subsequent metabolism of glucose and by density-dependent inhibition of hexose transport .... of h (defined as the ratio of the Km after deprivation to that before ...
J. Cell Sd. 68, 257-270 (1984)
257
Printed in Great Britain © The Company of Biologists Limited 1984
ALTERATIONS INDUCED BY GLUCOSE DEPRIVATION AND TUNICAMYCIN IN THE KINETIC PARAMETERS OF HEXOSE TRANSPORT IN HYBRID CELLS M. K. WHITE, M. E. BRAMWELL AND H. HARRIS Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford 0X1 3RE, U.K.
SUMMARY Matched pairs of malignant and non-malignant hybrid cells were compared in their response to glucose deprivation and to tunicamycin. Glucose deprivation induced an increase in the maximum velocity in the malignant cells, but not in the non-malignant cells. The Michaelis constant of hexose uptake 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 nonmalignant cell lines. It is therefore possible that the Michaelis constant of hexose uptake is affected by the extent of glycosylation of one or more of the cell membrane glycoproteins
INTRODUCTION
In 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 and a reduction in the Michaelis constant for hexose transport. In the present paper we describe experiments designed to probe this phenomenon further. We have examined the effect of glucose starvation on the parameters of hexose transport and the effect of blocking the dolichol pyrophosphate-mediated glycosylation of glycoproteins by tunicamycin. Glucose deprivation has frequently been shown to result in an enhancement of hexose uptake in cultured cells, and this phenomenon has been called 'deprivation derepression' (Martineau, Kohlbacher, Shaw & Amos, 1972). When fibroblasts in culture are deprived of glucose for 16-24 h, large increases in the apparent rates of glucose uptake have been reported (Martineau et al. 1972; Demetrakopoulos & Amos, 1976; Christopher, Kohlbacher & Amos, 1976). However, subsequent work has shown that this effect is largely due to changes in glucose metabolism rather than glucose transport (Salter & Cook, 1976; Musliner, Chrousos & Amos, 1977). The enhancement of glucose transport, as measured by uptake of the non-metabolizable analogue, 3-0-methyl-D-glucose, appears to be about twofold (Salter & Cook, 1976). Most previous studies on the effects of glucose deprivation have been done with confluent cultures of fibroblastic cells. These have low rates of hexose uptake, and the enhancement seen on glucose deprivation is due at least in part to 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 much smaller enhancement of hexose uptake (Kalckar & Ullrey, 1973; Kletzien & Perdue, 1975; Musliner et al. 1977). In the present study, the complications produced by subsequent metabolism of glucose and by density-dependent inhibition of hexose transport were avoided by measuring uptake with the non-metabolizable analogue, 2deoxy-D-glucose, and by using rapidly growing cell cultures. Numerous studies have described alterations in the patterns of glycosylation of membrane proteins in malignant cells. It was therefore of interest to explore whether changes in the Km of the hexose transport system could be brought about by interference with the normal process of glycosylation of membrane proteins. Tunicamycin, which interferes with the dolichol pyrophosphate-mediated glycosylation of asparaginyl residues in glycoproteins (Hubbard & I vatt, 1981), has been shown to inhibit several transport systems in chick embryo fibroblasts (Olden, Pratt, Jowarski & Yamada, 1979). In the present paper we describe the effects of tunicamycin on the parameters of hexose transport in both malignant and non-malignant cells.
MATERIALS
AND
METHODS
Cell lines Mouse PG19: hypoxanthine-guanine phosphoribosyl transferase-deficient (HGPRT) derivative of a spontaneous 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 YACIR lymphoma cells with CBAT6T6 fibroblasts (Evans et al. 1982). YACIRXCBAT6T6 clone 1G1T2: malignant hybrid produced by fusion of YACIR lymphoma 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 derivative and human fibroblast line S1814 (Klinger, 1980). HeLa D98F908 A3 x S 1814 clone 5AMC3: malignant hybrid between a HeLa derivative and human fibroblast line S1814 (Klinger, 1980).
Glucose deprivation Monolayer 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 lXlO 5 and 3xlO s 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 the wells 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's modified 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 a Glucose Test Combination assay kit (Boehringer Mannheim, East Sussex, U.K.). After a further 24 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 described above, except that the MEM —G was supplemented with 5 % foetal calf serum that had been dialysed for 24h at 4°C against 200vol. of phosphate-buffered saline (PBS), changed once after 12 h. The dialysed foetal calf serum was sterilized by Millipore filtration. This medium contained no detectable glucose.
Growth in the presence of glucose In 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 cultures PG19 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 second 24 h. Tunicamycin Cells were distributed into tissue-culture wells as above and grown for 24 h at 37 °C. Some of the wells 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 again measured. In experiments where the effect of tunicamycin on the Km and Vm^x of deoxyglucose uptake 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 three different inbred strains of mouse, chick embryo fibroblasts, human fibroblasts, and one 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 after deprivation shows that the cells grew during the experimental period. The kinetic parameters of deoxyglucose uptake before and after starvation are also given in Table 1 and Hanes (1932) plots of the kinetics of uptake for the cell line 2B1 Col 1 are shown in Fig. 1. There was no large change in Vm^ on glucose deprivation. Values for an induction ratio 7V (denned as the ratio of the Vmix after glucose deprivation to that before glucose deprivation) are given in Table 2, and changes in Vm»x significant at the P
