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Binding of tumor-promoting and biologically inactive phorbol esters to artificial membranes
135
Cancer Letters, 17 (1982) 135-140 Elsevier Scientific Publishers Ireland Ltd.
BINDING OF TUMOR-PROMOTING AND BIOLOGICALLY PHORBOL ESTERS TO ARTIFICIAL MEMBRANES
INACTIVE
MICHEL DELEERS and WILLY J. MALAISSE Laboratory (Belgium)
of
Experimental
Medicine,
Brussels
Free
University,
Brussels
B-l 000
(Received 11 June 1982) (Revised version received 11 August 1982) (Accepted 16 August 1982)
SUMMARY
Multilamellar liposomes formed of phospholipids bind [ 20-3H (N)] phorbol12,13dibutyrate, the dissociation constant and maximal binding being comparable to those found in fibroblasts or epidermal cells. The relative capacity of distinct phorbol esters to compete with the labeled ligand was similar to their relative tumor-promoting capacity. It is proposed that the specific binding of phorbol esters to biological material may be accounted for by their insertion in the phospholipid domain of membranes.
INTRODUCTION
Several, but not all, phorbol esters are potent tumor-promoting agents and also exert early effects upon different cell types [2]. The primary site of action of these phorbol esters upon target systems is not fully elucidated. It was reported that phorbol esters bind to specific receptors in chicken embryo fibroblasts and mice epidermal cells, and proposed that phorbol ester-receptor binding is involved in the biological response to these agents [ 1,10,14]. This view appears at variance with the postulate that phorbol esters are primarily located in the phospholipid domain of the plasma membrane and that their biological effects are secondary to a disturbance in the physico-chemical properties of such a domain. The latter hypothesis is compatible with the recent demonstration that phorbol esters interact with phospholipids in artificial lipid monolayers or bilayers and that, under suitable conditions, the influence of phorbol esters upon the properties of such artificial membranes parallels the tumor-promoting capacity of Address all correspondence to: Dr. W.J. Malaisse, Laboratory of Experimental Medicine, 115, Boulevard de Waterloo, B-1000 Brussels, Belgium. 0304-3835/82/0000-0000/$02.75 o 1982 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland.
136
these agents [ 5,6,8,9,12]. In order to reconcile the receptor and phospholipid theories, we have measured the binding of phorbol esters to liposomes. The results of this study indicate that it is possible to mimic in the artificial system several aspects of the specific binding process previously characterized in intact cells or subcellular particulate fractions [ 1,10,14]. MATERIALS
AND METHODS
The experimental conditions used in the present work were selected to simulate, as closely as possible, those used for studying the binding of phorbol esters to biological membranes [ 10,141. Multilamellar liposomes formed of dimyristoylphosphatidylcholine (DMPC) or egg yolk phosphatidylcholine (EYPC) were prepared as described elsewhere in a 50 mM Tris-HCl buffer (pH 7.4) to yield a final concentration of 2 mg of lipid/ml. Aliquots of this suspension (250 ~1 each) were placed in polyethylene microcentrifuge tubes and incubated for 30 min at 37°C in the presence of [20-3H(N)]phorbol 12,13dibutyrate ([jH] PDB; New England Nuclear, Boston, MA) and, as required, phorbol 12-myristate 12-acetate (PMA), phorbol 12,13didecanoate (PDD), 4e-phorbol 12,13didecanoate (4e-PDD) or phorbol 12,13diacetate (PDA). Lipids and phorbol esters were purchased from Sigma Chemical Co. (St. Louis, MO). The specific radioactivity of [ 3H] PDB was kept constant (2.96 Ci/mmol) in all cases, except when measuring the non-specific binding of [ 3H] PDB, in which case unlabeled PDB was added to the tubes to yield a final concentration of 50 PM. This non-specific binding was measured for each condition under study. The specific binding was taken as the difference between total and non-specific binding each measured in quintuplicate. All phorbol esters were added from stock solutions prepared in dimethylsulfoxide, the final concentration of which (0.8-1.6%, v/v) was kept constant within each individual experiment. After incubation, the tubes were centrifuged for 30 min at 4°C and 3000 X g. The supernatant solution was then removed and the bottom of the tube containing the pellet of liposomes cut, placed in a counting vial containing 6 ml of scintillation fluid (Lumagel; Lumac, Schaerberg, Holland) and eventually examined for its radioactive content. RESULTS
A major fraction (57-87s) of the radioactivity added to each tube was recovered in the liposomal pellet. The non-specific binding represented 78.7-94.8s of the total binding. The amount of specifically bound radioactivity increased as the concentration of [‘HI PDB was raised (Fig. 1, left panel). Such a specific binding was slightly .higher in DMPC than in EYPC liposomes, both types of liposomes being incubated above transition temperature. Incidentally, when the liposomes were formed of dipalmitoylphosphatidylcholine (DPPC) and incubated at 37”C, i.e. below transition temper-
c
1.2‘0
E 0 E & P t : m q
0
0.8 -
0.4
-
P
01
8 0
gj
I
20 PDB
I
30 Total
L
40
I
50
(“M)
Fig. 1. Specific binding of [‘H]PDB to DMPC ( o ) or EYPC (0) liposomes. Left panel: the amount of [ ‘H]PDB specifically bound is plotted as a function of the initial concentration of [ ‘H]PDB. Right panel : Scatchard plot of specific binding of [ ‘H]PDB to DMPC liposomes. Mean values are derived from 20 individual measurements. The S.E.M. averaged 13% of the mean value.
