Characterization of the phorbol 12, 13-dibutyrate (P(Bu)2) induced binding between human lymphocytes

CELLULAR

IMMUNOLOGY

81,

373-383 (1983)

Characterization of the Phorbol 12,13-dibutyrate (P(Bu)*) Induced Binding between Human Lymphocytes MANUEL

PATARROYO, MIKAEL JONDAL,

Department

of Tumor

Biology,

Received

April

Karolinska

JOHN GORDON,

Institute&

26, 1983; accepted

S-104

AND EVA KLEIN

01 Stockholm.

Sweden

July 14, 1983

The mechanisms, cell surface structures, and cell types involved in the phorbol 12,13dibutyrate (P(Bu)2)-induced binding between human lymphocytes were studied. Induction of cell aggregation by 20 min treatment with P(Bu), required Ca2+, an intact membrane, functional microfilaments. and the possible participation of an esterase or, less likely, a protease. Trypsin-sensitive cell surface structures were needed and neuraminidase (NANase) treatment slightly increased the intercellular binding. Retinoic acid, an anti-tumor promoting agent, was inhibitory. Calmodulindependent processes, microtubules, phospholipid methylation, intracehular levels of cyclic adenosine monophosphate, and cellular secretion did not seem to be involved. Cell conjugation between 24 hr P(Buh-treated and untreated cells required participation of trypsin-sensitive cell surface structures in each of the interacting cells and NANase treatment of one partner slightly increased the intercellular binding. Thymocytes, T cells, mature B and Epstein-Barr virus-transformed B cells aggregated while pre-B, early B, and intermediate B lymphocytes derived from representative malignancies did not. The lack of aggregation was not due to the absence of phorbol ester receptors. It is concluded that the P(Bur)-induced intercellular binding is mediated by cell surface proteins, depends on certain enzymatic activities and metabolic events and involves certain cell types.

INTRODUCTION Phorbol esters, such as tetradecanoyl phorbol acetate (TPA) and phorbol dibutyrate (P(Bu)z), are a family of compounds derived from croton oil. They were originally described as tumor promoters in mouse skin (1). Later on, these molecules were shown to induce a variety of cell membrane changes, to mimic transformation and to modulate differentiation (2). The effective concentrations are very low (nanomolar) suggesting that these compounds mimic the action of naturally occurring substances (2). Recently, specific receptors were demonstrated on fibroblast (3, 4) and human lymphocytes (5). TPA is mitogenic for human lymphocytes (6) modulates differentiation of T- and B-leukemia cells (7-10) and induces adhesion to substrate ( 11) and aggregation (12) of human lymphoblastoid cells. Studies on binding between neural cells ( 13) embryonal carcinoma cells ( 14) hepatocytes ( 15), and slime mold ( 16) have been reported. The phenomenon appears to represent intercellular recognition and involves membrane molecules, now partially characterized (15, 17-19). This cell surface interaction has been implicated in the “social” organization of the cells, their differentiation and growth regulation (20). The nature of the intercellular binding of lymphocytes is poorly understood even though interactions between various cell subpopulations have been demonstrated (2 1. 373

374

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ET

AL.

22). Lectin-treated lymphocytes are known to aggregate. However, these sugar binding proteins are polyvalent and may act through a passive bridging between the cells. On the other hand, it is also likely that they induce membrane changes that permit intercellular binding (23). In a previous publication (24) we described an increase of binding between Daudi cells (a Burkitt lymphoma derived line) and blood lymphocytes after TPA treatment of the former. Later on Pi was shown to induce morphological changes and aggregation of human blood lymphocytes within few minutes (25). This intercellular binding was due to the induction of a membrane change and was shown to be energy and temperature dependent and to require divalent cations but not protein synthesis (26). The cell-to-cell binding was interpreted as a way of intercellular recognition between elements of the immune system, achieved by the expression of a cell-binding phenotype and mediated through cell-binding molecules. In the present paper we further analyze P(Bu)z-induced intercellular binding by studying the participation of various cellular mechanisms and structures as well as the cell types involved. MATERIALS

