Synthesis of a brain-specific protein (S100 protein) in a lectin-resistant mutant of a rat glial cell line (C6)

M6moires originaux

BIOCHIMIE, 1983, 65, 609-618.

Synthesis of a brain-specific protein (S1 O0 protein) in a lectin-resistant mutant of a rat glial cell line (C6). Alexander MARKSV, John LAW and James MAHONY (Regu le 6-9-r982, acceptd apr~s rdvision le 9-H-X983.)

Banting a n d Best, D e p a r t m e n t Research, U n i v e r s i t y o f Toronto, Toronto, Ontario M 5 G I L 6 C a n a d a

R6sum6.

Summary.

La synth~se de la protdine SIO0 augmente spontandment dans les cellules gliales de rat (clone C6) vers la fin de la phase exponentielle de croissance en monocouche. De plus la synth~se de cette protdine peut gtre augmentde par traitement des cellules C6 avec une lectine, la concanavaline A succinylde (succinyl ConA ). Pour dtudier la relation entre ces deux inductions de synth~se de la protdine SIO0 nous avons isold un type de cellule rdsisrant d~ l'action ldthale de la ConA d~partir d'une population de cellules C6. Les cellules rdsistantes (C6-ConA R) poss~dent moins de rdcepteurs membranaires pour la succinyl ConA que les cellules C6.

The synthesis of SIO0 protein increases toward the end of the exponential phase of growth of clonal rat glial cells C6 in monolayer culture. Moreover the synthesis of this protein can be increased by treatment of C6 cells with the lectin succinylated concanavalin A (succinyl ConA ). I n order to study the relationship between these two inductions of $700 protein we have isolated a cell line resistant to ConA from a population of C6 cells. The resistant cells (C6-ConA R) have less succinyl ConA receptors than C6 cells.

Contrairement d~ la souche C6, la synth~se de la protdine S 1O0 n' augmente pas dans les cellules C6-ConA R apr~s traitement avec la succinyl ConA. Cependant dans les deux types cellulaires la synth~se de la protdine SIO0 augmente vers la fin de la phase exponentielle de croissance.

Ces rdsultats sugg~rent d'une part que l'induction de synth~se de la protdine SIO0 dans les cellules C6 par la succinyl ConA ddpend d'une liaison entre la lectine et ses rd~epteurs membranaires et d'autre part que les dtapes initiales de l'induction de synth~se de la protdine $100 par la lectine sont diffdrentes des drapes initiales de l'induction de synth~se de cette protdine qui a lieu vers la fin de la phase exponentielle de croissance en

of

Medical

I n contrast to C6 cells, the synthesis of SIO0 protein does not increase in C6-ConA ~ cells after treatment with succinyl ConA. However in both cell types the synthesis of SIO0 protein increases toward the end of the exponential phase of growth. These results suggest firstly that the induction of $700 protein in C6 cells by succinyl ConA is mediated by an interaction of the lectin with its membrane receptors and secondly that the initial steps in the induction of SIO0 protein by the lectin are different from the initial steps in the induction of this protein which occurs toward the end of the exponential phase of growth in monolayer culture.

monocouche. Mots clds, cellules C6 / r6sistance ~ ta ConA / protdine Sloo.

Key words : C6 cells / ConA resistance / Sloo protein.

Abbreviations used : ConA, concanavalin A ; succinyl ConA, succinylated ConA ; C6-ConA R, C6 cells resistant to ConA ; c A M P , cyclic A M P ; P B S , phosphate buffered saline (750 m M NaC1, lO rnM sodium phosphate p H 7.4) ; FCS, fetal calf serum ; B S A , bovine calf serum; H Y P , iodohydro-xybenzylpindolol ; SRBC, sheep red blood cells. Trivial name : SIO0 protein is a brain protein soluble in 100 percent ammonium sulfate. V To whom all correspondence should be addressed.

