alterations induced by glucose deprivation ...hexose transport in hybrid cells 261 of h (defined as...

J. Cell Sd. 68, 257-270 (1984) 257Printed in Great Britain © The Company of Biologists Limited 1984

ALTERATIONS INDUCED BY GLUCOSEDEPRIVATION AND TUNICAMYCIN IN THE KINETICPARAMETERS OF HEXOSE TRANSPORT IN HYBRIDCELLS

M. K. WHITE, M. E. BRAMWELL AND H. HARRISSir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford0X1 3RE, U.K.

SUMMARY

Matched pairs of malignant and non-malignant hybrid cells were compared in their response toglucose deprivation and to tunicamycin. Glucose deprivation induced an increase in the maximumvelocity in the malignant cells, but not in the non-malignant cells. The Michaelis constant of hexoseuptake was largely unchanged by glucose deprivation except in the case of one melanoma derivative,PG19 G—, which showed a large increase in Michaelis constant when deprived of glucose.Tunicamycin increased the Michaelis constant of hexose uptake in both malignant and non-malignant cell lines. It is therefore possible that the Michaelis constant of hexose uptake is affectedby the extent of glycosylation of one or more of the cell membrane glycoproteins

INTRODUCTIONIn an earlier paper (White, Bramwell & Harris, 1983) it was shown that there was

a systematic association between the ability of cells to grow progressively in vivo anda reduction in the Michaelis constant for hexose transport. In the present paper wedescribe experiments designed to probe this phenomenon further. We have examinedthe effect of glucose starvation on the parameters of hexose transport and the effectof blocking the dolichol pyrophosphate-mediated glycosylation of glycoproteins bytunicamycin. Glucose deprivation has frequently been shown to result in an enhance-ment of hexose uptake in cultured cells, and this phenomenon has been called'deprivation derepression' (Martineau, Kohlbacher, Shaw & Amos, 1972). Whenfibroblasts in culture are deprived of glucose for 16-24 h, large increases in theapparent rates of glucose uptake have been reported (Martineau et al. 1972;Demetrakopoulos & Amos, 1976; Christopher, Kohlbacher & Amos, 1976). How-ever, subsequent work has shown that this effect is largely due to changes in glucosemetabolism rather than glucose transport (Salter & Cook, 1976; Musliner, Chrousos& Amos, 1977). The enhancement of glucose transport, as measured by uptake of thenon-metabolizable analogue, 3-0-methyl-D-glucose, appears to be about twofold(Salter & Cook, 1976). Most previous studies on the effects of glucose deprivationhave been done with confluent cultures of fibroblastic cells. These have low rates ofhexose uptake, and the enhancement seen on glucose deprivation is due at least in partto a reversal of the reduction in hexose uptake associated with density-dependent

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

inhibition of growth. Rapidly multiplying cultures of fibroblastic cells show a muchsmaller enhancement of hexose uptake (Kalckar & Ullrey, 1973; Kletzien & Perdue,1975; Musliner et al. 1977). In the present study, the complications produced bysubsequent metabolism of glucose and by density-dependent inhibition of hexosetransport were avoided by measuring uptake with the non-metabolizable analogue, 2-deoxy-D-glucose, and by using rapidly growing cell cultures.

Numerous studies have described alterations in the patterns of glycosylation ofmembrane proteins in malignant cells. It was therefore of interest to explore whetherchanges in the Km of the hexose transport system could be brought about by inter-ference with the normal process of glycosylation of membrane proteins.Tunicamycin, which interferes with the dolichol pyrophosphate-mediated glycosyla-tion of asparaginyl residues in glycoproteins (Hubbard & I vatt, 1981), has been shownto inhibit several transport systems in chick embryo fibroblasts (Olden, Pratt, Jowar-ski & Yamada, 1979). In the present paper we describe the effects of tunicamycin onthe parameters of hexose transport in both malignant and non-malignant cells.

MATERIALS AND METHODS

Cell lines

MousePG19: hypoxanthine-guanine phosphoribosyl transferase-deficient (HGPRT) derivative of aspontaneous melanoma arising in a C57BL mouse (Jonasson, Povey & Harris, 1977).

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

YACIRXCBAT6T6 clone 1G8: non-malignant hybrid produced by fusion of YACIRlymphoma cells with CBAT6T6 fibroblasts (Evans et al. 1982).

YACIRXCBAT6T6 clone 1G1T2: malignant hybrid produced by fusion of YACIRlymphoma cells with CBAT6T6 fibroblasts (Evans et al. 1982).

