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Biochimica el Biophysica Acta, 5 1 0 ( 1 9 7 8 ) 2 9 2 - - 2 9 7 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 78068

AN ALTERED RESPONSE OF VIRALLY TRANSFORMED 3T3 CELLS TO OUABAIN

KENNETH

D. B R O W N * a n d J O S E P H F. L A M B

Department of Physiology, University of St. Andrews, St. Andrews, Fife (U.K.) (Received December 5th, 1977)

Summary T h e e f f e c t o f o u a b a i n on K ÷ t r a n s p o r t was e x a m i n e d in 3T3 and virally t r a n s f o r m e d 3T3 cells. A 10 m i n e x p o s u r e to o u a b a i n (10 _3 M) p r o d u c e d a p p r o x i m a t e l y 40% i n h i b i t i o n o f t h e u n i d i r e c t i o n a l K ÷ influx in all cell lines. In 3 T 3 cells t h e r e s p o n s e was n o t significantly altered b y u p t o 70 m i n e x p o s u r e to the drug. In c o n t r a s t , t h e c o n t i n u e d e x p o s u r e o f t r a n s f o r m e d cells to o u a b a i n p r o d u c e d a t i m e - d e p e n d e n t increase in t h e K ÷ influx. This increased influx was s h o w n to be a c c o m p a n i e d b y an increase in t h e K ÷ efflux. T h e results suggest t h a t , in t r a n s f o r m e d cells, o u a b a i n p r o d u c e s b o t h an i n h i b i t i o n o f Na+-K ÷ e x c h a n g e and a s t i m u l a t i o n o f K+-K ÷ e x c h a n g e . T h e latter was s h o w n to b e m o r e readily reversible t h a n t h e f o r m e r .

Introduction The physical and biochemical properties of the plasma membrane of normal and virally t r a n s f o r m e d cells are d i f f e r e n t . T r a n s f o r m e d cells s h o w greater a g g l u t i n a b i l i t y with c o n c a n a v a l i n A [ 1 ] , a n d c o n t a i n altered p r o t e i n s [ 2 ] , g l y c o p r o t e i n s and glycolipids [3,4]. T r a n s f o r m e d cells o f t e n s h o w increased rates o f m e m b r a n e t r a n s p o r t [ 5 , 6 ] . R e c e n t l y , several r e p o r t s have a p p e a r e d in w h i c h K ÷ t r a n s p o r t in n o r m a l a n d t r a n s f o r m e d cells was c o m p a r e d [ 7 - - 1 2 ] . In such studies it is desirable to s e p a r a t e the t o t a l K ÷ influx into its active and passive c o m p o n e n t s . F o r this p u r p o s e , o u a b a i n , an i n h i b i t o r o f ( N a ÷ + K÷) A T P a s e [ 1 3 ] , is usually used. During t h e c o u r s e o f e x p e r i m e n t s designed to investigate t h e e f f e c t s o f cell p o p u l a t i o n d e n s i t y on K ÷ t r a n s p o r t in 3 T 3 and virally t r a n s f o r m e d 3T3 cells [ 1 4 ] we o b s e r v e d a qualitative d i f f e r e n c e in t h e r e s p o n s e o f t h e t r a n s f o r m e d cells to o u a b a i n .

* To whom correspondence should be sent at the present address: Membrane Physiology Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K.

