Regulation of glucose transport by interleukin-3 in growth factor-dependent and oncogene-transformed bone marrow-derived cell lines

Leukemia Research Vol. 21, No. 7, pp. 609-618, 1997. 0 199’7 Elsevier Science Ltd. All riehts reserved Printed in &eat Britain 014%212m7 $17.M) + 0.00

Pergamon PII: SO145-2126(97)0001~7

REGULATION OF GLUCOSE TRANSPORT BY INTERLEUKIN3 IN GROWTH FACTOR-DEPENDENT AND ONCOGENE-TRANSFORMED BONE MARROW-DERIVED CELL LINES Nuzhat Ahmed and Michael V. Berridge Malaghan Institute of Medical Research,Wellington School of Medicine, P.O. Box 7060, Wellington South, New Zealand (Received 29 July 1996.Accepted 18 January 1997) Abstract-Growth factors maintain cell viability and promote cell growth by stimulating glucose transport into cells and by progressing cells through the cell cycle. In the short term, effects on glucose transport involve transporter activation, while in the longer term increased gene expression is involved. This study aimed to investigate growth factor regulation of glucose transport in an interleukin (IL13-dependent bone marrow-derived cell line and its oncogenetransformed counterparts. 32D clone 3 (32Dcl3) cells and cells transfected with temperature-sensitive (ts) ras and abl oncogenes, were treated with and without IL-3 and their ability to take up 2-deoxy-o-glucose compared. Transformed cells, which are not dependent on IL-3 for growth at the permissive temperature of 32”C, exhibited a two- to six-fold higher proliferative response, enhanced tyrosine kinase activity and c-myc expression than control cells optimally stimulated with IL-3. Compared with control 32Dcl3 cells, P-deoxy-D-glucose uptake was also 3676% higher in transformed cells. The increased glucose uptake in transformed cells was consistent with 2.5-fold higher affinity of the glucose transporters for glucose. IL-3 stimulated glucose uptake in both control and oncogene-transformed cells. With control and ras-transformed cells, enhanced glucose uptake in response to IL-3 was associated with increased affinity of glucose transporters for glucose but with abl-transformed cells, no significant affinity changes were observed. IL-3 also increased glucose transoorter exoression in both control and oncogene-transformed cells, suggesting that increased transporter expression as well as changes in transporter affinity for glucose can affect glucose uptake. 0 1997 Elsevier Science Ltd

Key words: Oncogenes,

interleukin-3,

glucose

Introduction

transporters.

factor-dependent cells, increased glucose transport in the short term has been associated with increased affinity of glucose transporters for glucose without an apparent increase in transporter expression at the plasma membrane [2,3]. Introducing transforming oncogenes such as V-SK, vrus and v-abl into these cells imposes a growth factorindependent state on them and in some situations this can result in a tumorigenic phenotype. This raises the possibility that oncogenes, in overcoming growth factor dependence, may also promote glucose transport by activating transporter molecules in the plasma membrane, and evidence for this has been presented (N. Ahmed and M. V. Berridge, manuscript submitted). In fact, enhanced glucose uptake and utilization is a well established and characteristic feature of tumour cells [4], and activated oncogenes including v-src and v-abl [5,6] and v--s [7] are known to promote glucose transport. Although increased glucose transport was shown to be

Hormones, growth factors and cytokines such as insulin, platelet derived growth factor (PDGF), insulin-like growth factor 1 (IGF-l), colony stimulating factor-l (CSF-1) and interleukin (IL)-3 promote glucose transport into cells. Acute responses are observed within minutes and involve transporter translocation and activation, while longer term exposure involves gene expression and protein synthesis [l]. With growth Abbreviations: IL-3, interleukin-3; Gluts, glucose transporters; 320~13, 32D clone 3; PDGF, platelet derived growth factor; CSF-1, colony stimulating factor-l; IGF-I, insulinlike growth factor 1; ts, temperature-sensitive; 2DOG, [3H]2-deoxy-o-glucose; PMSF, phenylmethylsulfonylfluoride; M7T, 3-[4,5-,dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide. Correspondence to: N. Ahmed, Malaghan Institute of Medical Research,Wellington School of Medicine, P.O. Box 7060, Wellington South, New Zealand. 609

