KINETIC PARAMETERS OF HEXOSE TRANSPORT IN ...Mouse PG19: hypoxanthine-guanine phosphoribosyl transferase-deficient (HGPRT") derivative of a spontaneous melanoma arising in a C57B1

J. Cell Sci. 62, 49-80 (1983) 49Primed in Great Britain © The Company of Biologists Limited 1983

KINETIC PARAMETERS OF HEXOSE TRANSPORT IN

HYBRIDS BETWEEN MALIGNANT AND NON-

MALIGNANT CELLS

M. K. WHITE, M. E. BRAMWELL AND H. HARRISSir William Dunn School of Pathology, University of Oxford, Oxford 0X1 3RE,England

SUMMARY

Matched pairs of isogeneic hybrid cells, in which one member of the pair was malignant and theother not, were used to examine the linkage between malignancy and functional alterations in hexosetransport. The kinetic parameters of uptake of 2-deoxy-D-glucose were measured in a range of suchhybrids, both human and murine. Some other malignant cell lines were also examined and werecompared with non-tumorigenic derivatives of tumour cells selected by exposure to the lectin,wheat-germ agglutmin. In every case, malignancy, as denned by the ability of cells to growprogressively in vivo, was found to be linked to a decrease in the Michaelis constant of hexoseuptake. Independent measurement of the transport and phosphorylation reactions involved inhexose uptake revealed that this decrease was determined by the membrane transport system. Thedifference in Michaelis constant between malignant and non-malignant cells was observed with3-0-methylglucose, a hexose that is transported into the cell but not further metabolized. Theactivity of hexokinase in cell homogenates was higher than the level that would be required to copewith transport and showed no correlation with tumorigenicity. Measurement of the uptake ofD-glucose itself, by a rapid nitration centrifugation method, gave results similar to those obtainedwith 2-deoxy-D-glucose.

INTRODUCTION

Hybrids generated by the fusion of malignant with non-malignant cells provide asegregating genetic system in which malignancy, defined as the capacity of cells toproduce progressive tumours in a suitable host, can be correlated with biochemical orcytological parameters measured in vitro (Harris, 1971; Straus, Jonasson & Harris,1977; Watt, Harris, Weber & Osborn, 1978; Stanbridge& Wilkinson, 1978). Initiallysuch hybrids are usually non-malignant, but on continued cultivation in vitro theymay give rise to segregants in which malignancy has re-appeared (Klein, Bregula,Wiener & Harris, 1971; Wiener, Klein & Harris, 1971, 1974a; Stanbridge, 1976;Sager & Kovac, 1978). Hybrids in which malignancy is suppressed and malignantsegregants derived from them thus constitute isogeneic matched pairs, which providea screen to test whether or not a particular cellular property co-segregates with malig-nancy. In this way, Bramwell & Harris (1978a) showed that malignancy is systematic-ally linked to a structural alteration in the carbohydrate moiety of a particular mem-brane glycoprotein. Further characterization of this glycoprotein provided circum-stantial evidence that it had some role in glucose transport (Bramwell & Harris,19786; Bramwell, 1980; Bramwell & Atkinson, 1982; Gingrich, Wouters, Bramwell& Harris, 1981o,6).

50 M. K. White, M. E. Bramwell and H. Harris

In the present work, isogeneic matched pairs of hybrids were examined to seewhether any functional alteration in hexose transport was associated with malignancy.Non-malignant variants derived from tumour cells by selection for resistance to thelectin wheat-germ agglutinin (WGA) were also examined.

There has been little previous work, and no formal kinetic study, on the relation-ship between glucose uptake and tumorigenicity. However, many investigations ofhexose uptake have compared virus-transformed cells with untransformed controls.Hatanaka, Huebner & Gilden (1969) were the first to report that the uptake ofglucose was enhanced on viral transformation. In this study, and several other earlystudies, transformation was associated with a large reduction in the apparent Km forglucose uptake (Hatanaka & Hanafusa, 1970; Hatanaka, Todaro & Gilden, 1970;Hatanaka, 1971; Hatanaka, Gilden & Kelloff, 1971). However, since glucose was thehexose used in these studies, uptake rates reflect both transport and subsequentmetabolism, so that it is difficult to interpret the apparent Km values (for discussionsee Plagemann & Richey, 1974). Many other studies have used 2-deoxy-D-glucose,an analogue of glucose that is transported into cells, phosphorylated by hexokinase,but not further metabolized (Renner, Plagemann & Bernlohr, 1972; Weber, 1973).Kinetic analysis of the enhancement of uptake of this sugar on viral transformationhas given conflicting results. Some investigators have reported a reduction in theKm of uptake (Hatanaka, Augl & Gilden, 1970; Hatanaka & Hanafusa, 1970;Hatanaka, 1971; Hino & Yamomoto, 1971;Bader, 1972; Gazdaretal. 1972; Bradley& Culp, 1974; May, Somers & Kit, 1974). However, it has been suggested that thisfall in Km is due to a failure to ascertain properly the initial rates of uptake(Plagemann, 1973). More careful analysis of deoxyglucose uptake kinetics has shownthat transformation results in an increase in the VmiX for hexose uptake, the Km

remaining constant (Isselbacher, 1972; Plagemann, 1973; M. J. Weber, 1973; Klet-zien&Perdue, 19746, 1975; Royer-Pokoraef a/. 1978). This conclusion is supportedby studies of the uptake of 3-O-methyl-D-glucose, which is transported into cells butnot further metabolized (Renner et al. 1972; Weber, 1973). Again, a kinetic analysisindicated that the Vm^x was increased on viral transformation, but the/Cm was unaffec-ted (Venuta & Rubin, 1973; Weber, 1973; Kletzien & Perdue, 19746). Viral trans-formation in vitro thus appears to result in an increased Vmex of the membrane hexosetransport system and is apparently due to an increase in the number of carriermolecules in the cell membrane (Salter & Weber, 1979; Salter, Baldwin, Lienhard& Weber, 1982).

In the present study the relationship between tumorigenicity and hexose transportwas explored, initially by measuring the kinetic parameters of uptake of 2-deoxy-D-glucose. Significantly lower Km values were found for all tumorigenic cells, whetherhybrid or not, compared to their non-tumorigenic homologues. Further studies ofthis phenomenon with 3-O-methyl-D-glucose confirmed that the reduction in Km wasdue to a change in the transport of hexose across the cell membrane. Finally, similarKm values were obtained with D-glucose, where the uptake was measured by a rapidassay method in which subsequent metabolism did not significantly complicate therate measurements.

Hexose transport in hybrid cells 51

Preliminary reports of this work have been published (White, Bramwell & Harris,1981, 1982).

MATERIALS AND METHODS

2-Deoxy-D-[l-3H]glucose, 3-O-methyl-D-[l-3H]glucose, D-[U-l4C]glucose, L-[l-14C]glucose,inulin-[HC]carboxylic acid and 2-deoxy-D-[l-14C]glucose were obtained from Amersham Interna-tional Ltd, Bucks, U.K. L-[l-3H]glucose was obtained from New England Nuclear, Boston,Massachusetts, U.S.A. Unlabelled 2-deoxy-D-glucose was from Calbiochem-Behring Corporation,La Jolla, California, U.S.A.; and L-glucose and 3-O-methyl-D-glucose were from Sigma ChemicalCompany Ltd, Poole, Dorset, U.K.

Cell lines

MousePG19: hypoxanthine-guanine phosphoribosyl transferase-deficient (HGPRT") derivativeof a spontaneous melanoma arising in a C57B1 mouse (Jonasson, Povey & Harris, 1977).

SEWA: polyoma virus-induced osteosarcoma arising in an A/Sw mouse (Sjogren, Hell-strom & Klein, 1961).

A9HT: malignant derivative of the A9 cell line selected by passage in vivo (Wiener, Klein& Harris, 1973).

YACIR: HGPRT" derivative of a Moloney virus-induced lymphoma arising in an A/Snmouse (Fenyo, Klein, Klein & Swiech, 1968).

TA3 Hauschka: spontaneous mammary carcinoma arising in an A/Ha mouse (Hauschka,1953).

TA3HaB: HGPRT" derivative of TA3 Hauachka (Wiener, Klein & Harris, 19746).

MSWBS: methylcholanthrene-induced sarcoma of the A/Sw mouse (Klein & Klein, 1958).

P3/NS/l-Ag4: HGPRT" derivative of a myeloma arising in a BALB/c mouse (Kohler,Howe & Milstein, 1976).

3T3A31 clone 9: fibroblast-like embryonic culture from BALB/c mouse (Antoniades,Stathakos & Scher, 1975).