293 Materials and Methods Cell culture p r o c e d u r e s . Swiss 3T3 and SV3T3 cells were originally obtained from Flow Laboratories Ltd. P y 3 T 3 cells were kindly supplied by Dr. E. Steinhardt, Institute o f Virology, University of Glasgow. Each cell line was restarted fr o m a frozen supply at a p p r o x i m a t e l y 10-week intervals. Cells were grown in Dulbecco-Vogt modified Eagle's medium [15] with 10% calf serum at 37°C in 5% CO2. Stock cultures were grown in R o u x bottles and subcultered every three to four days. For experimental purposes, 5 - 1 0 S cells were seeded in plastic culture dishes (Nunc, 9 cm diameter) and used two days later during exponential growth. M e a s u r e m e n t o f K ~ uptake. Cells were incubated at 37°C for periods of time in 86Rb÷-labelled Krebs solution [16] and then washed in ice-cold Krebs solution to remove extracellular activity. The washed cells were detached from the dish by incubation at 37°C with 2 ml of a 0.025% solution of trypsin in Krebs solution minus calcium and magnesium. 10 ml Krebs solution were added to the dish and a suspension of single cells was form ed by aspiration. A 1 ml sample o f the cell suspension was used to determine the cell n u m b e r and mean cell volume (Coulter Counter) as described previously [17]. The 86Rb ÷ c o n t e n t o f the remaining suspension was measured by detection of Cerenkov radiation in a liquid scintillation c o u n t e r [ 18]. M e a s u r e m e n t o f K ~ washout. Cells were incubated at 37°C for 3 h in 86Rb +labelled Krebs solution and t hen washed in ice-cold Krebs solution to remove extracellular activity. 10 ml Krebs solution were added to the dish which was then incubated at 37 ° C. After 5 min the collecting solution was quickly poured into a scintillation vial for 86Rb÷ counting and a further 10 ml of inactive Krebs solution were added to the dish. This pr oc e dure continued for a total of 30 min. The cells were t hen trypsinised and the cell number, mean cell volume, and remaining cellular 86Rb ÷ activity determined.

Results 86Rb + was used as a K ÷ tracer in this work. Preliminary experiments using 42K÷, and reports from o t h e r laboratories [7,19], justified the substitution of 86Rb ÷ for the short half-life 42K÷ isotope. The time-course o f total K ÷ upt ake in 3T3, P y 3 T 3 and SV3T3 cells is shown in Fig. 1. The results indicate t hat K ÷ uptake was linear within 10 min in all cell lines and 10 min was, therefore, chosen as a suitable incubation period for m e a s u r e m e n t o f the initial rate of K ÷ uptake (influx). In this e x p e r i m e n t the 3T3 cells were at a density o f 1.5 • 104 cells/cm 2 and the transformed cells at 2.8 • 104 cells/cm 2. Under these conditions there was no difference in the K ÷ influx b etween the cell lines. It is well k n o w n t h a t ouabain causes an inhibition o f Na ÷-K÷ exchange across cell membranes [13]. In 3T3 cells a ouabain c o n c e n t r a t i o n o f 10 -3 M was required to p r o d u c e a 40% r educt i on of the K ÷ influx. The response was n o t significantly altered by up to 60 min p r e t r e a t m e n t with the drug (Fig. 2). This result demonstrates, in agreement with others [ 20,21], t hat cultured mouse cells retain the low sensitivity o f the species to ouabain. When the e x p e r i m e n t was

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~-) a n d S V a T 3 (A ':~) cells. Fig. 1. T h e t i m e - c o u r s e o f K + u p t a k e b y 3 T 3 ( o o), P y 3 T 3 (D-T h e K + c o n c e n t r a t i o n of t h e K r e b s s o l u t i o n w a s 5.4 m m o l / 1 a n d the 8 6 R b + a c t i v i t y w a s a p p r o x i m a t e l y 0.2 p C i / m l . Fig. 2. T h e e f f e c t o f o u a b a i n on t h e K + i n f l u x in 3 T 3 (o o), P y 3 T 3 (m I) and SV3T3 (A A) cells. T h e g r o w t h m e d i u m w a s r e m o v e d a n d t h e cells w e r e i n c u b a t e d at 3 7 ° C in 10 m l K r c b s s o l u t i o n w i t h or w i t h o u t o u a b a l n ( 1 0 -3 M) f o r 0 - - 6 0 rain. T h i s s o l u t i o n w a s t h e n r e p l a c e d by 10 ml 8 5 R b + - l a b e l l e d K r e b s s o l u t i o n , w i t h or w i t h o u t o u a b a i n , a n d t h e K + i n f l u x m e a s u r e d o v e r 10 m i n . E a c h p o i n t r e p r e s e n t s t h e v a l u e f o r t h e K + i n f l u x in t h e p r e s e n c e o f o u a b a i n e x p r e s s e d as a p e r c e n t of t h e c o n t r o l v a l u e s m e a s u r e d in t h e a b s e n c e o f o u a b a i n . A r e p r e s e n t a t i v e e x p e r i m e n t o f f o u r s e p a r a t e e x p e r i m e n t s p e r f o r m e d is p r e s e n t e d .