610

A. Marks, J. Law and J. Mahony

Introduction. S100 p r o t e i n is a n a c i d i c c a l c i u m b i n d i n g p r o t e i n w i d e l y d i s t r i b u t e d in t h e n e r v o u s s y s t e m of v e r t e b r a t e s w h o s e c o n f o r m a t i o n determined by its immunological cross-reactivi t y h a s b e e n r e m a r k a b l y well c o n s e r v e d d u r i n g e v o l u t i o n E1-6]. S 100 p r o t e i n a c c u m u l a t e s d u r i n g the enzymatic and ~europhysiological muturat i o n of t h e m a m m a l i a n b r a i n E7-11]. B a s e d on these observations the protein has been used a s a m a r k e r of c e l l u l a r d i f f e r e n t i a t i o n i n c u l t u r e d cell l i n e s d e r i v e d f r o m t h e m a m m a l i a n n e r v o u s s y s t e m [11-14]. I n p a r t i c u l a r i t h a s b e e n f o u n d t h a t S100 p r o t e i n a c c u m u l a t e s in a r a t g l i a l cell l i n e (C6) w h e n t h e cells r e a c h c o n f l u e n c e i n m o n o l a y e r c u l t u r e [16, 17]. T h i s i n d u c t i o n is m e d i a t e d b y a n i n c r e a s e in S 1 0 0 p r o t e i n mRNA [18]. S u p p o r t for t h e n o t i o n t h a t a c c u m u l a t i o n of S100 p r o t e i n i n C6 cells r e p r e s e n t s a n i n d e x of d i f f e r e n t i a t i o n in vitro c o m e s from the observation that individual astrocytes of t h e r a t c e r e b e l l u m b e g i n t o a c c u m u l a t e S 1 0 0 protein after they cease proliferating during the t h i r d w e e k of p o s t n a t a l d e v e l o p m e n t [19]. T h e s i g n a l s t r i g g e r i n g t h e i n d u c t i o n of S100 p r o t e i n a t h i g h cell d e n s i t i e s in m o n o l a v e r c u l t u r e s of C6 ceils a r e n o t k n o w n . A n i n v o l v e m e n t of cell c o n t a c t h a s b e e n p o s t u l a t e d on t h e b a s i s of i n d i r e c t e v i d e n c e [16, 18, 20]. E x p e r i m e n t a l a t t e m p t s t o s t u d y t h e b a s i s of S100 p r o t e i n i n d u c t i o n i n C6 cells h a v e u t i l i z e d a n u m b e r of p h a r m a c o l o g i c a l p r o b e s . T r e a t m e n t with norepinephrine, cyclic AMP (cAMP) and t h e l e c t i n s u c c i n y l a t e d c o n c a n a v a l i n A (succ i n y l ConA) h a v e b e e n s h o w n t o s t i m u l a t e S100 p r o t e i n s y n t h e s i s i n C6 cells [21, 22] w h e r e a s treatment with antimicrotubular drugs inhibi t e d t h e s y n t h e s i s of t h e p r o t e i n [22-25]. In the present communication we h a v e c o m p a r e d t h e effect of s u c c i n y l C o n A on C6 cells a n d a s u b c l o n e of C6 cells r e s i s t a n t t o c o n c a n a v a l i n A (C6-ConA~) in o r d e r t o a s s e s s t h e u s e f u l n e s s of s u c c i n y l C o n A as a p r o b e t o s t u d y S100 protein, i n d u c t i o n in C6 cells.

Materials and Methods.

1. Preparation of succinyl ConA. Succinyl ConA was prepared by double derivatization with succinic anhydride [26]. Specifically 100 mg of ConA (Grade IV, Sigma, St Louis, Mo) were dissolved in 25 ml H~O and filtered (Whatman GF/C, Mandel Scientific, Rockwood, Ontario). The solution

BIOCHIMIE, 1983, 65, no 11-12.