PG19-G: derivative of PG19 selected for ability to grow in a low concentration of glucose(Bramwell, 1980).

SV-3T3-B: SV40 virus-transformed derivative of the BALB/c 3T3 cell line (Todaro, Habel& Green, 1965).

SV-3T3-BT: SV-3T3-B tumour explanted from a nude mouse.

PG19-WGAR clone C2: derivative of PG19 resistant to 10/jg/ml WGA (Chan, 1980).

A9HTWC: non-malignant WGA-resistant A9HT derivative (Chan, 1980).

A9HTWD: malignant WGA-resistant A9HT derivative (Chan, 1980).

Rat

NRKTG3: HGPRT" rat kidney cell line (NRK) (Marshall, 1980).

3.B77.Sc4: NRK cell line transformed by Rous sarcoma virus strain B77 (Marshall, 1980).

HumanMRC5: fibroblast strain from lung of male foetus of 4 months gestation (Jacobs, Jones &Bailie, 1970).

S1814: fibroblast strain from male foetus (Klinger, 1980).


DS1: fibroblast strain from spontaneous male abortus (Sir William Dunn School of Pathol-ogy, unpublished).

GM1604: foetal fibroblast cell strain (Der & Stanbridge, 1981).

WI38: fibroblast strain from lung of female foetus of 3 months gestation (Hayflick & Moor-head, 1961).

RT112/84: cell line derived from carcinoma of bladder (Marshall, Franks & Carbonell,1977).

HeLa (Flow): cell line derived from carcinoma of cervix (Gey, Coffman & Kubicek, 1952)supplied by Flow Laboratories, Irvine, Scotland, U.K.

HeLa spinner: derivative of HeLa selected for growth in spinner culture (Sir William DunnSchool of Pathology, unpublished).

HeLaD98F908A3: HGPRT" HeLa derivative (Klinger, 1980).

HeLa D98AH2: HGPRT" HeLa derivative (Der & Stanbridge, 1981).

H29/219: cell line derived from a carcinoma of the rectum (Marshall et al. 1977).

H.Ep.2: cell line derived from a carcinoma of the larynx (Moore, Sabachewsky & Toolan,1955).

RPMI2650: cell line derived from a carcinoma of the nasal septum (Moore & Sandberg,1964).

HT55F: cell line derived from a carcinoma of the colon (donated by Professor J. F. Watkins,University of Wales, unpublished).

H T U 5 : cell line derived from a carcinoma of the colon (donated by Professor J. F. Watkins,unpublished).

Daudi: lymphoid cell line derived from a patient with Burkitt's lymphoma (Klein et al.1967).

Measurement of deoxyglucose uptakeCells growing as a monolayer. The cells were brought into suspension in Eagle's minimal essential

medium supplemented with 10% foetal calf serum or 5 % foetal calf serum plus 5 % newborn calfserum (5-15 (XlO4) cells per ml) and evenly distributed into 48 16-mm diameter tissue-culture wells(Coster, Cambridge, Mass., U.S.A.). A sample (2 ml) of cell suspension was added to each well 24 hbefore assay. The adherent cell monolayers were washed twice with 500/il of phosphate-bufferedsaline (PBS), and 500/il of a solution of 2-deoxy-D-[l-3H]glucose (0-1-5 mM, l-210mCi/mmol)in PBS added at 20CC. After 1-10min at 20°C, the radioactive medium was aspirated and themonolayer washed twice with 600/*1 of PBS. The cell monolayers were dissolved in 440 /̂1 0-4M-NaOH, neutralized with l0f.ll glacial acetic acid and then mixed with lOmlUnisolve 1 (Koch-Light,Colnbrook, Berks, U.K.). The activity of tritium in these samples was determined with a Packardscintillation counter. In each experiment six wells were assayed for each concentration of 2-deoxy-D-glucose. Uptake was usually measured at six different concentrations.

Non-specific retention of radiolabel was measured with L-[l-3H]glucose under parallel con-ditions. Measurements were made on six wells and averaged. The number of cells per well wasdetermined by detaching the cells with trypsin, transferring them to 10 ml of Isoton (CoulterElectronics, Dunstable, Beds, U.K.) and counting 500/il samples with a Coulter counter (CoulterElectronics). Measurements were made on six wells and averaged.

The velocity of sugar uptake, V\, at each substrate concentration, S;, and the variance of eachvelocity estimate, VarV,, were calculated by linear regression through the origin of the plot of2-deoxy-D-glucose uptake against time. The Km and Vmtx values and their standard errors werecalculated by a weighted linear regression of S/V against 5, essentially as described by Cornish-Bowden (1976). The final weight applied to each Sj/V, was [Ki2/VarVi] [ V 2 ™ / ^ + Si)2). Thevalidity of this approach was confirmed by inspection of residuals in V (Cornish-Bowden, Porter &Trager, 1978) and by comparison of the results with those obtained by non-parametric analysis ofthe same data (Porter & Trager, 1977).

Cells in suspension. This method was used with cells that grew in suspension culture or ascites


in vivo and also with lymphocytes. The cell number was determined in a haemocytometer andviability estimated by dye exclusion. The cells were then distributed into microfuge tubes (3-20(X 105) cells per tube), pelleted by centrifugation for 4s in a Beckman Microfuge B and washed withPBS. The uptake measurements were started by resuspending the cells in 300/d of 2-deoxy-D-[l-3H]glucose. The same concentrations and specific activities of radiolabel were used as for cellsin monolayers and incubation times ranged from 1-5 min. Cells were.collected by centrifugation(4s) and rapidly washed in ice-cold PBS. Non-specific retention of label was again measured withL-[l-3H]glucose. The kinetic parameters were calculated as for cells in monolayers.

Measurement of 3-O-methyl-D-glucose uptakeUptake of 3-O-methyl-D-glucose into cells growing as monolayere was measured in the same way

as the uptake of 2-deoxy-D-glucose, but with the following modifications: (a) 3-O-methyl-D-[l-3H]glucose (0-1-5 mM, 10-500 mCi/mmol, 50/jCi/ml) was added to the cells in a volume of250/il at 20CC; (b) uptake was terminated by washing the cells rapidly twice with ice-cold PBS; (c)shorter incubation times were used (< 2 min).

Measurement of hexokinase activityThe activity of hexokinase in homogenates of cultured cells was determined essentially as

described by Kletzien & Perdue (1974a). Homogenates were prepared by ultrasonication of washedcell suspensions in lml of PBS (107— 10s cells). The cells were given four 5-s pulses with anMSE100 W ultrasonic disintegrator at 0°C. Each pulse was separated by a 25 s interval.

The reaction mixture for determining hexokinase activity consisted of lOmM-HEPES buffer(pH7-4), 10mM-ATP, 10mM-MgCl2l 10mM-KCl, 3mM-NADP+, 12mM-glucose, 2 units ofglucos*-6-phosphate dehydrogenase and 10— 50/d of cell homogenate in a total volume of 1-5 ml.The reaction mixture was incubated at 20 °C and the reduction of NADP+ was measured by theincrease in absorbance at 340 nm. Reaction mixture without homogenate was used as a blank.

The protein content of each sample was measured by the method of Lowry, Rosebrough, Farr& Randall (1951), with bovine serum albumin as a standard.

Measurement of D-glucose uptakeUptake of D-glucose during very short incubations (5-30 s) was measured by the silicone oil

filtration centrifugation technique described by Werdan et al. (1980). In order to measure theuptake of glucose by this method, it is necessary for the cells to be in suspension. Monolayers of cellswere detached with PBS containing 0-2% (w/v) EDTA (disodium salt). After cell number andviability were determined, cells were resuspended in uptake buffer at a density of between 5 X 10s

and 1 X 106 cells per ml. Uptake buffer consisted of 137 mM-NaCl, 5-4 mM-KCl, 4 8 mM-NaHCO3,OlmM-EDTA, 0 0 0 1 % (w/v) phenol red, 20mM-HEPES and 25/iCi/ml3H2O (AmershamInternational Ltd), pH7-4.

The uptake incubation was done in 400/xl polyethylene tubes (Beckman). These tubes contained20^1 of 1 M-HCIO4 with an overlay of 70^1 of silicone oil. The silicone oil was a mixture of AR20and AR200 oils in a ratio of 1:1-5 (Wacker Chemie, Munich). A total of 250[A of cell suspensionwas layered onto the silicone oil in each tube and the uptake incubation was started by adding10 fA of D-[U-MC]glucose (7-260 mCi/mmol) to give a final concentration of 0-1 mM-5mM. After5-30 a at 20 °C, the incubation was ended by centrifuging the cells for 10 s through the layer ofsilicone oil into the perchloric acid. This was done in a Beckman microfuge B. The activity of 14Cwas used as a measure of the amount of D-glucose associated with the cells, and the activity of 3Has a measure of the volume of material passing through the silicone oil. The activity of each isotopein both the supernatant layer and the perchloric acid was determined with a Packard scintillationcounter preprogrammed to give 14C and 3H disintegrations per minute for doubly labelled samples.