610

N. Ahmed and M. V. Berridge

associatedwith increasedmRNA and protein expression in somecells [5,7,8] and decreasedtransporterturnover in others [9], changesin transporter affinity for glucose were not determined in these studies. In previous investigations, increasedaffinity of glucose transporters for glucose had been demonstratedin tumour cells and in tumorigenic revertants of suppressedhybrid cells compared with non-malignant cells [lo], but in these studiescells of widely different backgroundswere used, and where viral transformation was studied, parental lines were not employed. Oncogenesactivate signal transductionpathwaysused by growth factors or impose autocrine growth control that can overcome the growth factor requirements of cells [ 111.However, in some situations, growth factors can suppress oncogene signalling [12] suggesting interactive rather than identical pathways. Furthermore, recent evidencehas demonstratedthat overexpressionof c-myc, which is constitutively induced in oncogenetransformedcells, can induce apoptosisin serum-starved fibroblasts [13), and in myeloid cell lines [14], and that this effect is dominant to the suppressionof apoptosisby growth factors [15]. Together, theseresults demonstrate that interfering with oncogene imposed mitogenic signalling can detrimentally affect cell survival and proliferation. Preliminary results in our laboratory indicated that growth factors could negatively affect the growth of oncogene-transformedcells (N. Ahmed and M. V. Berridge, unpublished observation) prompting us to investigate early effects of IL-3 on proliferative responses and glucose transport in a growth factordependentcell line. In the presentstudy, we have usedthe IL-3-dependent murine bone marrow-derived cell line, 32D clone 3 (32Dc13), and its temperature-sensitive(ts) oncogenetransformed counterparts 32Drus and 32Dabl to investigate IL-3 regulation of glucose transport and the mechanism of transporter activation by IL-3. We show that IL-3 promotes glucose transport in both growth factor-dependent and oncogene-transformed cells. In control 32D cells and in 32Drus, increased glucose transport was associated with increased transporter affinity (K,,,) for glucose, but with 32DabZ,transporter affinity remained unaffected. In control and oncogenetransformed cells, increased glucose transport was also associatedwith increasedGlut-l expressionin the cells. Materials and Methods Cells and cell culture

32Dc13 originally derived from long-term bone marrow cultures of C3WHeJ mice [161, and 32Dts-rus, 32Dts-abl and 32Dsrc were gifts from Dr S. T. Anderson, Department of Pathology, University Of Colorado Health ScienceCenter. Cells were maintained

in RPM1 1640 medium (Gibco-BRL, Grand Island, NY, U.S.A.) supplementedwith 25 &ml penicillin, 25 pg/ ml streptomycin and 10% (v/v) fetal bovine serum. In the case of 32Dc13 cells, 10% WEHI-3-conditioned medium was usedas a sourceof IL-3 for routine culture, but in all experimental situations, pure recombinant murine IL-3 obtained from Dr J. D. Watson, Genesis R&D Corporation, Auckland, New Zealand, was used. Cells were cultured at the permissive temperature of 32°C in a humidified incubator maintained at 5% COz. Inactivation of the ts-oncogenes was performed by shifting cells to 40°C for 16-20 h. Cell viability was determined by Trypan Blue exclusion using a haemocytometer. [3H]Thymidine incorporation assay

Proliferative responseswere measuredby incubating 5 x lo4 cells in 0.1 ml of culture medium in 96-well microtitre plates for periods of 16-20 h before adding 0.5 uCi [3H]thymidine (Amersham, U.K.) for 3 h. Incorporation of radioactivity into DNA was determined using an automatedcell harvesterand liquid-scintillation counting. Tyrosine kinase activity

This assaywas performed using the Protein Tyrosine Kinase Assay System (Gibco-BRL, Grand Island, NY). The ability of cells to transfer phosphate from [32P]yATP to a synthetic peptide (RR-SRC) was measuredin crude cell lysates as describedby the manufacturer. Evaluation of c-myc expression

MYC protein was extracted from cells according to the methods described as follows. Briefly, cells were washed twice with PBS (phosphate-buffered saline; 137 mM NaCl, 0.3 mM KCl, 8.1 n-M Na2HP04, 1.5 mM KH2PO4, 0.7 mM CaC12. 0.5 mM MgC12, pH 7.2), resuspendedat 1 x lo7 cells/ml in lysis buffer [20 n&I Hepes, pH6.8, 250mM NaCl, 5 mM KCl, 5 mM MgC12, 0.5% Triton X- 100, 0.1% Na deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] at 4”C, and sonicated.Cellular debris was pelleted by centrifugation at 15 OOOgfor 15 min, and the supematantwas frozen and stored at -70°C. Western Immunoblotting procedureswere performed and c-MYC protein detected with anti-MYC monoclonal antibody (D2 85, Santa Cruz) and visualized by enhanced chemiluminescence detection kit (Amersham). 3-[4,5-dimethytthiazol-2-yl]-2,Sdiphenyl bromide (MlT) reduction

tetrazolium

The metabolic activity of cells was measured by incubating 5 x lo4 cells in 0.1 ml of culture medium in 96-well microtitre plates for periods of 16 h in the presenceof different concentrationsof recombinant IL-3