was saturated with solid sodium acetate at 4 oC and stirred with 30 mg of finely crushed succinic anhydride for 60 rain at 4 oC. The reaction mixture was dialyzed against 2 × 6 1 of H,O overnight at 4 oC, lyophilized and subjected to a second derivatization with succinic anhydride at room temperature for 90 rain. The reaction mixture was dialyzed exhaustively against H,O, lyophilized, dissolved in 10 ml of phosphate buffered saline (PBS) (150 mM NaC1, 10 mM sodium phosphate p H 7.4) and 5 ml of the solution chromatographed on a Sephadex G75 (Pharmacia, Dorval, P.Q.) column, 45 x 2.5 cm diameter, equilibrated with PBS. The column was first washed with 300 ml of PBS, then with 300 ml of PBS containing t00 mM dextrose. Seven ml fractions were collected and absorbance at 280 nm measured. The fractions containing protein were pooled separately under each peak, dialyzed exhaustively against H~O and lyophilized. Succinyl ConA was separated into two fractions b y affinity chromatography on Sephadex G75, Peak I which did not bind to the dextran and was Muted with PBS and Peak I I which was competitively Muted with 100 mM dextrose. Both fractions stimulated SI00 protein synthesis in C6 cells but failed to agglutinate SRBC when used in concentrations up to 5 mg/ml. ConA was similarly Muted in two fractions. Both fractions were Muted in the same positions when pooled and rechromatographed separately after exhaustive dialysis. Both fractions agglutinated SRBC in concentrations as low as 2 ;< 10~4 mg/ml (data not shown). The physical basis for the heterogeneity of our lectin preparations was not readily apparent since fractions of both high and low affinity for the dextran were composed of a mixture of proteins with sim.ilar subunits with molecular weights ranging from 12,000-20,000 (data not shown). Our results indicate t h a t a fraction of lectin molecules which fail to bind to dextran can still interact with glycoprotein receptors on cell surfaces, suggesting t h a t the affinity of these molecules for membrane glycoprotein receptors is higher than their affinity for dextran. Nonetheless, only the fraction of succinyl ConA which was bound to Sephadex G75 and was Muted with 100 mM dextrose was used in further studies. Antibodies to succinyl ConA were prepared in rabbits b y four weekly intramuscular injections of 1 mg succinyl ConA emulsified in complete Freund's adjuvant.

2. Growth of cells, incorporation of ?~S]-methionine into proteins. C6 cells were grown in alpha MEM E27] (Gibco, Burlington, Ont.) supplemented with 4 per cent fetal calf serum (FCS) at 37 oC and 5 per cent CO2. Cultures were initiated at 5 × 103/cm * and grown for 10 days with changes of medium on alternate days. At this time the cultures were fully confluent and in the stationary phase of growth. Monolayers were harvested b y scraping with a rubber policeman ; cell counts and DNA measurements were performed as described E17, 18, 22-25]. Cell viability was determined b y T r y p a n Blue exclusion.

Synthesis of $100 protein in C6 cells For measurements of S100 protein synthesis in the cells, the cells were incubated with 8.5 l~Ci/ml of [asS]-methionine (500 Ci/mmol), (New England Nuclear, Lachine, P.Q.) in c~-MEM without methionine, supplemented with 1.5 per cent FCS, for 3 hours at 37 oC. Monolayers were washed and harvested as described above. Measurements of incorporation of [35S]-methionine into proteins and of relative synthesis of S 100 protein were performed as described previously [17, 18]. Relative synthesis of S100 protein -- (radioactivity in S100 protein/radioactivity in total proteins) × 100. To study the effects of succinyl ConA, cultures were initiated at 5 × 10~/cm" in 175 cm ~ tissue culture dishes (Falcon Plastics, Oxnard, Ca.) and grown as described above. On day 3 after subculture the medium was changed ; experimental cultures were treated for different periods of time with succinyl ConA dissolved in H,O and filtered through Millipore GS filters, 0.22 ~/diameter, prior to use. Following treatment with the lectin, the ceils were incubated further for 5 hours with 8.5 {~Ci/ml of psS]-methionine in ~-MEM without methionine; supplemented with 1.5 per cent FCS ; in the presence of the lectin. Relative synthesis of S100 protein was assayed as described above.

3. Selection of C6 cells resistant to ConA. Cells resistant to ConA were selected by plating l0 S C6 cells in a 25 cm 2 tissue culture dish in the presence of ConA, 88 ~g/ml. One of the three surviving colonies was picked with a Pasteur pipette, grown up and recloned. The m u t a n t cell line, C6-ConA~, was grown in the same culture conditions and treated experimentally by the same protocols as those described above for the parental C6 cells.