A significant amount of extracellular material passed through the silicone oil with the cells. Thecontribution of this to the total uptake was determined in two ways: (1) tubes were assayed witheither L-[l-MC]glucose (61-4mCi/mmol, final concentration 0-1 mM) or inulin-[14C]carboxylicacid (7-8 mCi/mmol, final concentration 1 £JM), both of these beine excluded from the intracellularspace; (2) uptake of D-glucose was extrapolated to zero time. L-[1- C]gluco«e measurements werefound to give the most accurate estimate of extracellular space. The amount of glucose taken up into


the intracellular space was calculated by subtracting the extracellular glucose from the total amountof glucose in the perchloric acid fraction. This was then divided by the volume of the intracellularspace to give the amount of glucose taken up per unit volume of intracellular space. This wasexpressed in picomoles per nanolitre. A more detailed account of these calculations is given byWerdanefa/. (1980).

Six nine-point time courses of D-glucose uptake against time at different D-glucose concentrationswere measured. Rates of uptake and their variances were again calculated by linear regressionthrough the origin, and the weighted linear regression method was applied to these data to determineKm and Vmu , as described for 2-deoxy-D-glucose.

Effect of fluoride on glucose uptakeWerdan et al. (1980) demonstrated that D-glucose uptake measurements made by this method

may not be affected by loss of intracellular isotope due to glucose metabolism. To test this, YACIRcells were preincubated with 1 mM-sodium fluoride for 20min at 20 °C. Under these conditionsfluoride gave a 90 % inhibition of glycolysis as determined by lactate production (measured by themethod of Everse, 1975), with no significant loss of viability. Uptake of D-glucose was measuredwith D-[U-HC]glucose (0-l ITIM, 300mCi/mmol) by the method described and compared to that inuntreated YACIR cells.

Measurement of 2-deoxy-D-glucose uptake by silicone oil filtration centrifugationThe uptake of 2-deoxy-D-glucose into YACIR cells was measured with 2-deoxy-D-[l-14C]glucose

(O'lmM, 58-SmCi/mmol) and compared to D-[U-14C]glucose (0-1 min, 300mCi/mmol) uptakeinto the same batch of cells.

Measurement of L-glucose uptake by silicone oil filtration centrifugationThe silicone oil filtration centrifugation method was modified in the following ways to measure

L-glucose uptake: (a) L-[l-HC]glucose (61-4mCi/mmol) was added to give a final concentration of0-1 mM; (b) the length of incubation was increased to between 60 and 180 min, as the uptake is veryslow; (c) between three and six tubes were assayed in each experiment, and the rate of uptakecalculated by dividing the mean amount of L-glucose taken up by the time of incubation; the uptakeof D-glucose (O'l mM, 300 mCi/mmol) was measured in the same batch of cells for comparison.

RESULTS

Uptake of 2-deoxy-D-glucose

The time courses of deoxyglucose uptake into cells were usually found to be linearfor 5—10 min, thus allowing the rate of uptake to be calculated by linear regression.The variation of these rates with deoxyglucose concentration obeyed Michaelis-Menten kinetics for most cell lines. This allowed the determination of the kineticparameters of deoxyglucose uptake in a large range of cell lines. Tables 1 and 2 givethe Km and Vmix values for the non-human tumour cells and the non-malignant cellstested. Included in these tables are the parental tumour cells and the diploid cells thatwere fused to give the hybrids investigated.

The Vmax values are highly variable ranging from 4-5 to 495 nmoles/106 cells perh. Comparison of Tables 1 and 2 shows that there is no correlation betweentumorigenicity and a high Vmmx in vitro. Where replicate determinations were made,there was variability in the replicate V™,, values, but less than that between differentcell types. Variation in Vm̂ t is presumably due to factors such as cell density and otherconditions of culture. Some cell types (e.g. YACIR, BALB/c fibroblasts and A/Snfibroblasts) give a reproducible Vmax value.


Table 1. Kinetic constants of hexose uptake in malignant mouse cells

Cell type

PG19

YAC1R

A9HT

SEWA

SEWA

TA3HaB

TA3

TA3

P3/NS/l-Ag4

MSWBS

PG19-G

Cell density(cells/cm2)

8-8 X104

2-5 x 10s

1-2X105

1-5 x 10s

10 x 10s

7-8 x 104

9-7 x 104

Suspension culture

2-1 X 10s

Suspension culture

Ascites

2 0 X 105

Suspension culture

Ascites

Suspension culture

Suspension culture

1-5 X 105

No. ofconcentrations

assayed

6466646

56

5

64

5

5

6

5

6

4

6

Km (miu)

1-104 ±0-0660-608 ±0-1140-992±0-1191-364 ±0-0740-779 ± 0-0840-834 ±0-1621-126 ±0-079

1-154 ±0-2441-101 ±0-033

1-170 ±0-079

0-520 ±0-0730-683 ±0-008

0-369 ±0-100

0-762 ± 0-078

0-377 ±0-042

0-870 ±0-024

1-495 ±0-229

1-378 ±0-268

0-846 ±0-049

Vv majc

(nmol/106 cellsperh)

28-6 ± 0-9961 ± 7-8

108-2 ± 7-883-2± 3-081-7± 4-6

132-5 ±10-860-8 ± 2-6

46-9 ± 6-344-7 ± 0-8

131-4 ± 6-3

200-0 ±20-3494-7 ± 2-6

299-2 ±18-4

291 -2 ±16-7

122-8 ± 5-3

134-4 ± 2-1

47-7 ± 3-2

161-4 ±18-5

50-7 ± 1-8

There is a clear difference in/Cm between the tumour cells and the normal cells. TheKm values of the tumour cells range from 0-37 to 1-49 HIM while those of the non-malignant cells range from 1-61 to 3-69mM. There is no unique Km value for eithermalignant or non-malignant cell types. For example, there are differences in Kmbetween fibroblasts derived from different inbred strains of mouse, which is probablydue to genotypic variation since replicate determinations were made with cells fromdifferent animals. There is reasonable agreement between replicate determinations onthe same cell type indicating the reproducibility of the/C value. The small variationbetween replicates can probably be accounted for by experimental variation.

The question of whether the reduced Km value observed for the tumour cells islinked to malignancy can now be explored by comparing matched pairs of hybrids asdescribed above.

Deoxyglucose uptake by lymphoma X fibroblast hybrids

The hybrids described in this section were derived from clone 1G. This wasproduced by the fusion of YACIR lymphoma cells with CBAT6T6 fibroblasts and hasbeen described previously (Jonaason et al. 1977). The clone originally was non-tumorigenic but, after several weeks cultivation in vitro, generated malignant

M. K White, M. E. Bramwell and H. Ham's

N - b ~ m w - c o c o b v, N W ~ N U ~ W N 2 W v l N

b N - 3 ~ 2 ~~~~ m-. * - ? 8; $ $ 8 s 3 2 0 G Z ?" 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

+I ti +I +I ti +I ti +I ti ti +I +I +I ti ti +I +I ti +I ti +I +I ti +I


segregants, which overgrew the cultures. Early passages of clone 1G were reclonedand the secondary clones tested for tumorigenicity. Two of these secondary clones,1G1 and 1G8, were selected for further study. Clone 1G8 produced no tumours withinocula of 5 X 105 cells per mouse, while clone 1G1 produced tumours in about 90%of the animals with this inoculum. Three such tumours derived from clone 1G1 wereexplanted and grown in vitro (clones 1G1T1, T2 and T3). The kinetic constants ofdeoxyglucose uptake for clone 1G8 and these three tumours are given in Table 3.Hanes (1932) plots of the uptake kinetics of clones 1G8 and 1G1T2 are shown in Figs1 and 2. The Km for deoxyglucose uptake in the three tumours is approximately onethird of that of the non-tumorigenic hybrid 1G8.

When the deoxyglucose uptake kinetics of clone 1G1 were measured, it was foundthat the reciprocal Hanes plots were markedly non-linear (Fig. 3) although thetumours derived from 1G1 gave linear plots (e.g. see Fig. 2). This curvature wasreproducible and there are several possible explanations for it: (a) in addition tofacilitated diffusion, deoxyglucose might enter clone 1G1 cells at a significant rate bynon-carrier-mediated diffusion; (b) clone 1G1 cells might possess two uptake systemswith different Michaelis constants; (c) clone 1G1 might be a mixed population of cellswith differing Michaelis constants for deoxyglucose uptake.

