Glucose transport in developing Ehrlich ascites tumor cells: Parallel changes in the rate of glucose uptake and cytochalasin B binding activity during tumor development and methotrexate treatment

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 223, No. 2, September, pp. 458-466, 1983

Glucose Transport in Developing Ehrlich Ascites Tumor Cells: Parallel Changes in the Rate of Glucose Uptake and Cytochalasin B Binding Activity during Tumor Development and Methotrexate Treatment’ T. W. CHAN, K. P. FUNG, Y. M. CHOY, Department

of Biochemistry,

The Chinese University Received March

AND

C. Y. LEE2

of Hong Kong, Shdin,

N.!l’., Hmg Kong

17, 1933

The ability of Ehrlich ascites tumor cells to take up glucose increased progressively during the course of tumor development. Simultaneously as the rate of uptake rose, the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface also increased. In its stereospecificity requirement toward competing sugars and in its sensitivity to phloretin and diethylstilbestrol, this class of binding sites resembled the putative glucose carriers identified in various other cell systems and may represent the glucose transporter in Ehrlich ascites cells. Work with methotrexate (MTX) substantiated this view. Methotrexate arrested tumor growth, inhibited glucose uptake, and reduced the number of cytochalasin B binding sites. In both MTX-treated and untreated cells, the magnitude of changes in number of cytochalasin B binding sites closely paralleled and sufficiently accounted for the magnitude of changes in glucose uptake. Qualitative changes in the turnover and affinity for substrate of the putative glucose carrier need not be invoked. Ehrlich ascites tumor cells depend primarily on glycolysis for the provision of energy. To maintain growth potential, it is essential that they possess a highly efficient system for sugar transport and metabolism. Indeed, the cytotoxic effect of methotrexate (MTX)3 has been shown to be at least in part attributable to its ability to inhibit the rate of cellular glucose consumption (1) although the main action is probably exerted through the inhibition of DNA synthesis and the induction of a “purineless state” in target cells (2). Thus, Kaminskas and Nussey (3) showed that MTX markedly reduced glycolysis in cultured Ehrlich ascites tumor cells; Kaminskas (1) further demonstrated that this reduction was the result of decreased hexose uptake

although the inhibition of sugar phosphorylation has also been implicated. The transport of hexoses across the plasma membranes of a variety of cell types including human red blood cells (4), adipocytes (5), Novikoff rat hepatoma cells (6), and cultured Ehrlich ascites tumor cells (‘7) has been demonstrated to be carrier mediated. Since the initiation of cell growth and differentiation in a variety of systems can be correlated with rates of hexose uptake (8, 9), it is of interest to investigate the changes in glucose transport and alterations in the characteristics of the glucose carriers during the course of tumor development as well as the effect of MTX thereon. We have studied these changes in Ehrlich ascites tumor cells. The results are presented in this article.

1 Dedicated to Dr. Choh Hao Li on the occasion of his 70th birthday. ’ To whom correspondence should be addressed. * Abbreviations used: MTX, methotrexate; PBS, phosphate-buffered saline. 0003-9861/83 $3.00 Copyright All rights

0 199.3 by Academic Press. Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Ehrlich ascites tumor, Ny Klein maintained by weekly intraperitoneal 458