r e p e a t e d using P y 3 T 3 and S V 3 T 3 cells it was f o u n d t h a t the initial i n h i b i t i o n (40%) o f the K + influx was f o l l o w e d b y an increase in the K + influx (Fig. 2). The chloride influx was n o t increased after a 6 0 min i n c u b a t i o n with 10 -3 M ouabain. This result suggests that the e f f e c t o n the K ÷ influx was n o t due to m e m b r a n e damage. The e f f e c t o f ouabain o n the efflux o f K ÷ from 3 T 3 and transformed 3 T 3 cells was also investigated. The e f f e c t o f various bathing solutions o n the rate o f loss o f S6Rb ÷ from preloaded P y 3 T 3 and 3 T 3 cells is s h o w n in Table I. ExtracelluTABLE I THE EFFECT OF OUABAIN ON THE RATE OF LOSS OF K ÷ FROM 3T3 AND PY3T3 CELLS T h e w a s h o u t o f 8 6 R b + f r o m p r e l o a d e d cells w a s f o l l o w e d i n t o e a c h o f t h e f o l l o w i n g s o l u t i o n s : ( 1 ) c o n t r o l K r e b s s o l u t i o n , ( 2 ) K r e b s s o l u t i o n + 10 -3 M o u a b a i n , (3) K+-free K r e b s s o l u t i o n , ( 4 ) K+-free K r e b s solut i o n + 10 -3 M o u a b a i n . T h e a m o u n t o f 8 6 R b + lost d u r i n g six s u c c e s s i v e 5 r a i n p e r i o d s is e x p r e s s e d as a f r a c t i o n o f t h e a m o u n t o f 8 6 R b + p r e s e n t at t h e b e g i n n i n g o f e a c h 5 m i n p e r i o d . T h e f r a c t i o n a l loss o f 8 6 R b * f r o m 3 T 3 cells w a s n o t s i g n i f i c a n t l y a f f e c t e d b y a n y o f t h e c o n d i t i o n s t e s t e d (P > 0 . 1 0 ) a n d t h e m e a n v a l u e s ( + S . E . ) a r e s h o w n . T h e f r a c t i o n a l loss o f 8 6 R b + f r o m P y 3 T 3 cells i n t o s o l u t i o n s 1, 3 a n d 4 w a s n o t s i g n i f i c a n t l y d i f f e r e n t ( P > 0 . 1 0 ) a n d t h e m e a n v a l u e s (-+S.E.) a r e s h o w n . T h e f r a c t i o n a l loss o f 8 6 R b + f r o m P y 3 T 3 cells w a s s i g n i f i c a n t l y i n c r e a s e d (P < 0 . 0 1 ) b y t h e p r e s e n c e o f o u a b a i n in t h e collecting solution. Cell line

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rninufes Fig. 3. R e v e r s i b i l i t y o f t h e r e s p o n s e o f v i t a l l y t r a n s f o r m e d cells to o u a b a i n . S V 3 T 3 cells w e r e i n c u b a t e d w i t h 10 m l K r e b s s o l u t i o n w i t h or w i t h o u t o u a b a i n ( 1 0 -3 M) f o r 6 0 m i n . All d i s h e s t h e n r e c e i v e d 10 rnl K r e b s s o l u t i o n m i n u s o u a b a i n f o r p e r i o d s r a n g i n g f r o m 0 to 1 2 0 rain. T h e K + i n f l u x w a s m e a s u r e d b y i n c u b a t i o n w i t h 8 6 R b + - l a b e l l e d K r e b s s o l u t i o n f o r 10 rain. E a c h p o i n t r e p r e s e n t s t h e v a l u e s f o r t h e K + i n f l u x a f t e r o u a b a i n t r e a t m e n t e x p r e s s e d as a p e r c e n t o f t h e value o b t a i n e d f o r t h e c o n t r o l . T h e value at 0 r a i n w a s m e a s u r e d w i t h o u a b a i n p r e s e n t in t h e 8 6 R b + - l a b e l l e d K r e b s s o l u t i o n .

