glut-1 and hexokinase expression: relationship with 2-fluoro-2 … · glut-1 and hexokinase...

[CANCER RESEARCH 59, 4709–4714, September 15, 1999]

Glut-1 and Hexokinase Expression: Relationship with 2-Fluoro-2-deoxy-D-glucoseUptake in A431 and T47D Cells in Culture

Luigi Aloj, 1 Corradina Caracó,2 Elaine Jagoda, William C. Eckelman, and Ronald D. Neumann3

Nuclear Medicine [L. A., R. D. N.] and Positron Emission Tomography [C. C., E. J., W. C. E.] Departments, Clinical Center, NIH, Bethesda, Maryland, 20892-1180

ABSTRACT

Uptake of 2-[18F]-2-deoxy-D-glucose (FDG) has been used as a markerof increased glucose metabolism to visualize, stage, and monitor progres-sion of human cancers with positron emission tomography. Many humantumors have been shown to overexpress the Glut-1 glucose transportprotein. The aim of this study is to define whether a quantitative relation-ship exists between the amount of Glut-1 expressed by cells and theirability to accumulate FDG. We characterized the expression of the knownfacilitative and sodium-dependent glucose transporter isoforms in sixdifferent cancer cell lines used in our laboratory (A431, MDA-MB-231,T47D, CaCo II, MCF7, and HepG2). A431 and T47D cells express,respectively, the highest and lowest amount of Glut-1 mRNA by Northernblot of all of the cells analyzed, and no other glucose transporter isoformswere detectable by this method in both cell lines. Both total and plasmamembrane-associated Glut-1 protein levels were higher in A431 than inT47D cells, consistent with the higher Glut-1 mRNA levels. However,T47D cells accumulate FDG more rapidly than do A431 cells. 3-O-Methyl-glucose transport is higher in A431 cells. Although hexokinase I and IImRNA levels are higher in A431 cells than in T47D cells, the ability ofmitochondrial preparations to phosphorylate FDG is higher in T47D cells.Our data indicate that in these cultured cells, FDG uptake correlatesbetter with FDG phosphorylating activity of mitochondrial preparationsrather than the level of expression of the Glut-1 or hexokinase I and IIgenes.

INTRODUCTION

Cancer cell growth is heavily dependent on increased glucosemetabolism. The association between neoplastic growth and increasedglucose metabolism has been known for many years (1). This propertyof tumors has also promoted the development of drugs aimed atblocking glucose metabolism for therapeutic purposes (2). The mo-lecular mechanisms by which increased glucose metabolism is sus-tained, however, are still not fully understood and are the subjects ofvery intense research efforts.

In recent years, the development of PET4 has provided a tool tostudy increased glucose metabolismin vivo by measuring uptake ofthe glucose analogue FDG. Di Chiroet al. (3) first demonstrated thatFDG is more avidly accumulated in human brain tumors than insurrounding brain and that tumor recurrence is associated with in-creased FDG uptake, whereas posttreatment changes, such as radia-tion necrosis, are not (4). The concept was rapidly extended andapplied to tumors in other areas of the body and has been used inmany studies to diagnose, monitor, and evaluate treatment response ofcancers (5).

Two steps are required to accumulate FDG in cancer cells: (a)facilitated diffusion through a glucose transport protein; and (b)subsequent phosphorylation by one of the hexokinase isoforms, FDG-6-P. FDG-6-P is not transported out of cells nor undergoes glycolyticbreakdown; it is metabolically trapped inside cells. Identification ofwhich of these two steps is rate limiting in the process of FDG uptakeshould provide a better understanding of how glucose metabolism isregulated in cancer cells. This information may also be useful fordeveloping therapeutic strategies aimed at blocking increased glucoseconsumption in tumors.

Several studies have focused on the expression of glucose trans-porters and hexokinase activity to define the role of these two classesof genes in the regulation of FDG uptake. Five subtypes of the humanfacilitative glucose transporters have been described (6). Althoughmost of these subtypes have, to some extent, been detected in differenthuman cancers and cancer cell lines, Glut-1 is the only subtype thathas been detected in nearly all cell lines tested (6) and has also beenshown to be overexpressed in many human cancers (7). The overex-pression of Glut-1 in cancers has lead to speculation that this proteinmay be regulating glucose metabolism and FDG uptake in cancercells. On the other hand, several hexokinase subtypes have also beendescribed. Hexokinase I and II have been found to be expressed intumors. Some authors suggest that these proteins, hexokinase II inparticular, are indeed regulating glucose metabolism in cancer cells(8), and some clinical studies show that the FDG phosphorylation stepmay be rate limiting in the FDG uptake of cancer (9). Some studiesalso suggest that the lack of glucose-6-phosphatase activity in tumorsplays a role in determining FDG retention by preventing dephospho-rylation of FDG-6-P to FDG (10).