Human

MRC5: fibroblast strain from lung of male foetus of 4 months gestation (Jacobs, Jones & Bailie,1970).

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

HeLa D98 F908 A3 X S1814 clone 2B1 Col 1: non-malignant hybrid between a HeLa derivativeand human fibroblast line S1814 (Klinger, 1980).

HeLa D98F908 A3 x S 1814 clone 5AMC3: malignant hybrid between a HeLa derivative andhuman fibroblast line S1814 (Klinger, 1980).

Glucose deprivationMonolayer cultures of cells were harvested by trypsinization, resuspended in Eagle's modified

minimal essential medium (MEM), and distributed into 96 16-mm tissue-culture wells (Costar,Cambridge, Mass, U.S.A.) in a volume of 1 ml per well. Between lXlO5 and 3xlOs cells were

Hexose transport in hybrid cells 259

added to each well. After 24 h growth at 37 °C, a deoxyglucose uptake assay was done with 48 of thewells by the method described by White, Bramwell & Harris (1983).

The medium was aspirated from each of the remaining 48 wells and replaced by 1 ml of Eagle'smodified minimal essential medium without added glucose (MEM—G) supplemented with 5%foetal calf serum. MEM—G prepared in this way contained 40/ig/ml D-glucose as measured by aGlucose Test Combination assay kit (Boehringer Mannheim, East Sussex, U.K.). After a further24 h growth at 37 °C, these wells were assayed for deoxyglucose uptake.

Experiments in which glucose was completely absent from the medium were done as describedabove, except that the MEM —G was supplemented with 5% foetal calf serum that had beendialysed for 24h at 4°C against 200vol. of phosphate-buffered saline (PBS), changed once after12 h. The dialysed foetal calf serum was sterilized by Millipore filtration. This medium containedno detectable glucose.

Growth in the presence of glucoseIn these control experiments, glucose was present at the normal concentration during the second

24 h of growth, that is, MEM supplemented with 5 % foetal calf serum replaced MEM—G.

Addition of glucose to glucose-starved culturesPG19 G - can be grown in M E M - G supplemented with 5 % foetal calf serum (Bramwell, 1980).

The starvation protocol could thus be reversed by changing the medium to MEM during the second24 h.

Tunicamycin

Cells were distributed into tissue-culture wells as above and grown for 24 h at 37 °C. Some of thewells were then assayed for deoxyglucose uptake. Tunicamycin (Calbiochen-Behring Corporation,La Jolla, Calif., U.S.A.) was added to the remaining wells to give a concentration of 0-1-5/ig/ml.After a further 24 h growth at 37 °C, in the presence of tunicamycin uptake of deoxyglucose was againmeasured. In experiments where the effect of tunicamycin on the Km and Vm^x of deoxyglucoseuptake was to be investigated, the tunicamycin concentration was used at a concentration of 2 fig/ml.

RESULTS

Effect of glucose deprivation on non-malignant cells

The effect of glucose deprivation was investigated in fibroblasts from threedifferent inbred strains of mouse, chick embryo fibroblasts, human fibroblasts, andone mouse and one human hybrid cell line in which malignancy was suppressed.The results are given in Table 1. Comparison of the cell density before and afterdeprivation shows that the cells grew during the experimental period. The kineticparameters of deoxyglucose uptake before and after starvation are also given inTable 1 and Hanes (1932) plots of the kinetics of uptake for the cell line 2B1 Col1 are shown in Fig. 1.

There was no large change in Vm^ on glucose deprivation. Values for an inductionratio 7V (denned as the ratio of the Vmix after glucose deprivation to that beforeglucose deprivation) are given in Table 2, and changes in Vm»x significant at theP<0-05 level are indicated. The /v values of the non-malignant cells ranged from0-68 to 1-31. Thus none of the non-malignant cell lines showed a change in Vmaxof more than about 30%.

Similarly, glucose deprivation, produced no large change in the Km of deoxyglucoseuptake. However, some cell lines did show a small but significant reduction. Values


of h (defined as the ratio of the Km after deprivation to that before deprivation) varybetween 0-6 and 1-0, thus indicating that the Km fell by up to 40% on glucosedeprivation.

S/V

(mM(jimol/106 cells p«r h)"1)

24-

20-

16-

4-

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

Fig. 1. The effect of glucose deprivation on the kinetics of deoxyglucose uptake by a non-malignant hybrid cell line, 2B1 Col 1.