AND

METHODS

Cell aggregation. A T-enriched fraction obtained from heparinized blood of healthy donors after Ficoll (Pharmacia, Uppsala, Sweden)-Isopaque (Nyeggard & Co., Oslo, Norway) separation (27) and passage through a nylon wool column (28) resuspended in RPM1 1640 medium (Grand Island Biological Co., Grand Island, N.Y.) with 5 a Hepes without fetal calf serum was used for most intercellular binding studies unless otherwise stated. The intercellular binding was measured as previously described (26). Briefly, 0.3 ml of 10 X lo6 cell/ml in plain RPM1 with 5 mM Hepes in a siliconized cuvette (CU 312-5, Peyton Associated Ltd., Seaborough, Canada) was stirred with a bar revolving at approx 300 rpm. After a 3-min delay to allow warming of the cells to 37°C P(Bu)* was added to obtain a final concentration of 30 nM. After 20 min, the percentage of aggregated cells was determined in a hemacytometer (400X). The suspension was also inspected before treatment and a control sample was incubated without P(Bu*). At least 5 X lo2 cells were counted in each sample. The Piinduced intercellular bining (% Pi aggr.) was calculated as percentage aggregated cells in the P(Buh-treated sample minus percentage aggregated cells in the control sample, both read after 20 min. The effect of various drugs and enzymes was tested. The cells were pretreated with the drugs for 1 h at 37°C and thereafter P(Bu2) was applied. The lymphocytes were not washed thus the drugs were present also during the P(Bu,) treatment. In contrast, the enzyme-treated cells (for 30-45 min at 37’C) were washed three times prior to phorbol ester treatment. The effect of the treatment (inhibition) was expressed as % Pi

aggr. in the control sample - % Pi aggr. in the treated sample % Pi aggr. in the control sample

The mean value of P(Bu)2-induced aggregation was 22.8% of the lymphocytes. Reagents. Phorbol 12,13-dibutyrate (Sigma Chemical Co., St. Louis, MO.) was suspended in dimethyl sulfoxide (DMSO) and stored at -20°C (0.2 mM). P(Bu)2

CHARACTERISTICS

OF INTERLYMPHOCYTIC

BINDING

375

was added to cells to a final concentration of 30 nM. The final concentration of DMSO, as solvent, was always less than 0.5%. Ethylene glycol bis(Saminoethy1 ether)NJ’-tetraacetic acid (EGTA, diluted in Hz0 and used at 10 n&f), cytochalasin B (in DMSO, used at 20 PM), colchicine (in DMSO, used at 10 PM), antipain (in DMSO, used at 50 pug/ml), chymostatin (in DMSO, used at 400 &ml), phenylmethylsulfonyl fluoride (PMSF, in DMSO, used at 1 mM), soya bean trypsin inhibitor (SBTI, in medium, used at 300 pg/ml), t-aminocaproic acid (EACA, in medium, used at 50 m&f), N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK, in DMSO, used at 1 mM), N-tosyl-L-lysyl-chloromethyl ketone (TLCK, in DMSO, used at 1 mit4), N-benzoyl-L-tyrosine ethyl ester (BTEE, in DMSO, used at 200 PM), N-benzoylL-tyrosineamide (BTA, in DMSO, used at 200 PM), N-p-tosyl-L-phenylalanine benzyl ester (tPABE, in DMSO, used at 200 PM), N-p-tosyl+arginine methyl ester (TAME, in DMSO, used at 3 n-&f), dibutyryl cyclic adenosine 3’, S-monophosphate (in H20, used at 1 mM), prostaglandin E, (PGEr, in ethanol, used at 0.5 &Y), and retinoic acid (RA, in DMSO, used at 50 PM) were purchased from Sigma. Trifluoperazine (in H20, used at 10 PLM), 3-deazaadenosine plus homocysteine (in H20, used at 100 PM), and ZK627 11 (in H20, used at 5 PM) were obtained from Dr. Bertil Fredholm (Karolinska Institute). Monensin (diluted in ethanol, used at 1 &ml) was a kind gift of Dr. Hans Wigzell (Karolinska Institute). Dimethyl sulfoxide was obtained from KEBO AB, Stockholm, Sweden. Trypsin was obtained from Statens Bakteriologiska Laboratorium and used at 0.25%. Neuraminidase (NANase) from Vibrio Cholerue (BDH Chemicals Ltd., Poole, England) was used at 12.5 unit/ml. The viability of the cells was not affected by these treatments. Scoring of lymphocyte conjugates after enzyme treatment. The T-enriched suspensions were treated for 24 hr with Pi. The cells were washed twice and separated into two aliquots and one aliquot was treated with trypsin or neuraminidase. Thereafter, the cells were washed three times and labeled with the vital dye carboxyfluorescein diacetate (CPD) as previously described (26). For conjugate formation labeled and unlabeled cells were mixed in a 1:25 ratio (approx 2 X 1O6 cells in the total mixture) and centrifuged for 5 min at 1500 rpm. The pelleted cells were incubated at 37°C for 20 min, resuspended by shaking, and counted in a Leitz Orthoplan microscope with a Ploem type “Opak illuminator” system at 320x. The proportion of conjugates between labeled and unlabeled cells was determined. [-‘H]P(Bu)~ binding studies. [20-3H]-P(Bu)2 (15.3 Ci/mmol, 0.5 ml, 0.05 mCi; 6.7 PM) was purchased from New EngIand Nuclear (Dreieich, West Germany), transferred from toluene to DMSO, and kept in stock solutions of 1 PMat -20°C. The [3H]P(Bu)z binding assay was carried out as described previously (26). Briefly, one million lymphocytes in 0.2 ml of RPM1 1640 with 30 nM [3H]P(Bu)2 were incubated in Falcon II microplates (Falcon Labware, Division Becton-Dickenson, Oxnard, Calif.) for 20 min at 37°C. Thereafter, the samples were passed rapidly through glass fiber filters. The cell-containing filters were washed three times with phosphate-buffered saline, dried, and transferred to scintillation tubes with 5 ml of Aquasol (New England Nuclear). The total binding represents the mean of quadruplicate determinations with 30 nM [3H]P(Bu)2. Nonspecific binding was measured on samples treated with 3 wCLM cold P(Bu)z and 30 nA4 [‘H]P(Bu)~. The specific binding is expressed as the value of nonspecific binding substracted from the value of total binding Leukemic cells. Blood or spleen from patients presenting with chronic lymphocytic leukemia (CLL) were the source of neoplastic cells for study. Ficoll-Isopaque-separated

