kleihues et al., 1995 ),for example to glial fibrillary acidic protein (GFAP),myelin associated glycoprotein (MAG), myelin basicprotein (MBP), S100 beta or vimentin. GFAP and/orS100 positive cells are frequently termed astrocytomas,MBP- or MAG-positive cells; Oligodendrogliomasand cells that stain for both sets of markers are mixedgliomas. While these names imply a known and well-defined lineage relationship of these tumors withnormal glial cell type or their progenitor cells, such arelationship has not actually been demonstrated andthe cell types of origin remain controversial. In stud-ies addressing this question, investigators have trans-fected glial progenitor cells with known mutationsin oncogenes and tumor suppressor genes and have

been able to induce a malignant transformation thatyielded tumor growth in mice, suggesting that com-mitted glial progenitor cells may indeed be the most

likely cell type of origin ( Dai et al., 2001 ).Gliomas exhibit many of the characteristic featuresof systemic cancers which include mutations in thetumor suppressor genes P16 and P53, and amplificationand overexpression of certain oncogenic growth factorreceptors including EGF-R or PDGF-R ( Von Deimling et al., 1995 ). As with other cancers, angiogenesis orthe induction of new blood vessels in response to therelease of vascular endothelial growth factor is com-mon ( Plate and Risau, 1995 ). Furthermore, the releaseof matrix degrading enzymes that facilitate the remod-eling of the tumor associated extracellular space is com-mon and facilitates cell invasion ( Giese et al., 1994 ).

A glioma diagnosis is almost always fatal as currenttreatment options are limited ( Butowski et al., 2006 ).By the time a tumor is detectable, it has frequentlyseeded tumor cells throughout the nervous system,and upon surgery these cells can quickly give rise torecurrent malignancies. The diffuse pattern of cellularinvasion illustrated in Fig. 26.1 not only makes com-plete surgical resection impossible, but also limits focaltreatments such as exogenous beam irradiation as cellsremote from the tumor will escape the radiation. Uponrecurrence, many gliomas become even more malig-nant. Recurrence is believed to result from cells thathave invaded surrounding brain areas. Surprisingly,little is known about the underlying mechanisms.For example, pathways of cell migration are poorlyunderstood as are molecules involved in chemotaxisand path finding. These aspects of glioma biology arepromising areas for future research as they may yieldmore effective therapeutic tools. An important aspectof tumor biology that has been well studied in recentyears and which will be discussed in greater detail in

FIGURE 26.1 Primary glioma at autopsy. A. Poorly definedmargins are characteristic of cerebral gliomas, like the one shown inthis example (arrows). B. Although gliomas rarely metastasize out-side the brain, they often present secondary tumors in other partsof the brain, often distant from the site of the primary tumor. Thesesecondary tumors are highlighted in B by white ovals. Copyrightedimages: University of Alabama at Birmingham, Department ofPathology.


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this chapter pertains to biophysical and biomechanicaladaptations that support the migration and invasionof gliomas into normal brain tissue. Some of thesefindings may pertain to other migratory cells in the

brain and even to other cancers.

III. GLIoMA MIGRATIon AndInVAsIon

As illustrated in Fig. 26.1, the boundaries betweena primary glioma and normal brain tissue are oftendifficult to delineate at a macroscopic level. At amicroscopic level, thousands of glioma cells will havediffusely invaded the surrounding areas of brain tissue,and, over time, they will have spread to very distantsites. Wherever possible, invading glioma cells appearto take advantage of other structures in the brain tomigrate. For example, they frequently migrate along

nerve fiber bundles or, as illustrated in Fig. 26.2A,along blood vessels. Whether the spaces along thesestructures are more favorable for migration, or whetherthere are other guidance cues, or a more slippery extra-cellular matrix, is not entirely clear. Without question,however, the narrowness of the extracellular space pro-vides a significant impediment to cell migration. At theelectron microscopic level, invading cells appear elon-gated, wedge shaped, and with an overall shrunkenappearance ( Fig. 26.2B). This has led to the hypothesisthat glioma cells may dynamically adjust their cell vol-ume as they invade. As illustrated in cartoon form inFig. 26.2C, and further discussed below, recent findings

support this hypothesis and suggest an important rolefor Cl channels and transporters in this context.