611

OncogeneandIL-3 regulationof glucosetransport a

T

o-

I 32Dc13

ras

abl

src

32Dcl3

ras

abl

C

123456 Fig. 1. Effects of ts-rus, ts-abl and v-src on the proliferative responses,tyrosine kinase activity and c-myc expressionin exponentially growing 32D cells at permissive and non-permissive temperatures.Parental 32D clone 3 cells and cells transformedwith ts-rus, tsabl or v-src were grown exponentially in RPM1 1640culture medium. With 32Dc13cells, WEHI- conditioned-medium was used as a sourceof IL-3. [3H]Thymidine incorporation (A), tyrosine kinase activity (B) were determinedas describedat permissive (32°C n ) and non-permissive (4O”C, q ) temperatures.Values are the meanfS.E.M. of at least three independent experiments, each involving triplicate determinations.(C) Showsthe expressionof c-MYC protein in 32Dc13,32Dts-rusand 32Dts-abl cells at 32°C (1, 3 and 5) and at 40°C (2, 4 and 6). a, significantly different from control cells grown at the permissive temperature,P < 0.02; b, significantly different from cells grown at the permissive temperature,P < 0.05.

at 32°C. After 16 h, 0.01 ml MTT (5 mg/ml) was added and the cells were further incubated at 32°C for 2 h. The reaction was stopped by adding 0.1 ml lysis buffer (10% SDS, 45% DMF, pH 4.7) and formation of the blue formazan precipitate measured at 595 nm.

treatment of cells with or without 20 @ml of IL-3 for 2 h or 4 h. Glucose transport measurements were normalized against measurements in the presence of cytochalasin B (10 PM). Under these conditions 80% inhibition of the transport was obtained.

Glucose transport assay Glucose transport was measured by the zero tram method using [3H]2-deoxy-o-glucose (2DOG), 200 PM, 0.5 uC!i (Amersham, U.K.) as described previously [2]. The effect of IL-3 on glucose transport was determined by culturing cells in serum-free (SF) RPMI 1640 containing 2 rig/ml of IL-3 for 16 h. Under these conditions cell viability was maintained but cell proliferation ceased. Cells were then washed with SFRPM1 1640, resuspended in the same medium with or without IL-3 for various times. Kinetic analysis (K, and V,,) of 2DOG uptake was investigated by varying the 2DGG concentration between 0.1 and 2 mM after

Fractionation of cells and Western blot analysis Cells were collected and homogenized in Hepesbuffered sucrose (10 mM Hepes pH 7.4, 250 mM sucrose, 1 mM EGTA, 2 mM MgCl,, 1 mM PMSF, and 5 @ml leupeptin, pepstatin A and aprotinin) by passing 10-15 times through a ball bearing homogenizer (8.020 mm bore, EMBL, Heidelberg, Germany) with a 8.008 mm ball bearing until ca 95% of cells were broken. The cell homogenate was centrifuged at 900g for 10 min to remove nuclei and unbroken cells. The postnuclear homogenate was then fractionated on a linear o-45% Nycodenz gradient (Nycomed AS Pharma, Oslo, Norway) in Hepes-buffered sucrose. Fractions

612

N. Ahmed and M. V. Berridge

b b b

b

32Dcl3

ras

abl

32Dcl3

ras

abl

src

Fig. 2. Effects of ts-ras, ts-abl and V-SK on the MTT reduction and [3H]2DOG uptake in exponentially growing 32D cells at permissiveand non-permissivetemperatures.Parental32D clone 3 cells and cells transformedwith ts-ras, ts-abl or V-SICwere grown exponentially in RPM1 1640 culture medium. With 32Dc13cells, WEHI- conditioned medium was used as a sourceof IL-3. MTI reduction (A), [3H]2DOG uptake (B) were determined as described at permissive (32°C n ) and non-permissive (40°C I@ temperatures.Values are the mean k S.E.M. of at least three independentexperiments,each involving triplicate determinations.a, significantly different from control cells grown at the permissivetemperature,P < 0.05; b, significantly different from cells grown at the permissive temperature,P < 0.02.

were collected starting from the bottom of the gradient, stored at -20°C and analysed for marker enzyme activities and protein content. Alkaline phosphodiesterase 1 activity [17] was used as a marker for plasma membrane while cytochrome c reductase [17] and NADH-dependent M’IT reduction [ 181 was used as a microsomal marker. Respectivefractions containing the peak marker enzyme activities were pooled. Marker enzyme activity profiles from 12 different experiments with 32D cells showed the plasma membranefractions to be 80-90% pure. Western blot analysis on the subcellular fractions were performed using antibodies raised against synthetic peptides corresponding to the carboxyl terminus of Glut-l (Eastacres Biologicals, Southbridge, MA) and Glut-3 (Dr G. W. Gould, University of Glasgow, Scotland) proteins. Normal rabbit serumwas used as a control. Transporter subtypes were visualized using a chemiluminescence kit (ECL, Amersham Life Science) and high performance luminescence detection film (Amersham Life Science). Densitometric analysis used a Macintosh LC 630 computer and the public domain NM image program. Density of autoradiographic bands was linear over tbe range used.

