The effect of macrofixation on derepressed sugar transport systems of chick embryo fibroblasts

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Experimental Cell Research 108 (1977) 239-243

T H E E F F E C T OF M A C R O F I X A T I O N ON D E R E P R E S S E D SUGAR T R A N S P O R T SYSTEMS OF CHICK EMBRYO FIBROBLASTS E. R. PHILLIPS, M. K. RAIZADA and J. F. PERDUE L a d y Davis Institute f o r Medical R e s e a r c h o f the Jewish General Hospital, Montreal, Quebec, Canada H 3 T 1E2

SUMMARY The large molecular weight polyaldehyde (macrofixative) obtained by periodate oxidation of dextran has been shown to react with the external cell surface. Chick embryo fibroblasts (CEF), which had been treated with macrofixative (MFx), were examined for the effect on the rates of sugar transport. The low "basal" rate of sugar uptake, seen in confluent (high density) cultures of CEF was unaffected by such treatment. Low density (rapidly growing) cultures, oncogenically transformed cultures, and glucose-starved cultures have rates of sugar uptake that are significantly higher than the "basal" level. Macrofixation was found to inhibit the induction of higher rate of glucose transport under all of these conditions. The results indicate that a difference may exist between the sugar transport system in resting (confluent) cells, and that in "derepressed" cells.

A previous report [1] from this laboratory has described the preparation of a macromolecular polyaldehyde by periodate oxidation of dextran. This substance has been termed "macrofixative (MFx)"; and it appears to react only with biological materials of the extracellular space. Treatment of living cells with MFx serves to stabilize the plasma membrane to fragmentation during hypotonic lysis. Such treatment does not interfere with cellular metabolism as represented by the ability of chick embryo fibroblasts (CEF) to transport and phosphorylate glucose; but it does inhibit cell multiplication [1]. Although the antibodyinduced redistribution properties of plasma membrane-associated viral envelope antigens of avian tumor virus-infected CEF were not impaired by MFx treatment [1], it was thought that such a substance might have some effect on plasma membrane

dynamics, e.g. translational mobility or metabolic turnover of membrane macromolecules. Because it also seemed possible that membrane dynamics could play a role in the increased rate of glucose uptake by CEF under growth or culture conditions which "derepress" the sugar transport system [2], the effects of MFx on the rate of glucose transport in CEF placed under these conditions were investigated. These conditions include: 16-24 h of glucose starvation; culture at low density (phase of rapid growth); and oncogenic transformation by avian sarcoma virus. Each of these conditions results in substantial elevation of sugar uptake over that of high density (confluen0 cultures of uninfected CEF [2]. It was previously observed that an increase in the rate of glucose transport in CEF, induced by prolonged incubation in PBS, was abrogated by MFx treatment; although the Exp Cell Res 108 (1977)

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Phillips, Raizada and Perdue

pre-existing rate of sugar uptake was not d i m i n i s h e d b y p r o l o n g e d m a c r o f i x a t i o n [1]. In more general terms, "basal rates" of glucose transport were not affected by macr o f i x a t i o n . I n this c o m m u n i c a t i o n , w e d e s c r i b e t h e e f f e c t s o f m a c r o f i x a t i o n on: (a) glucose deprivation-induced enhancement o f s u g a r t r a n s p o r t p r i o r to a n d s u b s e q u e n t to m a c r o f l x a t i o n ; (b) t h e h i g h r a t e o f gluc o s e t r a n s p o r t o b s e r v e d in s p a r s e ( r a p i d l y g r o w i n g ) c u l t u r e s ; (c) t h e high r a t e o f s u g a r transport of CEF oncogenically transformed by avian sarcoma virus.

1.5 ml of PBS or MFx for 1 h at 23°C and again washed twice with PBS at 23°C. They were then subjected to further treatment as described for each experiment.