cell type, was implantation

GLUCOSE

TRANSPORT

IN DEVELOPING

in albino mice (strain ICR). Methotrexate was a product of Lederle. Cytochalasin B, 3-O-methyl glucose, 2-deoxy-D-glucose, L-glucose, phloretin, and diethylstilbestrol were purchased from Sigma. All other sugars used were from British Drug House. [BH]Cytochalasin B (7.2 Ci/mmol) and 2-deoxy-D[aH$&cose (37.3 Ci/mmol) were obtained from New England Nuclear and used without further purification. Ehrlich ascites tumor. Mice weighing 30-35 g were inoculated intraperitoneally with lo7 ascites tumor cells harvested from 7-day-old tumors in 0.2 ml phosphate-buffered saline (PBS), pH 7.4, on Day 0. The effect of methotrexate was examined in groups of at least 10 mice each. In the test groups, MTX in normal saline (0.4 mg/kg body wt) was administered on Days 2, 4, and 6 or on Days 4, 5, and 6 postimplantation. In the control group, only saline was injected. The mice were killed by cervical dislocation. Tumor cells were collected by exhaustive drainage. Pooled cells were washed five times with half-isotonic saline to remove blood cells and harvested by centrifugation. The final cell suspension was prepared in PBS enriched with 3.2 mrd CaCIZ, 1.5 mM MgSO,, 6.1 mM pyruvate, 6.7 mM fumarate, and 6.1 mM glutamate (incubation buffer). Cell count was determined with a hemocytometer. Uptake Of 2-O!f?Ozy-D-QhX%?. To determine the rate of glucose uptake by Ehrlich ascites tumor cells, the procedure of Kaminskas (1) was used with minor modifications. Tumor-bearing mice were killed on Days 2,4,5,7,9, and 11. Tumor cells from each group of at least 10 mice were harvested as described. Cell count was adjusted to l@/ml in incubation buffer. Aliquots (1 ml) were placed in stoppered flasks and equilibrated to 37°C in a shaking water bath. One milliliter of 2-deoxy-D-[*H]glucose (1 pCi/rmol) was then added to give final concentrations of 0.25, 0.33, 0.5, 1.0, and 2.5 mM. The contents of the flasks were rapidly mixed and the incubation continued at 37“C. Samples of 200 pl were removed after 15, 30, 60, 90, 120, 150, and 180 s and added with mixing to test tubes containing 3 ml of ice-cold PBS supplemented with 15 mM 2-deoxy-D-glucose. Cells were collected by centrifugation at 8OOg for 1.5 min at 4°C and washed once with 3 ml of the same buffer. The supernate was removed by aspiration. Cell lysis was accomplished by adding 2 ml distilled water and mixing with a Vortex mixer. One milliliter of lysate was then mixed with 7 ml of a Triton X-toluene scintillant (10) and counted in a Beckman LS 7000 liquid scintillation counter. The initial rate of uptake (QUO) of 2-deoxy-D-glucose at each sugar concentration used was measured graphically. The kinetic parameters of the uptake process, V,, and K,,,, representing, respectively, the maximal uptake rate and apparent half-saturation constant, were determined by correlating v0 with sugar

EHRLICH

ASCITES

TUMOR

CELLS

459

concentration according to the double-reciprocal formulation of Lineweaver and Burk (11) after correction for nonspecific diffusion (12). Equilibrium binding of cgtochulasin B. Equilibrium binding of cytochalasin B to Ehrlich ascites tumor cells was performed according to Cuppoletti et al (7) with slight modifications. Tumor cells harvested as described were suspended in PBS. For measuring total cytochalasin B binding, 1 ml of incubation mixture contained 10’ cells, 0.02 &i [3Hkytochalasin B, and 2 X 10m9-5 X 10e6 M cytochalasin B. To assess the glucose-sensitive binding, D-glUCOSe was also added to the incubation mixture to a final concentration of 500 mM. The mixtures were allowed to equilibrate for 20 min at room temperature. Bound ligand was separated from free ligand by centrifugation at 25,OOOg for 20 min at 4°C in a Beckman 52-21 refrigerated centrifuge. An aliquot (0.5 ml) of the supernate was removed and its radioactivity counted. The tubes were then drained and the residual supernate was carefully removed. One milliliter of distilled water was added to lyse the cells, after which 0.5 ml was used for radioactivity measurement. The cytochalasin B bound was calculated as percent of total. Inasmuch as the glucose-sensitive cytochalasin B binding sites have been shown to represent the functional glucose carriers in various cell types studied (5,7,13-15), the putative glucose carriers on Ehrlich ascites tumor cells were determined by Scatchard analysis (16) of the difference between cytochalasin B bound in the presence of 500 mM D-glucose and the absence of D-glucose. The specificity of the glucose carriers was also examined. In these experiments, 7-day-old tumor cells were used. Equilibrium binding by cytochalasin B was performed in the presence or absence of 500 mM D-mannose, D-galactose, D-fructose, L-glucose, D-arabinose, lactose, or sucrose. Other inhibitors of cytochalasin B binding examined included phloretin (5 X lo-’ M) and diethylstilbestrol(1 X lo-’ M). Binding data were analyzed by Scatchard plots as for D-glUcase. Serum and ascitic fluid glucose was determined using Kit 510-DA supplied by Sigma Chemical Company. Cell-free ascitic fluid was obtained by centrifuging the peritoneal exudate from tumor-bearing mice at 8OOg for 5 min. Protein was removed with a 0.5 vol of 0.3 N barium hydroxide and zinc sulfate. Sera were similarly deproteinized. Detailed analytical procedures are described in Sigma Technical Bulletin No. 510. RESULTS

Figure 1 shows the effect of MTX on the growth of Ehrlich ascites tumor cells. It is clear that early administration of MTX was essential for suppressing cell prolif-

460

CHAN ET AL.