Table 2. Induction ratios for glucose deprivation

Cell typeTumori-genicity IK P<5% P<5%

A/Sn fibroblasta P4Balb/c fibroblasts P6CBAT6T6 fibroblasts P4Chick embryo fibroblasts P4 —Clone 1G82B1 Col 1MRC5 fibroblasts P35

PG19 +PG19G- +Clone 1G1T2 +5Amc3 +H.Ep.2 +

0-812 ±0-1531-004±0-1160-803 ± 0-0930-815 ±0-0620-689 ±0-0690-675 ± 0-050-599 ±0-054

0-863 ±0-1472152± 0-2101006±0-1721-278 ±0-1671-104±0-195

0-770±0-lll1-058 ±0-0861-310 ±0-109 +

+ 1-257 ±0-073 ++ 0-684 ±0-046 ++ 0-950 ±0-059+ 0-687 ±0-033 +

2-214 ±0-213 ++ 7-557 ±0-441 +

3-166±0-223 +l-606±0-128 +2-004 ±0-197 +


Effect of glucose deprivation on malignant cells

The effect of glucose deprivation, was investigated in five malignant cell types.These cells also continued to grow during the period of glucose deprivation. Thekinetic parameters of deoxyglucose uptake before and after glucose deprivation arelisted in Table 3, and examples of Hanes plots for two of the malignant cell lines areshown in Figs 2 and 3.

The Vmtx of all the malignant cell lines was increased by glucose deprivation(/v= 1-6—7-6). Thus, in the malignant cells, the response to glucose deprivation ismuch more marked than in the non-malignant cells.

There was no significant effect of glucose deprivation on the Km of deoxyglucoseuptake (A = 0-86-1-28) for the malignant cell lines, except in the case of themelanoma derivative PG19 G— (A = 2-2). This finding is of interest because this cellline also shows an exceptional pattern of membrane glycoprotein glycosylationassociated with the glucose deprivation (Bramwell, 1980). The possibility ofglycoprotein glycosylation affecting the Km of hexose transport is discussed later.

Since PG19 G— can be maintained in low glucose medium (Bramwell, 1980), thereverse experiment could also be done. Table 4 shows that a shift from glucosedeprivation to normal levels of glucose reverses -the effects described above on bothKm and Vmax . Both Km and VmiX fell on addition of glucose.

S/V(mM(/imol/106 cells per h)"1)

60

50

40

30

20

10

(+Glucose)T

(-Glucose)

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

Fig. 2. The effect of glucose deprivation on the kinetics of deoxyglucose uptake by themurina melanoma cell line, PG19.


s/v(mM(/*mol/106 cells per h) ')

120-

100-

80-

60-

40-

20-

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

Fig. 3. The effect of glucose deprivation on the kinetics of deoxyglucose uptake by themurine melanoma cell line PG19 G—, which was selected for growth in low levels ofglucose.

Effect of complete absence of glucose

Preliminary experiments showed that complete removal of glucose from themedium for 24 h resulted in extensive cell death in most cell lines. For this reason,complete deprivation of glucose was not used routinely. However, results were ob-tained for one malignant and one non-malignant cell line (Table 5). The cell densitymeasurements before and after removal of glucose show that little growth of PG19cells occurred in the absence of glucose, and there was some cell death in the fibroblastpopulations. The effect of complete deprivation of glucose on the kinetic parametersof both cell lines is essentially the same as that obtained with the usual protocol, i.e.a 2-4-fold increase in Vmax with no change in Km for PG19, and little effect on eitherVmax or/Cm for CBAT6T6 fibroblasts.

Effect of growth in normal concentrations of glucose

Table 6 gives the results of control experiments in which cells were not deprivedof glucose during the second 24 h of growth. Comparison of the kinetic parameters onday 1 with those on day 2 for CBAT6T6 fibroblasts and PG19 cells shows that thereis no significant alteration in either Km or Vmax . These controls show that in the presentexperiments factors such as cell density do not alter the kinetic parameters of uptakeduring the second day of growth.


S/V

(mM(/*mol/10 cells per h)~

60-

50-

40-

30-

+Tunicamycin

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

Fig. 4. The effect of tunicamycin on the kinetics of deoxyglucose uptake by the H.Ep.2cell line.

Effect of tunicamycin

Tunicamycin at concentrations between 0-5/ig/ml and 5/ig/ml has been repor-ted to inhibit glycosylation of N-linked glycoproteins (Tkacz & Lampen, 1975).This was confirmed in our hands by measuring the binding of lectins toglycoproteins extracted from cells, and also by the incorporation of tritiatedglucosamine into glycoproteins, in a variety of cell lines. Preliminary experimentsshowed that tunicamycin inhibited deoxyglucose uptake by various cells in culturewhen uptake was measured at a single deoxyglucose concentration (Ol miw). Dose-response curves for this effect were measured for A/Sn fibroblasts and H.Ep.2 cellswith tunicamycin concentrations ranging from 0-1 to 5/ig/ml. These showed that50% inhibition of deoxyglucose uptake occurred at about 2/ig/ml of the antibiotic.Maximal inhibition (about 70%) occurred at about 5/ig/ml. Above this concentra-tion, extensive cell death occurred.