376

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ET AL.

blood (27) or wire-mesh-passed finely minced tissue (29) was used. Surface (Sm) and cytoplasmic (C) immunoglobulins were determined in direct immunofluorescent tests on 2 X lo6 cells in suspension and methanol fixed ceil smears, respectively (30), using 25 ~1 of DAKOPATTS fluorescein-labeled rabbit antibody (diluted 1: 10) to human CL,6, y, and (Yheavy chains and K and X light chains. Cells were incubated in serumfree medium for 2 hr prior to staining for SmIg. B,- (31), BZ- (32), BBr-, and LB1 (33)-reactive cells were determined by indirect immunofluorescent tests (34) using monoclonal antibodies which were the generous gift of Dr. Stuart Schlossman and Dr. Edward Clark. la-, OKT3-, and OKMl-positive cells were also determined by indirect immunofluorescence using antibodies supplied by New England Nuclear (Ia) and Ortho-Diagnostics (OKT3, OKMl). The intensity of staining for SmIg, B, , BZ, LB1 , and BB, was judged as being either very weak (+), weak (+), moderate (++), or strong (++-t-). RESULTS Eflect of Various Drugs on the Binding

between Blood Lymphocytes

Table 1 shows the list of compounds with description of their actions. These were selected in order to see whether the aggregation is dependent on extracellular Ca’+, membrane organization, function of microfilaments and microtubules, phospholipid methylation, calmodulindependent processes, the CAMP system, and cellular secretion. Inhibition occurred after pretreatment and in the presence of EGTA, DMSO, retinoic

TABLE

1

Effect of Various Drugs on P(Bu)Jnduced

Dw EGTA

Concentration 10 mM

Binding between Lymphocytes Inhibition of binding (%)

Mode of action Chelation of Ca2’

95 (3)”

Dimethyl sulfoxide

10%

Affects membrane organization

90 (2)

Retinoic acid

50 pM

Anti-tumor promoting

81 (3)

20 pM

Inhibition of microfilaments

43 (4)

Inhibition of methyltransferases

14 (2)

Cytocholasin B 3Deazaadenosine + homocysteine Trifluoperazine

100 /.tM looti 10 /.JM

Inhibition of calmodulindependent

processes

10 (3)

Dibutyryl CAMP

In&

Increase of intracellular CAMP

5 (1)

ZK 62711

5pM

Inactivation of phosphodiesterases

5 (1)

Colchicine

10 PM

Inhibition of microtubules

-2 (2)

Prostaglandin E,

0.5 pu

Activation of adenylic cylcase

-5

(1)

Monensin b

1 adml

Inhibition of cell secretion

-7

(2)

’ Number of experiments in parenthesis. b No inhibition was observed in the presence of 5-10 pg./ml of monensin.