4. Binding of [135I] labeled ligands to cells. Succinyl ConA was labeled with 1~5I using carrierfree Na [1~5I] (Amersham, Oakville, Ontario) and chloramine T [28]. [1~I] succinyl ConA was separated from free 12~I by gel filtration on a 10 ml Sephadex G25 (Pharmacia) column. Cells were harvested from confluent monolayers by scraping, washed twice by centrifugation at 4oC with PBS, and incubated in 2.4 per cent gluteraldehyde in PBS for 30 rain at 20°C with shaking. The cells were washed twice with PBS, resuspended in PBS and stored at 4oC until use. Binding was performed in plastic tubes in a total volume of 0.25 ml of 0.4 per cent BSA in PBS with 5 × 105 gluteraldehyde-fixed C6 or C6ConA R cells, and [1~5I] succinyl ConA. The reaction mixture was incubated for 20 min at 20oC, the cells washed twice by centrifugation and the cell pellet counted in a gamma counter. Binding of [1'5I] Iodohydroxybenzylpindolol (['25I] HYP) (New England Nuclear 2 200 Ci/mmol) was adapted from procedures described in the literature [29, 30]. The steric isomers of propranalol (--) and (+_) were kindly donated by Drs. P. Seeman and M. Lucas. Ceils were harvested from confluent mono-

B I O C H I M I E , 1983, 65, no 11-12.

611

layers by scraping with a rubber policeman and suspended in Hanks' balanced salt solution (HBSS, Gibco). Binding reactions were performed in a total of 0.5 ml of HBSS with 4 × 10~ of C6 or C6-ConA ~ cells, (--) propranalol (.02 - - . 1 aM) and 74,000 cts/min of [ ~ I ~ H Y P . The reagents were mixed, the tubes counted in a gamma counter to determine input radioactivity and incubation continued for 60 rain at room temperature. The reaction was terminated by the addition of ( + ) propranalol to a final concentration of 0.1 mM. The cells were washed once by centrifugation with HBSS containing 0.1 mM propranaim and counted in a ,(-counter. Bound radioactivity in the cell pellet was expressed as the percentage of input radioactivity. DNA determinations on the cell pellet were performed as described E22].

5. lmmunofluorescent studies. C6 cell monolayers were grown in 10 cm ~ dishes as described above and incubated with succinyl ConA, 500 ~g/ml, for 60 rain. The ceils were washed extensively with HBSS and incubated with rabbit antisuecinyI ConA antiserum, diluted 1:20, for 20 rain at room temperature. The cultures were rinsed and further incubated with fluorescein-conjugated goat anti-rabbit immunoglobulin antibody (Hyland), diluted 1:5, as described above. After another wash with HBSS, a cover slip was mounted over the cultures with a drop of glycerol : saline (1:1). The cultures were examined immediately with a Leitz microscope equipped with phase contrast and fluorescent illumination, and photographed.

Results.

1. M u t a n t cells are succinyl ConA.

resistant

to

ConA

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A c l o n e of C o n A - r e s i s t a n t cells, C 6 - C o n A R was selected w i t h o u t mutagenesis b y plating p a r e n t a l C6 cells in t h e p r e s e n c e of C o n A (88 a g / m l ) . P a r e n t a l cells f a i l e d t o f o r m c o l o n i e s w h e n p l a t e d in t h e p r e s e n c e of c o n c e n t r a t i o n s of C o n A g r e a t e r t h a n 25 a g / m l , w h i l e C6C o n A R cells p l a t e d e f f i c i e n t l y a t c o n c e n t r a t i o n s of C o n A u p t o 90 ~zg/ml (figure 1). I n c u b a t i o n of l o w d e n s i t y (day 3 a f t e r s u b c u l t u r e ) c u l t u r e s of C6 cells w i t h s u c c i n y l C o n A , 500 a g / m l , c a u s e d r e t r a c t i o n of c e l l u l a r p r o c e s s e s a n d r o u n d i n g u p of cells. C 6 - C o n A ~ cells s h o w e d n o a l t e r a t i o n s in m o r p h o l o g y on i n c u b a t i o n w i t h t h e l e c t i n (figure 2).