lar o u a b a i n increased t h e rate o f S6Rb* loss f r o m the t r a n s f o r m e d cells b u t n o t f r o m the u n t r a n s f o r m e d cells (Table I). Similar results were o b t a i n e d using 42K+ (data n o t shown). T h e increased K ÷ loss f r o m the t r a n s f o r m e d cells was abolished b y t h e r e m o v a l o f K ÷ f r o m the b a t h i n g solution" (Table I). This result indicates t h a t the e f f e c t o f o u a b a i n o n t h e K ÷ fluxes o f t r a n s f o r m e d cells is d u e t o the e s t a b l i s h m e n t o f K+-K + exchange. Such e x c h a n g e was n o t p r e s e n t in u n t r e a t e d cells since the e f f l u x o f K ÷ was n o t a f f e c t e d b y t h e removal o f extracellular K ÷ (Table I). It appears t h e n t h a t the K ÷ influx o f o u a b a i n - t r e a t e d t r a n s f o r m e d cells is initially r e d u c e d b y t h e i n h i b i t i o n o f Na÷-K ÷ e x c h a n g e and t h e n rises a f t e r the o n s e t o f K÷-K ÷ e x c h a n g e . We wished t o test w h e t h e r the inhibition o f Na+-K ÷ e x c h a n g e c o n t i n u e d a f t e r the e s t a b l i s h m e n t o f K+-K ÷ exchange. T o this end, P y 3 T 3 cells were t r e a t e d with 10 -3 M o u a b a i n and t h e intracellular Na ÷ and K ÷ c o n c e n t r a t i o n s were m e a s u r e d b y flame p h o t o m e t r y [ 1 6 ] . Over a 60 min p e r i o d the [Na+]i rose e x p o n e n t i a l l y f r o m 20 t o 70 mmol/1 intracellular w a t e r and the [K+]i fell e x p o n e n t i a l l y f r o m 200 t o 130 mmol/1 intracellular water. T h e r e f o r e , in t h e t r a n s f o r m e d cells, o u a b a i n causes a s i m u l t a n e o u s r e d u c t i o n o f Na+-K + e x c h a n g e and s t i m u l a t i o n o f K*-K ÷ exchange. In a similar e x p e r i m e n t using 3T3 cells [Na*]i rose f r o m 14 t o 72 mmol/1 o f intracellular w a t e r and [K*]i fell f r o m 168 t o 102 mmol/1 intracellular water. T h e reversibility o f these effects has been e x a m i n e d . S V 3 T 3 cells were t r e a t e d with o u a b a i n for 60 min and t h e n t r a n s f e r r e d t o a c o n t r o l Krebs solution. T h e K ÷ influx was m e a s u r e d at 15-min intervals up to 120 rain a f t e r t h e r e m o v a l o f ouabain. T h e K ÷ influx decreased during t h e first 60 rain and t h e n r e m a i n e d c o n s t a n t at a value 40% b e l o w t h a t o f t h e u n t r e a t e d c o n t r o l s (Fig. 3). Thus, t h e o u a b a i n - i n d u c e d K÷-K ÷ e x c h a n g e is m o r e readily reversible t h a n t h e i n h i b i t i o n o f Na+-K ÷ exchange.