Protein determination Protein was determined using a microplate adaptation

of the Bradford method [ 191.

Results Esfects of oncogenes on proliferative responses, tyrosine kinase activity, c-myc expression and metabolic activity of 320 cells

The proliferative responsesof 32Dc13cells grown in WEHI- conditioned medium were comparedwith those of their oncogene-transfectedcounterparts, 32Dts-r-as, 32Dts-abl and 32Dsrc which was included as a nontemperature-sensitivecontrol. Figure 1A shows that at the permissive temperature, [3H]thymidine incorporation was elevated in all oncogene-transfer&d cells, the effect with ts-abl being much greaterthan with ts-ras or v-src. Elevated [3H]thymidine incorporation in oncogene-transformed cells was consistent with enhanced c-MYC expression as shown in Fig. 1C. The tyrosine kinase activity of ts-abl transfectedcells was also threefold higher than control 32Dc13cells optimally stimulated with IL-3, while in r-us-transformedcells the activity remained unaltered. In contrast, with MIT reduction which is a measureof the metabolic activity of cells [ 181, only ts-abl-transformed cells showed a significantly elevated response(Fig. 2A). Cell doubling time was similar in the oncogene-transformedcells (16 18 h) which progressedthrough the cell cycle about 5 h faster than parental 32D cells (20-24 h) at 32°C. Exposing ts-ras and ts-abl-transformed cells to the non-permissive temperature (40°C) for 16 h greatly reduced r3H]thymidine incorporation, but with 32D cells transformed with v-src which is not ts, and with

613

OncogeneandIL-3 regulationof glucosetransport Table 1. Effects of IL-3 on kinetics of glucose uptake by control and oncogene-transformed32D cells V,,

Km (n-J@

(A) 16 h serum-starvation with 2 q/ml IL-3

32Dc13 32Dras 32Dabl

+IL-3

-IL-3

Cell type

2.67 f 0.45 2.32 f 0.03 3.25 +_ 1.20

32Dabl

32Dabl

+IL-3

4.58 f 0.27 4.45 f 0.12 6.25 + 1.24

3.51 + 0.05 2.89 f 0.02 8.80 f 0.01

3.03 f 0.05* 0.74 * 0.20* 0.99 f 0.01

7.00 f 0.21 3.20 f 0.50 2.05 f 0.09

3.92 + 0.05 2.87 f 0.06 2.69 f 0.48

8.33 + 0.57 1.80 f 0.48 0.92 f 0.14

(C) Exponentially growing cells

32Dc13 32Dras

-IL-3

1.78 f O.OO* 1.48 f 0.09* 4.32 f 1.17

(B) 2 h serum-starvation without IL-3

32Dc13 32Dras

(ntnoY106cells/min)

4.00 f 0.26 1.68 f 0.55.t 1.56 + 0.07t

10.00 * 1.77 4.18 f 0.78 2.48 f 0.38

Cells were starvedof serumfor 2 h without IL-3 (B), or for 16 h (A) in the presenceof 2 @ml of IL-3 before being treatedwith or without IL-3 (20 rig/ml) for 4 h (A) or 2 h (B). All data are the meanf S.E.M. of at least three separateexperiments,each performed in triplicate. *Significantly different from cells in the absenceof IL-3, P < 0.025. tsignificantly different from control 32Dc13cells, P < 0.025

parental 32D cells, [3H]thymidine incorporation was only slightly reduced at 40°C. Decreased proliferative responses of ts-rus and ts-abl-transfected cells at the non-permissive temperature were correlated with loss of c-MYC expression and tyrosine kinase activity. At the non-permissive temperature c-MYC expression and tyrosine kinase activity were drastically reduced in rus and abl-transfected cells, whereas with control cells both remained unchanged (Fig. 1B and C). On the other hand, M’IT responses of ts-rus and ubl-transformed cells were reduced by 40-60% at the non-permissive temperature, MTT responses were elevated by 1.8-2.5-fold in control and src-transformed cells at 40°C. Thus, enhanced metabolic activity at 40°C could account for the muted loss of MTT responses observed with ts-rus and ts-ubltransfected cells at the non-permissive temperature. Effects of ts-ras, ts-abl and v-src on 2DOG uptake by 320 cells 2DOG uptake was elevated by 36-76% in ts-rus, tsubl and v-src-transformed cells (Fig. 2B) and was consistent with 2.5-fold higher affinity of the transporters for glucose (Table 1). With each cell type, exposure at the non-permissive temperature for 16 h increased 2DOG uptake above that observed at the permissive temperature. Because this effect was observed with both control and src-transformed cells, the results indicate a heat stress effect on glucose transport that overrides the effects of oncogene loss (see Fig. 2B) in ts-rus and tsubl-transformed cells.