IV. Cl TRAnsPoRT And CELLVoLUME REGULATIon In

GLIoMA CELLs

As extensively discussed in Chapter 15 in this book,all eukaryotic cells have developed powerful mecha-nisms to maintain a constant cell volume even whenextracellular osmotic conditions change. Glioma cells arenot an exception; when exposed to a 50% hyposmoticchallenge they regulate their volume back to baselinewithin just a few minutes. As illustrated in Fig. 26.3A,this regulatory volume decrease (RVD) is inhibited bydrugs known to block Cl channels including NPPB,Cd2 and DIDS with almost complete inhibition byNPPB and Cd 2 (Ernest et al., 2005 ). The remainingvolume regulatory response is inhibited by drugs that

block the K -Cl cotransporters ( Ernest et al., 2005 )Furthermore, volume regulation is supported when Clis replaced by halide ions such as I or Br with knownpermeability to Cl channels, but not when gluconatesubstitutes for Cl (Ernest, 2007). These studies suggestthat RVD in glioma cells utilizes Cl as osmolyte, whichis released from the cell through Cl channels.

An important question is whether Cl may simi-larly act as an osmolyte during cell volume changesassociated with cell invasion, a process that is verydifferent from cell volume changes elicited by osmoticchallenges. Under hyposmotic conditions, a gradi-ent for Cl efflux is favored by the dilution of extra-cellular ions with water, whereas under isosmoticconditions, this is not the case, unless the cell has asufficiently high [Cl ]i. Hence, the hypothesized cellshrinkage of invading cells requires that intracellu-lar Cl be accumulated so that an outward directed

FIGURE 26.2 Invading glioma cells in situ . A. Confocal imagesof invading D54MG cells stably expressing EGFP (green). Theinvading glioma cells adhere to blood vessels (red). Cells oftenexhibit an elongated wedge-shaped appearance as shown in thelower panel. B. The elongated shape is quite apparent in this elec-tron micrograph that captured an invading glioma cell (*, recog-nized because of the abundance of ribosomes and other organellesthat incorporate lead citrate and give a darker appearance) extend-ing between normal brain cells, likely astrocytes (based on theirlarge nuclei and abundance of electron dense, glycogen depositsthroughout the cytoplasm). C. Cell shrinkage requires water effluxwhich is driven by the concomitant efflux of Cl and K throughtheir respective ion channels. Glutamate is shown as a possiblemotogenic stimulus acting via AMPA receptors that raise intracel-lular [Ca 2 ] which may in turn activate BK channels. (Panel B isreproduced with permission from Soroceanu et al., 1999 .)

Iv. C TRAnsPoRT And CEll volumE REGulATIon In GlIomA CElls


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electrochemical gradient for Cl is maintained. Usingthe Cl sensitive fluorescence indicator SPQ, intracel-lular [Cl ] was measured in glioma cells and found to

be around 100 mM (Ernest, 2007), a value far greaterthan that typically observed in mature central neurons(710 mM) , glial cells (3040 mM) or mature primaryafferent neurons ( 45 mM) (see Chapters 7, 19 and22 in this volume). These findings were recently con-firmed by patch-clamp studies at the single cell levelin which the reversal potential of Cl currents wasused as an indirect indicator of [Cl ]i. Since gliomacells in culture lack ligand-gated Cl channels, suchas the GABA A receptor-channel widely expressed inneurons, recombinant ligand-gated non-inactivatingCl channels (GABA-rho) were introduced into glialcells, and stable cell-lines expressing GABA-gatedCl channels were created. Gramicidin-perforatedpatch recordings allowed determination of the rever-sal potential of the GABA-induced currents ( EGABA).