0
10 PHORBOL
102 ESTER
Fig. 2. Dose-response curves for the inhibition of [ ‘H]PDB specific binding by nonradioactive phorbol esters. EYPC liposomes were incubated in the presence of 54 nM [3H]PDB and increasing concentrations of either PMA (e), PDD Co), 4a-PDD (A) or PDA (+). Each value refers to the point-moving mean derived from quintuplate measurements made at each concentration of phorbol ester. All results were expressed in percent of the control value found in the absence of the non-radioactive phorbol ester. The S.E.M. for individual measurements averaged 13%. Covariance analysis indicated that the regression lines differed from one another in their elevations (P < 0.01 or less) but not in their slopes.
138
ature, the apparent specific binding of [ 3H] PDB was much lower and actually insufficient to allow for precise dose-response measurements. A S&chard plot of the data obtained with DMPC liposomes displayed a single slope corresponding to a Kd for [ 3H] PDB of 14 nM (Fig. 1, right panel). At saturation, 2.9 pmol of [3H]PDB were bound per mg of lipid or 1.97 pmol per mol of lipid. Figure 2 illustrates the results of experiments designed to study the structureactivity relationships for inhibition of [ 3H] PDB binding by distinct phorbol esters. In the presence of 54 nM [ 3H] PDB (total concentration), the slopes of the dose-response curves were fairly similar. However, the concentrations of phorbol esters required to cause 50% inhibition of binding spanned a range of 2-3 orders of magnitude, averaging 0.12 PM for PMA, 0.30 PM for PDD, 2.2 PM for 4ar-PDD and 42 PM for PDA. Taking into account the Kd for [‘H]PDB, the calculated Ki amounted to 25 nM for PMA, 62 nM for PDD, 0.46 PM for 4+PDD and 8.7 PM for PDA. These values are not corrected for the partitioning of the unlabeled phorbol esters into the liposomal material. DISCUSSION
The results obtained in our artificial system are surprisingly close to those collected with biological membranes. The Kd for [3H]PDB amounted to 12 nM in the present system and 25 nM in a particulate fraction derived from chicken embyro fibroblasts. The maximal binding (saturation value) amounted to 2.9 pmol/mg of lipid, as compared to 1.4 pmol/mg of protein in fibroblasts. These 2 values are in fair agreement since the lipid/protein ratio of biological membranes is close to l/2 [ 111. The non-specific binding (79-95s) was somewhat higher in the present study than in natural membranes (57-79%), this difference being conceivably attributable, in part at least, to the trapping of some free [‘H]PDB in the pellet of liposomes. The Ki for PMA, PDD and PDA were either identical to or 2-5 times higher than those reported by Driedger and Blumberg in the crude particulate fraction prepared from chicken fibroblasts [lo]. The fact that our Ki values tended to be higher than those recorded by the latter authors is probably attributable to the fact that, in the present system, most of the [3H] PDB added to each tube was eventually bound to the liposomal pellet. Under these ,conditions, the Ki is indeed overestimated [ll]. Incidentally, it was not easy to reduce the total binding to less than lo%, as recommended in binding experiments, without either losing in precision (if the amount of liposomes added to each tube was decreased) or augmenting considerably the cost of the experiments (if the volume of incubation medium in each tube was increased). In comparing the present results to those obtained with biological membranes, it should be kept in mind that the lipid composition of our liposomes is not identical to that of living membranes. However, the results obtained with DMPC and EYPC liposomes, respectively, suggest that changes in the
139