Measurement of sugar transport This assay was carded out as previously described [1], by incubation of CEF in 1 mM 2-deoxyglucose (2-DG), containing 1.2/xCi/ml [aH]2DG (New England Nuclear) for 5 min at 37°C. RESULTS

Effect of MFx on hexose starvationinduced derepression of sugar transport

C o n f l u e n t c u l t u r e s o f C E F w e r e t r e a t e d , as d e s c r i b e d , w i t h 2.5 % M F x o r P B S . T h e cell MATERIALS AND METHODS l a y e r s w e r e w a s h e d t w i c e w i t h P B S a n d inc u b a t e d at 37°C in 4 ml o f g l u c o s e - f r e e T E M Cell culture Chick embryo fibroblasts (CEF) were prepared from c o n t a i n i n g d F C S o r in 4 ml o f g l u c o s e - c o n I 1-day-old chick embryos and cultured as previously t a i n i n g T E M w i t h 4 % F C S . T r i p l i c a t e reported [3]. Oncogenically transformed CEF were p l a t e s o f t r e a t e d cells w e r e a s s a y e d f o r produced by infection with the BratisL "a strain (B~7) of Rous sarcoma virus (RSV) as previously described s u g a r t r a n s p o r t a c t i v i t y a t t i m e d i n t e r v a l s [4]. Confluent cultures of virus-transformed or unin- a f t e r c o m m e n c e m e n t o f g l u c o s e s t a r v a t i o n . fected CEF were obtained by growing the cells in Temin-modified Eagle's medium (TEM), containing T h e r e s u l t s o f this e x p e r i m e n t a r e s h o w n in 4 % fetal calf serum (FCS), for 4--6 days or until the fig. 1. P B S - t r e a t e d c e l l s w h i c h w e r e s t a r v e d cells completely covered the 60 mm in diameter dish. The cultures were fed 16-24 h prior to use with fresh f o r g l u c o s e d e m o n s t r a t e d a n i n c r e a s i n g r a t e TEM (with or without glucose) made 4 % in FCS or o f s u g a r t r a n s p o r t f o r u p to 24 h. T h i s s u g a r dialysed FCS (dFCS). d e p r i v a t i o n - i n d u c e d i n c r e a s e in u p t a k e w a s s u b s t a n t i a l l y i n h i b i t e d in M F x - t r e a t e d cells. Macrofixative Dextran was oxidized to the polyaldehyde form in a C E F w h i c h w e r e t r e a t e d w i t h P B S o r M F x manner slightly simplifiedfrom the previously reported a n d i n c u b a t e d in g l u c o s e - c o n t a i n i n g m e procedure [1]: 2.2 g of sodium metaperiodate (Sigma Chemical Co., St Louis, Mo), 10 ml of 10% (w/v) d i u m m a i n t a i n e d a c o n s t a n t b a s a l l e v e l o f dextran (Rheomacrodex, Pharmacia Fine Chemicals, s u g a r t r a n s p o r t . T h i s i n h i b i t i o n o f 2 - D G u p Uppsala), 2 ml of 2 M acetate buffer, pH 5.5, and 28 ml deionized water were combined and stirred at room t a k e w a s d u e to a d e c r e a s e in t h e Vmax(200 temperature for 3 h. Excess periodate was destroyed n m o ! e s / m i n / m g f o r P B S - t r e a t e d a n d 27 by the addition of 1 ml of glycerol and the solution was dialysed against 5-6 one liter changes of Dulbecco's n m o l e s / m i n / m g f o r M F x - t r e a t e d C E F ) a n d phosphate-buffered saline (PBS) for 12-24 h each. The w a s n o t d u e to c h a n g e in K m (Kin f o r P B S resulting macrofixative was assumed to be approx. 2.5 %--the concentration of dextran in the reaction t r e a t e d a n d M F x - t r e a t e d C E F w a s 1.6 m M ) . mixture--and was used at this concentration in all experiments. The equilibration of each solution with nitrogen described in the previous report was omitted Effect of MFx on hexose-deprivation without apparent effect. MFx was used within one induced glucose transport week of preparation or discarded. Confluent cultures of CEF were washed once with glucose-free TEM and incubated Macrofixative treatment f o r 16-18 h in g l u c o s e - f r e e T E M c o n t a i n i n g Cells grown on 60 mm diameter culture dishes were washed twice with cold (4°C) PBS, incubated with 4 % d F C S , w a s h e d , t r e a t e d w i t h P B S o r