The effect of tunicamycin on the kinetic parameters of deoxyglucose uptake wasmeasured at a tunicamycin concentration of 2/ig/ml, and the results are given inTable 7. In the three cell types investigated, inhibition of deoxyglucose uptake wasassociated with an approximately twofold increase in Km, and, also, in the caseof A/Sn fibroblasts and H.Ep.2, a decrease in Vrruu • The effect of tunicamycinon the kinetics of deoxyglucose uptake in H.Ep.2 cells is shown in Fig. 4.

These observations support the thesis that glycoprotein glycosylation is necessary


for the normal functioning of membrane transport proteins (Olden et al. 1979) andsuggest that the Michaelis constant for hexose uptake may be modulated by thecarbohydrate moiety of one or more glycoproteins.

DISCUSSION

Earlier work in this laboratory demonstrated an association between malignancy anda reduction in the Michaelis constant for hexose transport across the plasma membrane(White, Bramwell & Harris, 1981, 1982, 1983). Depriving cell cultures of glucose hasrevealed a second difference in hexose uptake between malignant and non-malignantcells. Malignant cells showed a large increase in the V^a of deoxyglucose uptake afterglucose deprivation, whereas non-malignant cells did not. This difference co-segregated with malignancy in a matched pair of mouse and human somatic cell hybrids.

Variations in the Vnum of membrane transport processes are usually ascribed toalterations in the number of transporter molecules present on the cell surface. Malig-nant cells thus appear to be able to mobilize more transport molecules in response todeprivation of glucose, whereas this effect is not seen in non-malignant cells. It hasalso been observed in this laboratory that expression of an antigen defined by themonoclonal antibody M/27 behaves in the same way (Gingrich, Wouters, Bramwell& Harris, 1981a,fc); and there is evidence to link this antigen with glucose transport(Gingrich et al. 19816; Banyard & White, 1984). The inducibility of hexose uptakecapacity in malignant cells may confer a selective advantage in vivo. Although theeffect has so far been studied in only a few cells, it is nonetheless very interesting andworthy of further work.

Demetrakopoulas, Linn & Amos (1978) reported that early-passage normal diploidcells were able to maintain their intracellular ATP content when starved of a carbonsource, whereas malignant cells suffered a dramatic rapid drop in ATP content. It ispossible that this effect may be related to the observations reported here, since it hasbeen reported that agents that deplete intracellular ATP levels (e.g. dinitrophenol)can mimic the induction of hexose uptake caused by glucose starvation (Kalckar,Ullrey & Laursen, 1980; Bader, Brown & Ray, 1981). These data thus support thehypothesis that ATP content may regulate hexose transport.

The inhibition of hexose uptake by tunicamycin suggests that glycoproteinglycosylation can affect the Km of hexose uptake. In the light of previous findings(Bramwell & Harris, 1978a,b; Atkinson & Bramwell, 1980a,b), it is possible thatdifferences in glycoprotein glycosylation between malignant and non-malignant cellsare associated with the difference in Km of hexose transport. Further support for thishypothesis is provided by the shift in Km caused by glucose deprivation in the cell linePG19 G —. This cell line is unique in that the extent of glycosylation of a membraneglycoprotein that has been implicated in glucose transport (Bramwell & Harris,I978a,b) is modulated by the glucose concentration in the growth medium (Bram-well, 1980). It has been shown that glucose starvation may alter the pattern ofglycoprotein glycosylation in other systems (Stark & Heath, 1979; Bailey, Burke,Sinclair & Mukherjee, 1981; Rearik, Chapman & Kornfeld, 1981).


We thank Dr H. P. Klinger, Department of Genetics, Albert Einstein College of Medicine,N.Y., U.S.A. for the human hybrid cell lines, and Mrs S. Humm and Mr S. Greig for technicalassistance. The work was supported by the Cancer Research Campaign of which M.E.B. is theJames Hanson Fellow. M.K.W. was in receipt of a Medical Research Council Scholarship forTraining in Research Methods.

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{Received 9 January 1984-Accepted 16 January 1984)

alterations induced by glucose deprivation ...hexose transport in hybrid cells 261 of h (defined as...

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