To shed more light on this issue, we have taken a comprehensiveapproach to studying the role of the Glut-1 protein and of hexokinaseactivity in FDG uptake in cultured cancer cells. We have characterizedthe expression of all of the known facilitative and sodium-dependentglucose transporters in six different cell lines. Among these, we havechosen two cell lines expressing only the Glut-1 isoform; one cell linehas severalfold higher levels of Glut-1 mRNA than the other. Thesetwo cell lines have been tested for their ability to: (a) express Glut-1protein as determined by Western blot; (b) accumulate DG whengrown in nude mice as xenografts; (c) accumulate FDG in culture inthe presence or absence of glucose; (d) transport 3-OMeG, a glucoseanalogue that is transported into the cell but not appreciably phos-phorylated; (e) synthesize mRNA for hexokinase subtypes I and II;and (f) phosphorylate FDG in mitochondrial extracts. Such an ap-proach has not been used in previous studies on this issue. Further-more, experiments conducted in cultured cancer cells allow us tocontrol potentially confounding variables, such as blood flow andnecrosis, which are present when studying these biochemical param-eters in animal tumor models or in human tumors.

MATERIALS AND METHODS

Cell Culture. A431 cells were originally obtained from Dr. G. Todaro(NIH). This is a human epidermoid carcinoma cell line that has been shown toyield s.c. tumors with high efficiency in immunologically incompetent mice(11). T47D cells were obtained from Dr. T. Moody (NIH). This cell line isderived from a pleural effusion of a patient with metastatic breast cancer (12).

Received 12/9/98; accepted 7/16/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Present address: Centro di Studio per la Medicina Nucleare, Consiglio Nazionaledelle Ricerche, c/o Dipartimento di Scienze Biomorfologiche e Funzionali, Universitádegli Studi di Napoli “Federico II,” Via S. Pansini 5, 80131 Napoli, Italy.

2 Present address: Istituto di Medicina Sperimentale e Biotecnologie, Consiglio Na-zionale delle Ricerche, Localitá Burga, Piano Lago Mangone, 87050 Cosenza, Italy.

3 To whom requests for reprints should be addressed, at Nuclear Medicine Department,Building 10, Room 1C401, 10 Center Drive, MSC 1180, Bethesda, MD 20892-1180.

4 The abbreviations used are: PET, positron emission tomography; FDG, 2-fluoro-2-deoxy-D-glucose; FDG-6-P, FDG-6-phosphate; DG, 2-deoxyglucose; 3-OMeG, 3-O-methyl-D-glucose; RT-PCR, reverse transcription-PCR; SGLT, sodium-dependent glucosetransporter; %ID/g, percentage of the injected dose per gram of tissue.

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Both of these cell lines were grown in a humidified atmosphere containing 5%CO2 and 95% air at 37°C in DMEM with 4.5 g/l glucose, supplemented with10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). HepG2,CaCo II, MCF7, and MDA-MB-231 were originally obtained from the Amer-ican Type Culture Collection (Rockville, MD) and were cultured according tothe instructions provided by the supplier. For all cells, the medium wasexchanged every 2 to 3 days, and cells were passaged after trypsinization whenconfluent. Cells that were overconfluent (i.e., confluent for more than 2 days)were discarded, and fresh cultures were reestablished by thawing cells storedin liquid nitrogen. All experiments herein described were repeated on at leastthree batches of frozen cells to ensure reproducibility.

Radioactive Glucose Analogues.[18F]FDG was prepared in the PETDepartment by standard means (13). All batches were quality controlled toassess purity of the compound and were suitable for patient use (;1000Ci/mmol, end of synthesis). [14C]3-OMeG (50 mCi/mmol) and [3H]DG (30Ci/mmol) were obtained from DuPont NEN (Boston, MA).

