Effects of hexachlorocyclohexane isomers on concanavalin a ‘capping’ in bovine lymphocytes

501

Biochimica et Biophysica Acta, 586 (1979) 501--511 © Elsevier/North-Holland Biomedical Press

BBA 28999

E F F E C T S OF H E X A C H L O R O C Y C L O H E X A N E ISOMERS ON CONCANAVALIN A 'CAPPING' IN BOVINE LYMPHOCYTES

CHEUNG H. KWONG and GERALD C. MUELLER

McArdle Laboratory for Cancer Research, The University of Wisconsin, Madison, WI 53706 (U.S.A.) (Received October 31st, 1978) (Revised manuscript received March 5th, 1979)

Key words: Hexachlorocyclohexane isomer; 'Capping'; Concanavalin A-receptor complex; Lectin; Lymphocyte membrane; (Bovine)

Summary The effects of a,/~, 7, and 5 isomers o f hexachlorocyclohexane on concanavalin A 'capping' in bovine lymphocytes were evaluated,7 and 5 hexochlorocyclohexane inhibited 'capping' whereas the ~ and ~ isomers were without effect. In addition, 7-hexachlorocyclohexane has been shown to antagonize the maintenance of preformed 'caps' and cause the rapid dispersal of the concanavalin A-receptor complexes over the surface of cells by a temperature-dependent mechanism. The possible role of a 7-hexachlorocyclohexane-sensitive process in the organization o f microflow patterns in the lectin-activated l y m p h o c y t e membrane is discussed.

Introduction The interaction of mitogenic lectins with surface glycoproteins of cultured lymphocytes results in a rapid activation of membrane metabolism. Among the earliest responses are the changes in the lipid fluidity [1] and the acceleration of the synthesis of phosphatidylinositol which are demonstrable within the first 10 min of the lectin treatment [2]. Pretreatment of the cells with the chloro congeners of inositol, 7- and 5-hexachlorocyclohexane, blocks both the activation o f phosphatidylinositol metabolism [3] and the subsequent lectin-induced growth response of the cells [4]. Delaying the addition of ~,-hexachlorocyclohexane for as little as 10 min largely obviated the inhibitory effects of this agent on both systems. These results suggested initially that the activation of phosphatidylinositol metabolism may be required in the triggering of the mitogenic response [2--4]; however, the possibility remained that 5- and ~,-hexa-

502 chlorocyclohexane may be affecting the function of a system indirectly which is c o m m o n to both the lectin activation of phosphatidylinositol metabolism and the lectin triggering of the mitogenic response. To explore this realm further, the effect of hexachlorocyclohexanes on the 'capping' of concanavalin A in bovine l y m p h o c y t e s was studied. As shown in many laboratories [5--8], the binding of concanavalin A to certain glycoproteins of the l y m p h o c y t e surface activates a rapid, energy-dependent movement of membrane constituents through which the tetravalent concanavalin A, along with the associated membrane proteins, are concentrated in a polar, cap-like structure. While this process is not fully understood, it is thought to involve a coordinated action of the microfilament system which is guided in part by intracellular components and in part by the concanavalin A that is b o u n d to cell surface glycosides. Although gross capping is not essential for mitogenesis [9,10], the p h e n o m e n o n is nonetheless a good indicator of membrane activation which appears to be a critical event in the triggering of cells by mitogenic lectins. Using this endpoint it has been found that 3'- and 5-hexachlorocyclohexane are highly effective in preventing concanavalin A capping whereas the and/3 isomers are inactive *. A surprising aspect of these studies, however, was the observation that the delayed addition of 3'- or 5-hexachlorocyclohexane mediated a temperature-dependent, cytochalasin-sensitive dispersal of preformed concanavalin A caps. This finding suggests that the active hexachlorocyclohexane isomers may not prevent capping by inhibiting membrane movement, b u t rather by disorganizing the microflow patterns which are required for both the formation and the maintenance of the concanavalin caps. The implications of these findings for the cell biology of the mitogenic response as well as the role that the activation of phosphatidylinositol metabolism plays in the response are discussed. Materials and Methods

