Cell Research 97 (1976) 6-14

Experimental

DENSITY-DEPENDENT UPTAKE

INTO

INHIBITION GLIOMA CELLS

A. EDSTRGM, Department

of Zoophysiology,

OF 2-DEOXY-D-GLUCOSE

AND NEUROBLASTOMA

IN CULTURE M. KANJE

University

and E. WALUM

of Gijtehorg,

S-40033

Goteborg

33, Sweden

SUMMARY The transport of 2-deoxy-D-glucose (2-DC) into cultured human glioma (138 MG) and mouse neuroblastoma (C 1300) cells has been studied in relation to the growth curves of the cells. An initial increase in the uptake of 2-DG into exponentially growing 138 MG cells, could be attributed to the number of days the cells were kept in culture rather than to their increased density. As the 138 MG cells reached confluency after 3 days the 2-DG uptake became density-dependent inhibited. In still denser cultures the cell growth was inhibited. This was accompanied by morphological ‘normalization’ of the cells and increased uptake of 2-DG. Uptake of 2-DG into C 1300 cells was density-dependent inhibited throughout the cell growth cycle. As the ccl! density increased from I.5 x IO3 to 130x IO3 cells/cm2 the rate of uptake/cell decreased to one-fourth. At the latter cell density the cells entered stationary phase, without any major changes in morphology. The results suggest that spontaneously occurring tumour cells, such as glioma and neuroblastoma cells, can regulate the sugar transport in relation to cell density. This could be due to newly-acquired differentiated properties of the cells or to true contact-inhibitory phenomena.

It has been suggested that the uncontrolled formed by chemical carcinogens, SV40 or division of malignant cells results from in- murine sarcoma virus, they lost growth regcreased availability of critical nutrients in- ulation and no change in the rate of gluside the cell due to surface structural cose transport could be detected as the cells changes that affect their transport across became confluent [7, 8, 28, 381. the cell membrane [22]. The specific transGlioma 138 MG [32] and neuroblastoma port of some glucose-analogues has been Cl300 [2], both cell lines of tumour origin, reported to be 3-5 times faster into virus- exhibit several properties of normal glia and transformed fibroblasts than into their nor- nerve cells. Glioma cells can be induced to mal counterparts [23,27,40,43,46]. In con- synthesize the glia-specific protein S-100 trast, Roman0 & Colby [35] claimed that [12]. Other specific nerve proteins like SV40 virus transformation of 3T3 cells was 14-3-2 [3, 201 and enzymes involved in not accompanied by an increase in the rate neurotransmitter metabolism [ 1,6, 361have of 2-deoxyglucose transport. Cultured fi- been demonstrated in neuroblastoma cells. broblasts become growth inhibited at con- After treatment with dibutyryl cyclic AMP fluency [41]. This is preceded by a drastic (db-CAMP) or prostaglandin E, (PGE,), the decrease in the rate of 2-deoxyglucose cells attain the morphology characteristic of transport. When fibroblasts were trans- differentiated glia and nerve cells [ 13, 33, fiptl

Cell Res 97 (1975)

Glucose uptake into glioma and neurohlastoma cells

7

the present experiments been used to study the effects of cell growth and density on the rate of sugar transport into 138 MG and Cl300 cells. METHODS Standard culture conditions

I. Abscissa: time (min); ordinate: uptake (dpm x 103/cell). Uptake of 2-deoxy-o-l-[3H]glucose (I &i/ml incubation medium) into 138 MG (0-O) and Cl300 (O-O) cells as a function of time. Cells were mated 48 h prior to experiments at a concentration of 35~ IO3cells/cm* (138 MG) and 30x IO3cells/cm2 (C1300). Uptake/cell was assayed in duplicate dishes.

Fig.