CHARACTERISTICS

OF

INTERLYMPHOCYTIC

377

BINDING

acid, and cytochalasin B. Trifluoperazine and deazaadenosine plus homocysteine had only a minor effect. Compounds without inhibitory activity were dibutyryl CAMP, ZK627 11, prostaglandin E, , colchicine, and monensin. Eflect of Protease Inhibitors Lymphocytes

and Amino Acid Derivatives

on the Binding

between

The results are shown in Table 2. Cells pretreated with the protease inhibitors tosylphenylalanyl-chloromethyl ketone or tosyl-lysyl-chloromethyl ketone did not aggregate. On the other hand phenylmethylsulfonyl fluoride, t-aminocaproic acid, antipain, chymostatin, and soya bean trypsin inhibitor did not affect the phenomenon. Among the non-alkylating substrate analogs that inhibit protease and esterases, benzoyl tyrosine ethyl ester, but not benzoyl tyrosine amide, was inhibitory. Two other amino acid derived esters, tosyl phenylalanine benzyl ester and tosyl arginine methyl ester had a low and no effect, respectively. The results indicate that the high inhibitory capacity requires esterification of certain amino acid derivatives and suggest that an esterase rather than a protease may be involved in the intercellular binding. Effect of Trypsin and Neuraminidase We showed previously that P(Bu)z-treated lymphocytes bind untreated autologous lymphocytes (25). Thus, only one of the partners had to be exposed to the phorbol ester. The binding capacity was abolished if both or one of the partners were pretreated with trypsin. Neuraminidase treatment slightly increased the P(Bu)z-induced aggregation of lymphocytes. Treatment of one partner was also sufficient to obtain the enhancing effect (Table 3).

TABLE

2

Effect of Protease Inhibitors and Amino P(Bu)Jnduced Binding between

Acid Derivatives Lymphocytes

Concentration Protease inhibitor N-Tosyl-L-phenylalanyl-chloromethyl N-Tosyl-L-lysyl-chloromethyl Phenylmethylsulfonyl fluoride t-Aminocaproic acid Antipain Chymostatin Soya bean trypsin inhibitor Aminoacid derivative N-Benzoyl-L-tyrosine N-p-Tosyl-L-phenylalanine N-pTosyl+arginine N-Benzoyl-L-tyrosineamide ’ Number

of experiments.

ethyl

ketone ketone

ester benzyl ester methyl ester

on

Inhibition binding

of (%)

ImM 1mM 1mM 50 mM 50 &ml 400 &ml 300 &ml

loo (2)” 97 (2)

200 pM 200 pM 3mM 200 JLM

85 (3) 15 (2) 3 (2) 3 (2)

6 (2) 5 (2) 1 (2) 0 (2) -4 (2)

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ET AL.

TABLE 3 Effect of Trypsin or Neuraminidase Treatment on P(Bub-Induced Binding between Lymphocytes” After 20 mm P(Bu), treatment Pretreatment of the lymphocytes for 30 min

Alteration of the intercellular binding (W)

Trypsin (0.25%) Neuraminidase (12.5 U/ml)

Inhibition: 84.3 (3)” Enhancement: 35.0 (2)

After 24 h P(Bub treatment CPD labeled cells

Nonlabeled cells

Untreated P(Bub treated P(Bu)r-trypsin treated P(Bu), treated Untreated P(Bu), treated P(Bu),-NANase treated NANase treated

Untreated Untreated Untreated Trypuntreated Untreated Untreated Untreated Untreated

% Conjugates 1.7 (3) 19.3 (3) 1.7 (3) I .7 (3) 1.7 (3) IS.3 (3) 19.3 (3) 5.3 (3)

’ After treatment with the enzyme the cells were washed three times. ‘Number of experiments.