Table 3. Kinetic constants of hexose uptake in malignant and non-malignant lym-phoma X fibroblast hybrids

HybridTumori-genicity


assayedCell density(cells/cm2) Km (mM)

* IIIU


Clone 1G8Clone 1G8Clone 1G8

Clone 1G1

Clone 1G1T1Clone 1G1T2Clone 1G1T2Clone 1G1T3

CloneCloneCloneCloneCloneCloneCloneCloneClone

1G1A1G1E1G1F1G1G1G1G1G1H1G1H1G1J1G1J

Clone lG8aClone lG8bClone lG8c

Clone lG8bTlClone lG8bT2

445

6

6566

665444656

555

66

5-76-44-6

4-75-51-18-9

1-68-92-18-98-82-19-31-9

7-51-56-0

5-26-2

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

104

104

104

3-214 ±0-2553-500 ±1-0203-202 ±0-182

Non-linear reciprocal

104

104

105

104

0-725 ±0-0981-088 ±0-3360-888±01151-063 ±0-157

Non-linear reciprocal105

104

105

104

104

105

104

105

104

105

104

104

104

1-461 ±0-2081-198 ± 0 1 6 51-087 ±0-170l-312±0-1550-666 ±0-1071051 ±0-0970-938 ±0-1981-353 ±0-203

0-851 ±0-0681-413 ±0-2630-835 ±0-047

1-281 ±0-1681-349 ± 0 1 1 0

140-7 ± 8-9294-5 ± 57-2203-3+ 8-8

plot

106-1 ± 7-686-9+13-9

156-2 ± 8-9242-6 ±21-9

plot37-7 ± 3-2631 ± 4-813-5 ± 1-448-0 ± 4-641-6± 3-622-9 ± 0-957-6 ± 6-537-1 ± 3-4

74-8 ± 2-962-8 ± 8-5

1461 ± 4-2

135-9 ± 6-8179-5 ± 8-8

CEI.62

58 M. K. White, M. E. Bramwell and H. HarrisSV

(mM (/imol/10 cells per h)~60-

50-

40-

30-

20-

10

- 5 - 4 - 3 - 2 - 1 0

S(rnM)

Fig. 1. Reciprocal plot of 2-deoxy-D-glucose uptake in the non-malignant hybrid YACIRX CBAT6T6 fibroblast clone 1G8. The x intercept is -Km. Bars equal two standarderrors. Standard error of 5/Vis (S./V?) X VarV,.

sV

(fimol 106 cells per h)~')60-

50-

40-

30-

20-

10-

- 5 - 4 - 3 - 2 - 1 0 1 2 3 4 5S(mM)

Fig. 2. Reciprocal plot of 2-deoxy-D-glucose uptake in the malignant hybrid YACIR XCBAT6T6 fibroblast clone 1G1T2.


SV

(^mol/10° cells per h)"1)60-

50-

40-

30-

20-

10-

- 5 - 4 - 3 - 2 - 1

Fig. 3. Reciprocal plot of 2-deoxy-D-glucose uptake in the malignant hybrid YACIR XCBAT6T6 fibroblast clone 1G1.

The first possibility was tested by measuring the amount of L-glucose (at a con-centration of 0-1 mM) that was taken up by 1G1 cells in 10 min. It was found that theL-glucose uptake was less than 2 % of that of deoxyglucose at the same concentration.This non-specific uptake cannot therefore account for the degree of curvature ob-tained. In order to test whether clone 1G1 was a mixed population of cells, this clonewas recloned to give tertiary clones 1G1A,E,F,G,H and J. When these clones wereassayed, they each gave linear reciprocal plots with the exception of clone 1G1A,which still gave a curvilinear plot. The kinetic constants of the tertiary clones are givenin Table 3. These results support the view that clone 1G1 is a mixed populationcontaining non-malignant cells and malignant segregants. Thus, on recloning, or onselection of the malignant subpopulation by growth in vivo (clones 1G1T1, T2 andT3), linear reciprocal plots with low Km values are obtained.

As no tumours were produced by direct injection of clone 1G8, it was of interestto attempt to select malignant segregants from this clone in order to compare theiruptake kinetics with those of 1G8. To this end, subclones were selected for theirability to grow in agarose, in the hope that such subclones might be enriched for cellscapable of progressive growth in vivo. From three dishes, each seeded with 105 cellsin agarose as described by Steinberg & Pollack (1979), five primary colonies wereobtained, of which three survived isolation and further subculture (clones lG8a, band c). These gave 80-100 % take incidences at 5 X 105 cells per mouse. Hence, threetumorigenic derivatives of 1G8 were prepared, each arising by a separate segregation


event. Table 3 shows the Km and Vmax values for these clones and for tumours derivedfrom them (clones lG8bTl and T2). Both the clones themselves and the tumoursproduced by them have Km values two to three times lower than that of the non-tumorigenic clone 1G8 from which they were derived.

In this cross, the low Km for deoxyglucose uptake characteristic of the malignantparent is associated with tumorigenicity in the hybrids. Tumorigenic derivativesobtained directly by the inoculation of hybrid cells into the animal or indirectly byselection in semi-solid medium systematically show a reduction in Km compared to thenon-tumorigenic hybrids from which they were derived.

Deoxyglucose uptake by melanoma X fibroblast hybrids

Clone 7 and clone 8 are non-tumorigenic hybrids produced by fusion of the PG19melanoma derivative and diploid fibroblasts homozygous for the T13H translocation(Jonasson et al. 1977). The kinetic constants of these clones and a tumour producedfrom clone 8 are given in Table 4. The hybrids in which tumorigenicity is suppressedshow/Cn values that are much higher than the parental PG19 tumour cells; the tumourderived from clone 8 shows a marked reduction in Km compared with that of clone 8itself.

Deoxyglucose uptake by a melanoma derivative selected for resistance to wheat-germagglutinin

Several derivatives of PG19 cells have been produced by selection for their abilityto grow in the presence of the cytotoxic lectin WGA (Bramwell & Harris, 1978a).One such line (C) produced no tumours in 17 animals with inocula of 5 X 104 cellsper animal. This line was recloned and one of the secondary clones, PG19WGARclone C2, was assayed. This non-tumorigenic clone had a Km approximately 2-5-foldhigher than the PG19 cells from which it had been derived (Table 4). The productionof non-tumorigenic clones by selection with WGA provides an additional test for the

Table 4. Kinetic constants of hexose uptake in melanoma X fibroblast hybrids andin a lectin-resistant melanoma derivative

No. of K™,Tumori- concentrations Cell density (nmol/106 cells

Cell type genicity assayed (cells/cm2) Km (mM) per h)

PG19XT13HT13HClone 7 - 6 2 - l x lO 5 2-348 ±0-339 1950 ±16-4

PG19XT13HT13HClone 8 - 4 5-3 x 104 3-587 ±0-572 160-5 ± 18-4

6 7-9 xlO4 2-533 ±0-466 132-5 ± 16-2

PG19XT13HT13HClone 8T1 + 6 2 1 x 10s 1-480 ±0-051 204-3 ± 3-8

PG19WGARClone C2 - 5 2 0 x 105 2-400 ±0-230 173-5 ±11-5


association of markers with malignancy (Bramwell & Harris, 1978a). In this case, lossof tumorigenicity of the PG19 melanoma is associated with loss of the low Km charac-teristic of tumour cells.

Deoxyglucose uptake by fibrosarcoma X lymphocyte hybrids

The tumorigenicity of the malignant L-cell derivative A9HT can be suppressed byfusion with mouse diploid lymphocytes (Wiener et al. 1974a). Assay of two suchsuppressed clones (A9HT X C57B1 lymphocyte clones 3 and 4) showed their Kmvalues to be of the order of 2 HIM (Table 5), while the parental tumour cell had a Kmof the order of 1 mM (Table 1). A tumour derived from another such clone (A9HTX C57B1 lymphocyte clone 2T1) resembled the parent tumour cell A9HT in its Kmvalue (Table 5).