phospholipid composition of the membrane may not represent a major source of variability in the binding of phorbol esters. Nevertheless, the data obtained with DPPC liposomes indicate that a sufficient fluidity of the lipid bilayer is required to ensure optimal insertion of the phorbol esters into the phospholipid matrix. It could be argued that our artificial membranes contained no specific receptors and, hence, that the terms ‘specific’ and ‘non-specific’ binding were used here abusively. However, the aim of the present study was precisely to test whether artificial membranes devoid of ‘specific’ receptors may behave phenomenologically in the same manner as natural membranes. Our data demonstrate that such is indeed the case. This finding is compatible with the view that the insertion of phorbol esters into the phospholipid domain of living membranes represents a primary event in the biological response to these tumor-promoting agents. In this perspective, it is remarkable that the relative binding capacity of distinct phorbol esters was not vastly different from their tumor-promoting capacity [3], with the following hierarchy: PMA > PDD % 4rx-PDD or PDA. The differences in binding may well be attributed to the configuration of each phorbol ester. We have indeed shown, by conformational analysis, that the spatial configuration of tumor-promoting phorbol esters (e.g. PDD) is much more adequate than that of biologically inactive agents (e.g. 401-PDD) for insertion into a phospholipid domain [ 41. ACKNOWLEDGEMENTS
We thank M. Mahy for technical help and C. Demesmaeker for secretarial assistance. This work was supported in part by a grant from the Belgian Ministry of Scientific Policy. REFERENCES 1 Ashendel, C.L. and Boutwell, R.K. (1981) Direct measurement of specific binding of highly lipophilic phorbol diester to mouse epidermal membranes using cold acetone. Biochem. Biophys. Res. Commun., 99, 543-549. 2 Blumberg, P.M. (1980,198l) In vitro studies on the mode of action of the phorbol esters, potent tumor promoters. CRC Crit. Rev. Toxicol., 8,153-197,199-234. 3 Boutwell, R.K. (1974) The function and mechanism of promoters of carcinogenesis. CRC Crit. Rev. Toxicol., 2,419433. 4 Brasseur, R., Deleers, M., Ruysschaert, J.M. and Malaisse, W.J. (1982) Conformational analysis of phorbol esters at a simulated lipid-water interface. Abstracts 12th International Congress of Biochemistry (Perth), in press. 5 Deleers, M., Castagna, M. and Malaisse, W.J. (1981) Phorbol esters parallel effects on tumor promotion, insulin release and calcium ionophoresis. Cancer Letters, 14, 109-114. 6 Deleers, M., Defrise-Quertain, F., Ruysschaert, J.M. and Malaisee, W.J. (1981) Interaction of phorbol esters with lipid bilayers: thermotropic changes in fluorescence polarization, phase transition and calcium ionophoresis. Res. Commun. Pathol. Pharmacol., 34,423-439.
140 7 Deleers, M. and Malalsse, W.J. (1980) Ionophore-mediated calcium exchange diffusion in liposomes. Biochem. Biophys. Res. Commun., 95,650-657. 8 Deleers, M. and Malaisse, W.J. (1982) Influence of phorbol esters on ionophoremediated calcium exchange-diffusion in liposomes. Chem. Phys. Lipids, 31 000-000. 9 Deleers, M., Ruysschaert, J.M. and Malaisse, W.J. (1982) Interaction between phorbol esters and phospholipid in a monolayer model membrane. Chem.-Biol. Interact., 42, in press. 10 Driedger, P.E. and Blumberg, P.M. (1980) Specific binding of phorbol ester tumor promoters. Proc. Natl. Acad. Sci. U.S.A., 77,567-571. 11 Jacobs, S., Chang, K.-J. and Cuatrecasas, P. (1975) Estimation of hormone receptor affinity by competitive displacement of labeled ligand: effect of concentration of receptor and of labeled l&and. Biochem. Biophys. Res. Cornmum., 66, 687-692. 12 Jacobson, K., Wenner, C.E., Kemp, G. and Papahadjopoulos, D. (1975) Surface properties of phorbol’esters and their interaction with lipid monolayers and bilayers. Cancer Res., 35, 2991-2995. 13 Katschalski, E., Silman, I. and Goldman, R. (1971) Effect of the microenvironment on the mode of action of immobilized enzymes. Adv. Enzymol., 34,445-536. I4 Solanki, V. and Slaga, T.J. (1981) Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice. Proc. Natl. Acad. Sci. U.S.A., 78, 2449-2553.