Materials. Concanavalin A (grade IV), fluorescein isothiocyanate (approximately 10% fluorescein isothiocyanate on celite, Isomer I), a-methyl-D-mannoside, cytidinediphosphodiglyceride (CDPdiglyceride) and myoinositot were purchased from Sigma Chemical Co., St. Louis, MO. 12-O-Tetradecanoylphorbol13-acetate was provided b y Dr. R.K. Boutwell, McArdle Laboratory, University of Wisconsin, Madison, WI. a-Hexachlorocyclohexane was purchased from the Aldrich Chemical Co., Milwaukee, WI, and the /3, 3' and 5 isomers were gifts of Professor Paul Lichtenstein, Entomology Department, University of Wisconsin, Madison, WI. Dimethylsulfoxide, (spectrophotometric grade, Aldrich Chemical Co.) was used as the vehicle for 12-O-tetradecanoylphorbol-13-acetate and the hexachlorocyclohexanes; the final concentration of dimethylsulfoxide in all cultures was 0.5%. Lymphocytes. L y m p h o c y t e s were isolated from bovine retropharyngeal lymph nodes of freshly slaughtered cattle (Oscar Mayer and Co., Madison, WI) as described previously [11]. The cells were suspended in a modified Eagle's HeLa medium containing 10% bovine serum [12] and gentamicin and incu* See F i g . 2 f o r t h e s t r u c t u r a l f o r m u l a s o f t h e h e x a c h l o r o c y c l o h e x a n e

isomers.

503 bated at 37°C in a spinner bottle in a 5% CO2/95% air atmosphere for 1--3 days prior to use in the capping and concanavalin A binding studies. A 100 ml aliquot of the cell culture was centrifuged at 150 X g for 5 min and the cell pellet resuspended in 20 ml of fresh medium. The clumped and dead cells were allowed to settle for 5 min and the upper portion containing the disperseci cells was removed. These cells were then pelleted, washed once in phosphate-buffered saline (buffered saline [ 13] ), and resuspended in buffered saline at 20 • 106 cells/ml. In the concanavalin A binding studies it was necessary to remove the remaining erythrocytes by treating the cells with hypotonic shock [14]. Typically, cells from 100 ml of cell culture were pelleted, resuspended in 2 ml buffered saline or medium and 6 ml of double
504 capped cells. The percentage of cap-forming cells for each sample is expressed as means of triplicate counts (100 cells/determination) ± S.E. Concanavalin A binding studies. In order to determine the amount of concanavalin A b o u n d to cells, l y m p h o c y t e s ( 2 0 . 106/ml) were incubated with 12SI-labeled concanavalin A prepared according to McConahey and Dixon [17]. 50 pg/ml 12SI-labeled concanavalin A was required to saturate the binding sites of 2 0 . 1 0 6 bovine lymphocytes in 1 ml buffered saline. At the appropriate times after 12SI-labeled concanavalin A addition, the cells were pelleted (150 × g, 5 min) and washed with 5 ml buffered saline containing the drugs tested. After a second centrifugation, 1 ml buffered saline was added and the radioactivity of the 12SI-labeled concanavalin A retained in the cell pellets was determined by a 7 counter. Nonspecific adsorption of concanavalin A was the a m o u n t of 12SI-labeled concanavalin A b o u n d to cells in the presence of 40 mM a-methyl-D-mannoside. This c o m p o u n d competes with the cell membrane glycoprotein for the sugar binding sites on concanavalin A. Results