DG Uptake in A431 and T47D Xenografts.Before being implanted innude mice, cells were trypsinized, washed in culture medium, and resuspendedin PBS at a density of 23 107/ml. Approximately 100ml of the cellsuspensions were s.c. injected in the flank of Balb-cnu/nuathymic nude mice(4–6 weeks of age; weight, 17–23 g). Xenografts were allowed to grow for10–14 days. Final tumor weight was between 0.5 and 1 g at thetime of thebiodistribution experiments. Sixty min after i.v. injection of 3–5mCi/mouse of[3H]DG, the mice were killed, and the tumors were excised, weighed, andsolubilized in 1 ml of Solvable (Packard, Meriden, CT) overnight at 50°C.Twenty ml of scintillation fluid (Acquasol; Packard) were then added to eachvial. Radioactivity in the tumor samples and in aliquots of the injectionsolution were then quantitatively determined in a Packardb-counter that hadbeen calibrated with quenched3H standards. Uptake was expressed as %ID/gof tumor tissue normalized to a 20-g mouse.

RNA Analysis. Total RNA was extracted from cells grown in culture and,in the case of A431 and T47D cells, xenografted tumors using RNAzol B(Tel-Test, Inc., Friendswood, TX), following the manufacturer’s suggestedprocedure. Fifteenmg of total RNA were denatured by heating at 65°C for 15min and loaded onto a 1% agarose formaldehyde gel containing ethidiumbromide (14). The samples were electrophoresed for 2 h at 100 V. Theethidium signal was captured using a Molecular Dynamics 595 Fluorimager(Sunnyvale, CA), and the gel contents were subsequently transferred to a nylonmembrane using a Turboblotter apparatus (Schleicher and Schuell, Keene,NH). After UV cross-linking, the membrane was hybridized with32P-labeledprobes obtained by random priming of the following cDNA templates. Plas-mids containing the sequences for the human Glut-1, -2, -3, -4, and -5 isoformswere kindly provided by Dr. Charles Burant (University of Chicago, Chicago,IL). The human SGLT 1 and 2 isoform cDNA plasmids (15) were kindlyprovided by Dr. Matthias Hediger (Harvard University, Boston, MA). Thehumanb-actin and hexokinase I cDNA plasmids were purchased from theAmerican Type Culture Collection (Rockville, MD). A 461-bp fragment of thehuman hexokinase II coding sequence, corresponding to nucleotides 3108 to

3568 of the human hexokinase II mRNA sequence (GenBank accessionnumber Z46376), was amplified by RT-PCR from commercially availablepoli-A RNA of human myocardium (Clontech, Palo Alto, CA) and cloned intoa pCR2 vector (Invitrogen, San Diego, CA). After hybridization, the mem-branes were washed twice in 23 sodium chloride-sodium citrate with 0.1%SDS at 70°C, exposed overnight on imaging plates, and analyzed using a FUJIBAS-1500 Phosphorimager (Stamford, CT). All probes were tested on com-mercially available Northern blots (Clontech) prior to use in the experimentsreported. The intensities of the different bands were normalized to theb-actinsignal or to the 18S band values of the ethidium stain.

Protein Analysis. Cells grown in 15-cm diameter plates were rinsed fivetimes in ice-cold PBS, scraped off the plate, and collected in centrifuge tubes.After centrifugation, the pellet was resuspended in 1 ml of 20 mM Hepes, 1 mMEDTA, and 255 mM sucrose (HES, pH 7.4) containing protease inhibitors(Compleat; Boehringer Mannheim, Indianapolis, IN). The suspension washomogenized with 20 strokes in a Teflon homogenizer on ice, and aliquotswere either processed to obtain plasma membrane fractions or total extractswere stored at270°C prior to use. Plasma membrane preparations wereobtained by centrifugation over a sucrose cushion as described (16). Proteinconcentrations were determined using a Coomassie-Plus kit (Pierce ChemicalCo., Rockford, IL). SDS-PAGE was performed as described (17) on precast10% acrylamide Tris-glycine gels (Novex, San Diego, CA). Samples were notboiled prior to loading. After electrophoresis, the proteins were transferred topolyvinylidene difluoride membranes using a Novex X-cell II apparatus. Themembranes were incubated with dilutions of the following antibodies, accord-ing to the technical sheets supplied with the products, in Tris buffered salinecontaining 0.05% Tween 20 (TBS-T) for 1 h. Polyclonal antibodies raisedagainst the NH2 terminus of the human Glut-1 protein were purchased fromAlpha Diagnostics (San Antonio, TX). A mouse monoclonal antibody (M7-PB-E9) against thea1 subunit of the human Na1K1-ATPase (Affinity Biore-agents, Golden, CO) was used as a control. After several washes in TBS-T, themembranes were incubated with a dilution of an appropriate fluorescein-labeled secondary antibody (Amersham, Arlington Heights, IL) for an addi-tional hour, and washed several times in TBS-T; finally, the fluorescent signalwas detected with a Molecular Dynamics 595 Fluorimager.