[Cl ]i was estimated from EGABA (Habela et al., 2009 ).These studies indicated an intracellular [Cl ] of105 mM in glioma cells, a value close to that deter-mined using SPQ. Hence, glioma cells maintain asteep outward directed gradient for Cl . In most cells,Cl is actively accumulated via the Na -K -2Cl cotransporter (NKCC1), which is a widely expressedCl importer (Chapters 2, 16 and 19 in this volume).Western blot and immunostaining analyses of sev-eral glioma cell-lines, including those obtained fromacute patient biopsies, demonstrated prominentexpression of NKCC1 and absence of NKCC2 (seeFig. 26.3C and 26.3D as well as Ernest and Sontheimer, 2007). Gliomas also express KCC1 and KCC3 ( Ernest et al., 2005 ). Consistent with NKCC1 being princi-pally responsible for the accumulation of intracellu-lar Cl above electrochemical equilibrium in gliomas,pharmacological inhibition of the cotransporter with

bumetanide causes a significant drop in intracellular

FIGURE 26.3 Volume regulation in glioma cells. A. On exposure to a 50% hypotonic challenge, glioma cells swell and regulate their vol-ume back to baseline or even below the baseline level. This regulatory volume decrease is partially inhibited by NPPB or DIDS and is almostcompletely inhibited by NPPB and Cd 2 . B. Glioma cells maintain an elevated intracellular [Cl ] which is accumulated via the bumetanide-sensitive Na -K -Cl cotransporter, NKCC1. Pharmacological inhibition of the cotransporter by 20 M bumetanide causes a decrease in [Cl ]i.Exposure to 40 M DIOA causes an increase in [Cl ]i above control levels, presumably by inhibition of KCC mediated Cl efflux. C. Western

blot of lysates from two glioma cell-lines D54-MG and U251, and from samples obtained from one patient (GBM50), show prominent expres-sion of NKCC1 but absence of NKCC2. Rat kidney lysates were used as control for NKCC2. D. Immunostaining also shows prominent mem-

brane associated labeling for NKCC1 in representative U251 glioma cells. Antibodies directed against NKCC1 and NKCC2 were from AlphaDiagnostics, and were used at a dilution of 1:500. (Panels A and B are reproduced with permission from Ernest et al. (2005) , and panel C isreproduced with permission from Ernest and Sontheimer (2007) .)


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Cl (Fig. 26.3B). As we will be discussing below, highintracellular [Cl ] is possibly required for immatureneurons and glioma cells to migrate. High Cl mightfacilitate water extrusion and cell shrinkage, processesnecessary for migrating cells to navigate through con-fined extracellular spaces.

V. ChAnGEs In CELL VoLUME oFInVAdInG CELL REqUIRE Cl EFFLUX

VIA ClC ChAnnELs

As illustrated in Fig. 26.2C migratory glioma cellsappear to undergo profound changes in cell volume asthey invade surrounding tissues. We hypothesize thatthese spontaneously occurring changes in cell volumeare driven by efflux of Cl and obligatory water. Thenotion that a favorable outward Cl gradient is estab-lished by NKCC1 was experimentally tested in a recent

study ( Habela et al., 2009 ). Glioma cells were stablytransfected with GABA-rho channels, and subjectedto volume measurements using a Coulter Counter.Application of GABA induced opening of Cl chan-nels which caused progressive cell volume decrease.This cell shrinkage was not observed in untransfectedcells, or in the absence of GABA. This suggests thatopening of Cl channels causes efflux of Cl associatedwith obligatory water loss, and cell shrinkage. Theseexperiments also suggest that Cl efflux is sufficient toinduce a volume decrease in glioma cells. Interestingly,these studies were made by inserting a ligand-gatedCl channel which could be activated on demand, butglioma cells express a significant resting Cl conduc-tance ( Ransom et al., 2001 ). Indeed, when recordedusing perforated patch-clamp technique to avoid dis-turbing cytosolic Cl , glioma cells exhibit a resting Cl conductance sensitive to NPPB and DIDS. These cur-rents are outwardly rectifying, show time-dependentinactivation at positive potentials and exhibit the fol-lowing anion permeability sequence: I Br Cl .However, although the currents could be potentiated