341. In spite of their tumour origin both 138 MG and Cl300 cells show densitydependent inhibition of growth [37, 44, 481. It is not known if spontaneously occurring tumour cells in the nervous system, such as glioma and neuroblastoma, have the ability to regulate their glucose uptake in relation to cell growth and density. Glioma cells (138 MG) in sparse and rapidly growing cultures have been shown to take up 3-O-methyl-D-glucose (3-OMG) by a saturable low affinity transport system with a K, of 20 mM and a V,,, of 500 nmole/mg protein/min. The transport is mediated by a Na+-independent passive carrier shared by 3-OMG and D-glUCOSe [14]. Under similar conditions Cl300 cells take up 2-deoxy-D-glucose using a carriermediated, saturable, passive transport system with a Michaelis constant (K,) of 0.8 mM and a V,,, of 18 nmolelmg protein/ min. The uptake is competitively inhibited by D-glucose [45]. The glucose-analogue, 2-deoxy-D-glucase (2-DG), which is transported into the cell and phosphorylated by hexokinase but not further metabolized [24, 39, 401, has in

Established lines of human glioma (138 MG) and mouse neuroblastoma (Cl300, clone Nb 41A3) cells were used. The culturing conditions of 138 MG cells have been described previously [14]. Under standard conditions the Nb 41A3 cells were grown in 60 mm Falcon tissue culture dishes containing 4.0 ml of F 10 medium [ 171,supplemented with 10% calf serum, 5 % foetal calf serum, 100U of penicillin and 50 pg streptomycin per ml. During these conditions an adequate oxygen supply can be expected [47]. The medium was changed every second day. Subcultures were made from late logarithmic phase cultures by means of 0.15 % trypsin in a phosphate-buffered salt (PBS) solution.

Cell growth curves At appropriate times triplicate dishes were counted for cells with a Celloscope 401. The cells were washed 5 times, each time with 5 ml of Hanks’ balanced salt solution (HBSS) [I81 and suspended in 2.0 ml of trypsin solution prior to counting. In parallel experiments, dishes were washed 5 times with HBSS, the cells collected by scraping and assayed for total protein according to Lowry et al. [26].

Uptake of 2-DG The cells were passed through two 30 set washes with 5 ml warm (37°C) glucose-free HBSS and subsequently incubated for various periods of time at 37°C in o-glucose-free HBSS containing r3H12-DG (I &i/ml). After incubation the HBSS was removed by suction, immediately followed by three rapid washes, each time with 5 ml ice-cold 0.9% NaCI-solution containing lO-4 M H&I,. The cultures were left to drv in the ai; for about-2 h and then extracted twice, each time with 1 ml 10% TCA. The extracts were counted for radioactivity in scintillation fluid [9] with a Packard Tri-Carb (3375) liquid scintillation spectrometer. Quench corrections were used for calculation of dpm. The method has been carefully standardized to ensure high reproducibility of the results. The procedure has been described in detail elsewhere [ 141. The uptake of 2-DG into both 138 MG and Nb 4lA3 cells was linear for at least 2 min (fig. 1). By using an incubation time of 30 set the initial rate of 2-DG uptake was measured in the various experiments.

Chemicals An aqueous solution of 2-deoxy-o-l-[SH]glucose (27 Ci/mmole. 1 mCi/ml) was purchased from the RadioExprl Cell Res 97 (1975)

8

Edstriitn,

Kunje and Walum

Figs 3. 3. Bar 200 Frn. Fig. 2. Human glioma cells (13X MC) ((11 3 days and (h) IO days after inoculation of 35x IO” cells/cmY. Cells were grown on plastic tissue culture dishes in Ham’s F10 medium containing 10% calf serum, fixed in absolute methanol and stained with Giemsa solution. Fin. 3. Mouse neuroblastoma cells (Cl300) ((I) 2 days

and (h) 10 days after inoculation of 30X IO” cells/cm’. Cells were grown on plastic tissue culture dishes in Ham’s FIO medium containing 10% calf serum and S ‘7r foetal calf serum, fixed in absolute methanol and R, round; D. differenstained with Giemsa solution. tiating; F. flattened cells.

chemical Centre, Amersham. Ham’s F IO medium and foetal calf serum IFCSt were obtained from Flow Laboratories Ltd., Irvine, Scotland, calf serum from Wallenberg Laboratories, Uppsala, Sweden. and penicillin and streptomycin from Glaxo, London.