107

1

e

4

e

e

6 10

DAYS

FIG. 1. Effect of methotrexate on the proliferation of Ehrlich ascites tumor cells. Tumor-bearing mice were inoculated with 0.4 mg/kg body wt methotrexate on Days 4, 5, and 6 (0) or Days 2,4, and 6 (0) posttransplantation; (0) no treatment. Cells were harvested on the indicated days and counted. Values are presented as means + SE for three experimental groups.

eration. Thus 0.4 mg/kg body wt injected on Days 2, 4, and 6 postimplantation reduced the tumor size on Day 7 from about 2 X 10’ to 5 X lo7 cells; a similar dose administered on Days 4, 5, and 6 was much less effective. Withdrawal of MTX allowed the tumor to resume growth so that by Day 9, there was little apparent difference between the sizes of the tumors in treated and untreated animals. In later experiments with MTX, both administration schedules were used to allow a comparison of the effect of the two regimen. Uptake of deoxyglucose by Ehrlich ascites tumor cells during tumor development was studied. Figure 2A shows a typical time course of incorporation of [3H]deoxyglucose when 7-day-old cells were exposed to various concentrations of deoxyglucose. The rate of uptake was rapid in the first 10 s; it then fell gradually with time. When the initial rate was plotted against the concentration of deoxyglucose (Fig. 2B, curve A), considerable deviation from normal Michaelis-Menten kinetics was seen. Similar observations have been made with the transport of deoxyglucose

(12) and nucleosides (17) in Novikoff rat hepatoma cells, and it has been suggested that at high enough concentrations, deoxyglucose can enter the cells at a significant rate by simple diffusion. The extent of the diffusion-mediated uptake can be estimated graphically by drawing a line (Fig. 2B, curve B) through the origin and parallel to the linear portions of the uptake curves (12). The uptake rate due to the transport reaction was obtained by subtracting the estimated diffusion rates from the overall rates (Fig. 2B, curve C). The corrected rates showed that the uptake of deoxyglucose by Ehrlich ascites tumor cells is a saturable process, and follows Michaelis-Menten kinetics. Lineweaver-Burk plots of the facilitated uptake rates yielded typical straight lines from which the kinetic parameters V,,, and Km of the transport process can be determined. Figure 2C shows that the values of V,,,,, increased linearly with the progress of tumor growth, rising from 45 nmol/min/5 X lo6 cells on Day 2 to 215 nmol/min/5 X lo6 cells on Day 11 posttransplantation. By contrast, the value of Km remained unchanged throughout the course of the experiment at about 0.8 mM. These observations can be interpreted to indicate that while the nature of the hexose carrier molecules appeared to be relatively unaffected, the number of such carrier molecules increased progressively with tumor development. Alternatively, the turnover rate of the glucose transport system might have increased as the tumor grew. To differentiate between these two possibilities, we measured the equilibrium binding of cytochalasin B by Ehrlich ascites tumor cells. Figure 3A shows the results of a representative experiment using cells from ‘I-day-old tumors. The presence of 500 mM glucose in the incubation medium inhibited the binding of cytochalasin B in a competitive manner. This glucosesensitive binding site for cytochalasin B, comprising approximately 60% of the total cytochalasin B-binding sites, has been identified as the glucose transporter in several cell types including cultured Ehrlich ascites tumor cells (4, 5, 7). When the difference between cytochalasin B bound