Deoxyglucose uptake by fibrosarcoma derivatives selected for resistance to wheat-germ agglutinin

Selection of A9HT cells for resistance to WGA produced some derivatives that hadlost tumorigenicity and others that had retained it (Chan, 1980). Clones A9HTWCand A9HTWD were both resistant to WGA at a concentration of 25^ig/ml, butwhereas clone WC produced no tumours with inocula up to 6-6 X 106 cells per animal,clone WD remained as tumorigenic as the original A9HT cell line. Table 5 shows thatthe tumorigenic clone WD had a low .Km (0-731 ± 0-087 mM), whereas that of the non-tumorigenic clone WC was more than twice as high (1-685 ± 0-151).

Deoxyglucose uptake by polyoma virus-induced osteosarcoma (SEWA) X fibroblasthybrids

As previously described (Wiener et al. 1971; Jonasson et al. 1977), hybrids be-tween SEWA and diploid cells are highly unstable and generate malignant segregantsat a high frequency, so that the tumorigenicity of the hybrid clones is variable.

Table 5. Kinetic constants of hexose uptake in fibrosarcoma X lymphocyte hybrids andin lectin-resistant fibrosarcoma derivatives

Cell type

No. ofTumori- concentrations Cell densitygenicity assayed (cells/cm2) Km (miu)

(nmol/106

cells per h)

A9HT X C57B1lymphocyte clone 3

A9HTXC57B1lymphocyte clone 4

A9HT X CS7B1

lymphocyte clone 2T1

A9HT WC

A9HT WD

6 2-6 x 10s 2-240 ±0-160 247-6 ±13-2

6 2-5 xlO5 2-080 ±0-120 451-3 ±20-8

5 2-OxlO5 l-123±0-046 119-4 ± 2-8

6 1-8 xlO5 1-685 ±0-151 133-9 ± 7-6

6 1-8 xlO5 0-731 ±0087 120-4 ± 7-9


Table 6. Kinetic constants ofhexose uptake in osteosarcoma X fibroblast hybrids

Hybrid clone

BlBDlDJ2C1T2C1T1FT3J3D3


•3x 10=•2X10=•6X105

1-7 xlO5

'•0x10=1-6x10=1-4 x 10=2-5 x 105

1-3x10=2-0x10=


assayed

56 (5666 ;6666

Km (mM)

•058 ± 0-086)-877 ± 0-0351-390 ±0-0401-909 ±01271-548 ±0-040'•023 ±0-2581-493 ±0-072i-746±0-310

Non-linearNon-linear


167-6± 11-3109-1 ± 2-6115-2± 2-0243-3 ±12-6276-9 ± 4-2295-9 ±23-5377-1 ±12-3249-1 ±29-9

reciprocal plotsreciprocal plots

Table 7. Effect of continuous culture in vitro on the kinetic parameters ofhexoseuptake in osteosarcoma X fibroblast hybrids

Hybridclone

Cl

F

No. of daysin continuous

culture

379

16226

1340


assayed

66645665


2-1 X 105

1-4 x 10s

16X10=2-0x10=2-9x10=1-8x10=2-4 x 10=6-3 xlO4

Km (mM)

2-986 ±0-1402-499 ± 0-2671-896 ±0130l-156±0-1081-420 ±0-3763-218 ±0-5292-220 ±0-0521-363 ±0-092


445-3 ±16-3243-6 ± 19-1354-6 ±17-2129-5 ± 9-4462-6 ±39-0372-1 ±37-6723-0± 11-0240-6 ± 7-4

Hybrids that initially fail to produce tumours become tumorigenic again on cultiva-tion in vitro. Fusion of SEWA with Rb7BnR/Rb7BnR fibroblasts gave nine hybridclones (clones B, Bl, Cl, D, Dl, D3, F, J2and J3). These hybrids were assayed andgave Km values ranging from 0-8 to 2-9 mM (Tables 6 and 7); in some cases thereciprocal plots were curvilinear, as described for YACIR X CBAT6T6 clone 1G1.These clones were obviously heterogeneous, presumably due to varying degrees ofsegregation. Two of the clones (Cl and F), which initially gave high Km values, weregrown continuously in vitro and their Km values determined at intervals. The results(Table 7) show that, on continued cultivation, the Km values fell progressively. CloneF, tested in vivo, initially produced no tumours, but later became tumorigenic. Inaddition, the morphology and cell volume of both clones were altered during thisperiod of cultivation. Thus, these clones appear to be rapidly segregating populationsand this segregation is associated with a fall in Km .

Three tumours produced by inoculation of early passages of these clones were alsoassayed (C1T1, C1T2 and FT3). These tumours gave lower Km values than the cellsinoculated, indicating a selection for lower Km in vivo (Table 6).


Table 8. Kinetic constants of hexose uptake in human fibroblasts

Passage Cell densityCell type no. (cells/cm2)


assayed Km (mm)(nmol/106 cells

perh)

MRC5S1814DS1GM1604WI38

P35P6P4

P27

1-7X4-2 x4-7 x5-0 x5-3 x

104

104

104

104

104

65665

3-210±0-1822-769 ±0-1693-886 ±0-1462-577 ±0-2372-358 ±0-205

552334283169159

•o±•8±•4±•4±•8±

17-914-98-1

14-5120

Table 9. Kinetic constants of hexose uptake in human cancer cell lines

No. Of Vn»*Tumori- concentrations Cell density (nmol/106 cell

Cell type genicity assayed (cells/cm2) Km (miu) per h)

RT112/84HeLa flow

HeLa spinner

HeLa D98F908A3

HeLa D98AH2

H29/219

H.Ep.2

RPMI2650

HT55F

HT115

Daudi

++

+

+

+

+

+

ND*

ND

ND

-

• ND, not determined.

6

56

4

5

6

6

66

6

5

5

5

1-6 xlO5

1-1 x 105

8-6 x 104

1-3 x 105

7-8 x 104

7-4 x 104

7-6 xlO4

1-1 XlO5

1-1 xlO5

7-1 xlO4

3-4 xlO4

5-8 xlO4

1-4 xlO5

Suspension culture

1-630 ±0-1231-271 ±0-1000-984 ±0-219

1-517 ±0-190

1-260 ±0-046

1-285 ±0-033

1-161 ±0-120

1-278 ±0-0511-598 ±0-097

1-303 ±0-151

2-382 ±0-174

0-600 ±0-1401-447 ±0-162

2-342 ±0-150

44-1 ±105-1 ±133-5 ±

83-2 ±

105-3 ±

95-8 ±

2-04-9

13-5

7-9

2-4

1-8

332-2 ±20-0

114-7 ±253-0±37-9 ±

301-0±

16-2±141 ±

43-3 ±

2-18-1

2-9

15-4

2-111

11

Deoxyglucose uptake by malignant and non-malignant human cells

Tables 8 and 9 give the kinetic constants of deoxyglucose uptake for five humanfibroblast cell lines and ten human cell lines of malignant origin. Included in thesetables are the parental cell lines for two series of hybrids that were examined. Figs 4and 5 give examples of the Hanes plots of the human cell lines. As was the case withthe mouse fibroblasts, there is significant variation in the kinetic constants for thedifferent types of human fibroblast. The Km values obtained for the human fibroblastsrange from 2-3 to 3-9 mM. These values are much higher than those obtained for thehuman tumorigenic cell lines, which range from 0-9 to 1-6 mM. The tumorigenicity


Sv

(/imol/106 cells per h)~

24-

20-

16-

4 -

- 5 - 4 - 3 - 2 - 1 0 1 2 3 4 5

S(ITIM)

Fig. 4. Reciprocal plot of 2-deoxy-D-glucose uptake in DSl human foetal fibroblasts.

SV

M (/.mol/106 cells per h)"1) 60-

50-

40-

30-

20-

10"

- 5 - 4 - 3 - 2 - 1 0 1 2 3 4 5S(mM)

Fig. 5. Reciprocal plot of 2-deoxy-D-glucose uptake in the human carcinoma cell lineHeLaD98AH2.


of the human cell lines was assessed by their ability to grow progressively in nudemice. Tumorigenicity in human cells is thus associated with a reduction in the Km fordeoxyglucose uptake.

Four different sublines of the cervical carcinoma cell line HeLa were assayed.These are similar both in Km and Vmax, indicating that the kinetic constants haveremained largely unchanged since these sublines diverged from the original HeLa celllines (Gey etal. 1952).

Two of the cell lines in Table 9 have unexpectedly high Km values (Daudi andHT55F). Daudi has failed to give tumours when injected into nude mice, whileHT55F has not yet been tested for growth in vivo.