Cell Uptake Experiments. For experiments with FDG, cells were plated ata density of 1–200,000 cells/well in 12-well multiwell plates (Costar, Cam-bridge, MA) 2 or 3 days prior to the uptake experiments. These conditionsallowed for the cells to be almost confluent at the time of the assay. On the dayof the experiment, the wells were rinsed once in cold uptake medium; subse-quently, 1 ml of uptake medium containing;20 mCi of [18F]FDG was addedto each well. Uptake medium consisted of DMEM supplemented with 0.5%FBS with or without 5.5 mM (100 mg/ml) glucose in the medium. FDG wasallowed to accumulate into cells in the incubator, with times ranging from 10to 40 min. Triplicate wells were rinsed twice in cold PBS, and the cells werelysed with 1M NaOH to collect cell-associated radioactivity. Uptake of FDGand 3-OMeG between 5 s and 3 min was performed under similar conditions.Cells were plated in 35-mm-diameter dishes. Each plate received;20 mCi of

Fig. 1. Northern blot analysis of Glut-1 expression. A431 cells show higher Glut-1mRNA levels than do T47D cells. The same blot was hybridized with ab-actin probe ascontrol.

Fig. 2. Western blot analysis of total and plasma membrane-enriched fractions of A431and T47D cells. Total and plasma membrane Glut-1 levels are higher in A431 extractsthan in T47D extracts. The plasma membrane marker,a1 subunit of the human Na1K1-ATPase, is enriched in the plasma membrane fractions of both cell lines. Comparableamounts of total protein are loaded in each lane, as shown by amido black staining of theblot.

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GLUT-1 AND HEXOKINASE IN CANCER CELLS



[18F]FDG and 2–4mCi of [14C]3-OMeG, and uptake was evaluated fordifferent times. Radioactivity not incorporated into cells was washed awaywith five washes in ice-cold PBS with 100 mM phloretin as described (18).Cell-associated counts were recovered in 1 mM NaOH and measured. In someplates, the glucose transport inhibitor cytochalasin-B (Sigma) was added to theuptake medium at a concentration of 50mM. Fluorine-18 radioactivity wasimmediately determined using a Packard 5600 gamma counter (Packard,Meriden, CT). After 48 h, 20 ml of scintillation fluid (Acquasol; Packard) wereadded to the same vials, and14C radioactivity was quantitatively determined ina Packardb-counter that had been calibrated with quenched14C standards.Protein concentrations for all samples were determined. The uptake of the twocompounds was expressed as the cytochalasin-B-inhibitable percentage ofavailable tracer/mg of cell protein.