Exp CellRes 108(1977)

Effect of MFx on glucose transport in CEF

241

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Figs 1.-4. Abscissa: time (hours); ordinate: 2-DG uptake in nmoles/mg protein/5 min. Limits are S.E.M. Fig. 1. Confluent CEF grown in TEM, with 4 % FCS, were treated with PBS or MFx, washed and placed in glucose-free TEM containing 4 % dFCS. 2-DG Transport was assayed at 0, 6, 12 and 24 h. (©--©, PBStreated; /x__/x, MFx-treated.) Control specimens which were not deprived of glucose were also prepared. ( 0 - - 0 , PBS-treated; &--&, MFx-treated.)

i 3

~ 6

~ 9

, ,. 12

Fig. 2. Confluent CEF were cultured in glucose-free MEM with 4 % dFCS for 20 h, washed and treated with PBS or 2.5 % MFx. At timed intervals, uptake of 2-DG was determined ( A - - A , PBS-treated; /x__/x, MFxtreated.) Control cultures not starved for glucose were also examined ( 0 - - 0 , PBS-treated; O - - O , MFxtreated).

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20 15

24

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Fig. 3. CEF were transferred at 300x10 a per 60 mm diameter plate, 18 h later cells were treated with P B S ( 0 - - 0 ) or MFx (A__/x), washed, and returned to culture. Specimens were examined at 24 h intervals.

Fig. 4. BrT-transformed CEF were grown to confluency and treated with PBS ( e - - e ) or MFx ( A _ A ) . Thereafter triplicate samples were tested for 2-DG uptake at 0 and 12 h.

MFx and incubated in glucose-containing TEM with 4% FCS. 2-DG uptake was determined at various time intervals after the return to glucose-containing medium. The results from a representative experiment are shown in fig. 2. Returning glucosestarved CEF to glucose-containing medium resulted in a time-dependent decrease in the rate of sugar transport to that observed in CEF not starved for glucose. MFx treatment caused an apparently immediate loss

of approx. 50 % of the glucose deprivationinduced transport followed by a gradual loss of the remainder.

Effect of MFx on the rate of sugar transport of low density cultures of CEF CEF were seeded onto 60 mm culture plates at 300 000 cells/plate and allowed to attach for 14-18 h. These low density cultures were then treated with MFx or PBS and reExp Cell Res 108 (1977)

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Phillips, Raizada and Perdue

Table 1. The influence of macroflxation on sugar transport capacity under various conditions which result in enhancement of this function Conditions of culture at times of MFx

No. of independent experiments

High density, confluent Low density, non-confluent 5 High density, BTT-transformed 2 High density and starved for glucose after MFx 2 High density, glucose-starved 1

% Inhibition Range

-

Mean

0

28-88

47

DISCUSSION

55-76

60

48-74

61

A previous report on macrofixative treatment of confluent cultures of CEF indicated that such treatment had no effect on the processes of transport and phosphorylation [1], This finding was in concert with the observation that macrofixed cells retained the ability to organize cell surface antigen-antibody complexes into redistribution patterns characteristic of living cells [1]. On the other hand, macrofixative was found to have a striking inhibitory effect on cell multiplication. Although no loss in the capability of resting cells to take up sugar was observed, there was some suggestion of an MFxinduced inhibition of enhanced rates of glucose transport. It has been shown that basal levels of sugar transport in CEF may be substantially elevated by a number of experimental conditions including: a low culture density (the cells are in a state of rapid growth [5]); prolonged glucose starvation [2]; serum starvation followed by serum readdition [6]; and transformation by oncogenic viruses [4]. The effect of macrofixative on the enhanced hexose uptake induced by some of these conditions was investigated. Although appreciable variation was encountered in these experiments (see table 1), the results clearly indicated that macrofixation decreased the rate of sugar transport in cells which were derepressed for transport. Previous work from this and