by cell swelling, this was not required for current acti-vation. Because Cl channel inhibitors still lack speci-ficity, attributing the inhibitory effect of NPPB andDIDS to a specific ion channel is not yet possible. Asa first step towards the molecular identification of theCl channels expressed in glioma cells, Western blotsusing lysates obtained from gliomas isolated frompatients were probed with antibodies directed againstepitopes of cloned Cl channels. These studies dem-onstrated the presence of ClC-2, ClC-3 and ClC-5proteins in all gliomas examined ( Olsen et al., 2003 ).Further, immunolabeling studies showed that ClC-3

staining was predominant in invading processes ofisolated glioma cells. In an effort to further identify theCl channels functioning in gliomas, cells were treatedwith antisense oligonucleotides to known membersof the ClC Cl channels super family. These stud-ies showed prominent expression of currents attrib-utable to ClC-2 and ClC-3, respectively ( Olsen et al.,2003). ClC-2 currents, known to be sensitive to Cd 2inwardly rectifying, and potentiated by a negative pre-pulse to 120 mV, were selectively eliminated inglioma cells treated with ClC-2 antisense oligonucle-otides ( Fig. 26.4A). As expected, these currents wereunaltered by ClC-3 antisense oligonucleotides.

ClC-3 channels giving rise to outwardly rectifyingcurrents that show time-dependent inactivation andare sensitive to NPPB were greatly reduced in gliomacells treated with ClC-3 antisense oligonucleotides(Fig. 26.4B). These data are consistent with both ClC-2and ClC-3 channels being functionally expressed ingliomas. However, functional expression of ClC-3

channels in the plasma membrane is controversial, asdiscussed in detail in Chapter 12 of this volume. ClC-3 knockout mice primarily show CNS pathology asso-ciated with loss of synaptic vesicles in hippocampalneurons ( Stobrawa et al., 2001 ). Thus, whether ClC-3protein is able to generate functional channels in theplasma membrane has been questioned. Immuno-gold electron microscopy of human gliomas, however,shows immunoreactivity associated with both plasmamembrane and intracellular vesicles ( Fig. 26.4C)Further, ClC-3 in cultured glioma cells colocalizes tothe -subunit of cholera-toxin, which binds to lipidraft domains, arguing for membrane localization ofthe Cl channel (see merged signals in Fig. 26.4D).

Figure 26.5A shows that currents with the biophysi-cal signature of ClC-3 are inhibited in a dose-dependentfashion by chlorotoxin (Cltx), a peptide isolated fromthe venom of the scorpion Leiurus quinquestriatus(DeBin et al., 1993 ). This toxin might inhibit Cl chan-nels with some specificity (McFerrin and Sontheimer,2005). Importantly, as shown in Fig. 26.5BC, whencells were challenged to cross a transwell barrier thatmimics the spatial constraints of the extracellularspace in the brain, cell migration across the barrier wasinhibited when the Cl conductance was blocked withNPPB (Ransom et al., 2001 ), Cd 2 or Cltx ( Soroceanuet al., 1999 ). Of all these drugs, NPPB and Cltx werethe most effective inhibitors of cell migration in thetranswell assays. Cltx in both its native and recom-

binant form inhibited transwell migration in a dose-dependent fashion ( Deshane et al., 2003 ). Further, afluorescently labeled Cltx showed binding to the cellsurface of human malignant glioma cells in patient

biopsies. Based on these data we proposed ClC-3 as a

v. CHAnGEs In CEll volumE of InvAdInG CEll REquIRE C Efflux vIA C C CHAnnEls


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prime candidate for mediating the Cl fluxes requiredto accomplish the cell shrinkage needed for gliomacell migration. The data further suggest that ClC-3may be a biological target of Cltx and that the lattermight be a potent inhibitor of glioma cell migration.

VI. MEChAnIsM oF ChLoRoToXInACTIon on GLIoMA InVAsIon

The finding that Cltx inhibited Cl channels ( Fig. 26.5A) and was effective in preventing cell invasionin transwell assays ( Fig. 26.5B) prompted further studies

on its mechanism of action; Cltx has a potential clini-cal use as an anti-invasive drug. While biophysicalstudies suggested that Cltx inhibits Cl channels inglioma cells, it took up to 15 minutes to achieve itsmaximal effect. This long delay questioned a directaction on the channels; channel-specific toxins typi-cally act almost instantaneously. Using a His-taggedrecombinant Cltx, Deshane and collaborators wereable to isolate a protein complex and analyzed it bymass-spectroscopy ( Deshane et al., 2003 ). These stud-ies showed that matrix-metalloproteinase-2 (MMP-2),a 72 kD protein that is highly expressed on the surfaceof invading glioma cells, could be the primary bindingsite for Cltx. However, the isolated protein complex