Effects o f hexachlorocyclohexane on concanavalin A capping As shown by studies in other laboratories [5,18] on l y m p h o c y t e s and other cells, tetravalent concanavalin A mediates a temperature and energy
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MINUTES Fig. 1. E f f e c t of 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e o n t h e k i n e t i c s o f c o n c a n a v a l i n A c a p p i n g . L y m p h o c y t e s ( 2 0 " 1 0 6 / m l ) w e r e i n c u b a t e d at 3 7 ° C w i t h 5 0 p g / m l f l u o r e s c e i n - l a b e l e d c o n c a n a v a l i n A in t h e p r e s e n c e ( e ) or a b s e n c e (~) o f 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e ( 1 0 n M ) . 1 2 - O - T e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e w a s a d d e d c o n c u r r e n t l y w i t h f l u o r e s c e i n - l a b e l e d c o n c a n a v a l i n A. F o r m a l d e h y d e w a s a d d e d at t h e i n d i c a t e d i n t e r v a l s a f t e r f l u o r e s c e [ n - l a b e l e d c o n c a n a v a t i n A a d d i t i o n a n d t h e p e r c e n t o f c a p p e d cells evaluated under fluorescence microscopy.

505

maintained by the presence of 10 -s M 12-O-tetradecanoylphorbol-13-acetate. 12-O-Tetradecanoylphorbol-13-acetate is best known as a potent tumor-promoting agent in mouse skin [19]; however, it is also an effective l y m p h o c y t e comitogen when added to cultures treated with concanavalin A or phytohemagglutinin [11]. While its mechanism of action in l y m p h o c y t e s is yet unclear, the rapid acceleration of phospholipid synthesis and amino acid transport [20,21] suggest that it has a primary action on the cell membrane; this subject, as well as the manner in which 12-O-tetradecanoylphorbol-13-acetate increases capping will be discussed elsewhere. In the present report 12-O-tetradecanoylphorbol-13-acetate has been used primarily as a tool to facilitate the capping response which has been triggered b y fluorescein-labeled concanavalin A. When 7-hexachlorocyclohexane, an agent which interferes with the mitogenic action of lectins in these cells [3,4] was added to the system, the yield of capped cells was greatly reduced. A comparison of the a, fi, ~, and 5 isomers of hexachlorocyclohexane (Fig. 2) shows that this is a stereospecific p h e n o m e n o n with the ~, and 5 isomers being highly active inhibitors whereas the a and fi isomers are essentially inactive. The IDs0 for the ~,- and ~i-hexachlorocyclohexane was 50 pM and 20 pM, respectively. Thus a- and ~-hexachlorocyclohexane, which differ in configuration of only one chlorine atom from 7- and 5-hexachlorocyclohexane, respectively, were shown to be inactive. Similar doseresponse curves are obtained when the cells were not treated with 12-O-tetradecanoylphorbol-13-acetate (data not shown); however, the level of capping was much lower. A surprising feature of these studies was the observation that the addition of

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HEY~I~HL~YC LOHEXANE (mM) Fig. 2. Dose-response relationship o f the effects of various h e x a c h l o r o c y c l o h e x a n e isomers o n cap format i o n in l y m p h o c y t e s . H e x a c b l o r o c y c l o h e x a n e s were added 15 m i n prior to the fluore~ein-lAheled c o n canavalln A addition. Cells were i n c u b a t e d at 3 7 ° C t h r o u g h o u t t h e e x p e r i m e n t . A f t e r e x p o s u r e to fluorescein-labeled coneanavalln A (50 # g l m l ) and 1 2 - O - t e t r a d e e a n o y l p h o r b o l - 1 3 - a e e t a t e ( I 0 nM) for 20 m i n t h e l y m p h o c y t e s w e r e fixed w i t h f o r m a l d e h y d e ~-d the percent capped cells determined, u------.---~ ~; ~, .G; a - - - - - - - - o , 7 ; o o, 6.