GLUCOSE

0

30

TRANSPORT

60 TIME Irec)

90

120

IN DEVELOPING

0

0.5

1.0

EHRLICH

1.5

I DEOXYCLUCOSEI

2.0

2.1

ASCITES

TUMOR

CELLS

461

0

ImMi

FIG. 2. Uptake of 2-deoxy-D-glucose by Ehrlich ascites tumor cells during tumor development. (A) Representative experiment showing initial rate of uptake by cells harvested from 7-day-old tumors at various final concentrations of 2-deoxy-D-glucose: 0.25 (A); 0.33 (W); 0.5 (A); 1.0 (0); and 2.5 mM (0). Cells were incubated with 2-deoxy-D-glucose and 2-deoxy-D-[sH$lucose (1 pCi/rmol) at 3’7°C. Aliquots of 200 pl were withdrawn at the indicated time intervals and resuspended in icecold PBS supplemented with 15 rnM 2-deoxy-D-glucose. Cells were collected by centrifugation and lysed with distilled water, and their radioactivity was determined. (B) Initial rates of 2-deoxy-nglucose uptake by ‘I-day-old cells as a function of substrate concentration. Total uptake, determined graphically from Fig. 2A, was used to construct curve A (0). The rate of simple diffusion (curve B) was determined by drawing a line through the origin parallel to the linear portion of curve A (see Ref. (12)). The rate of uptake due to facilitated diffusion (0) was determined by subtracting these values from the rates of total uptake. It is seen that the facilitated uptake process follows Michaelis-Menten kinetics. The kinetic parameters V,, and K,,, were obtained from LineweaverBurk plots. (C) Changes in V,, (0) and Km (0) for uptake of 2-deoxy-D-glucose during tumor development.

in the presence of 500 mM D-glucose and the absence of glucose from Fig. 3A was analyzed by the Scatchard plot, a linear plot was obtained (Fig. 3B) from which it was estimated that for ‘I-day-old cells, the density of cytochalasin B binding sites (&) was about 215 pmol/lO’ cells; the Kd value was 2.7 X lop7 M. Figure 3C shows that as the tumor developed the number of cytochalasin B binding sites increased. When compared with the B,, value for Day 2 cells, the difference was highly significant (P < 0.001). The apparent dissociation constant, however, remained unchanged. Cytochalasin B binding in the presence of other sugars and agents known to inhibit glucose transport was also examined. The

data in Table I show that 2-deoxy-D-glucase, 3-@methyl-D-glucose, D-palactose, Dmannose, phloretin, and diethylstilbestrol were all capable of competitively inhibiting the binding of cytochalasin B to the D-glucose-sensitive site, where the apparent dissociation constant was 1.5-4.0 X lo-? M. Other sugars tested but found to be ineffective included L-glucose, D-fructose, Darabinose, L-fucose, sucrose, and lactose (data not shown). The effect of methotrexate on the uptake of 2-deoxyglucose and the glucose carriercytochalasin B binding system in 7-dayold Ehrlich ascites tumor cells was also studied. Administration of 0.4 mg MTX/ kg body wt on Days 4, 5, and 6 posttran-

462

CHAN ET AL.

I.6 -

I.6 _

I.4 _

‘!2 -

B

\. . 1. \ . 3.0,:

, , .1

I CB I I lo-EM

j

50 loo 150 MO BOUND C8 I pmol )

2.0’4 1.0 ;;y 0

2

4

6 DAYS

8

10

FIG. 3. Capacity of Ehrlich ascites tumor cells to hind cytochalasin B during tumor development. (A) Representative experiment showing binding of cytochalasin B by Ehrlich ascites cells harvested from ?-day-old tumors as a function of free ligand concentration. Each milliliter of incubation mixture contained lo7 cells, 0.02 &i [SH]eytochalasin B, and various concentrations of cytochalasin B. After incubation, the supernatants were separated from the pellets by centrifugation and the radioactivities in each fraction determined. Cytochalasin B bound was calculated as a percentage of total binding in the absence (A) and in the presence (0) of 500 mM glucose. The difference represents the glucose-sensitive portion. (B) Scatchard analysis of the glucose-sensitive binding of cytochalasin B to cells harvested from 7-day-old Ehrlich ascites tumors. Control data from Fig. 3A were used to calculate the bound and free ligand. The line was the best fit according to linear least-squares analysis. In this particular experiment, it was estimated that the glucose-sensitive sites bound cytochalasin B with a Kd of 2.4 X lo-’ M and a &, of 215 pmol/lO’ cells. (C) Changes in B0 (0) and Kd (0) values for glucose-sensitive binding of cytochalasin B during tumor development. Values are presented as means + SE for triplicate determinations in three separate experiments. Statistical analysis for significance of difference relative to Day 2 values was performed using the Student t test. *, P value for the significance of difference < 0.01; **, P value for the significance of difference < 0.001.