Deoxyglucose uptake by human carcinoma X human fibroblast hybrids

Hybrids between 6-thioguanine-resistant human cervical carcinoma derivativesand human diploid fibroblasts have been obtained from two sources. Five hybridsbetween HeLa D98AH2 cells and GM1604 fibroblasts were obtained from E. J.Stanbridge (Der & Stanbridge, 1978, 1981) and four hybrids between HeLaD98F908A3 and S1814 fibroblasts were obtained from H. P. Klinger (Klinger,1980). Both investigators have shown that in these crosses tumorigenicity is stablysuppressed but that rare malignant segregants can be isolated. Tumorigenicity inthese hybrids was tested by inoculation into nude mice (Stanbridge, 1976; Klinger,1980).

Table 10 gives the kinetic constants of deoxyglucose uptake for these hybrids.Comparison of the hybrids in which malignancy is suppressed with their tumorigenicderivatives shows that there is a reduction in Km associated with tumorigenicity.Thus, in these crosses, a low Km value is associated with tumorigenicity.

Effect of viral transformation on deoxyglucose uptake

Since many investigators have compared the hexose uptake of transformed cells invitro with that of their non-transformed counterparts, it was of interest to comparesuch cells in the present assay.

Two pairs of transformed and non-transformed cells were investigated: (a) the ratkidney cell line, NRKTG3, and its Rous sarcoma virus (RSV)-transformed counter-part, 3.B77.Sc4 (Marshall, 1980); (b) the mouse fibroblastic cell line, 3T3 A31 clone9 and its simian virus 40 (SV40)-transformed derivative, SV-3T3-B. In addition, acell line derived from a tumour produced in a nude mouse by SV-3T3-B cells wasassayed (SV-3T3-BT). These results are presented in Table 11. The following con-clusions can be drawn: (1) transformation of NRKTG3 by RSV results in a 2-7-foldincrease in V ^ with no significant alteration in the/Cm; (2) transformation of 3T3 bySV40 results in a two-fold increase in VmiX with no significant alteration in Km; (3) thetumour cell line, SV-3T3-BT, has a significantly lower Km (and Vmax) than the SV-3T3-B cell line from which it was derived.

These data support the observations of other workers (Isselbacher, 1972;Plagemann, 1973; Weber, 1973; Kletzien & Perdue, 19746, 1975; Royer-Pokora etal. 1978) that viral transformation by both RNA and DNA tumour viruses is

is T

able

10.

Gn

etic

con

stan

ts o

f he

xose

upt

ake

in h

uman

car

cino

ma

xfib

robl

ast

hybn

bIs

5;

No.

of

v- 9

Tum

ori-

C

ell

dens

ity

conc

entr

atio

ns

(nm

ol/1

06 c

ells

-'

Hyb

rid

pare

nts

Hyb

rid

geni

city

(c

ells

/cm

2)

assa

yed

Km

(m

~)

Pe

r h)

%

G

M16

04 x

HeL

a D

98 A

H2

4-4-

4 -

8.9

x lo

4 6

1.88

2 f 0

.120

86

.5 f

4.3

?

541E

-

7.7

X 1

0' 6

4.1

13 f 0.

208

217.

4 f

9.3

b

541M

+

7.5

X 1

0' 6

1.49

1 k 0

.049

18

3.1 f

3.9

CG

04

+ 7.

8 x

lo4

6 1.

175 f 0.

078

158.

5 f

6.4

# C

GL

3 7.

7 x

lo4

6 1.

228 f 0

.079

17

3.8 f

5.2

S

+ %

C

C

S181

4 X

HeL

a D

98 F

908A

3 1 A

cn2

-

6.6

X 1

0' 5

2.04

7 f 0.

101

335.

4 f 1

1.5

0

8.8

x lo

4 5

0.89

9 f 0

.061

11

6.5 f

3.3

3

lAcn

lTG

+

4

2BlC

oll

-

6.9

x lo

4 5

3.48

4 + 0

.241

26

5.5 f 1

5.1

5Am

c3

+ 1.

0 X

105

6

1.45

1 f 0

.070

12

4.2

k

4.3

3 2

0 3 h.


Table 11. Effect of viral transformation on the kinetic constants of hexose uptake

No. of V™concentrations (nmol/106

assayed Km (min) cells per h)Cell typeTransformedmorphology


NRK TG33.B77.Sc4 +3T3A31clone9SV-3T3-B +SV-3T3-BT +

7-7 x 104

1-3 x 105

l-7x 105

2-6 x 105

3-3 xlO4

665S6

2-056 ±0-2661-67010-0801-691 ±0-0921-966 ±0-0921-334 ±0082

120-318-8323-818-6122-6 ±2-8248-2 ±9-0101-8±4-2

associated with an increase in Vmax for hexose uptake, but with no alteration in Km .These findings emphasize the now well-documented difference between transforma-tion in vitro and tumorigenicity (Boone, 1975; Stiles, Desmond Jr, Sato & Saier,1975; Gee & Harris, 1979; Straus e* al. 1977; Stanbridge & Wilkinson, 1978).

Uptake of 3-O-methyl-D-glucoseThe uptake of 2-deoxy-D-glucose is determined by a coupled reaction in which the

transport of the hexose is linked to its subsequent phosphorylation. In any givensituation, the rate of uptake may be influenced predominantly by either the transportstep or the phosphorylation reaction (Waley, 1963; Wohlhueter & Plagemann, 1980;Pasternak et al. 1982). The purpose of the experiments described in this section wasto measure transport independently of the phosphorylation reaction. This can be donewith the analogue 3-O-methyl-D-glucose, which is transported into cells but is notmetabolized further (Renner et al. 1972; Weber, 1973). Uptake of 3-O-methyl-D-glucose by most of the cell types investigated was linear for 2 min at 20 °C. Time pointsmeasured at 3 min or longer deviated from linearity. Over the linear portion of theuptake curves, reciprocal plots obeyed Michaelis—Menten kinetics. The kinetic con-stants of the 12 cell types investigated are given in Table 12.

Vman values range from 107 to 413 nmol/106 cells per h and show no correlation withtumorigenicity. There is, however, a clear relationship betweenKm and tumorigenic-ity. Malignant cell types have/Cm values between 1-7 and 3-8 min while non-malignantcell types range from 4-4 to 8-5 miw. The hybrid in which malignancy is suppressed,clone 1G8, has aKm that is two-fold higher than the malignant segregant clone 1G1T1and five-fold higher than the malignant segregant clone 1G1T2 (Fig. 6). Thesuppressed hybrid, lAcn2, has a Km that is three-fold higher than the malignantsegregant lAcnlTG, and the suppressed hybrid, 2BlColl, has zKm that is two-foldhigher than the malignant segregant 5Amc3. The non-malignant WGA-resistantmelanoma derivative PG19WGARC2 has zKm that is 3-5-fold higher than its malig-nant parent PG19.

These data are therefore in accord with those obtained with 2-deoxy-D-glucose.Since 3-O-methyl-D-glucose is not metabolized, the reduction in A'm seen in malignantcells must be due predominantly to an alteration in the transport of hexose across thecell membrane.

Tab

le 1

2. Ki

netic

cons

tant

s of

3- 0

-met

hyl

glu

cose

upt

ake

No.

of

Tum

ori-

C

ell

dens

ity

conc

entr

atio

ns

v- C

ell t

ype

geni

city

(c

ells

/cm

2)

assa

y ed

Km

(m

)

is (n

mol

/ lo6

cells

per

h)

a P

G19

+

1-7

X 1

05

5 2.

114

+ 0.1

72

10

6-6

f 5.

1 1.

4 X

105

4

2.22

1 f

0.46

0 11

6.6 f 1

8.2

s! -

6.6

X 1

0'

5 4.

419 f 0.

520

325.

1 f

26.8

C

BA

T6T

6 fi

brob

last

s: P

3 P4

9.6

x 10

' 5

4.86

8 + 1

.214

42

0.3

+ 90.

8 is

A/S

n fi

brob

last

s W

-

1.2

x 10

5 6

6.56

4 f 1

.000

30

0.4 f

39.7

t?

A9H

T

+ 1.

9 x

105

5 3.

674 f 0.

228

202.

1 f 8

.2

m a

YA

CIR

x C

BA

T6T

6 fi

brob

last

s:

Clo

ne 1

G8

-

1.5

X 1

05

Clo

ne l

GlT

l +

1-9 x

1@

C

lone

1G

lT2

+

7.4

x 10

'

S181

4 x

HeL

a D

98 1

Acn

2 -

1.3

x 10

'

S181

4 x

HeL

a D

98 l

Acn

lTG

+

1.2

x 10

5 4

2.77

1 f 0

.241

10

7.6 f 7

.3

i.

'4

S181

4 X

HeL

a D

98 2

BlC

oll

- 1.

6 X

105

5

6.30

1 f

1.17

0 20

6.0 f

25.5

S181

4 X

HeL

a D

98 5

Am

c3

+ 1.