506 7-hexachlorocyclohexane 3 0 m i n after the initiation of the capping phenomenon b y fluorescein-labeled concanavalin A caused a rapid decrease in the number of capped cells (Fig. 3). Thus, while caps are known to slowly disperse when the temperature of cells is dropped (possibly due to the diffusion of the membrane glycoprotein in absence of an energy
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Fig. 3. E f f e c t s o f t e m p e r a t u r e a n d t h e d e l a y e d a d d i t i o n o f 7 - h e x a c h l o r o c y c l o h e x a n e o n t h e m a i n t e n a n c e o f p r e f o r m e d caps. F l u o r e s c e i n - l a b e l e d c o n c a n a v a l i n A a n d 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e w e r e i n t r o d u c e d c o n c u r r e n t l y t o t h e b o v i n e l y m p h o c y t e c u l t u r e s and i n c u b a t e d at 3 7 ° C . T h e t e m p e r a t u r e w a s s h i f t e d f z o m 3 7 ° C t o 0 ° C b y t r a n s f e r z i n g t h e l y m p h o c y t e c u l t u r e s f r o m a 3 7 ° C w a t e r b a t h t o a 0 ° C ice/ w a t e r b a t h . 7 - H e x a c h l o r o c y c l o h e x a n e , w a s a d d e d 3 0 r a i n a f t e r f l u o r e s c e i n - l a b e l e d c o n c a n a v a i i n A addit i o n t o give a final c o n c e n t r a t i o n o f 0 . 2 raM. F o r m a l d e h y d e w a s a d d e d at t h e end o f t h e r e a c t i o n t o fix t h e cells and t h e p e r c e n t c a p p e d cells w e r e d e t e r m i n e d , o o, 3 7 ° C ; • _- O°C; ~ . . . . . . m 37°C plus 7 - h e x a c h l o r o c y c l o h e x a n e t r e a t m e n t ; n . . . . . . o O°C p l u s 7 - h e x a c h l o r o c y c l o h e x a n e t r e a t m e n t .

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Fig. 4. E f f e c t o f c y t o c h a l a s i n B o n t h e d i s p e r s i o n o f c o n c a n a v a l i n A caps. F l u o r c s c e i n - l a b e l e d c o n c a n a valln A a n d 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e ( 1 0 n M ) w e r e a d d e d to l y m p h o c y t e c u l t u r e s at t i m e z e r o a n d t h e c u l t u r e s i n c u b a t e d a t 3 7 ° C t o establish t h e c a p p e d cells. A f t e r 3 0 m i n , c y t o c h a l a s i n B ( 1 0 # g / m l ) w a s a d d e d as i n d i c a t e d a n d t h e cells i n c u b a t e d a n a d d i t i o n a l 1 2 rain at w h i c h t i m e T - h e x a c h l o r o c y c l o h e x a n e (0.2 raM) w a s a d d e d ( . . . . . . ) to b o t h c o n t r o l a n d c y t o c h a l a s i n B - t r e a t e d c u l t u r e s .

!:$I-labeled concanavalin A. As shown in Fig. 5, the labeled concanavalin A was bound to the lymphocyte surface through an a-methyl-D-mannoside-sensitive linkage. When treatment with a-methyl-D-mannoside was delayed for 45 min approximately 20% of the specific cell-associated 12SI-labeled concanavalin A +=-METHYL MANNOSIDE

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Fig. 5. K i n e t i c s o f a n d s p e c i f i c i t y o f 1 2 $ i . l a b e l e d c o n c a n a v a l l n A b i n d i n g . B o v i n e l y m p h o c y t e s ( 2 0 • I 0 5 in 1 m l b u f f e r e d saline) w e r e i n c u b a t e d a t 3 7 ° C w i t h 1 0 0 p g / m l 1 2 5 i . l a b e l e d c o n c a n a v a l i n A in t h e prese n c e o r a b s e n c e o f 4 0 m M a - m e t h y l - D - m a n n o s i d e . T h e r V - m e t h y l - D - m a n n o s i d e w a s a d d e d at 0 t i m e ( e ) o r 4 5 m i n (4) a f t e r ! 2 $ I - l a b e l e d c o n c a n a v a l i n A a d d i t i o n . Cell-associated r a d i o a c t i v i t y w a s d e t e r m i n e d at t h e i n d i c a t e d t i m e s , o, c u l t u r e s r e c e i v i n g n o ~ - m e t h y l - D - m a n n o s e . Specific b i n d i n g is o b t a i n e d b y subtracting the c p m of cell-bound concanavalin A of l y m p h o c y t e s receiving ~-methyl°D-mannoside at zero t i m e f r o m t h e c p m o f cell*bound c o n c a n a v a l i n A in a given l y m p h o c y t e c u l t u r e . D a t a r e p r e s e n t t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s -+S.E.