2. M u t a n t cells bind less succinyl ConA parental C6 cell.

than

B i n d i n g of s u c c i n y l C o n A t o t h e cell s u r f a c e of C6 cells w a s first d e m o n s t r a t e d b y i n d i r e c t

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FIG. 1. - - Mutant dones are resistant to ConA. A clone of ConA-resistant cells, C6 ConA~, was selected after plating parental C6 cells in the presence of ConA (88 Izg/ml). Parental and mutant cells were assayed for colony formation in the presence of increasing concentrations of ConA. The cells were plated in 25 cm 2 dishes. After 8 days the colonies were stained with methylene blue and counted. For C6 cells 200 cells were plated in the absence of ConA and 5 × 10~ cells in the presence of ConA concentrations shown in the abcissa. No colonies grew up in the presence of any of the concentrations of ConA used. For C6 ConAR cells 250 cells were plated both in the absence of ConA and in the presence of increasing c o n c e n t r a t i o n s of ConA. T h e p l a t i n g efficiencies of C6 a n d C 6 - C o n A ~*cells in t h e p r e s e n c e of C o n A a r e e x p r e s s e d r e l a t i v e l y to t h e i r r e s p e c t i v e p l a t i n g efficiencies in t h e a b s e n c e of C o n A w h i c h were t a k e n as 100 p e r cent. T h e a c t u a l p l a t i n g efficiencies of C6 a n d of C 6 - C o n A R cells in t h e a b s e n c e of C o n A w e r e 27 p e r c e n t a n d 20 p e r cent, r e s p e c t i v e l y . R e s u l t s a r e a v e r a g e s of t w o e x p e r i m e n t s . T h e t w o v a l u e s o b t a i n e d a t e a c h C o n A c o n c e n t r a t i o n v a r i e d < 10 p e r c e n t from the mean.

immunofluorescence. W h e n the cells were e x a m ined with a focus in the p l a n e of the centre of the cell, the lectin was localized in a ring circumscribing the cellular b o u n d a r y . T h e r e was no intracellular fluorescence. T h e lectin could be visualized over the cell surface when the plane of the focus was c h a n g e d to the b o t t o m or to the t o p of the cell, t h u s confirming its localization on the cell surface. I n control reactions, cells which h a d n o t been t r e a t e d w i t h succinyl ConA prior to exposure to antisuccinyl ConA antiserum, did n o t fluoresce. P a r e n t a l C6 cells fluoresced b r i g h t l y a f t e r

BIOCHIMIE, 1983, 65, no 11-12.

i n c u b a t i o n w i t h succinyl ConA a n d staining with t h e specific antisera. I n c o n t r a s t C6ConA ~ cells showed only a fine g r a n u l a r fluorescence (figure 3). T h e s e results could be due to a decreased n u m b e r of ConA receptors ill C6-ConA ~ cells a n d / o r an increased m o b i l i t y of these receptors in the m u t a n t cells. I n order to confirm t h a t the m u t a n t cells in fact b o u n d less succinyl ConA, equal n u m b e r s of g l u t e r a l d e h y d e - f i x e d C6 a n d C6-ConA cells were i n c u b a t e d w i t h [125I~ succinyl ConA. M u t a n t cells b o u n d a p p r o x i m a t e l y five-fold less of the r a d i o a c t i v e lectin t h a n p a r e n t a l cells a n d t h e binding was specifically displaced in b o t h cell lines b y excess u n l a b e l e d succinyl ConA (figure 4). I n order to d e t e r m i n e the specificity of this decreased binding we e x a m i n e d the interaction of the ceils with a n o t h e r r a d i o a c t i v e ligand [12aIIHYP, which has been r e p o r t e d to bind to adrenergic receptors on the surface of C6 cells [291. The a m o u n t of this ligand b o u n d to b o t h cell lines was a p p r o x i m a t e l y the same, w i t h C6-ConA ~ ceils a c t u a l l y binding slightly m o r e [I~5IIHYP t h a n p a r e n t a l cells (figure 5). In our hands, the o b s e r v e d binding of [12q]HYP was only slightly displaced b y (-) propranalol. Therefore, this m e a s u r e in all likelyhood r e p r e s e n t e d an e s t i m a t e of non-specific binding of this ligand to the cell surface a n d did not reflect the n u m b e r of specific ~ adrenergic receptors [291. This result suggests t h a t nonspecific binding of ligands to the cell surface is the s a m e in the p a r e n t a l a n d in the m u t a n t cells. Therefore, the decreased binding of succinyl ConA to C6-ConA ~ cells is indicative of a selective binding defect in C6-ConA ~ ceils.