296

Discussion Ouabain has been shown to bind to cells in two ways: (1) a saturable, specific binding to Na ÷ p u m p sites which is sensitive to [K÷]o and (2) a nonspecific uptake which is not affected by [K÷]o and does not saturate [22]. The slow onset and rapid reversal on the K÷-K÷ exchange suggests that this effect is n o t caused by ouabain a t t a c h m e n t to the specific binding sites. We propose t h a t the K+-K ÷ exchange arises from a non-specific interaction of ouabain with the transformed cell's membrane. In an a t t e m p t to obtain further evidence on this question we tried to measure the characteristics of [3H]ouabain binding to 3T3 and P y 3 T3 cells. It is difficult to obtain accurate measurements of specific ouabain binding for cells which are insensitive to the drug [22]. We were unable to separate the two c o m p o n e n t s of binding because of the high level of non-specific uptake at 10 -3 M ouabain. We were also unable to det ect differences in the time-course of total ouabain binding to normal and transformed cells. Thus the nature of the interaction with ouabain responsible for the stimulation o f K%K ÷ exchange in t r a n f o r m e d cells remains unknow n. It is possible that a change in the properties of m e m b r a n e lipids after viral transformation [4] is involved. This interpretation is s uppor t ed by a preliminary observation that treatm e n t of SV3T3 cells with ouabain for 60 min at 18°C did not induce K÷-K ~ exchange. Under these conditions the K ÷ influx was reduced 38% by ouabain, demonstrating that inhibition of Nat-K ÷ exchange was maintained at the reduced temperature. Spaggiare et al. [7] r e por t ed a value of 50% for the inhibition of the unidirectional K ÷ influx (measured over 10 min) in SV3T3 cells. However, t hey did n o t measure the K ÷ influx in cells treated with the drug for longer periods of time. Kimelberg and Mayhew [8,9] have measured the K ÷ " u p t a k e " in 3T3 and SV3T3 cells exposed to ouabain for 60 min. However, in their experiments S6Rb+ was present during the whole period of exposure to ouabain, whereas in the experiments repor t e d here 86Rb÷ was present only during the final 10 min of incubation. The problems associated with the use of long uptake times have already been n o t e d [7]. Because of the substantial isotope backflux these u p t a k e data are n ot comparable to unidirectional influx measurements. It is, therefore, impossible to determine w he t he r the SV3T3 cells of Kimelberg and Mayhew display the anomalous response to ouabain seen in our cells. If so, their conclusion that the transformed cells have a larger ouabain-sensitive K ÷ u p t ak e than normal cells may not be justified. In our experiments, the values for the ouabain-sensitive K ÷ influx were similar when normal and transformed cells were co mp ar e d at low cell densities. Previous results on this question have been variable. The ouabain-sensitive K ÷ influx has been r e por t ed to be increased [10], unaltered [12,14] or decreased [7,11] after virus-transformation. Similar variability has been observed for the effect of virus-transformation on (Na ÷ + K÷)-ATPase activity [ 8 , 9,2 3 - - 2 7 ] . T u p p e r [28] has recently evaluated the effect of subcultivation on the ouabain-sensitive K ÷ influx in 3T3 and SV3T3 cells. The influx changed little over 50 passages of the transformed cell. In contrast, over a similar n u m b e r o f passages, the influx in the 3T3 cells increased 5-fold. Thus, the

297 ouabain-sensitive K ÷ influx in 3T3 cells may be less, greater or equivalent to that in the transformed cells. It is proable that this finding accounts for much of the variation in previous studies. At the m o m e n t we cannot say whether stimulation of K÷-K÷ exchange by ouabain is a characteristic feature of virally transformed cells. The experiments need to be repeated using other cell lines, preferably more sensitive to ouabain so that lower concentrations of the drug may be used. Negative selection on a cloned population of transformed 3T3 cells permits the recovery of revertants whose growth [29] and transport [30] characteristics are similar to those of normal cells. It will be interesting to examine the response of such cells to ouabain.

Acknowledgements We thank Iain Laurie and Mary Paisely for technical assistance. This work was supported by a grant from the Cancer Research Campaign.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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