P(Bu)rInduced

Intercellular

Binding

in Different Cell Types

Pi did not induce aggregation of ah types of lymphocytes. T cells from blood were used in experiments in which the characteristics of the phenomenon were studied. Thymocytes behaved similarly. The Epstein-Barr virus (EBQtransformed B-cell line, LCL, gave high proportion of aggregated cells. Among the eight chronic lymphocytic leukemias which were classified according to various immunologic markers, the one which carried the OKT3 antigen (Bjo) had the highest percentage of aggregated cells. No aggregation was induced in pre-B, early B-, and intermediate B-CLL cells while it occurred in more differentiated cells such as mature B-CLL cells (Table 4). Interestingly and in contrast to P(Bu)*, the non-tumor promoter agent 4a-phorbol 12, I 3didecanoate (PDD) could not induce binding between human blood lymphocytes (data not shown). Binding of [3H]P(Bu)2

to Trypsinized Cells and the B-CLL

(Bje) Cells

Neither the total nor the specific binding of [3H]P(Bu)~ was altered in trypsinized cells or nonaggregating CLL cells. Compared to normal lymphocytes, the leukemic cells bound a higher number of phorbol ester molecules (Table 5). DISCUSSION In the present study of intercellular binding, P(Bu)* was used instead of TPA because the former compound is more easily removed and is used in phorbol esterreceptor studies (3).

Tissue

CLL

CLL

CLL

MA-+’ M(D)X+ M(D)K+ MDA+(+) M(D)X++ M(D)K+++ DMK++

’ The number of + refers to intensity of fluorescence.

MP (LCL) EBV-transformed B cells

Blood Blood Blood Blood Blood Blood Blood Spleen

2

0 35 P 0 0 0 5 MA 47 Mu 62 Mx

1

Bt

BB,

0 0

LB,

31+ 84+ 94+ 92++ 95++ 94+++ 95++

0 0 0 5+ 88++ 0

21+ 41+ 61+ 73+ 55+ 21+

0 0

0 0

47+ 91+++ 76++ 43+

0 0

-

90 91 47 79 -

2 59

5

la

-

-

1

11 -

I

1

OKMI

93 93 14 3 -3 -

75

OKT3

22

41

0 1 2 13 14 29

59 59 1

44 91 95 93 98 95 92

B,

Bjii Ric Dan Bje SW Ber Kam Per

1

Surface Ig

% P(Bu)&duced aggregation

29 29

CLL

Diagnosis

Binding Between Different Cell Types

Thymocytes

Nylon wool passed (enriched T cells)

Name

Cytoplasmic k

P(Bu)Jnduced

TABLE 4

k

z

=! 0

3

$

2

2

3 z ti 9

Ii

Q

380

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TABLE 5 Binding of [‘H]P(Bu)~ to Untreated and Trypsinized Cells and to Nonaggregating CLL Cells (Bje)

Expt

Cell”

Total binding @pm) b

I

Enriched T cells Trypsinized enriched T cells’

3550 3341

524 399

3026 2948

0.30 0.29

II

Enriched T cells CLL cells (Bje)

3908 5604

741 797

3167 4807

0.32 0.48

Nonspecific binding (cpm) b

Specific binding (cpm) b

Specific binding (pmol/ 1O6 cells)

(1The cells were incubated with 30 nM [‘H]P(Bu)z for 20 min at 37°C. b Mean of quadruplicate samples. c The cells were treated with the enzyme for 30 min, washed three times and incubated with [“H]P(Bu)z in the presence of cyclohexamide.