Cancer Letters, 17 (1982) 135-140 Elsevier Scientific Publishers Ireland Ltd.
BINDING OF TUMOR-PROMOTING AND BIOLOGICALLY PHORBOL ESTERS TO ARTIFICIAL MEMBRANES
INACTIVE
MICHEL DELEERS and WILLY J. MALAISSE Laboratory (Belgium)
of
Experimental
Medicine,
Brussels
Free
University,
Brussels
B-l 000
(Received 11 June 1982) (Revised version received 11 August 1982) (Accepted 16 August 1982)
SUMMARY
Multilamellar liposomes formed of phospholipids bind [ 20-3H (N)] phorbol12,13dibutyrate, the dissociation constant and maximal binding being comparable to those found in fibroblasts or epidermal cells. The relative capacity of distinct phorbol esters to compete with the labeled ligand was similar to their relative tumor-promoting capacity. It is proposed that the specific binding of phorbol esters to biological material may be accounted for by their insertion in the phospholipid domain of membranes.
INTRODUCTION
Several, but not all, phorbol esters are potent tumor-promoting agents and also exert early effects upon different cell types [2]. The primary site of action of these phorbol esters upon target systems is not fully elucidated. It was reported that phorbol esters bind to specific receptors in chicken embryo fibroblasts and mice epidermal cells, and proposed that phorbol ester-receptor binding is involved in the biological response to these agents [ 1,10,14]. This view appears at variance with the postulate that phorbol esters are primarily located in the phospholipid domain of the plasma membrane and that their biological effects are secondary to a disturbance in the physico-chemical properties of such a domain. The latter hypothesis is compatible with the recent demonstration that phorbol esters interact with phospholipids in artificial lipid monolayers or bilayers and that, under suitable conditions, the influence of phorbol esters upon the properties of such artificial membranes parallels the tumor-promoting capacity of Address all correspondence to: Dr. W.J. Malaisse, Laboratory of Experimental Medicine, 115, Boulevard de Waterloo, B-1000 Brussels, Belgium. 0304-3835/82/0000-0000/$02.75 o 1982 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland.
136
these agents [ 5,6,8,9,12]. In order to reconcile the receptor and phospholipid theories, we have measured the binding of phorbol esters to liposomes. The results of this study indicate that it is possible to mimic in the artificial system several aspects of the specific binding process previously characterized in intact cells or subcellular particulate fractions [ 1,10,14]. MATERIALS
AND METHODS
The experimental conditions used in the present work were selected to simulate, as closely as possible, those used for studying the binding of phorbol esters to biological membranes [ 10,141. Multilamellar liposomes formed of dimyristoylphosphatidylcholine (DMPC) or egg yolk phosphatidylcholine (EYPC) were prepared as described elsewhere in a 50 mM Tris-HCl buffer (pH 7.4) to yield a final concentration of 2 mg of lipid/ml. Aliquots of this suspension (250 ~1 each) were placed in polyethylene microcentrifuge tubes and incubated for 30 min at 37°C in the presence of [20-3H(N)]phorbol 12,13dibutyrate ([jH] PDB; New England Nuclear, Boston, MA) and, as required, phorbol 12-myristate 12-acetate (PMA), phorbol 12,13didecanoate (PDD), 4e-phorbol 12,13didecanoate (4e-PDD) or phorbol 12,13diacetate (PDA). Lipids and phorbol esters were purchased from Sigma Chemical Co. (St. Louis, MO). The specific radioactivity of [ 3H] PDB was kept constant (2.96 Ci/mmol) in all cases, except when measuring the non-specific binding of [ 3H] PDB, in which case unlabeled PDB was added to the tubes to yield a final concentration of 50 PM. This non-specific binding was measured for each condition under study. The specific binding was taken as the difference between total and non-specific binding each measured in quintuplicate. All phorbol esters were added from stock solutions prepared in dimethylsulfoxide, the final concentration of which (0.8-1.6%, v/v) was kept constant within each individual experiment. After incubation, the tubes were centrifuged for 30 min at 4°C and 3000 X g. The supernatant solution was then removed and the bottom of the tube containing the pellet of liposomes cut, placed in a counting vial containing 6 ml of scintillation fluid (Lumagel; Lumac, Schaerberg, Holland) and eventually examined for its radioactive content. RESULTS
A major fraction (57-87s) of the radioactivity added to each tube was recovered in the liposomal pellet. The non-specific binding represented 78.7-94.8s of the total binding. The amount of specifically bound radioactivity increased as the concentration of [‘HI PDB was raised (Fig. 1, left panel). Such a specific binding was slightly .higher in DMPC than in EYPC liposomes, both types of liposomes being incubated above transition temperature. Incidentally, when the liposomes were formed of dipalmitoylphosphatidylcholine (DPPC) and incubated at 37”C, i.e. below transition temper-
c
1.2‘0
E 0 E & P t : m q
0
0.8 -
0.4
-
P
01
8 0
gj
I
20 PDB
I
30 Total
L
40
I
50
(“M)
Fig. 1. Specific binding of [‘H]PDB to DMPC ( o ) or EYPC (0) liposomes. Left panel: the amount of [ ‘H]PDB specifically bound is plotted as a function of the initial concentration of [ ‘H]PDB. Right panel : Scatchard plot of specific binding of [ ‘H]PDB to DMPC liposomes. Mean values are derived from 20 individual measurements. The S.E.M. averaged 13% of the mean value.