splantation reduced the V,,, of the uptake brought about a more pronounced drop in process significantly (P < 0.005) by about the rate of glucose uptake and a bigger 25% (Table II). This decrease could be reduction in the number of glucose carriers. shown to be due principally if not exclu- The magnitude of changes in the latter two sively to a reduction in the number of glu- parameters was again comparable (35 cose carrier molecules per cell, as evidenced vs 40%). by the 25% reduction in the B,, value in It has been demonstrated that the denMTX-treated cells when compared with sity of glucose carriers in cultured chick that of untreated cells. The apparent Km embryo fibroblasts is closely regulated by value for hexose transport was unaffected the level of glucose in the culture medium. by MTX; correspondingly, no significant It was suggested that glucose acts as a difference was observed for the apparent “repressor” for carrier synthesis (18). We dissociation constants for the binding of therefore measured the concentration of cytochalasin B (P < 0.1). Table II also glucose in the serum and the ascitic fluid shows that MTX administered on Days 2, of mice during the course of tumor devel4, and 6 posttransplantation, which was opment. Figure 4 shows that the serum more effective in reducing tumor size, level of glucose in tumor-bearing mice fell

GLUCOSE

TRANSPORT TABLE

IN DEVELOPING

EHRLICH

I

ligand*

D-Glucose 2-Deoxy-D-glucose 3-O-Methyl-D-glucose D-Galactose D-Mannose Phloretin Diethylstilbestrol

TUMOR

463

CELLS

DISCUSSION

The present study shows that the rate of glucose uptake by Ehrlich ascites tumor cells increased progressively as the tumor developed. When present at high concentrations, glucose can enter cells at a significant rate by simple diffusion (Fig. 2B and Ref. (12)). The negligibly low level of glucose in the ascitic fluid (Fig. 4), however, would require that, in vivo, Ehrlich ascites cells obtain glucose through a facilitated transport process. Such a process has been shown to follow simple Michaelis-Menten kinetics and may adequately be described by the kinetic parameters V,,, and Km (7, 20). The data presented in Fig. 2C show that the V,,, for glucose uptake increased continuously with tumor growth but the Km remained constant throughout. Methotrexate, an antimetabolite demonstrated to inhibit glucose consumption by cultured Ehrlich ascites tumor cells (1) was also seen in the present study to decrease glucose uptake by lowering the I’,,, values without appreciably affecting the Km (Table II). Increased glucose uptake has been found to accompany phytohemagglutininstimulated proliferation of cultured bovine lymphocytes (21) as well as the virus-induced neoplastic transformation of several

APPARENT DISSOCIATION CONSTANT (I&) AND NUMBER OF BINDING SITES (B,) FOR CYTOCHALASIN B ON EHRLICH ASCITES TUMOR CELLS”

Competing

ASCITES

BO (pmol/lOr cells)

Kd (lo-7 M)

215 218 205 149 144 215 139

2.6 2.3 2.2 2.0 1.5 2.3 4.0

DD-ghCOSeand ligand-reversible binding of cytochalasin B was measured as described in the text. Cells from ‘I-day-old tumors were used. The difference in binding in the presence and in the absence of competing ligands was analyzed by Scatchard plot. Numbers represent the mean values of triplicate samples. * The final sugar concentration was 500 mM; those of phloretin and diethylstilbestrol were, respectively, 5 X 10m5and 1 X lo-’ M.

progressively as the tumor grew. There was essentially no measurable glucose in the ascitic fluid from Day 2 to Day 11 after inoculation. The concentration of glucose in normal peritoneal fluid is 95 mg/lOO ml (19). TABLE

II

EFFECT OF MTX ON TUMOR SIZE, 2-DEOXY-D-GLUCOSE UPTAKE, AND CYTOCHALASIN B BINDING OF EHRLICH ASCITES TUMOR CELLS’

~-DWX~-D-~~COSIZ

Tumor size” Treatment

None MTX (days 4,5, 6) MTX (days 2, 4, 6)

(Cell No. X 1O-8)

12.3 f 0.6 5.0 f 0.6 0.7 * 0.3

V

(nmol/min/Z

Glucose-sensitive cytochalasin B binding

uptake 6

lo6 cells)

157 f 5.8 118 k 4.3**

102 + 2.8””

(mM)

BO (pmol/lO’ cells)