Mitochondrial Phosphorylating Activity. Mitochondrial preparationsfrom A431 and T47D cells were obtained as described (19). Briefly, cellsgrown in 15-cm-diameter plates were rinsed several times with ice-cold PBSand recovered by scraping. After centrifugation at 7703 g, cells wereresuspended in 40 mM potassium phosphate buffer (pH 7.6) containing 255 mMsucrose. Cells were disrupted using a Polytron homogenizer for 20 s at highspeed. The suspension was centrifuged at 7703 g once more to separateundisrupted cells. The supernatant was centrifuged at 88003 g, and theresulting pellet was resuspended in 40 mM potassium phosphate buffer-sucroseand centrifuged once more at 88003 g. This final pellet was resuspended ina small volume of 40 mM potassium phosphate buffer (pH 7.6), and proteinconcentration was subsequently determined. The ability of the mitochondrialpreparations to phosphorylate FDG was assessed in a total volume of 50ml of40 mM potassium phosphate buffer containing 5 mM ATP, 5 mM MgCl2, and0.1 mg/ml of mitochondrial preparation. Samples containing cold FDG con-centrations ranging from 5mM to 50 mM were tested. Tubes were incubated at33°C, aliquots were taken at times ranging from 5 to 30 min, and the reactionwas stopped by adding 500ml of 0.3 M ZnSO4. FDG-6-P was precipitated byadding 500ml of a saturated solution of Ba(OH)2 by mixing and centrifugingfor 5 min at 14,000 rpm. in an Eppendorf microcentrifuge. Radioactivity in thepellets and in aliquots of the supernatants was then determined to calculate thephosphorylated fraction as described previously (20). In control tubes, mito-chondrial preparations were substituted with buffer alone to measure andcorrect for nonspecific phosphorylation. Initial reaction velocities were deter-mined for the different substrate concentrations, and Michaelis-Menten kinet-ics were calculated using PRISM software (Graphpad Software, Inc., SanDiego, CA).

RESULTS

Expression of mRNA for Glut-1, -2, -3, -4, and -5 and for the SGLTtypes I and II was assessed by Northern blot. A431, T47D, and HepG2cells expressed detectable levels of Glut-1 only. Glut-1 and Glut-3mRNAs were detected in MDA-MB-231 and MCF-7 cells. In CaCo IIcells, Glut-1, Glut-3, Glut-5, and SGLT-2 were detectable. A431 cells

expressed the highest amount of Glut-1 mRNA of all cells tested. Theintensity of the Glut-1 band was 11.86 5.6 (mean6 SD,n 5 3) timeshigher when normalized to theb-actin signal and 15.76 7.6 timeshigher when normalized to the 18S rRNA band on the ethidium stainthan in T47D cells, which had the lowest level. These two cell lineswere, therefore, selected for further analysis (Fig. 1). The level ofGlut-1 protein expression was determined in total extracts and inplasma membrane preparations of A431 and T47D cells (Fig. 2). TheGlut-1 band shows higher intensity in A431 than in T47D in both totalextracts and plasma membrane-enriched fractions. In plasma mem-brane fractions, the cell surface markera1 subunit of the humanNa1K1-ATPase is enriched for both cells lines, and levels of thisprotein appear higher in A431 in both fractions. The total amount ofprotein loaded is very similar for all samples, as shown by the amidoblack staining of the blot. Total Glut-1 protein levels in A431 were4.4 6 1.2 (mean6 SD, n 5 3) times higher than in T47D cells,similar to the results obtained for the mRNA. Plasma membrane-enriched fractions showed similar differences in Glut-1 protein con-tent, with A431 extracts containing 3.86 1.0 times higher levels thanT47D.

FDG uptake in the two cell lines is shown in Fig. 3. Both in theabsence (Fig. 3a) or presence (Fig. 3b) of 5.5 mM glucose in themedium, uptake of FDG between 10 and 40 min after addition of thecompound is linear for both cell lines and higher in T47D cells thanin A431. Absolute uptake values are 17–20 times higher when cellsare incubated in medium without glucose as expected. Uptake of FDG

Fig. 4. Initial uptake of FDG in A431 and T47D cells. Values are higher in A431 cellsup to the 40-s time point (n $3 per time point;bars,SD).

Fig. 3. FDG uptake in A431 and T47D cells in the presence of noglucose (a) or with 5.5 mM glucose (b) in the uptake medium (n $3per time point;bars, SD). Under both conditions, FDG uptake ishigher in T47D cells. Uptake values are 17–20 times higher in theabsence of glucose in both cell lines.

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in the two cell lines at early time points and in the presence of 5.5 mMglucose are shown in Fig. 4. A431 cells show more rapid initial uptakeof FDG, as values are higher for these cells up to the 40-s time point.T47D cells show higher uptake at later times. Time uptake curves for3-OMeG are shown in Fig. 5. Both cell lines show uptake of thiscompound that rapidly reaches equilibrium. Initial transport rates of3-OMeG are more rapid in A431 (0.966 0.30, percentage of avail-able tracer/mg protein/min, mean6 SD) than in T47D cells(0.426 0.12), calculated assuming linear uptake for the first 20 s afteraddition of the compound.