0
10 PHORBOL
102 ESTER
Fig. 2. Dose-response curves for the inhibition of [ ‘H]PDB specific binding by nonradioactive phorbol esters. EYPC liposomes were incubated in the presence of 54 nM [3H]PDB and increasing concentrations of either PMA (e), PDD Co), 4a-PDD (A) or PDA (+). Each value refers to the point-moving mean derived from quintuplate measurements made at each concentration of phorbol ester. All results were expressed in percent of the control value found in the absence of the non-radioactive phorbol ester. The S.E.M. for individual measurements averaged 13%. Covariance analysis indicated that the regression lines differed from one another in their elevations (P < 0.01 or less) but not in their slopes.
138
ature, the apparent specific binding of [ 3H] PDB was much lower and actually insufficient to allow for precise dose-response measurements. A S&chard plot of the data obtained with DMPC liposomes displayed a single slope corresponding to a Kd for [ 3H] PDB of 14 nM (Fig. 1, right panel). At saturation, 2.9 pmol of [3H]PDB were bound per mg of lipid or 1.97 pmol per mol of lipid. Figure 2 illustrates the results of experiments designed to study the structureactivity relationships for inhibition of [ 3H] PDB binding by distinct phorbol esters. In the presence of 54 nM [ 3H] PDB (total concentration), the slopes of the dose-response curves were fairly similar. However, the concentrations of phorbol esters required to cause 50% inhibition of binding spanned a range of 2-3 orders of magnitude, averaging 0.12 PM for PMA, 0.30 PM for PDD, 2.2 PM for 4ar-PDD and 42 PM for PDA. Taking into account the Kd for [‘H]PDB, the calculated Ki amounted to 25 nM for PMA, 62 nM for PDD, 0.46 PM for 4+PDD and 8.7 PM for PDA. These values are not corrected for the partitioning of the unlabeled phorbol esters into the liposomal material. DISCUSSION
The results obtained in our artificial system are surprisingly close to those collected with biological membranes. The Kd for [3H]PDB amounted to 12 nM in the present system and 25 nM in a particulate fraction derived from chicken embyro fibroblasts. The maximal binding (saturation value) amounted to 2.9 pmol/mg of lipid, as compared to 1.4 pmol/mg of protein in fibroblasts. These 2 values are in fair agreement since the lipid/protein ratio of biological membranes is close to l/2 [ 111. The non-specific binding (79-95s) was somewhat higher in the present study than in natural membranes (57-79%), this difference being conceivably attributable, in part at least, to the trapping of some free [‘H]PDB in the pellet of liposomes. The Ki for PMA, PDD and PDA were either identical to or 2-5 times higher than those reported by Driedger and Blumberg in the crude particulate fraction prepared from chicken fibroblasts [lo]. The fact that our Ki values tended to be higher than those recorded by the latter authors is probably attributable to the fact that, in the present system, most of the [3H] PDB added to each tube was eventually bound to the liposomal pellet. Under these ,conditions, the Ki is indeed overestimated [ll]. Incidentally, it was not easy to reduce the total binding to less than lo%, as recommended in binding experiments, without either losing in precision (if the amount of liposomes added to each tube was decreased) or augmenting considerably the cost of the experiments (if the volume of incubation medium in each tube was increased). In comparing the present results to those obtained with biological membranes, it should be kept in mind that the lipid composition of our liposomes is not identical to that of living membranes. However, the results obtained with DMPC and EYPC liposomes, respectively, suggest that changes in the
139