K., (lo-’ M)

0.64 * 0.12 0.58 + 0.06 0.52 It 0.05

221.3 + 7.9 162.8 + 7.2* 130.9 f 9.8***

2.48 2 0.07 2.17 f 0.11 2.20 z!z0.08

’ MTX was administered intraperitoneally at a concentration of 0.4 mg/kg body wt either on Days 2,4, and 6 or on days 4,5, and 6 posttransplantation. Uptake of Zdeoxy-D-glucose and cytochalasin B binding were measured as described in the text using cells harvested from treated or untreated ‘I-day-old tumors. B0 and K,, refer, respectively, to the total glucosereversible binding capacity and the apparent dissociation constant and were obtained from Scatchard analyses of the difference in binding in the presence and absence of 500 rn~ D-ghrcose. Values are expressed as means * SE of triplicate sample determinations from two separate experiments. ‘The data points for ‘I-day-old tumor from Fig. 1 are displayed here to facilitate comparison of the various MTX effects. Values are expressed as means I+_SE for three experimental groups. * P value for the significance of difference < 0.01. ** P < 0.005. ***p < 0.001.

CHAN

2

4

6

a

10

DAY

FIG. 4. Concentration of glucose in mouse serum (0) and ascitic fluid (0) during tumor development. Values are expressed as the means + SE for three determinations.

avian and mammalian cell lines including Rous sarcoma virus/chick embryo fibroblasts (22) and polyoma virus/mouse BHK cells (23). Normal mouse cells in culture, on the other hand, exhibited reduced transport rates when they attained confluency (24), as did rat 3T3-Ll fibroblasts upon conversion to adipocytes with dexamethasone/l -methyl - 3 - isobutylxanthine treatment (3). The changed glucose transport rate in each case was shown to be the result of changes in values of I’,,,,, rather than Km. In this respect, glucose transport in these experimental systems resembles that in Ehrlich ascites tumor. Transport rates changed because of changes in the number and/or operational efficiency of transport sites; the affinity between glucose and its transport component was not affected, at least not in the aforementioned circumstances. In nearly all eucaryotic cells studied, glucose transport occurs as a carrier-mediated process (see Ref. (25) for review). Cytochalasin B inhibits this process, and a set of high-affinity glucose-inhibitable binding sites for cytochalasin B has been

ET AL.

tentatively identified to be the glucose transporter (5,7,13-15). Studies performed on this glucose-reversible cytochalasin B binding activity in cell systems as divergent as human erythrocytes, Novikoff rat hepatoma cells, and barnacle muscle cells (7,12, 13, 15,26) revealed that they share a number of common characteristics: (i) a high affinity for cytochalasin B (Kd l-6 X lop7 M); (ii) stereospecific inhibition by sugar; thus D-glucose, 3O-methyl-D-glucase, D-galactose, and D-mannose inhibit binding while L-glucose and L-fucose are without effect; and (iii) sensitivity to phloretin and diethylstilbestrol. These properties were also observed in the present study with Ehrlich ascites tumor cells (Tables I and II). If the postulation that this class of cytochalasin B binding sites is identical to the glucose carrier is valid, the data presented in Fig. 3C may be interpreted to mean that, as the Ehrlich ascites tumor grew and glucose uptake increased, the number of glucose carrier molecules also increased. The increase in number of putative glucose carriers sufficiently accounted for the elevated rate of glucose uptake associated with tumor development. Figure 2C shows that between Day 2 and Day 9 posttransplantation, the V,,, value for glucose transport increased approximately 4.5-fold. During the same interval, the maximum number of cytochalasin B binding sites increased by slightly less than 4-fold. Similarly, in Table II, it may be seen that MTX suppressed the uptake rate of 2-deoxy-Dglucose in 7-day-old tumor cells to the same extent as cytochalasin B binding was inhibited. Furthermore, the fact that MTX was without effect on the Kd for cytochalasin B binding (Table II) corroborates our earlier assertion that qualitative changes in the transport process are not related to substrate binding. It may therefore be concluded that changes in glucose uptake by Ehrlich ascites tumor cells are principally the result of changes in the number of transport carriers. While alteration in the turnover rate of the glucose carriers cannot be absolutely ruled out in the present study, its contribution to the overall transport process is at best mini-