3. Succinyl ConA does not induce SIO0 protein synthesis in mutant cells. E x p e r i m e n t s were p e r f o r m e d w i t h 4 to 6 d a y old cultures of C6 cells in e a r l y e x p o n e n t i a l growth. The relative synthesis of S100 protein m u n t r e a t e d control cultures was at the basal u n i n d u c e d level (.011 per cent) characteristic of C6 cells in this p h a s e of growth. T r e a t m e n t w i t h succinyl ConA increased the relative synthesis of S100 protein 2 to 3-fold. T r e a t m e n t s w i t h u n c o n j u g a t e d ConA or s u c c i n y l a t e d bovine s e r u m a l b u m i n (succinyl BSA) h a d no effect on the relative synthesis of S100 protein in C6 cells ( d a t a not shown). T h e dose response curve of S100 protein induction b y succinyl ConA r e a c h e d a p l a t e a u at a lectin c o n c e n t r a t i o n s of a p p r o x i m a t e l y 500 ~ g / m l (figure 6A). The t i m e course of S100 protein induction b y the lectin

Synthesis of $100 protein in C6 cells

FIG. 2. - - Mutant cells do not round up in the presence of succinyl ConA. P a r e n t a l C6 cells (top) and C6-ConA x cells (bottom) were i n c u b a t e d in the presence of succinyl ConA (500 tzg/ml). Phase c o n t r a s t optics (× 10).

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613

A. Marks, J. Law a~d J. Mahony

614

FIG. 3. - - Demonstration of binding of succinyl ConA to C6

a~zd C6 Co~d ~ ~elts using immunofluo¢,escence. P a r e n t a l C6 cells (top) a n d C6-ConA a cells ( b o t t o m ) w e r e i n c u b a t e d w i t h succinyl ConA, r a b b i t a n t i - s u c c i n y l ConA a n t i s e r u m , a n d g o a t a n t i - r a b b i t a n t i s e r u m coupled w i t h fluorescein. F l u o r e s c e n t optics ( × 40).

B I O C H I M I E , 1983, 65. no 11-12.

Synthesis of SIO0 protein in C6 cells

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FIG. 4. 2Vtutanl cells bind less succinyl ConA. A. Glutaraldehyde fixed cells (5 X 10~) were incubated with [l~I]-succinyl ConA (90,000 cts) for 20 rain at 20 °C in the presence of increasing amounts of unlabeled succinyl ConA. B. Cells were incubated with increasing amounts of t~"-5II-succinyl ConA (9,000 cts/10 V1). Results of representative experiments are shown. • • parental C6 cells o . . . . O C6-ConAR cells. -

0

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showed a linear increase in S100 protein synthesis in the first 24 hours a n d a slower increase o v e r the n e x t 24 hours (figure 6B). W h e n the lectin was w i t h d r a w n after 24 h o u r s the synthesis of S100 p r o t e i n decreased r a p i d l y d e m o n s t r a t i n g t h a t a continuous e x p o s u r e of the cultures to the lectin was n e c e s s a r y to m a i n t a i n the induction (data n o t shown). I n c o n t r a s t to the a b o v e results w i t h the p a r e n t a l cells, there was no s t i m u l a t i o n of synthesis of S100 protein w h e n C6-ConA ~ cells were t r e a t e d w i t h succillyl ConA at 500 a g / m l for 48 hours (figure 6B).