It was previously shown that EDTA inhibited the binding between lymphocytes (26). EGTA, a chelating agent that binds with more selectivity Ca2’ than other cations, was also effective. Ca2+ participates in the activation of enzymes, transmission of signals, cell transformation, and binding between various cell types (35). Readdition of Ca2+ reversed most of the effect of EGTA (data not shown). Calmodulin is an intracellular protein that binds Ca2+ with high affinity (36). Trifluoperazine, which suppresses several calmodulin-dependent processes such as cellular secretion, motility and glycogen metabolism (37), had a slight suppressive effect on cell aggregation. DMSO, a dipolar solvent, had been shown to inhibit binding between natural killer cells and their targets (38). Moreover, DMSO was the solvent of many of the drugs used in the present study. Several concentrations were tested in the P(Bu)2-induced intercellular binding. Considerable inhibition was observed with concentrations higher than 5%. Therefore the final DMSO concentration was never higher than 0.5% when used as solvent. The inhibitory effect of DMSO might be explained by a disorganization of the lipid bilayer in the cell membrane. Retinoic acid is an anti-tumor promoting agent that inhibits several phorbol esterinduced phenomena such as induction of Epstein-Barr virus cycle in latently infected B cells (39) and induction of ornithine decarboxylase in mouse epidermis (40). In accordance, retinoic acid also inhibited P(Bu)2-induced cell aggregation. Microfilaments, that are constitutive elements of the cytoskeleton, are composed of actin, interact with various cell surface proteins, and participate in their mobility and redistribution (23). Cytochalasin B, which inhibits microfilaments, also inhibited P(Bu)2-induced cell aggregation. Colchicine, a drug that inhibits microtubules function, had no effect. Phospholipid methylation is a biochemical process that permits transmission of signals through the plasma membrane and involves the participation of two methyltransferases (41). This phenomenon is coupled to Ca” influx and the release of arachidonic acid and prostaglandins and it is inhibited by deazaadenosine plus homocysteine (42). These drugs had a minor effect on intercellular binding. Phospholipid methylation is usually followed by generation of CAMP. Three drugs known to increase intracellular levels of CAMP such as dibutyryl CAMP, prostaglandin

CHARACTERISTICS

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381

El (a stimulator of adenylate cyclase) and ZK62711 (a phosphodiesterase inhibitor) did not mimic or alter cell aggregation. Lymphocyte aggregation is observed in the absence or presence of serum (data not shown). However, intercellular binding might be mediated by a “natural polyvalent molecule” secreted in the medium as phorbol esters have been shown to induce secretion (43). In addition, lactoferrin, a protein found in the specific granules of polymorphonuclear leukocytes (PMN), has been shown to induce aggregation of these cells (44). However, monensin, a carboxylic ionophore known to inhibit cellular secretion (45,46), had no effect. Moreover, the PMN-produced protein has not been detected in lymphocytes or monocytes (47, 48) and T cells, which aggregate after phorbol ester treatment, lack receptors for lactoferrin (49). Several proteases have been detected in human lymphocytes, some of them associated with the cell membrane (50, 51). TLCK, TPCK, PMSF, EACA, antipain, chymostatin, and SBTI are known to inhibit trypsin, chymotrypsin, papain, cathepsin A and B, and plasmin, and plasminogen activator. The possible participation of proteases in the induction of intercellular binding by P(Bu), was initially suggested by the inhibitory effect of TLCK and TPCK. However, these drugs are alkylating agents that also influence other cellular products (52). Moreover, SBTI and antipain, also inhibitors of chymotrypsin and trypsin, had no effect. Thus the role of proteases in the aggregation was uncertain. The inhibitory activity medited by esters such as BTEE suggested the participation of an esterase. Recently, Kwong and Mueller (53) found that TPA stimulated cap formation in bovine lymphocytes. This was inhibited by BTEE but not by BTA. Studying the effect of 20 amino acid derivatives the authors concluded that the inhibitory activity requires ester&cation of the carboxyl group of the amino acid and that the amino acid must also have an aromatic side chain for high inhibitory capacity. The trypsin sensitivity of P(Bu)z-induced aggregation indicated participation of surface proteins, most likely as cell-binding molecules. The inhibitory effect by trypsinization of either the P(Buh-treated or the untreated cells indicates participation of surface proteins in both interacting cells. It can be assumed that active-binding molecule(s), on the stimulated cell, may be complementary to passive-binding molecule(s) on the partner cell. NANase slightly increased cell aggregation, suggesting that a decrease of surface sialic acid may facilitate the intercellular binding. P(Bu)*-induced intercellular binding has been shown to increase with time (25, 26). This finding suggests that the number of lymphocytes expressing the cell-binding phenotype may increase in parallel; however, increase in the strength of binding may be also relevant. Lymphocytes treated for several hours with P(Buh might exhibit a more stable cell-to-cell binding than cells treated for a few minutes. P(Bu), induced aggregation in certain but not all cell types. It is likely that cells at certain stages of differentiation may acquire the capacity to bind other cells when intercellular cooperation may be required for either differentiation or control of proliferation. Preliminary data indicate that cell types which do not aggregate after short time treatment with P(Bu)z do so after long-term treatment (Patarroyo et al., in press). P(Bu), has been shown to induce differentiation in such cells. The cell-binding capacity expressed by EBV-transformed B cells and enhanced by phorbol ester might permit interaction with regulatory T cells as well. The possibility that trypsinization could inhibit the binding of P(Bu)* molecules

