characterization of specific binding of [3h]phorbol 12,13 ......and has made possible experiments...

[CANCER RESEARCH 40. 3635-3641, October 1980]0008-5472/80/0040-OOOOS02.00

Characterization of Specific Binding of [3H]Phorbol 12,13-Dibutyrate

and [3H]Phorbol 12-Myristate 13-Acetate to Mouse Brain1

William G. Dunphy, K. Barry Delclos,2 and Peter M. Blumberg

Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT

[20-3H]Phorbol 12,13-dibutyrate ([3H]PDBU) and [20-3H]-phorbol 12-myristate 13-acetate ([3H]PMA) bound specifically

and with high affinity to one class of saturable binding sites inparticulate preparations from mouse brain. The dissociationconstants for binding of [3H]PDBU and [3H]PMA were 7 nw and

66 PM, respectively. At half-maximal specific binding, bindingof [3H]PDBU was 96% specific and binding of [3H]PMA was91 % specific. At saturation, 28 pmol of [3H]PDBU were bound

per mg protein. Nonradioactive phorbol 12-myristate 13-acetate blocked >95% of the specific [3H]PDBU binding. Con

versely, nonradioactive phorbol 12,13-dibutyrate blocked>95% of the specific [3H]PMA binding. The binding sites inmouse brain for [3H]PDBU and [3H]PMA thus appear to be

equivalent. In addition to phorbol 12-myristate 13-acetate,seven other biologically active phorbol-related diterpenes inhibited [3H]PDBU binding with potencies which correlated with

their biological activities. In the case of phorbol 12,13-diace-tate, inhibition of [3H]PDBU binding was shown to be competi

tive. Phorbol (20 /ig/ml) and 4a-phorbol 12,13-didecanoate

(20 jug/ml), two compounds devoid of biological activity, hadno effect on binding. Binding was sensitive to heat, papain, andphospholipase A2 but was insensitive to neuraminidase. Basedon the high level of specific phorbol ester binding in brain, wepostulate that the phorbol ester target plays a functional ratherthan information-transducing role in the cell.

INTRODUCTION

Tumor promoters are compounds which, although in themselves neither carcinogenic nor mutagenic, greatly acceleratetumor outgrowth in animals previously treated with a subthresh-

old dose of a carcinogen (4, 40, 41, 46). The phorbol estersand related diterpenes comprise the most potent class of tumorpromoter (23, 25). Of this class, PMA" is the most active. At

the cellular and biochemical levels, the phorbol esters likewisehave dramatic effects (2, 5, 11,21, 42). These include partialinduction in normal cells of the transformed phenotype (3, 9,14, 47, 49), alteration of cellular responsiveness to otherbiologically active compounds (12, 13), and modulation ofdifferentiation and differentiated cell functions (9, 27, 31, 35).Among effects on cells of the nervous system are blockage ofspontaneous neurite formation in mouse neuroblastoma cells

' This research was supported by Grant CA 22895 from the National Cancer

Institute.2 A predoctoral trainee in pharmacology of the NIH.3 Recipient of a Research Career Development Award from the NIH. To whom

requests for reprints should be addressed.' The abbreviations used are: PMA, phorbol 12-myristate 13-acetate;

[3H]PDBU, [20-3H]phorbol 12.13-dibutyrate; [3H]PMA. [20-3H)phorbol 12-myris

tate 13-acetate; PDBU, phorbol 12.13-dibutyrate.Received April 28. 1980; accepted July 11,1980.

(29) and inhibition of nerve growth factor-induced neurite out

growth in embryonic chick ganglion cells (28).This laboratory has presented evidence that the phorbol

esters bind in a specific saturable fashion (10, 16, 18). Bindingwas measured with [3H]PDBU. This derivative is less active

biologically than PMA but is more suitable for binding studiesbecause of its lower lipophilicity and consequently reducednonspecific binding. At saturation, the 100,000 x g pellet fromwhole-cell homogenates of chicken embryo fibroblasts specifically bound 1.4 pmol of [3H]PDBU per mg protein. Similar

preparations from mouse skin specifically bound 3.9 pmol permg protein (10). At a concentration of [3H]PDBU equal to its

Kd, 25 nw, specific binding to chicken embryo fibroblasts andmouse skin was, respectively, 46 and 54% of the total binding.

We proposed that the promoting activity of the phorbol estersis mediated through interaction at this binding site. Binding of[3H]PDBU to chicken embryo fibroblast and mouse skin prep

arations was inhibited by other biologically active phorbol esters. PMA, with a KÂ¡of 1 to 2 nM, showed the greatest affinity.Other derivatives had relative affinities corresponding to theirrank order of promoting activity. Significantly, highly inflammatory but weakly promoting or nonpromoting phorbol-relatedditerpene esters inhibited [3H]PDBU binding only weakly or not

at all.In other studies, Estensen ef al. (20) recently reported bind

ing of [3H]PMA to intact human peripheral blood lymphocytes.