4. Parental and mutant cells regulate SIO0 protein synthesis as a Junction of growth in monolayer culture. Cultures of C6 a n d C6-ConA ~ cells were i n i t i a t e d at a d e n s i t y of 5 × 103/cm ~ and a s s a y e d for D N A c o n t e n t a n d relative synthesis of S100 protein. C6-ConA ~ cells w e n t t h r o u g h

BIOCHIMIE, 1983, 65, n o 11-12.

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5. -B i n d i n g o f ~ I ~ s I ] H Y P to C5 and C6 Cond R cells.

Binding of E12~I~HYPwas performed in the presence of increasing amounts of (--) propranalol using 4 × 10~ cells. After washing, bound radioactivity in the cell pellet was expressed as a percentage of input radioactivity. Bound radioactivity was normalized per I~g of DNA in the cell pellet. Results of a representative experiment are shown. • • C6 cells o . . . . o ConA~ cells.

a longer lag p h a s e t h a n C6 cells (3 d a y s versus 1 day) before entering the logarithmic p h a s e of growth. C o n s e q u e n t l y C6 cells r e a c h e d the s t a t i o n a r y p h a s e of g r o w t h earlier t h a n C6ConA R cells, after 10 d a y s ill m o n o l a y e r culture. H o w e v e r , a f t e r 14 d a y s C6-ConA R cells f o r m e d a fully confluent m o n o l a y e r a n d r e a c h e d a s a t u r a t i n g cell d e n s i t y equal to t h a t of C6 cells (figure 7). T h e relative synthesis of S100 protein b e g a n to i n c r e a s e during e x p o n e n t i a l cell division ill b o t h cell lines a n d c o n t i n u e d to increase until the s t a t i o n a r y p h a s e of growth. T h e increase was a p p r o x i m a t e l y five-fold o v e r the b a s a l level o b s e r v e d in early l o g a r i t h m i c cultures. T h e r e was a delay in the induction of S100 protein in C6-ConA R cells corresponding to the longer lag p h a s e o b s e r v e d w h e n these cells were seeded at low density. I n p a r e n t a l C6 cells a decrease in the relative synthesis of S100 p r o t e i n was s o m e t i m e s o b s e r v e d after m a i n t a i n i n g cultures for several d a y s in the

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Succinyl ConA induces SIO0 protein in parental C6 cells but not in C6-ConA R cells.

Cells were t r e a t e d w i t h increasing c o n c e n t r a t i o n s of succinyl C o n A for 48 h o u r s (A) or for increasing l e n g t h s of t i m e w i t h 500 ~ g / m l of succinyl ConA (]3). R e s u l t s r e p r e s e n t a v e r a g e s of analysis of t w o c u l t u r e s t r e a t e d i n d e p e n d e n t l y . R e p l i c a t e s varied < 8 per c e n t f r o m t h e m e a n . •

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Parental and mutant cells regulate SIO0 protein synthesis as a function of growth in monolayer culture.

Cells w e r e p l a t e d a t 5 × 10 a cell/cm*. Culture dishes w e r e a s s a y e d for D N A c o n t e n t (A) a n d r e l a t i v e s y n t h e s i s of S100 p r o t e i n (B). R e s u l t s of 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 are shown. o--o

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B I O C H [ M I E , 1983, 65, n ° !1-12,

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Synthesis of $700 protein in C6 cells stationary phase of growth (figure 7). This effect has been noted previously [18] and may be due to a secondary effect caused b y fluctuations in the p H of the medium in prolonged stationary cultures [311.

Discussion.