FIGURE 26.4 Glioma cells express functional ClC-2 and ClC-3 channels. Using specific antisense oligonucleotides to ClC-2 ( A ) and ClC-3 (B ), it was possible to identify currents attributable to these channels, respectively. The Western blots illustrate effective reduction in cor-responding protein expression following antisense treatment, demonstrating specificity of the effects observed in the membrane currents.

C. Immuno-gold EM with antibodies to ClC-3 show clusters of channels at the cell surface (thin white arrow) as well as in intracellular vesicles(thick white arrow). D. Merged image of triple immunolabeling of cultured D54-MG (human glioma cell line). ClC-3 antibody (labeled withalexa 546) shows that this protein colocalizes with lipid rafts which are identified by immunolabeling of the beta subunit of cholera-toxin(fluorescein-conjugated cholera-toxin subunit). Nuclei were counterstained blue with DAPI. (Panels A and B were modified from Olsen et al. (2003); C is an unpublished image; and D is reproduced with permission from McFerrin and Sontheimer (2006) .)


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also contained ClC-3 channels and several regulatorsof MMP-2. To further investigate how Cltx may havedecreased Cl channel function, cell surface expres-sion was examined by biotinylation. These studiesshowed that upon application of Cltx, membraneassociated ClC-3 channels gradually disappeared andwere almost undetectable at the surface after 30 min-utes (McFerrin and Sontheimer, 2005). Further, it wasobserved that the plasma membrane channels endedup in intracellular caveolar vesicles. In the presence offilipin, a sterol-binding drug that prevents the forma-tion of caveolar vesicles, Cltx lost its effect on ClC-3channel internalization. This suggested that binding ofthe toxin induces the internalization of ClC-3 channelstogether with Cltx into caveolar raft vesicles. Thesefindings explain the intracellular trapping of Cltxobserved in other studies, including those in humans,as discussed below.

VII. CLInICAL UsE oF ChLoRoToXIn

In light of the specific binding of Cltx to culturedglioma cells, it was logical to explore the biologicalactivity of this molecule in animal models of malig-nant glioma. Using a radiolabeled peptide we dem-onstrated its specific binding to human gliomasxenografted into the cerebrum of immunocompro-mised mice ( Soroceanu et al., 1998 ). These studies werefollowed by screening of human tissues searchingfor specific binding of Cltx ( Lyons et al., 2002 ). Thesestudies, which examined over 100 samples, revealed

binding of Cltx to gliomas of all malignancy grades,as well as to tumors that share an embryological rela-tionship with them. The latter includes primarily can-cers originating from neuroectodermally derivedtissues such as melanoma or small lung cell carcinomas.

FIGURE 26.5 Inhibition of Cl channels with chlorotoxin retards glioma cell migration. A. Representative currents in response to volt-age steps, recorded before (control) and 15 minutes after application of 1 M chlorotoxin. Outwardly rectifying, inactivating Cl currents wererecorded by whole-cell patch-clamp in D54-MG glioma cells using 20 mV voltage steps ranging from 120 to 160 mV. B. To show that a chlo-rotoxin-sensitive Cl conductance is required for migration across a spatial barrier, D54-MG glioma cells were plated on the upper surface of aTranswell insert with 8 m pores and allowed to migrate for 4 hours towards vitronectin coated on the bottom of the filter insert (top left). Undercontrol conditions, most cells migrated successfully, indicated by crystal violet staining of cells (control). In the presence of 5 M chlorotoxin onlya few cells migrated through the filter. C. Chlorotoxin inhibits glioma cell migration. Doseresponse curve of D54-MG cells treated with His-Cltxor commercial Cltx peptide (Alomone) and analyzed by matrigel invasion assay at 24 h post-treatment. Half maximal inhibition (IC50) for Cltx was184 nM. Percent inhibition was calculated as the decrease in the number of migrated cells normalized to control. (Panel A reproduced with per-

mission from McFerrin and Sontheimer (2006); panels B and C are reproduced with permission from Deshane et al. (2003).)