Total RNA was extracted from A431 and T47D xenografts. Glut-1mRNA levels in the xenografts were determined and compared withlevels found in cultured cells (Fig. 6a). Levels of Glut-1 mRNA inA431 xenografts are higher than in T47D cells, similar to results

obtained in cells grown in culture. Uptake of DG was determined 60min after injection (Fig. 6b). Average uptake in T47D xenografts wassignificantly higher than in A431 (4.356 1.09, %ID/g6 SD,n 5 14versus3.586 1.10, %ID/g6 SD,n 5 19; unpairedt test,P , 0.05).

Northern blot analysis for hexokinase subtypes I and II is shown inFig. 7. Levels of hexokinase I mRNA are 5.26 2.0 (mean6 SD,n 5 3) times higher in A431 than in T47D cells grown in culture,when normalized forb-actin, and 7.06 2.8 times when normalizedfor 18S rRNA. Hexokinase II mRNA levels are 7.56 0.2(mean6 SD, n 5 3) times higher in A431 cells than in T47D cells,when normalized forb-actin, and 10.06 0.4 times when normalizedfor 18S rRNA. FDG phosphorylating activity, however, is higher inmitochondrial extracts of T47D cells (Fig. 8), because these prepara-tions show similar affinity (Km is 109 6 49 mM in T47D versus3286 145mM in A431; unpairedt test,P . 0.1), similar to previouslypublished values for FDG using yeast hexokinase (21), and higherVmax (62 6 4 versus30 6 3 nmol/min/mg protein; unpairedt test,P , 0.0001) than in A431.

DISCUSSION

Among the cell lines tested, A431 and T47D cells were chosenbecause: (a) both cell lines only express Glut-1 mRNA at detectablelevels and no other facilitative or SGLTs, therefore Glut-1 appears tobe the isoform mainly being used by these cells to transport FDG; and(b) the level of expression of Glut-1 is very different in these twocells, with the A431 expressing 10–20 times more Glut-1 mRNA thanT47D, a condition that should favor uptake of FDG in A431 cells ifGlut-1 levels were indeed the rate-limiting step. The level of Glut-1protein detected by Western blot (Fig. 2) is consistent with resultsobtained at the mRNA level, with A431 cells expressing;4 timesmore total protein than T47D. Furthermore, the amount of Glut-1associated with the plasma membrane preparations, which should be

Fig. 5. Initial transport rates of 3-OMeG in A431 and T47D cells. Uptake of 3-OMeGrapidly reaches equilibrium in both cell lines. Initial rates are higher in A431 cells (n $3per time point;bars,SD).

Fig. 6. a, Northern blot analysis of A431 and T47D cells grown in culture (c) and as xenografts (x).b, relative levels ofb-actin normalized Glut-1 mRNA (n5 3; bars,SD) inA431 and T47D cells grown in culture and as xenografts, as well as DG uptake values 60 min after injection in xenografts (n $13 xenografts;bars,SD).

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the only fraction of active protein, also shows higher levels in A431than in T47D, indicating that these two cell lines adequately representa high (A431)- and a low (T47D)-level plasma membrane Glut-1-containing cell.

The rate of incorporation of FDG in the two cell lines is higher inT47D cells than in A431, both in medium containing physiologicalglucose concentrations and in the absence of added glucose (Fig. 3),suggesting that the amount of Glut-1 protein is not rate limiting undereither of these conditions and that the lower levels of the transporterpresent in T47D cells are adequate to provide sufficient flux of FDGacross the plasma membrane. Another indication that Glut-1 levels arenot regulating FDG uptake is given by the results shown in Fig. 4.Intracellular FDG levels, shortly after addition of the uptake medium,are slightly higher in A431 than in T47D cells, indicating that in thevery early phase, the amount of transporter present allows more rapidflux into the cells. This situation, however, is rapidly resolved, and by90 s after addition of radioactivity, T47D cells have a higher accu-mulation of FDG, suggesting that events following transport intocells, presumably the ability to phosphorylate and trap FDG, rapidlycome into play to control how much of the compound is retained.Further evidence is provided by the data on the incorporation intocells of 3-OMeG that shows that this compound, which is not appre-

ciably phosphorylated by cells, very rapidly reaches equilibrium be-tween extracellular and intracellular space in both cell lines. A431cells show initial transport rates of 3-OMeG that are slightly higherthan in T47D cells (0.966 0.30 versus0.42 6 0.12% of availabletracer/min/mg protein; Fig. 5), consistent with the different amountsof Glut-1 expressed, again indicating that the ability to transport is notthe rate-limiting step in FDG retention.