3 X

10'

6

3.50

0 f 0

.554

15

5.8 f 1

7.8


(rriM (^mol/106 cells per h)"1

1G8

1G1T2

9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1

Fig. 6. Reciprocal plots of 3-0-methyl-D-glucose uptake in the hybrids YACIR XCBAT6T6 fibroblast clones 1G8 and 1G1T2.

(mM (pmol/nl per min)

MSWBS

-5 - 4 -3 -2 -1

Daudi

Fig. 7. Reciprocal plots of D-glucose uptake in the cell lines MSWBS, Daudi and SEWA.


The Km for 3-O-methyl-D-glucose in any one cell type is higher than the Km for2-deoxy-D-glucose. This has been reported by other investigators in other cell types(Renneretal. 1972; Weber, 1973; Kletzien & Perdue, 1974a,b,c; Graff, Wohlhueter& Plagemann, 1978). The presence of substituents on the 3' position oxygen ofglucose has been shown to decrease its affinity for the erythrocyte glucose transporter(Barnett, Holman & Munday, 1973). This might also account for the higher Km for3-O-methyl-D-glucose seen in cells in culture. The Vmax values for the two hexoses,2-deoxy-D-glucose and 3-O-methyl-D-glucose, lie in the same range and show somedegree of correlation in any one cell type. Indeed, parallel cultures of PG19 assayedfor the uptake of both hexoses on the same day gave a V ^ value for 2-deoxy-D-glucoseof 132-5 ± 10-8 (Table 1) and a VnViX value for 3-O-methyl-D-glucose of 116-6 ± 18-2(Table 12). These values are not significantly different.

Hexokinase activity

If, as our results show, the kinetics of hexose transport in the cells we have studiedare determined predominantly by the transport component of the coupled reactionand not the phosphorylation component, one would expect that the rate at which thecell is able to phosphorylate the transported sugar would substantially exceed the rateat which the sugar is transported across the cell membrane. In this section we reportmeasurements we have made on the hexokinase activities of homogenates of most ofthe cell types in which we have studied 2-deoxy-D-glucose uptake (Kletzien & Perdue,1974a). These activities are expressed as V ^ values of the enzyme per 106 cells andVmax values per milligram of protein. Since there is some variation between differentcell types in the amount of protein per cell, the two sets of values are not interchange-able.

Table 13. Hexokinase activities of malignant and non-malignant mouse cells

Hexokinase activityCell type (nmol/106 cells per h) (/imol/mg protein per h)

516

5-04

2-57

11-245-184-72

5-48

7-44

3-29

2-65

3-41

4-39

5-93

PG19

YACIR

SEWA

A9HT

TA3HaB

PG19-G

CBAT6T6 fibroblasts P2

A/Sn fibroblasts P6

A/Sw fibroblasts P2

A/Sn X BALB/c fibroblasts P7

PG19WGAR clone C2

9541302

470

2052

15181080

497

2108

1631

1374

882

1096

960


Mouse cells. The Vmax for hexokinase in mouse tumour cells and mouse fibroblastsare given in Table 13. (Where replicate values for a cell type are given, determinationswere done on samples derived from different cell cultures.) Comparison of thesevalues with the Vmax values for deoxyglucose uptake (Tables 1 and 2) shows that theVWx for hexokinase is between 5-5 and 42-fold higher than for deoxyglucose uptake.(TA3HaB is exceptional in having a hexokinase Vmax only 1-7-fold higher.) Further-more, there is no correlation between hexokinase activity and tumorigenicity. Valuesfor malignant cells vary between 470 and 2108nmol/l06 cells per h while values fornormal cells vary between 882 and 1631 nmol/106 cells per h. The non-malignantWGA-resistant PG19 derivative, PG19WGAR clone C2, has a similar hexokinaseactivity to its malignant parent, PG19.

Hybrid mouse cells and lectin-resistant derivatives of malignant mouse cell lines.Table 14 gives the hexokinase Vmax values for some of the hybrid mouse cells andthe lectin-resistant mouse cell lines in which 2-deoxy-D-glucose uptake has beenstudied. These values are much higher than the respective Vmax values for 2-deoxy-D-glucose uptake. There is no correlation between hexokinase activity and tumori-genicity.

Human cells. The hexokinase activity of five tumorigenic human cell lines andthree types of non-tumorigenic human fibroblast were assayed (Table 15). There isno correlation between hexokinase activity and tumorigenicity. The hexokinase Vmaxin the human cell types is 2 to 26-fold higher than the Vmax for deoxyglucose uptakein the same cells (Table 9).

Human carcinoma X fibroblast hybrids. As described above, the kinetics of2-deoxy-D-glucose uptake have been investigated in nine hybrids between the HeLa

Table 14. Hexokinase activities of hybrid mouse cells and lectin-resistant mouse cellderivatives

Cell type

YACIR X CBA6T6 fibroblastsClone 1G8Clone 1G1T1Clone 1G1T2Clone 1G1T3Clone lG8aClone lG8bClone lG8c

A9HT x C57B1 lymphocyteClone 3Clone 4Clone 2T1

A9HTWC

A9HTWD

Tumori-genicity

—++++++

-—+

-

+

(nmol/106

cells per h)

966120613561854138621062238

85815841080

528

1296

V™,(jUmol/mg

protein per h)

2-816133-676142-631-843-56

4-763-225-20

3-02

5-45

72 M. K. White, M. E. Bramivell and H. Harris

Table 15. Hexokinase activities of human cells

Hexokinase activityCell type Tumorigenicity (nmol/106 cells per h) (/imol/mg protein per h)

H.Ep.2 + 616 2-24H29/219 + 756 1-98RT112/84 + 990 4-46HeLa flow + 1302 3-58HeLaD98AH2 + 1020 2-93HT115 ND* 396 1-27DS1 P5 - 678 3-74MRC5 P36 - 2490 5-03GM1604 - 786 1-31

• Not determined.

Table 16. Hexokinase activities of human carcinoma X fibroblast hybrids

Hexokinase activityTumori- (nmol/106 (jimol/mg

Hybrid parents Hybrid genicity cells per h) protein per h)

GM1604xHeLaD98AH2 4-4-4541E541M +CG04 +CGL3 +

S1814xHeLaD98 F908 A3 lAcn2lAcnlTG +2BlColl5Amc3 +

carcinoma cell line and human diploid fibroblasts (Table 10). The hexokinase activityof each of these hybrids was measured and is given in Table 16. Again, the hexokinaseVma-x value of each cell type was larger than that for the uptake of 2-deoxy-D-glucose.There is an apparent association between tumorigenicity and hexokinase activity inthis set of cells when hexokinase activity is expressed in nmol/mg protein per h, butnot when it is expressed in nmol/106 cells per h. This disparity arises because thesuppressed hybrids have a higher protein content per cell (0-42-0-48 mg protein/106 cells) than the malignant segregants (0-22-0-35 mg protein/106 cells). The higherprotein content of the hybrids in which malignancy was suppressed was peculiar tothis set of hybrids.

While hexokinase measurements in homogenates may not reflect hexokinaseactivities in the intact cell, it is, in any case, clear that hexokinase activity as measureddoes not correlate with tumorigenicity and that, in general, the amount of hexokinaseavailable greatly exceeds what would be required to phosphorylate the hexose trans-ported across the cell membrane.

1032768708936960

529599840786

2-311-603-153-042-90

1-261-711-962-79

Tab

le 1

7. k

i'net

ic c

onsl

ants

of

glu

cose

upt

ake

by d

tffe

rent

cel

l t-y

pes

No.

of

v- 2

conc

entr

atio

ns

2 C

ell t

ype

Tum

orig

enic

ity

Km

(m

~)

(p

mol

/nl

per

min

) (n

mol

/l@

cel

ls p

er h

) as

saye

d 8 m

SEW

A

+ 1.

060 f 0

.205

17

.90 f 2

.23

890.

5 + 1

10.9

5

.. a Y

AC

IR

+ 1.

030 + 0

.096

6.

06 f 0

.37

109.

1 f

6.6

A9H

T

+ 0.

756 f

0.12

7 1.

12 f 0

.18

70

.6f

7.4

5

PG

19

3-

+ 0.

490 f 0

.026

0.

63 f 0

.04

49

.1+

3.

1 4

1.25

0 + 0

.380

2.

84 f 0

.60

212.

5 f

46.4

4

s 3.

a

MSW

BS

+ 0.

683

+ 0.0

83

0.84

k 0

.05

78.3

f

4.5

5 -

B

Dau

di

1.95

0 f 0

.340

2.