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In contrast, non-malignant tissues were universally neg-ative. In 2002, a synthetically manufactured Cltx wasapproved by the US Food and Drug Administration(FDA) to be examined in 18 patients in a phase I study.Like in the previous preclinical studies, this trial useda radiolabeled form of the peptide which was intro-duced through an intrathecal catheter. The radiolabelcould then be detected by whole-body gamma cam-era scans ( Fig. 26.6A), or with greater resolution, bySPEC imaging ( Fig. 26.6B). In this study, fluid sam-ples were collected to determine the pharmacokinet-ics of the molecule. The data from this clinical studywere published in 2006 ( Mamelak et al., 2006 ). Sampleimages like those shown in Fig. 26.6 were published in2005 (Hockaday et al., 2005 ). The safety and localiza-tion data gathered in the phase I trial justified furtheruse of Cltx in 59 patients, in a phase II clinical studywhich concluded recently. Preliminary data releasedfrom this trial showed a significant increase in mean

survival, following administration of three or six dosesof Cltx. Importantly, imaging studies such as thoseillustrated in Fig. 26.6 suggest that Cltx is retained atthe tumor for 58 days. This observation is in goodagreement with the internalization of Cltx togetherwith ClC-3 and MMP-2 into caveolar vesicles. Thetherapeutic efficacy of Cltx is therefore, in all likeli-hood, due to (1) the internalization of ClC-3 channelsand decreased cell migration and (2) the trapping ofthe radiolabel toxin which could have its own effect oncellular DNA.

VIII. CELL VoLUME ChAnGEsAssoCIATEd wITh CELL

PRoLIFERATIon

In addition to being highly invasive, primary brain

tumors also exhibit relentless growth, with mitoticindices suggesting that over 30% of high-grade glio-mas are in the active process of cell division. As cellsdivide, they give rise to two daughter cells of approx-imately half the volume of the parent cell. Yet, within

just a few hours, cell size and volume are restoredin both daughter cells. Surprisingly, little is knownabout cell volume changes occurring in dividing cellsin general (see Chapter 27 in this volume). In a recentstudy, we imaged complete cycles of cell divisionusing three-dimensional time-lapsed video micros-copy following individual cells from birth throughto the next cell division giving rise to new daughter

cells (Fig. 26.7A, B). In this study, cell volume wasobtained from 200 to 400 serial sections at each timepoint, allowing relatively accurate cell volume mea-surements for the entire cell cycle ( Fig. 26.7C). Wedemonstrated a reduction in cell volume prior to theM-phase of the cell cycle ( Fig. 26.7D), a phenomenonwhich we termed pre-mitotic volume condensation(Habela and Sontheimer, 2007 ). Regardless of thecell volume that a cell maintains during interphase,it condenses to a volume of approximately 6000-fLprior to entering into M-phase, approximately 6 h

before giving rise to two daughter cells of approxi-mately 3000-fL volume ( Fig. 26.7D). The condensedcells have already synthesized the cell membrane ofthe two daughter cells, as this is readily visible by thethickened membrane ( Fig. 26.8A). This finding wasentirely unexpected, as the common assumption has

been that cells grow in size continuously until divi-sion occurs. A contraction of the cytoplasmic volumewas not expected. Furthermore, the fact that the cell

FIGURE 26.6 The Cl channel inhibitor chlorotoxin localizes togliomas in vivo. A. A single dose of 131I-chlorotoxin given to a patientin a phase I clinical study shows tumor-specific localization in whole-

body scans performed over a 5 day period (modified from Shen et al., 2005). B. Overlay of MRI and SPECT images showing tumor-specificretention of chlorotoxin, 8 days after administration of the drug.Axial view of T1-Wc (left), coregistered (middle), and SPECT (right).(Reproduced with permission from Hockaday et al., 2005 .)