Effects of IL-3 on 2DOG uptake by control and oncogene-transformed 320 cells 32D cells transformed with ts-rus and ts-ubl showed 36-76% elevated 2DOG uptake compared with parental IL-3-dependent 32D cells (Fig. 2B). To determine the effects of IL-3 on 2DOG uptake by control and oncogene-transformed cells, cells were serum-starved for 16 h in the presence of 2 rig/ml IL-3 to maintain the viability of control cells [2]. Figure 3 shows that subsequent treatment of control 32D cells with 20 @ml IL-3 stimulated 2DOG uptake by 40% within 1 h. In contrast, IL-3 withdrawal caused the rate of 2DOG to decline, 40% loss of ability to transport glucose being observed by 4 h. Under these conditions cell viability was maintained at 90-95%. Treatment of control and oncogene-transformed cells that had been serum-starved with increasing concentrations of IL-3 for 1 h caused a 30-50% increase in glucose transport at IL-3 concentrations at or above 20 @ml (Fig. 4C) regardless of the transformed status of the cells. Under these conditions, effects of IL-3 on r3H]thymidine incorporation and on M’IT responses were more complex. Thus, whereas IL-3 stimulated [3H]thymidine incorporation of control 32D cells by more than three-fold and increased MTT responses by 40% (Fig. 4A and B), IL-3 inhibited [3H]thymidine incorporation of 32Dts-rus cells by 50% while stimulating their MTT responses by 40%. With tsubl-transformed cells, M’IT responses were unaffected by IL-3 whereas r3H]thymidine incorporation was stimulated two-fold at 20-50 rig/ml IL-3, though lesser

614

N. Abmed and M. V. Berridge

r

10

20

time (h) Fig. 3. Effects of IL-3 on [3H]2DOG uptakeby parental 32Dc13cells with time. Cells were serum-starvedfor 16 h in the presenceof 2 rug/mlIL-3, then treatedfor times up to 16 h with ( q ) or without (0) 20 @ml IL-3 prior to measuring[3H]2DOG uptake.Values are the meanf S.E.M. of three independentexperimentseach involving triplicate determinations.Control value ( n ) correspondsto the reading without IL-3 at time 0 h.

effects were observed at higher IL-3 concentrations of 100-200 ngknl (Fig. 4A). Increased glucose uptake in response to IL-3 (Fig. 4C), was associated with changes in the affinity of glucosetransportersfor glucosein control 32D cells and in ts-rus-transformed cells, but with ts-abl-transformed cells, no affinity changes were observed (Table 1). Initially these experiments were carried out using cells that had been serum-starvedfor 16 h in the presenceof 2 rig/ml IL-3 (Table 1A). Because under these conditions, the K, values of oncogene-transformed and control cells treated with IL-3 differed significantly from those of exponentially growing cells, we repeated the experiment under a preconditioning regimen designed to minimize stressbut maximize IL-3 response. Thus, serum-starvationfor 2 h generatedsatisfactoryIL3 responsesbut maintained K,,, values similar to those of exponentially growing cells (Table 1, Experiments B, C). Under these conditions, IL-3 treatment of control and ts-rus-transformed cells resulted in a 2.4-2.8-fold increase in affinity of glucose transporters for glucose comparedwith untreated cells but again, no significant

increasein affinity was observedwith ts-abl transformed cells (Table 1B). In both experiments,lower V,, values were observed with control and rus-transformed cells following IL-3 treatment,but with ubl-transformed cells V,,, was unchangedor slightly increased. Eflects of IL-3 on plasma membrane Glut-l expression in control and oncogene-transformed 320 cells

Immunoblot analysis of fractionated plasma membranesfrom each cell type showedthe presenceof Glut1. Although Glut-3 was also detected in crude membrane preparations, it was not detected following membrane fractionation. Total Glut-l expression in exponentially growing rus and ubl-transformed cells in the absence of IL-3 was 3.4-4-fold less than control 32Dc13cells optimally stimulated with IL-3. Treatment with IL-3 increased total Glut-l expression in both control and oncogene-transformed32D cells (Table 2). In parental 32D cells, plasma membraneGlut-l expression increased about two-fold compared with 1.5-2.2-

fold increasein ras and abl-transformed cells. Although no microsomal expression of Glut- 1 was detected in ras-

0

IL-3

100

(q/ml)

200

300

!Jo

100

110

‘, 120 ag

8

a L: 130

140

0 IL-3

100

200 (nglml)

T+ 300 IL-3

@g/ml)

32Dd3 32Df=) 32D(abl)

I -

Fig. 4. Effects of IL-3 on the proliferative responses, metabolic activity and [3H]2DOG uptake by parental 32Dc13 cells and cells transformed with ts-ras and ts-abl oncogenes. In (A) cells were stimulated with increasing concentrations of IL-3 in the absence of serum for 16 h, while in (B) and (C) cells were serum-starved for 16 h in the presence of 2 rig/ml IL-3, then treated with IL-3 at concentrations up to 200 rig/ml for 1 h. [3H]Thymidine incorporation (A), MTT reduction (B) and [3H]2DOG uptake (C) were determined as described in Materials and Methods. H, 32Dc13; 0, 32Dts-rus; q , 32Dts-abl. In all three experiments control value corresponds to the reading without IL-3 treatment. For (A), values are the mean f S.E.M. of three independent experiments each involving triplicate determinations, while for (B), two experiments were carried out each involving triplicate determinations. For (C), standard errors were < 10% of the average stimulated value.