508 was not released. This non
Failure o f myoinositol and CDPdiglyceride to antagonize the hexachlorocyclohexane effect on concanavalin A capping The stereospecificity of hexaehlorocyclohexanes in inhibiting concanavalin A capping suggests that these agents are acting at specific target sites and that their effects might be modified b y specific products of intermediate metabolism. In this connection it has been reported b y Hokin and Brown [23] that the addition of exogenous CDPdiglyeeride, a substrate of CDPdiglyeeride-inositol transferase, could protect the acetylcholine-stimulated phosphatidylinositol synthesis in microsomal preparations of cerebral cortex from the inhibition by 7-hexachlorocyclohexane. In an a t t e m p t to use this concept, we preincubated l y m p h o c y t e s with CDPdiglyceride (at a final concentration similar to that used in the Hokin and Brown study) for 15 rain prior to addition of 7-hexachlorocyclohexane. In similar studies, we also preincubated l y m p h o c y t e s with a high level o f myoinositol, which bears the same configuration as 6-hexachlorocyclohexane. Neither of these agents was able to protect against the effects of 7and 5-hexachlorocyclohexane on the fluorescein-labeled concanavalin A capping (Table III). Lack of effects o f hexachlorocyclohexanes on cell viability Since capping is a complicated energy
O F "y- A N D 6 - H E X A C H L O R O C Y C L O H E X A N E

ON THE BINDING OF CONCANAVALIN

A

TO BOVINE LYMPHOCYTES ~'- a n d 6 - h e x a c h l o r o c y c l o h e x a n e w e r e i n t r o d u c e d 15 m i n p r i o r t o t h e 125 I - l a b e l e d c o n c a n a v a l i n A a n d 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e ( T P A ) treatment. The l y m p h o c y t e s w e r e i n c u b a t e d f o r 4 5 r a i n a t 3 7 ° C a n d t h e a m o u n t o f c e l l - b o u n d 12 S i_labele d c o n c a n a v a l i n A d e t e r m i n e d . P o i n t s a r e m e a n s o f triplic a t e d e t e r m i n a t i o n s -+ S.E. C o n c e n t r a t i o n o f d r u g s u s e d : T P A ( 1 0 n M ) ; c o n c a n a v a l i n A ( 1 0 0 t t g / m l ) ; ~ , - h e x a c h l o r o e y c l o h e x a n e (0.1 r a M ) ; 5 - h e x a c h l o r o c y c l o h e x a n e (0.1 r a M ) .

Treatment

Specific binding of 12 5 I-labeled concanavalin A ( c p m / 1 0 7 cells)

None + TPA + TPA + ~-hexaehlorocyclohexane + TPA + 5-hexachlorocyclohexane

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T A B L E II E F F E C T O F 7- A N D 6 - H E X A C H L O R O C Y C L O H E X A N E N A V A L I N A IN B O V I N E L Y M P H O C Y T E S