48 f 0

.50

96.0

f

13.4

4

2

C57

BI

fibr

obla

sts

P6

-

2.33

2 f 0

.237

14

.80 f 1

.34

10

84

.4f

98-2

4


Uptake of D-glucose

D-Glucose is the physiological substrate of the hexose transport system of animalcells and therefore the ideal compound for the measurement of uptake rates. How-ever, it is rapidly metabolized to compounds such as lactate and carbon dioxide, whichmay leave the cell, so that measured uptake rates may be severe underestimates of thetrue rates (see Plagemann & Richey, 1974). However, this problem can be in largepart circumvented by the use of very short incubation times (less than 30 s). This canbe achieved by the silicone oil filtration centrifugation technique of Werdan et al.(1980). This method was used to measure the kinetics of D-glucose uptake in severalcell types. In most experiments, linear uptake was maintained for up to 30 s. Theuptake rates at different D-glucose concentrations were used to determine the Km andVmax values, as described above for 2-deoxy-D-glucose and 3-O-methyl-D-glucoseuptake. The results are presented in Table 17.

Comparison of the Km values obtained for D-glucose with those for 2-deoxy-D-glucose shows that there is good agreement. The tumorigenic cell types have Km

values in the range 0-49 to 1-25 ITIM while the non-tumorigenic cells have Km valuesin the region of 2 mM. Werdan et al. (1980) obtained a Km of 2mM for human diploidfibroblasts by this method. The Vmax values obtained by this method are of the sameorder as those obtained with 2-deoxy-D-glucose. It thus appears that, in our assay,D-glucose and 2-deoxy-D-glucose behave similarly. Indeed, with YACIR cells, thetime course of uptake of 2-deoxy-D-[l-14C]glucose was found to be identical to thatobtained with D-[U-14C]glucose at the same concentration (data not shown). Further-more, since 2-deoxy-D-glucose is not metabolized beyond the phosphorylation step,these data indicate that metabolic conversion does not cause D-glucose uptake ratesto be underestimated in the present assay. This conclusion is reinforced by experi-ments in which glycolysis was inhibited with sodium fluoride. YACIR cells, pre-incubated with fluoride under conditions in which glycolysis was inhibited by 90%,gave uptake time courses identical to those given by untreated cells.

The silicone oil filtration centrifugation method was also used to measure theuptake of L-[l-14C]glucose, which is not a substrate for the hexose transport systemand is thus only able to enter cells by the much slower process of simple diffusion. Theuptake of this sugar was so slow that incubations of 1—3 h were necessary to achievesignificant uptake. Table 18 shows the results obtained and, for comparison, the rate

Table 18. Relative rates of uptake of L-glucose and D-glucose

Cell type

SEWAPG19MSWBSA9HTDaudiYACIR

Rate of L-glucose uptake(fmol/nl per min)

0-73 ±0-181-93 ±0-180-43 ±0-640-46 ±0-060-20 + 0-050-20 ±0-05

Rate of D-glucose uptake(fmol/nl per min)

1600 ±62220 ±24110 ± 14124 ± 9123 ± 7546 ±44


of D-glucose uptake at the same concentration (0-1 mM) with the same cell suspension.It is clear that L-glucose enters cells at a rate that is less than 1 % of that of D-glucose.Simple diffusion does not therefore contribute significantly to hexose uptake.Furthermore, these data demonstrate the stereospecificity of the glucose transporterof the cell membrane and the suitability of L-glucose as a control for trapping ofextracellular fluid.

DISCUSSION

In the present study the kinetics of hexose uptake were studied in 86 different celllines, and, without exception, the ability of cells to grow progressively in vivo waslinked to a reduction in the Km for hexose transport. This change in Km reflects achange in the transport system itself and is not determined by the subsequent metabol-ism of the hexose. Previous studies have shown a similar inverse correlation betweentumorigenicity and the Km of glucose uptake, but these studies did not dissociatetransport from metabolism (Burk, Woods & Hunter, 1967; Hatanaka, 1971). Howmight the alteration in Km that we have described be brought about?

One possibility is that the hexose transport protein has undergone mutation in a waythat increases its affinity for hexose. Whitfield et al. (1982) have isolated a mutantfrom CHO cells that is resistant to the cytotoxic effects of 3-0-methyl-D-glucose andhas a twofold lower Km for hexose transport. This result demonstrates that a lowerKm can be generated by a mutational event, but it remains unlikely that simplemutations in the hexose transport protein would generate a wide range of moleculeswith different degrees of increased affinity for hexose, as evidenced by the wide rangeof malignant cells examined. Another possibility is that there might be polymorphicforms of the hexose carrier protein with different Km values for hexose transport.Malignancy would then be associated with an increased expression of low/Cm variantsof the hexose transporter. A shift towards low Km isoenzymes has been observed forseveral glycolytic enzymes in transformed cells (G. Weber, 1974). Changes in Km

values might also be produced by secondary changes in the hexose transporter protein,for example, by glycosylation (Bramwell & Harris, 1978a; Warren, Buck & Tuszyn-ski, 1978) or phosphorylation. And finally, changes in the hexose transport Km mightbe produced by more general modifications of the cell membrane, which might affectnot only the transport of hexose but also of other molecules. Such modificationsmight, for example, be brought about by alterations in the lipid composition of themembrane. Itaya, Hakamori & Klein (1976) have described changes in membranelipid composition associated with malignancy in somatic cell hybrids; Yuli, Wilbrandt& Shinitzky (1981) have shown that the hexose transport system is sensitive to thelipid composition of the cell membrane; Pilch, Thompson & Czech (1980) haveproduced evidence that, in the rat adipocyte, changes in the phospholipid micro-environment of the glucose transporter might be involved in its response to insulin.Of these various possibilities we are most attracted by the notion that changes in theglycosylation of cell membrane proteins might induce changes in the Km of the hexosetransport process. Numerous authors have shown that the pattern of glycosylation of


membrane proteins is altered in transformed cells (see, for example, Warren et al.1978), and Atkinson & Bramwell (1980a,6) have shown that cell surface sialic acidcontent and sialyltransferase activity co-segregate with malignancy in hybrids be-tween malignant and non-malignant cells. A systematic change in the glycosylationof a membrane protein that appeared to be involved in some way in glucose transporthas also been shown to co-segregate with malignancy in hybrid cells (Bramwell &Harris, 1978a,6; Bramwell, 1980; Bramwell & Atkinson, 1982). Some preliminaryexperiments that we have done with tunicamycin indicate that this antibiotic, inconcentrations that largely inhibit the dolichol phosphate-mediated glycosylation ofmembrane proteins, produces changes in the Km for hexose transport.

We have now to consider what relevance the reduction in Km for hexose transportmight have to the biology of cancer cells. Since a reduction in Km entails that thedifference between malignant and non-malignant cells becomes more pronounced atlower external hexose concentrations, it is not difficult to see how this might confera selective advantage on malignant cells in vivo. The cell density in a primary tumournodule is high and the blood supply precarious, so that the availability of hexose couldeasily be limiting. This has indeed been shown to be the case for some ascitic tumours(Del Monte & Rossi, 1963). A decrease in the A'm of the hexose transport system mightthus make the malignant cell a more effective scavenger of whatever hexose is availablethan the normal cells with which it must compete. This does not necessarily mean thatthe change in hexose transport is the primary determinant of the malignant state,although some authors have argued that an increase in the activity of one or moretransport proteins might control cell proliferation (Holley, 1972; Bhargava, 1977).

We thank Dr Harold P. Klinger, Department of Genetics, Albert Einstein College of Medicine,Bronx, N.Y., U.S.A. and Dr Eric J. Stanbridge, Department of Microbiology, College ofMedicine, University of California, Irvine, Calif., U.S.A. for the gift of human hybrid cell lines;Professor J. F. Watkins, Department of Medical Microbiology, The Welsh National School ofMedicine, Heath Park, Cardiff; Dr L. M. Franks, Imperial Cancer Research Fund Laboratories,Lincoln's Inn Fields, London and Dr C. J. Marshall, Chester Beatty Research Institute, Instituteof Cancer Research, Royal Cancer Hospital, Fulham Road, London for the gift of cell lines; Dr S.J. Goss for his advice on the statistical aspects of the work; and Mrs Ruth Hennion and Mr StephenGreig for skilful technical assistance. The work was supported by the Cancer Research Campaign,of which M.E.B. is the James Hanson Fellow. M.K.W. was in receipt of a Medical Research CouncilScholarship for Training in Research Methods.

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(Received 20 December 1982 -Accepted 20 December 1982)

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