0

100

* 150

‘ii 250 b 8 umo %

300

150

160

616

N. AhmedandM. V. Benidge Table 2. Expression of Glut-l in 32D cells in the presenceand absenceof ~-3

Cell type

IL-3

Fraction type

32Dc13 32Dc13

-

32Dc13 32Dc13

+ +

32DabZ

-

Microsomal Plasmamembrane Total Microsomal Plasmamembrane Total Microsomal Plasmamembrane Total Microsomal Plasmamembrane Total Microsomal Plasmamembrane Total Microsomal Plasmamembrane Total

32DabZ

-

32Dabl 32Dabl

+ +

32Dras

-

32Dras

-

32Dras

+

32Dras

+

Effect of IL-3 on the specific activity of Glut- 1 ( x 103)

Relative membrane distribution of Glut-l

2.31 0.95 3.26

0.71 0.29

3.86

0.66 0.34

1.98 5.84

0.13 1.35 1.48

0.08

0.74

0.20 0.80

3.10

0.92

3.84

ND 1.72 1.72 ND 2.62 2.62

ND 1.00 ND 1.oo

ND, not detected.Cells were starvedof serumfor 16 h in the presenceof IL-3 (2 rig/ml) before being treatedwith or without IL-3 (20 rig/ml) for 4 h. Plasma membraneswere fractionated on Nycodenz gradients and transporter subtypes analysed by Western blotting and ECL. Densitometric analysis was performed and relative Glut-l expression determined for each cell type. Specific activity of Glut-l is expressedas relative density/pg of protein, and relative membranedistribution is expressedas a fraction of the combined microsomal and plasma membranetransporterfor each cell type at the respective condition.

transformed cells, its expression increased in both control and abl-transfected cells in the presence of IL-3. These results suggest that in 32D cells, IL-3 stimulates glucose transport by increasing intracellular Glut-l expression and by translocation of these transporters to the cell surface. Discussion The results presented in this study provide support for the view that glucose uptake into cells is acutely regulated by growth factors in both oncogene-transformed and parental 32D cells and that in control and tsrus-transformed cells, this regulation involves a change in the activation state of plasma membrane glucose transporters. With control 32D cells that had been serum-starved for 16 h, changes in glucose uptake were observed within 0.5-l h of IL-3 addition and withdrawal and these results are similar to those described previously for Ba/F3 cells [3] and for a different clone of 32D cells [2]. Although, in oncogene-transformed cells glucose uptake was 36-76% greater compared to control cells optimally stimulated with IL-3, the increased glucose uptake in response to IL-3 in ts-rus and ts-abl-transformed cells suggests that these oncogenes do not constitntively activate glucose transport in 32D cells. Rather, an IL-3 effect could be superimposed

on the oncogene effect, the extent of the effect being quantitatively similar to that observed following IL-3 treatment of control cells. Our results also show that growth factor activation of glucose transport in oncogene-transformed cells differs mechanistically between rus and ubl-transformed cells in that affinity changes were evident with rus-transformed cells treated with IL3 but were not observed with &l-transformed cells. Control 32D cells exhibited a heat stress response when exposed at 40°C for 16 h. This heat stress effect was evident in the increased glucose uptake measured at the non-permissive temperature and in increased metabolic activity as indicated by enhanced MTT responses. In contrast, [3H]thymidine responses were slightly reduced in control cells at 40°C. This is due to an increase in the doubling time of the cells which was extended by 10 h. These results are consistent with unaltered c-MYC expression and unchanged tyrosine kinase activity of 32Dc13 cells at 40°C. The heat stress effect on glucose transport was observed independently of whether the cells had been transformed with ts-rus, tsubl or src. Because the proliferative responses, c-MYC expression and tyrosine kinase activity of 32Dts-rus and 32Dts-ubl were abrogated at the non-permissive temperature, it can be concluded that the heat stress effects on glucose transport are mechanistically distinct from the effects of oncogenes as determined by [3H]thymi-