ON THE INTERNALIZATION

OF CONCA-

W h e r e i n d i c a t e d , t h e l y m p h o c y t e s w e r e t r e a t e d a t 3 7 ° C w i t h 7- a n d 6 - h e x a c h l o r o c y c l c o h e x a n e f o r 1 5 r a i n p r i o r t o t h e a d d i t i o n o f 12 SI-labeled c o n c a n a v a l i n A a n d 1 2 - O - t e t ~ a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e (TPA). T h e cells w e r e t h e n i n c u b a t e d f o r a n a d d i t i o n a l 4 5 r a i n in t h e p r e s e n c e o f t h e s e r e a g e n t s . ~ - M e t h y l - D - m a n n o s i d e w a s t h e n a d d e d t o all c u l t u r e s . A f t e r 3 0 r a i n t h e cells w e r e w a s h e d a n d t h e r a d i o a c t i v i t y d u e t o c e l l - b o u n d c o n c a n a v a l i n A w a s d e t e r m i n e d . T h e i n t e r n a l i z e d c o n c a n a v a l i n A is d e f i n e d as t h e a m o u n t o f c e l l - b o u n d c o n c a n a v a l i n A m i n u s t h e n o n s p e c i f i c b i n d i n g o f t h i s r e a g e n t w h i c h o c c u r s in cells t r e a t e d a t z e r o t i m e w i t h ~ - m e t h y l - D - m a n n o s i d e . T h e l a b e l i n g o f c o n t r o l cells p r i o r t o ~ - m e t h y l - D - m a n n o s i d e t r e a t m e n t w a s 1 0 5 4 1 -+ 4 1 8 c p m / 1 0 7 cells. Treatment

Internalized concanavalin A ( c p m / 1 0 7 cells)

None + TPA

1726 1790 2019 1613

+ 7-hexachlorocyclohexane + TPA + 5-hexachlorocyclohexane + TPA

+ 91 + 122 + 236 ± 148

cyclohexane might be due to interference in the energy-yielding systems and thus affect capping only indirectly. To this end, the four hexachlorocyclohexane isomers were tested for their ability to influence the exclusion of trypan blue by the lymphocytes. As shown in Table IV, levels of the hexachlorocyclohexanes which maximally inhibit capping only minimally altered the percentage of cells excluding this dye. From these studies it was concluded that inhibition of capping by hexachlorocyclohexane is probably not due to the acute cytotoxicity of these agents and that loss of cell viability was not a factor in these experiments. Consistent with this interpretation is the previous report that 7-hexachlorocyclohexane at 1 mM did not substantially interfere with glucose utilization in human lymphocytes [ 3 ].

T A B L E III EFFECTS OF MYOINOSITOL AND CDP-DIGLYCERIDE TAGONISM OF CONCANAVALIN A CAPPING

ON HEXACHLOROCYCLOHEXANE

AN-

All l y m p h o c y t e c u l t u r e s r e c e i v e d f l u o r e s c e i n - l a b e l e d c o n c a n a v a l i n A ( 5 0 / a g / m l ) a n d 1 2 - O - t e t r a d e c a n o y l phorbol-13-acetate (10 nM). Myoinositol (50 raM) and CDP-diglyceride (0.15 raM), when added, were introduced to the cultures 30 rain prior to the addition of fluorescein-labeled concanavalin A; the inhibitors0 7" a n d 6 - h e x a c b l o r o c y c l o h e x a n e w e r e a d d e d as i n d i c a t e d 1 5 r a i n p r i o r t o t h e 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e . F o l l o w i n g a 2 0 r a i n e x p o s u r e t o t h e f i u o r e s c e i n - l a b e l e d c o n c a n a v a l i n A, t h e cells w e r e f i x e d in 2% f o r m a l d e h y d e a n d t h e p e r c e n t c a p p e d cells d e t e r m i n e d . Added factor

Inhibitor (raM)

C a p p e d cells (%)

None Myoinositol CDP-diglyceride None None My oinositol My oinositol C DP-diglyceride

None None None 5-hexachlorocyc!ohe~ane 7-hexacldorocyclohexane 6 -hexachlorocyclohexane 7-hexachlorocyclohexane 7-hexachlorocyclohexane

51 50 52 4 5 3 6 6

(0.05) (0.1) (0.05) (0.I) (0.1)

510 T A B L E IV E F F E C T S OF H E X A C H L O R O C Y C L O H E X A N E

T R E A T M E N T ON T R Y P A N B L U E E X C L U S I O N

L y m p h o c y t e s ( 2 0 . 1 0 6 ] r n l b u f f e r e d saline) i s o m e r s for 30 m i n at 3 7 ° C . A 50 #1 a l i q u o t containing 0.25% bovine serum albumin and e a c h c u l t u r e w a s d e t e r m i n e d w i t h i n t h e first b e r o f d y e - e x c l u d i n g c e U s / 1 5 0 cells c o u n t e d .