phospholipid composition of the membrane may not represent a major source of variability in the binding of phorbol esters. Nevertheless, the data obtained with DPPC liposomes indicate that a sufficient fluidity of the lipid bilayer is required to ensure optimal insertion of the phorbol esters into the phospholipid matrix. It could be argued that our artificial membranes contained no specific receptors and, hence, that the terms ‘specific’ and ‘non-specific’ binding were used here abusively. However, the aim of the present study was precisely to test whether artificial membranes devoid of ‘specific’ receptors may behave phenomenologically in the same manner as natural membranes. Our data demonstrate that such is indeed the case. This finding is compatible with the view that the insertion of phorbol esters into the phospholipid domain of living membranes represents a primary event in the biological response to these tumor-promoting agents. In this perspective, it is remarkable that the relative binding capacity of distinct phorbol esters was not vastly different from their tumor-promoting capacity [3], with the following hierarchy: PMA > PDD % 4rx-PDD or PDA. The differences in binding may well be attributed to the configuration of each phorbol ester. We have indeed shown, by conformational analysis, that the spatial configuration of tumor-promoting phorbol esters (e.g. PDD) is much more adequate than that of biologically inactive agents (e.g. 401-PDD) for insertion into a phospholipid domain [ 41. ACKNOWLEDGEMENTS
We thank M. Mahy for technical help and C. Demesmaeker for secretarial assistance. This work was supported in part by a grant from the Belgian Ministry of Scientific Policy. REFERENCES 1 Ashendel, C.L. and Boutwell, R.K. (1981) Direct measurement of specific binding of highly lipophilic phorbol diester to mouse epidermal membranes using cold acetone. Biochem. Biophys. Res. Commun., 99, 543-549. 2 Blumberg, P.M. (1980,198l) In vitro studies on the mode of action of the phorbol esters, potent tumor promoters. CRC Crit. Rev. Toxicol., 8,153-197,199-234. 3 Boutwell, R.K. (1974) The function and mechanism of promoters of carcinogenesis. CRC Crit. Rev. Toxicol., 2,419433. 4 Brasseur, R., Deleers, M., Ruysschaert, J.M. and Malaisse, W.J. (1982) Conformational analysis of phorbol esters at a simulated lipid-water interface. Abstracts 12th International Congress of Biochemistry (Perth), in press. 5 Deleers, M., Castagna, M. and Malaisse, W.J. (1981) Phorbol esters parallel effects on tumor promotion, insulin release and calcium ionophoresis. Cancer Letters, 14, 109-114. 6 Deleers, M., Defrise-Quertain, F., Ruysschaert, J.M. and Malaisee, W.J. (1981) Interaction of phorbol esters with lipid bilayers: thermotropic changes in fluorescence polarization, phase transition and calcium ionophoresis. Res. Commun. Pathol. Pharmacol., 34,423-439.
140 7 Deleers, M. and Malalsse, W.J. (1980) Ionophore-mediated calcium exchange diffusion in liposomes. Biochem. Biophys. Res. Commun., 95,650-657. 8 Deleers, M. and Malaisse, W.J. (1982) Influence of phorbol esters on ionophoremediated calcium exchange-diffusion in liposomes. Chem. Phys. Lipids, 31 000-000. 9 Deleers, M., Ruysschaert, J.M. and Malaisse, W.J. (1982) Interaction between phorbol esters and phospholipid in a monolayer model membrane. Chem.-Biol. Interact., 42, in press. 10 Driedger, P.E. and Blumberg, P.M. (1980) Specific binding of phorbol ester tumor promoters. Proc. Natl. Acad. Sci. U.S.A., 77,567-571. 11 Jacobs, S., Chang, K.-J. and Cuatrecasas, P. (1975) Estimation of hormone receptor affinity by competitive displacement of labeled ligand: effect of concentration of receptor and of labeled l&and. Biochem. Biophys. Res. Cornmum., 66, 687-692. 12 Jacobson, K., Wenner, C.E., Kemp, G. and Papahadjopoulos, D. (1975) Surface properties of phorbol’esters and their interaction with lipid monolayers and bilayers. Cancer Res., 35, 2991-2995. 13 Katschalski, E., Silman, I. and Goldman, R. (1971) Effect of the microenvironment on the mode of action of immobilized enzymes. Adv. Enzymol., 34,445-536. I4 Solanki, V. and Slaga, T.J. (1981) Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice. Proc. Natl. Acad. Sci. U.S.A., 78, 2449-2553.