Another interesting aspect of the differences in transport ratesbetween the two cell lines is that they are not as different as one wouldexpect, given the very different levels of the protein in the two cells.Although we did not measure kinetic properties of glucose transport inA431 and T47D cells, our results suggest that the Glut-1 protein may,under different conditions (i.e., in the two different cell lines), be ableto transport glucose analogues at different rates. Differences in theintrinsic activity of Glut-1 under some experimental conditions havebeen demonstrated in other cell systems (22).

The extracellular tracer concentration in the experiments in cells inculture is virtually constant for the duration of the experiment. Thissituation is very different from that found in the setting of a PETstudy, where tracer availability (i.e., the concentration of tracer in theblood) decreases with time. The experiments in xenografts show thatunder experimental conditions, which are similar to those found inhuman studies, uptake of the glucose analogue DG still appears to beindependent of the level of Glut-1 expression (Fig. 6). Under theseconditions of tracer delivery, membrane transport does not appear tobe rate determining, and uptake of DG is significantly higher in T74Dxenografts than in A431.

Thus far, all of the evidence presented points away from Glut-1levels as the controlling factor on FDG uptake. It appears that flux ofFDG, 3-OMeG, and DG across the plasma membrane is not therate-limiting factor in determining how much FDG is retained in A431and T47D cells. Indeed, it appears that even with relatively low levelsof Glut-1 protein in the plasma membrane, T47D cells are able tosustain higher FDG retention values than A431 cells. Both hexokinaseI and II mRNA levels are lower in T47D cells (Fig. 7); however,mitochondrial extracts of these cells, where active hexokinase ispresumed to be located (23), show that T47D cells have a higheractivity than A431 cells, indicating that: (a) mRNA levels of the twohexokinase isoforms do not necessarily correlate with the actualability to phosphorylate; and (b) phosphorylating activity in the mi-tochondria plays a more important role in determining how muchFDG is retained by cells and perhaps glucose metabolic rates.

Other studies have focused on the relationship of glucose trans-porter expression and the uptake of FDG. Most data gathered thus farhave focused on vast evidence that the Glut-1 protein is overexpressedin many human tumors (7). In some instances, there appears to be acorrelation between the level of expression of Glut-1 and FDG uptake(24); however, there is no direct evidence linking the amount of

Fig. 7. Northern blot analysis for hexokinase I and II mRNA. A431 cells have highermRNA levels for both hexokinase subtypes.

Fig. 8. Mitochondrial FDG phosphorylating activity in extractsfrom the two cell lines. Initial reaction velocities are plotted againstsubstrate concentration. Fitting of the data assuming Michaelis-Menten kinetics shows similarKm (mM) values and significantlyhigher Vmax (nmol/min/mg mitochondrial protein) values in T47Dextracts (n5 2 per concentration point;bars,SD).

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Glut-1 protein to the level of FDG uptake. The idea that glucosemetabolism in tumors may be controlled by the level of Glut-1expression is, therefore, appealing because it may provide a target forthe development of alternative therapeutic strategies. Recently, Tori-zukaet al.(25) have shown, by kinetic modeling of clinical PET-FDGstudies, that the phosphorylation step appears to be rate determining inthe uptake of FDG in primary breast cancers but not so in primarylung cancers, suggesting that there may be differences among cancersderived from different tissues in how FDG uptake and perhaps glu-cose metabolism is controlled by the cells. Our data, although limitedto the comparison of two cell lines, indicate that the amount of Glut-1does not regulate how much FDG, a surrogate marker for glucosemetabolism, is retained by cancer cells.

In principle, higher levels of glucose transporter protein do notguarantee increased FDG uptake by cancer cells, and in our two cellsystems, metabolic trapping via phosphorylation of FDG appearsmore likely as the rate-determining step in FDG uptake.

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1999;59:4709-4714. Cancer Res Luigi Aloj, Corradina Caracó, Elaine Jagoda, et al. Culture2-Fluoro-2-deoxy-d-glucose Uptake in A431 and T47D Cells in Glut-1 and Hexokinase Expression: Relationship with

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