w e r e i n c u b a t e d w i t h the i n d i c a t e d h e x a c h l o r o c y c l o h e x a n e of the cell c u l t u r e was a d d e d to 1 m l Eagle's H e L a m e d i u m 0 . 0 1 % t r y p a n blue. T h e p e r c e n t a g e of d y e - e x c l u d i n g cells in 5 m i n o f e x p o s u r e to t h e d y e . R e s u l t s are b a s e d o n the n u m -

Treatment

D y e - e x c l u d i n g cells (%)

None 0.20 mM 0.20 mM 0.05 mM 0.20 mM 0.05 mM 0.20 mM

96 96 95 92 89 93 87

~-hexachlorocyclohexane fl-hexachlorocyclohexane ~-hexachlorocyelohexane 3~-hexachlorocyclohexane 5-hexachlorocyclohexane 5-hexachlorocyclohexane

Discussion This study clearly documents the ability of certain hexachlorocyclohexanes, the chloro congeners of inositol, to modulate cap formation in bovine lymphocytes treated with fluorescein-labeled concanavalin A. A comparison of the isomers shows a striking sterospecificity with activity paralleling their ability to inhibit phosphatidylinositol metabolism in other systems [3,23]. One interpretation of this correlation is that a phosphatidylinositol c o m p o u n d may s o m e h o w be involved in the capping phenomenon. Unfortunately, direct attempts to implicate this pathway using the potential substrates, CDPdiglyceride and myoinositol to alter the response have failed. However, these compounds, due to their hydrophilic properties, may not have reached the active sites of the intact cells in adequate concentration or may not have had sufficient affinity for the target enzyme to oppose the action of the hexachlorocyclohexanes in the lipophilic environment of the cell membrane. Support for this view comes from Hokin and Brown who have shown previously [23] that the putative target of the hexachlorocyclohexanes is CDPdiglyceride-inositol transferase. This membrane-bound enzyme is especially sensitive to ~/- and 5-hexachlorocyclohexane, even in the presence of myoinositol, when the enzyme is not saturated with CDPdiglyceride. Thus, while the competition experiments did n o t provide direct p r o o f that 7- and 5-hexachlorocyclohexane function as inositol analogs in the modulation of cap formation, it remains quite possible in view o f the stereospecificity of the response, that these hexachlorocyclohexanes function as inositol analogs for this or a related pathway. The possibility also remains that these agents mediate their effects through interaction with some c o m p o n e n t of the enzyme system which normally metabolizes this t y p e of drug. However, there is no evidence available at this time. A particularily interesting aspect of ~-hexachlorocyclohexane action is its ability to disperse preformed caps. The dependence of this response on a temperature which supports energy metabolism and the operation of a cytochalasin B-sensitive system suggests that the membrane-associated contractile systems

511

(i.e. the microfilament system) which promote capping continue to operate in the presence of 7-hexachlorocyclohexane but operate in an uncoordinated manner with the resultant active dispersal of the cap components. If this, in fact, does happen, it implies the existence of microflow patterns and surface organization in the membranes of individual cells which may be specifically derranged by certain hexachlorocyclohexanes. In this connection it would be of interest to determine if the uptake of lipoproteins [24], transferrin [25] and epidermal growth factor [26] is derranged by ~-and 5-hexachlorocyclohexane since the uptake of these macromolecules appears to depend on some such mechanism. This is the subject of continued study. Acknowledgements This study was supported by grants CA-07175 and CA-23076 from the National Cancer Institute, USPHS, and NIH Training Grants CA-09020 and CA09230. G.C.M. is the recipient of a Research Career Award, CA-00685, from the National Cancer Institute. The authors thank Ms. Mary LeMahieu for her assistance in the preparation of the manuscript. 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

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