The signals which cause the induction of S 100 protein in C6 cells as a function of growth are not known. We have previously shown that treatment with the lectin succinyl ConA increased the relative synthesis of S100 protein in early logarithmic cultures of C6 ceils [221. In the present study, we used a genetic approach to confirm this observation. Firstly, we isolated a mutant cell line (C6-ConAR) which was resistant to ConA and cloned efficiently at concentrations of ConA which inhibited completely the growth of C6 cells (figure 1). Secondly, we found that C6-ConA ~ cells bound five-fold less succinyl ConA than did parental C6 cells (figure 4) and were unaffected by doses of succinyl ConA which caused rounding up of the parental cells (figure 2). Thirdly, we showed that treatment whith succinyl ConA did not increase the relative synthesis of S100 protein in C6-ConA ~" cells (figure 6). Taken together these results suggest that succinyl ConA interacts with ConA receptors on the surface of C6 cells. Paradoxically, treatment with unmodified ConA did not increase S100 protein synthesis in C6 cells. This discrepancy m a y be related to differences in valences between ConA and succinyl ConA. ConA is a tetravalent ligand which agglutinates SRBC and succinyl ConA behaves as a monovalent ligand since it fails to agglutinate SRBC [261. It is possible that the observed differences between the effects of ConA and succinyl ConA on S100 protein synthesis in C6 ceils are likewise due to differences in interactions of the two ligands with membrane receptors. Thus tetravalent ConA might cross-link these receptors whereas monovalent succinyl ConA would not [26]. A similar explanation has been suggested previously to account for the observed differences in the respective actions of the two lectins on mitogenesis in mouse lymphocytes [321. Defects in ConA-binding at the cell surface have benn observed also in several other ConA resistant mammalian cell lines. The defects include reduced ConA binding, lack of coope-

BIOCHIMIE, 1983, 65, n ° 11-12.

617

rativity among ConA receptors and decreased mobility of ConA receptors [33-38]. In order to assess the involvement of ConA receptors in mediating distal physiological responses it is important to relate defects in lectin binding to other phenotypic alterations in ConA-resistant mutant cell lines. In several instances ConA resistance was accompanied b y pleiotropic characteristics such as variation in cell and colony morphology, decreased adherance to the substratum, increased sensitivity to membrane active agents and temperature sensitivity to growth [33, 38]. Recently ConA resistant rat myoblast cell lines which fail to fuse at confluence have also been reported [34]. It is possible that some of these characteristics are an incidental consequence of membrane alterations in the mutant cells, since there is no evidence to indicate that any of the above pleiotropic changes in ConA resistant cell lines are a secondary manifestation of a ConA binding defect. In contrast to the ambiguous role of ConA receptors in the above cellular responses, we have shown that treatment with succinyl ConA specifically increases the relative synthesis of S100 protein in logarithmic cultures of C6 cells [22]. This suggests an involvement of ConA receptors in mediating this distal intracellular response. Therefore we examined C6ConA R ceils for a possible correlation between defective ConA receptors and alterations in S100 protein induction. Succinyl ConA did not increase the relative synthesis of S100 protein in C6-ConAR cells at concentrations and times of treatment which exerted a maximal effect in the parental cells (figure 6). Since C6-ConA ~ cells are defective in binding succinyl ConA (figures 3, 4) it is likely that the binding of succinyl ConA to specific surface receptors is the first step in the induction of S100 protein b y the lectin in logarithmic cultures of C6 ceils. However, since the synthesis of S100 protein is regulated normally as a function of growth in C6-ConA R cells (figure 7), the induction of S100 protein which occurs in late logarithmic growth must differ from the induction mediated b y succinyl ConA. While the induction of S100 protein b y succinyl ConA is dependent on an interaction of succinyl ConA with its membrane receptors, the induction observed as a function of growth m a y be independent of these receptors (figures 4, 7). Therefore the initial events mediating the two inductions are clearly different. However, it is possible that the inductions of S100 protein in C6 cells b y succinyl ConA and as a function of growth are mediated

A . M a r k s , J . L a w and J . M a h o n y

618

d i s t a l l y b y a commo~a p a t h w a y . T h e r e f o r e t h e l e c t i n m i g h t p r o v e to b e a u s e f u l p r o b e to elucidate the biochemical steps u n d e r l y i n g this pathway.

16. 17. 18. 19.

ACKNOWLEDGMENTS. This work was supported by d grants ¢rom the Medical Research Council and the National Cancer Institute of Canada. We wish to thank Dr. A. O. Jorgensen for her assislance with the immunofluorescence studies. -

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20. 21. 22. 23. 24.

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