382

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to cells was considered. The phorbol ester receptor has been associated with the cell membrane but its exact localization and structure is unknown. Trypsin treatment did not appear to inhibit the binding of [3H]P(Bu)2 to the cells. Moreover, nonaggregating cells bound phorbol ester molecules as well. The lack of effect of cyclohexamide (26), a protein synthesis inhibitor, as well as the rapid induction of intercellular binding together with the inhibitory effect of trypsin indicates that the participating surface proteins are preformed in the cell membrane. Moreover, energy and temperature dependence as well as the participation of microfilaments and esterase/protease is consistent with a redistribution and reorientation of the membrane proteins. Preliminary results indicate that P(Bu)* enhances redistribution of membrane glycoconjugates in human lymphocytes after a few minute treatment as measured by cap formation (Patarroyo et al., submitted for publication). This active membrane rearrangement may be responsible for the expression of the cell-binding phenotype after phorbol ester treatment. In a similar system, complex formation between two surface glycoproteins induced by thrombin was shown to permit, only after stimulation, the binding of fibrinogen and the aggregation of human platelets (54). Studies on the biological relevance of intercellular binding are in progress. It is possible that cell-to-cell binding may permit intercellular communication by transmission of molecules and signals through membranes. This phenomenon may then allow cellular cooperation for the control of proliferation and maturation of lymphoid cells. ACKNOWLEDGMENTS This project has been supported by Grants 1 ROI CA 25250-04 awarded by the National Cancer Institute, DHHS. We thank Dr. Bernard Weinstein for helpful suggestions concerning the [3H]P(Bu)z-binding studies. M. Patarroyo, M. Jondal, and J. Gordon are recipients of fellowships from the National University of Colombia, the Swedish Cancer Society, and EMBO, respectively. We are grateful to Dr. Peter Biberfeld and Dr. Hgkan Mellstedt for proving the leukemia samples.

REFERENCES 1. Diamond, L., O’Brien, T. G., and Baud, W. M., Advan. Cancer Rex 32, 1, 1980. 2. Weinstein, I. B., Lee, L. S., Fisher, P. B., Mufson, A., and Yamasaki, H., J. Supramol. Struct. 12, 195, 1979. 3. Driedger, P. E., and Blumberg, P. M., Proc. Natl. Acad. Sci. USA 77, 567, 1980. 4. Horowitz, A. D., Greenebaun, E., and Weinstein, I. B., Proc. Natl. Acad. Sci. USA 78, 2315, 1981. 5. Sando, J. J., Hilfiker, M. L., Salomon, D. S., and Farrar, J. J., Proc. Natl. Acad. Sci. USA 78, 1189, 1981. 6. Touraine, J. L., Hadden, J. W., Touraine, F., Hadden, E. M., Estensen, E., and Good, R., J. Exp. Med. 145, 460, 1977. 7. Delia, D., Greaves, M. F., Newman, R. A., Sutherland, D. R., Minowada, J., Kung, P., and Goldstein, P., ht. J. Cancer 29, 23, 1982. 8. Nagasawa, K., and Mak, T. W., Proc. Natl. Acad. Sci. USA 77, 2964, 1980. 9. Totterman, T. H., Nilsson, K., Claesson, L., Simonsson, B., and Aman, P., Hum. Lymphocyte Dl$er. 1, 13, 1981. 10. Totterman, T. H., Nilsson, K., Sundstrdm, C., and Solstrom, J., Hum. Lymphocyte DL&%r.1, 83, 1981. 11. Castagna, M., and Rochette-Egly, C., Cancer Lett. 6, 227, 1979. 12. Hoshino, H., Miwa, M., Fujiki, H., and Sugimura, T., Biochem. Biophys. Rex Commun. 95, 842, 1980. 13. Edelman, G. M., Science 192, 2 18, 1976. 14. Yoshida, C., and Takeichi, M., Cell 28, 217, 1982.

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BINDING

383

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