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OncogeneandL-3 regulationof glucosetransport

dine incorporation, c-MYC expression and the activity of tyrosine kinases. Interestingly, an intermediate situation was observed with M’IT responses,the heat stresseffect which was evident in control and 32Dsrctransformed cells being partially reversed in cells bearing ts oncogenes. Wertheimer et al. [20] have previously shown that Glut-l belongs to the glucoseregulated family of stress-inducible proteins, although heat only weakly induced Glut-l mRNA expression in the L8 myocytes and NM 3T3 fibroblasts used in that study. Others have successfully used temperature shifts to investigate the effects of ts oncogenes,including SK, &psand abl, on glucose transport without observing the complicating effects of heat stress observed in the present study [7,21,6]. The reasons for these differences are at present uncertain. Thus, although it is possible that heat effects on glucose transport are cell type specific, the study by Kan et al. [21] used a bone marrow-derived murine cell line, IC.DP, that is closely related to 32D, and major differences in stressresponses would not have been expected. Despite our inability to use the ts property of ras and abl in investigating the effects of these oncogenes on glucose transport in 32Dc13 cells, investigations were carried out at the permissive temperature, using parental cells as an appropriate negative control. Enhanced glucose uptake in exponentially growing ras and abl-transformed cells was associatedwith 2.5 fold increased affinity of glucose transporters for glucose without an increase in V,, (Table 1C). In fact, lower V,, values in oncogene-transformed celis is consistent with their lower Glut-l expression on the plasma membranes.Similar effects of ras and abl on the kinetics of glucose transport were observed following serum-starvation for 2 h. Increased affinity of glucose transporters for glucose in exponentially growing cells transformed by ras and abl indicates that in addition to their role in increasing glucose transporter mRNA and protein expression [7,6] these transforming oncogenes may also act by regulating the intrinsic glucose transporting activity of plasma membrane glucose transporters. IL-3 stimulated glucose transport in both control and oncogene-transformed 32D cells. Increased glucose transport in responseto IL-3 in parental 32D cells and in 32D cells transformed with ts-ras correlated with increased metabolic activity as determined by MlT reduction, which is an index of the metabolic status of cells [ 181.In contrast,even though glucosetransport was elevated by IL-3 in 32Dts-abl cells, no changesin MTT responseswere observed. Preliminary results suggest a similar situation with 32Dsrc. The failure of IL-3 to affect MIT responsesin abl and src-transformed cells suggests that the constitutively activated tyrosine kinases of these oncogenes impose growth factor

controls that are distinct from those in control and rastransformed cells. Increased glucose transport in control and oncogenetransformed 32D cells in response to IL-3 (Fig. 3C) could be explained by changes in the ability of transporter molecules to transport glucose and/or by increased expression of glucose transporter molecules on the plasma membrane. Comparison of the kinetic parameters, K, and Vmax, of cells treated with and without IL-3 for 2 h showed that with control and rastransformed cells, IL-3 treatment was associatedwith increased transporter affinity for glucose under both short term and long term preconditioning regimens. In contrast, with abl-transformed cells Km values did not change significantly. IncreasedGlut-l expression in the microsomal and plasma membranefractions in response to IL-3 treatment was observed with both ras and abltransformed cells and also in parental 32D cells. Thus, with control and oncogene-transformed32D cells, the similar effects of IL-3 on glucose uptake were differentially contributed to by transporter affinity changesand also by increasedGlut-l expression which is attributed to enhanced transporter synthesis and its translocation to the plasma membrane. These differencesmay be due to the way that the oncogeneproducts interact with IL-3 signalling. The ABL geneproduct is a survival factor and a negative regulator of death signals [22,23] whereasthe RAS geneproduct acts mainly as a survival and a proliferative factor [24]. Addition of IL-3 to cells transfected with ras or abl triggers different signalling cascades with different outcomes. In this context we have shown that IL-3 induces apoptosis in 32D cells transformedwith ts-ras but not ts-abl (Ahmed and Berridge, manuscript in preparation). Regulation of glucosetransport in hemapoietic cells is not well understood. Several studies using malignant and non-malignant cells in conjunction with growth factors have suggested the association of increased transporter affinity with enhanced glucose transport. Recently there have been suggestionsthat phosphorylation or altered phosphorylation of glucose transporter proteins may be involved in their regulation [2]. The possibility that such a mechanism may exist in growth factor-dependent as well as factor-independent transformed cells is presently under investigation. Acknowledgements-This work was supported by the

Wellington Division of Cancer Society and the Health ResearchCouncil of New Zealand.