irregular morphology (fig. 2~). The cells were evenly spread with few nuclear overlaps. In dense cultures the cells seemed more elongated and formed a pattern of regular arrays (fig. 26). Due to the tight packing morphological characteristics of individual cells were difficult to resolve. If an area free of cells was made by local jetpipetting in the cell layer, the morphology of the cells, subsequently invading this

RESULTS Morphology

Subconfluent 138 MC cells under standard culture conditions showed a fairly flattened, Exptl Cd/ Res 97 (1973-j

Glucose uptake into glioma and neuroblastoma

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Figs 4, 5. Abscissa: time in culture (days); or&tare: (Iefr) cell density (cellsx 10-3/cm*); (right) uptake (dpmx 103/min/cell). Uptake of 2-deoxy-D-l-[3H]glucose (I &i/ml incubation medium) into fig. 4) 138 MG cells; (fTg. 5) Cl300 cells as a function of cell growth. Cells were

plated at a concentration of fig. 4) 35~10~; (fig. 5) 30x lo3 cells/cmz. Number of cells/cm2 (O-e, fig. 4; O-O, fig. 5) were determined every day in duplicate dishes; uptake/cell of 2-DG (W-m, fig. 4; O--O, fig. 5) in triplicate dishes.

area, could be studied. These cells showed about 50-60 %, i.e. somewhat higher than a typical glia-like appearance with two or reported earlier [44]. From the first to the more distinct processes, similar to that of seventh day after plating the cell number increased almost exponentially with a populadb-CAMP- or PGE,-treated cells [13]. tion doubling time (Td) of approx. 50 h, C 1300cells under standard culture conditions were represented by round undifferen- which is identical to that reported previtiated cells, differentiating cells charac- ously [ 14,44,48]. At the fourth day the cells terized by an extensive outgrowth of neu- became confluent (50x lo3 cells/cm*), but rites and irregular extremely flattened cells no major inhibition of cell growth was de(fig. 3a). According to Schubert et al. [37] tectable. After 7 days (about 80x IO3 cells/ these cell types are freely convertible forms cm*) Td increased considerably, and after and not indicative of a heterogeneous cell 11 days (about 90 x lo3 cells/cm*) only little population. The differentiating phenotype further growth occurred. Fig. 5 shows the growth curve of Cl300 seemed to be more frequent in dense (fig. cells under standard culture conditions the 3 b) than in sparse (fig. 3 a) cultures. first 12 days after plating 30x lo3 cells/cm*. Uptake of2-DG in refarion to cell growth A plating efficiency of about 50% was inThe growth curve of 138 MG cells under dicated by the number of cells recovered standard culture conditions is shown in fig. after 24 h (about 15x lo3 cells/cm*). Cell 4. One day after inoculation of 35 x IO3cells/ growth then continued exponentially for 3 cm* the cell number decreased to 20x lo3 days, with a Td of approx. 25 h. Others have cells/cm* indicating a plating efficiency of reported Td values of Cl300 cells of 24-28 h Exptl Cell Res 97 (1975)

[6, 361. After 4 days the cell density was 80x lo” cells/cm’ and the growth markedly inhibited. At the 10th day the cultures entered stationary phase corresponding to a cell concentration of 130x IO” cells/cm2. At this concentration the cells were not completely confluent. The rate of 2-DG uptake into 138 MG cells increased by about 70% during the first 3 days of culturing to reach a maximum of 46.4kO.7 dpmx IO”/min/cell (n =7) (fig. 4). When 138 MG cells were plated for experiments they were subcultured from dishes containing approx. 80x lo3 cells/cm2, a cell density that corresponds to a minimum rate of 2-DG uptake. One day after inoculation the rate of 2-DG uptake was at the same low level. This indicates that the uptake of hexoses into 138 MG cells is slowly adapted to the lower cell density. The 2-DG uptake decreased rapidly as the cells at day 4 became confluent. The decrease continued until day 7 when a minimum (29.82 1.O dpmx 103/min/cell; n =7, p