GLUCOSE

TRANSPORT

IN DEVELOPING

mal. A similar conclusion has been reached by Wardzala et aL (27) for the effect of insulin on glucose transport in rat adipocytes. The high density of cytochalasin B binding sites on Ehrlich ascites tumor cells is remarkable. In 7-day-old cells, the maximum binding of 220 pmol/107 cells (Table II) translates to 1.3 X lo7 carrier molecules/ cell. This is in close agreement with the value estimated by Cuppoletti et al. in cultured Ehrlich ascites tumor cells (7) and is considerably higher (3- to loo-fold) than that reported for human erythrocytes, HeLa cells, and Novikoff rat hepatoma cells (13, 28, 29). This observation probably reflects the fact that, whereas the other cells are capable of utilizing respiration for the provision of metabolic energy, Ehrlich ascites tumor cells depend primarily on glycolysis, and a high-capacity system for glucose uptake is essential for the maintenance of growth. The continuous fall in serum glucose during tumor development (Fig. 4) is a reflection of this high demand for glucose and the efficiency of the glucose transport system in Ehrlich ascites tumor cells. Methotrexate arrests the growth of Ehrlich ascites tumor cells by inhibiting nucleotide synthesis and the induction of a “purineless” state in these cells (2). However, the cytotoxic efficiency is dependent on the schedule of administration. Early administration was found to be more effective (Fig. 1 and Table II), an observation consistent with the cell cycle-specific nature of MTX action (30) and the known relative preponderance of resting cells in aged tumors (31). MTX also reduced the number of cytochalasin B binding sites in Ehrlich ascites tumor cells (Table II). The efficacy of the drug in this respect roughly paralleled its efficacy in reducing tumor size, suggesting that MTX-induced depletion of nucleotides and the consequent inhibition of DNA, RNA, and protein synthesis might have constituted the primary chain of events leading to reduced carrier production. The increase in carrier number during tumor development is more difficult to explain. Hexose starvation might have been a cause. In this connection, it is in-

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teresting to note in Fig. 3C that T-day-old tumor cells showed a four-fold drop in glucose carrier content within 48 h upon transplantation to new mice and exposure to fresh peritoneal fluid. The glucose concentration in normal peritoneal fluid is 95 mg/lOO ml (19), that in ascitic fluid almost immeasurably low (Fig. 4). Amos et aL (18) showed that hexose deprivation stimulates glucose uptake 30-fold in chick embryo fibroblasts. Pastan and collaborators (32) identified two membrane proteins whose rate of synthesis was accelerated when chick cells were starved of glucose; the implication was that glucose transporters were involved. In contrast, Graff et al. (6) reported that Novikoff rat hepatoma cells are not subject to glucose transport regulation by starvation for hexose. The exact stimulus triggering the synthesis of glucose carriers during tumor development remains to be identified. It would be interesting to examine in vitro the effect of glucose on carrier synthesis and changes in insulin level in ascitic fluid as tumor growth progresses. ACKNOWLEDGMENT This work was supported by a grant from the World Health Foundation (H.K.). REFERENCES 1. KAMINSKAS, E. (1979) Cancer Res. 39. 90-95. 2. HRYNIUK, W. M. (1972) Cancer Res. 32,1506-1511. 3. KAMINSKAS, E., AND NUSSEY, A. C. (1978) Cancer Res. 38.2989-2996. 4. PINKOFSKY, H. B., RAMPAL, A. L., COWDEN, M. A., AND JUNG, C. Y. (1978) J. Biol. Chem 253,49304937. 5. KARNIELI, E., ZARNOWSKY, M. J., HISSIN, P. J., SIMPSON, I. A., SALANS, L. B., AND CUSHMAN, S. W. (1981) J. BioL C’hem 256.4772-4777. 6. GRAFF, J. C., WOHLHUETER, R. M., AND PLAGEMANN, G. W. (1981) B&him. Biophys. Acta 641, 320-333. 7. CUPPOLETTI,J., MAYHEW, E., AND JUNG, C. Y. (1981) Biochim Biqphys. A& 642,392-404. 8. RESH, M. D. (1982) J. Bid Chem 257,6978-6986. 9. SEFTON, B. M., AND RUBIN, H. (1971) Proc. Natl Acad Sci USA 68, 3154-3157. 10. PAVERSON, M. S., AND GREENE, R. C. (1965) And Chem 37, 854-857. 11. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sex 56,658-666.

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