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membrane thickens as the cell volume condenses sug-gests that, at this stage, cells have membrane foldsready to be unfolded once a cell division and separa-tion of two daughter cells has occurred. Upon divi-sion, to achieve normal volume, each daughter cellonly needs to reaccumulate water through the uptakeof Na and Cl , presumably via NKCC1. The vol-ume changes that may occur through the cell cycleare illustrated in Fig. 26.8B. Importantly, studies thatdirectly compared intracellular [Cl ] in M-phase cellsversus the bipolar interphase cells showed a 40%reduction in [Cl ]i in the condensed M-phase cells,suggesting that Cl efflux is mechanistically linkedto the cell volume reduction ( Habela et al., 2009 ).Closer examination also showed that cytoplasmic

condensation is accompanied by condensation ofnuclear chromatin and indeed, the two processesappear to occur in close synchrony ( Habela andSontheimer, 2007 ). The initial condensation of thecytoplasm and hence the chromosomal condensa-tion are mediated by the efflux of Cl through thesame ClC-3 channels that are involved in cell volumedecreases associated with invading cells since shRNAknock-down of ClC-3 impaired cell condensation(Habela et al., 2008 ). While pharmacological studieshave long suggested a role for Cl channels in celldivision, these studies are the first to ascribe a mech-anistic role to these channels in cell division; theymediate cytoplasmic condensation through water loss,a necessary step for cells to enter the M-phase.

FIGURE 26.7 3D time-lapse imaging of glioma cells division allows accurate determination of cell volume throughout the cell cycle pro-cess. A. 3D projections created from image z-stacks computed from 200 optical sections such as those shown in B, and rendered in 3D usingImagePro. This program also computed volumes in fL for each 3D rendered cell. B. Sections from the z-stack used to generate the correspond-ing projections shown in A. Images are in chronological order from 1 to 5. C. Volume measurements (in fL) at specific time points relative todivision are shown for four cells including the cell in A (green triangle symbols). Time points 1 through 4 correspond to projections 14 in B.For each cell, M-phase was set at t 0 minutes. Note the convergence in volumes immediately before M-phase, where volumes are tightlyclustered around 6000 fL. D. Cells assume a volume of 6000 fL as they reach M-phase, regardless of their volume during interphase ( n 14cells). (Reproduced with permission from Habela and Sontheimer, 2007.)

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IX. ConCLUsIons

Taken together, the data discussed in this chaptersuggest that Cl channels and transporters cooperateto support dynamic changes in cell volume that gov-ern cell proliferation and cell migration/invasion. Theoutward electrochemical gradient for Cl is estab-lished by the Na -K -2Cl cotransporter, NKCC1,and this gradient permits rapid Cl efflux throughCl channels and obligatory water movementacross the plasma membrane, ultimately leading tocell volume reduction. It is conceivable that similarmechanisms operate in other migratory cells, e.g.

developing neurons, stem cells, and other cell typeswhich migrate during development prior to settlingdown, maturing and forming tissues. Furthermore,a similar role for Cl channels in the control of cellgrowth and proliferation may widen the therapeu-tic potential for Cl channel blockers as anti-cancerreagents.

Ack le geme t

The author is grateful for the continued support by grants from the National Institutes of Health RO1NS-31234, RO1 NS-52634, NS-36692 and P50-CA97247.

FIGURE 26.8 A. Membrane thickening of M-phase cells following volume condensation are displayed by labeling cells expressing cytosoliceGFP (green) with membrane bound DiI (red). A. In interphase, the cell membrane associated DiI is a thin membrane layer surrounding thecytoplasm. In M-phase, this area is thickened suggesting a ruffled membrane. B. Changes in cell volume during the cell cycle illustrated incartoon form. As cells progress through G1/S, they increase their plasma membrane area and overall cell volume. As they progress to the M-phase they condense their cytoplasmic volume but maintain their cell membrane area which becomes thickened and folded. Cell division intotwo daughter cells divides membrane and cytoplasm equally between daughter cells. The acquisition of new membrane is accompanied byuptake of Na , Cl and water through NKCC1, which establishes the normal cell size/volume. (Reproduced with permission from Habela and Sontheimer, 2007 .)


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Chloride Transport in Glioma Growth