-

48

turned to culture in TEM-containing 4 % FCS. Triplicate plates were taken for determination of 2-DG uptake at timed intervals. The results from one such experiment are shown in fig. 3. PBS-treated CEF transport 2-DG at a very high rate immediately following treatment (14-18 h after plating). MFx treatment, however, greatly reduced this transport activity. In some experiments, this sensitivity to MFx treatment was also seen on the second day after transfer (one day after treatment). In several experiments, only the initial time point showed a significant sensitivity to MFx treatment and the MFx effect on low density cultures proved to be more variable than that of the other phenomena reported in this communication (see table 1).

Effect of MFx on the high rate of transport in RSV-transformed CEF Confluent cultures of Brr virus-transformed CEF were treated with MFx or PBS and replicate samples were assayed for 2-DG uptake at 0 and 12 h. PBS-treated cells demonstrated the high rate of transport characteristic of transformed cells [4], but MFx Exp Cell Res 108 (1977)

treatment appeared to dramatically reduce the rate of transport to levels more closely approximating that of normal cells (fig. 4). Not surprisingly, some variation was encountered in the effects observed in repetitions of the experiments. The results and reproducibility of these experiments are summarized in table 1.

Effect of MFx on glucose transport in CEF other laboratories has indicated that the higher rates of hexose transport in CEF produced by these experimental conditions were due to Vmaxincreases which have been interpreted as the incorporation or activation of additional transport systems. The Km remains constant and-~uggests that, on a functional level, there is no detectable difference between the hexose transport system of resting cells and those of cells which are derepressed by a variety of conditions. The observation that MFx treatment specifically decreases sugar transport in cells displaying an enhanced capacity to take up this nutrient suggests some biochemical or structural differences exist between the transport systems of resting and those of the stimulated cells. The effect of MFx could be: (a) the result of direct chemical action on "new" and derepressed sites; (b) secondary to some inhibitory action on separate "activating" or "coordinating" molecules, or; (c) the mechanical fixation of some membrane dynamics necessary for activity of additional transport sites. The first possibility suggests the susceptibility to aldehyde reaction and a consequent inactivation of the derepressed system, but not of the basal system. The second and

243

third possibilities suggest susceptible coordinating molecules or membrane dynamics which are necessary for the expression of the derepressed system but not for that of the basal system. A kinetic analysis of 2-DG transport in CEF derepressed for the synthesis of the transport systems indicates that MFx decreased the Vmax without altering the K m . This result is consistent with the first possibility and with recent observations of Christopher et al. [7] that the hexose transport system of glucose-starved CEF is sensitive to inhibition by 0.5 M N-ethylmaleimide whereas that of untreated cells is not. This workers was supported by a grant from the Medical Research Council of Canada.

REFERENCES 1. Phillips, E R, Kletzien, R F & Perdue, J F, Exp cell res 105 (1976) 51. 2. Kletzien, R F & Perdue, J F, J biol them 250 (1975) 593. 3. Temin, H M, Virology 10 (1960) 182. 4. Kletzien, R F & Perdue, J F, J biol them 249 (1974) 3375. 5. m Ibid 249 (1974) 3366. 6. - - Ibid 249 (1974) 3383. 7. Christopher, C W, Kohlbacher, M S & Amos, H, Biochemj 158 (1976) 439. Received November 30, 1976 Accepted April 5, 1977

Exp Cell Res 108 (1977)