References 1. Merrall, N. W., Plevin, R. and Gould, G. W., Growth factors, mitogens, oncogenesand the regulation of glucose transport. Cell Signalling, 1993, 5, 667. 2. Berridge, M. V. and Tan, A. S., IL-3 facilitates glucose

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transportin a myeloid cell line by regulating the affinity of the glucose transporterfor glucose: involvement of protein phosphorylation in transporter activation. Biochemical Journal, 1995, 305, 843. 3. Nefesh, I., Bauskin, A. R., Alkalay, I., Golembo, M. and Ben-Neriah, Y., IL-3 facilitates lymphocyte hexose transport by enhancing the intrinsic activity of the transport system.International Immunology, 1991, 3, 827. 4. Warburg, O., On the origins of cancercells. Science, 1956, 123, 309. 5. Flier, J. S., Mueckler, M. M., Usher, P. and Lodish, H. F.,

Elevated levels of glucose transport and transporter messengerRNA are induced by ras or src oncogenes. Science, 1987, 235, 1492. 6. Martin, G. S., Venuta, S., Weber, M. and Rubin, H., Temperature-dependentalterations in sugar transport in cells infected by a temperature-sensitivemutant of Rous sarcomavirus. Proceedings of the National Academy of Sciences U.S.A., 1971, 68, 2739. 7. Birnbaum, M. J., Haspel, H. C. and Rosen, 0. M., Transformation of rat fibroblasts by FSV rapidly increases glucose transportergene transcription. Science, 1987,235, 1495. 8. White, M. K. and Weber, M. J., Transformation by the src oncogenealters glucosetransportinto rat and chicken cells by different mechanisms.Molecular and Cellular Biology, 1988, 8, 138. 9. Shawver,L. K., Olson, S. A., White, M. K. and Weber,M. J., Degradationand biosynthesis of the glucose transporter protein in chicken embryo fibroblasts transformed by the src oncogene.Molecular and Cellular Biology, 1987, 7, 2112. 10. White, M. K., Bramwell, M. E. and Harris, H., Kinetic parameters of hexose transport in hybrids between malignant and nonmalignant cells. Journal of Cell Science, 1983, 62, 49. 11. McCubrey, J. A., Smith, S. R., Algate, P. A., Devente, J. E., White, M. K. and Steelman,L. S., Retroviral infection can abrogatethe factor-dependencyof hematopoieticcells by autocrine and non-autocrine mechanismsdepending on the presence of a functional viral oncogene. Oncogene, 1993, 8, 2905. 12. Watson, J. D., Jenkins, D. R., Eszes, M. and Leung, E., Effect of granulocyte-macrophagecolony-stimulating factor and interleukin 3 on the v-src oncogene.Inhibition of tyrosine kinase activity in the absenceof changesin gene expression.Journal of Immunology, 1988, 140, 501. 13. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L. Z. and

M. V. Berridge Hancock, D. C., Induction of apoptosisin fibroblasts by cmyc protein. Cell, 1992, 69, 119. 14. Askew, D. S., Ashmun, R. A., Simmons, B. C. and Cleveland, J. L., Constitutive c-myc expressionin an B-3dependent myeloid cell line suppressescell cycle arrest and acceleratesapoptosis.Oncogene, 1991, 6, 1915. 15. Askew, D. S., lhle, J. N. and Cleveland, J. L., Activation of apoptosis associated with enforced myc expression in myeloid progenitor cells is dominant to the suppressionof apoptosisby interleukin-3 or erythropoietin. Blood, 1993, 82, 2079.

16. Greenberger,J. S., Sakakeeny,M. A., Humphries, R. K., Eaves,C. J. and Eckner,R. J., Demonstrationof permanent factor-dependentmultipotential (erythroid/neutrophil/basophil) hematopoieticcell lines. Proceedings of the National Academy of Sciences U.S.A., 1983, 80, 2931. 17. Storrie, B. and Madden, E. A., Isolation of subcellular organelles.Methods in Enzymology, 1990, 182, 203. 18. Benidge, M. V. and Tan, A. S., Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT): subcellular localization, substrate dependence and involvement of mitochondrial electron transport in M’IT reduction. Archives of Biochemistry and Biophysics, 1994, 303, 474. 19. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72, 248. 20. Wertheimer,E., Sasson,S., Cerasi, E. and Ben, N. Y., The ubiquitous glucose transporter GLUT-l belongs to the glucose-regulated protein family of stress-inducible proteins. Proceedings of the National Academy of Sciences U.S.A., 1991, 88, 2525. 21. Kan, O., Baldwin, S. A. and Whetton, A. D., Apoptosis is regulated by the rate of glucose transport in an interleukin 3 dependentcell line. Journal of Experimental Medicine, 1994,180,917. 22. Evans, C. A., Owenlynch, P. J., Whetton, A. D. and Dive, C., Activation of the Abelson tyrosine kinase activity is associatedwith suppressionof apoptosis in hemopoietic cells. Cancer Research, 1993, 53, 1735. 23. McGahon, A., Bissonnette,R., Schmitt, M., Cotter, K. M., Green, D. R. and Cotter, T. G., BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood, 1994, 83, 1179. 24. Kaplan, J. B., Biological activity of human N-ras and Kras genes containing the Asn17 dominant negative mutation. Oncology Research, 1994, 6, 611.