Although structure-activity relationships were not determined,

the binding affinity of PMA and its mitogenic activity on thesecells showed good agreement.

In the present paper, we report on the binding of [3H]PDBU

to the particulate fraction from mouse brain. Brain has provedto be a tissue of interest because of its very high level ofphorbol ester binding activity, approximately 20-fold that of

chicken embryo fibroblasts. The resultant increase in the ratioof specific to total binding has greatly facilitated binding studiesand has made possible experiments that were not feasible inthe tissue systems examined previously. Among these experiments is the direct measurement of [3H]PMA binding for comparison with [3H]PDBU binding.

MATERIALS AND METHODS

Materials. Bovine serum albumin (Fraction V), papain (typeIV), neuraminidase (type VIII), phospholipase A2, and PDBUwere obtained from Sigma Chemical Co. (St. Louis, Mo.).Phorbol 12,13-diacetate, phorbol 12,13,20-triacetate, phorbol12,13-dibenzoate, phorbol 12,13-didecanoate, and phorbol13-acetate were from Chemical Carcinogenesis, Inc. (EdenPrairie, Minn.). PMA, 4a-phorbol 12,13-didecanoate, and phor

bol were prepared by published methods (6, 25, 45). Mezereinwas kindly provided by Dr. J. Douros (National Cancer Institute). [3H]PMA (specific activity, 9 Ci/mmol; Chemical Carci-

OCTOBER 1980 3635

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W. G. Dunphy et al.

nogenesis, Inc.) was the kind gift of Dr. L. Levine (BrandeisUniversity, Boston, Mass.). As determined by thin-layer chro-matography, the [3H]PMA was 96% pure. 12-Deoxyphorbol

13-isobutyrate 20-acetate was isolated from euphorbium resinas described (17). Resiniferatoxin was generously supplied byDr. E. Hecker (German Cancer Research Center, Heidelberg,West Germany) and Dr. F. J. Evans (University of London,London, England). Phorbol-related diterpenes were dissolvedin dimethyl sulfoxide and stored at -20Â°. [20-3H]PDBU (spe

cific activity, 1.38 Ci/mmol; radiochemical purity, 97%) wassynthesized in this laboratory as described (16).

Tissue Preparation. Whole brains from female CD-1 mice(Charles River Breeding Laboratories, Inc., Wilmington, Mass.)were homogenized in 50 mw Tris-CI (pH 7.4) with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at100,000 x g for 60 min. The resulting pellet was resuspendedin a small volume of 50 mM Tris-CI (pH 7.4) at a proteinconcentration of 20 to 30 mg/ml and stored frozen at â€”70Â°

until needed. Protein concentrations were determined by themethod of Lowry et al. (32). For the experiments described inthis paper, brain preparations were routinely refrozen after onethawing and used once subsequently. Refreezing had no effecton the affinity or specific binding activity of the preparations.

Binding of [3H]PDBU. Assays of [3H]PDBU binding were

carried out in 50 mM Tris-CI (pH 7.4) containing bovine serumalbumin (4 mg/ml), brain particulate protein, [3H]PDBU, and

other ligands as specified. Most assays were performed in 0.4-

ml Brinkman microcentrifuge tubes with a final volume of 0.25ml for the incubation mixture. In some instances, assays wereperformed in 1.5-ml Brinkman microcentrifuge tubes with afinal volume of 1 ml for the incubation mixture. In either case,the tubes were incubated at 39Â° for 30 min after additions

were made. Immediately after incubation, the 0.4-ml tubeswere centrifuged at 17,500 x g for 45 min at 4Â°in an HB-4

rotor (equipped with adapters to give the rotor a capacity of 24tubes) in a Sorvall RC-5 centrifuge. The 1.5-ml tubes werecentrifuged at 12,800 x g for 45 min at 4Â°in an Eppendorf

microcentrifuge. After centrifugation, a 0.1- or 0.5-ml aliquot

(depending on volume incubated) of supernatant was removedfrom each tube in order to determine the free concentration of[3H]PDBU. The remaining supernatant was completely removed

and discarded. The tip of the tube containing the pellet was cutoff and transferred to a counting vial, and the radioactivity wasdetermined.

Binding of [3H]PMA. Assays for competition of [3H]PMA

specific binding by nonradioactive phorbol esters were performed in 1.5-ml Brinkman microcentrifuge tubes with a 1-mlincubation volume containing 50 HIM Tris-CI (pH 7.4), bovineserum albumin (4 mg/ml), brain particulate protein, [3H]PMA,

and other ligands as specified. The tubes were incubated at39Â° for 30 min and then centrifuged in an Eppendorf micro-centrifuge at 12,800 x g for 45 min at 4Â°.After centrifugation,

a 0.75-ml aliquot of supernatant was removed from each tubeto determine the free [3H]PMA concentration. The tip of the

tube containing the pellet was cut off and placed in a countingvial, and the bound radioactivity was determined. This assaywas suitable for measuring competition of [3H]PMA binding bynonradioactive ligands, since [3H]PMA could be used at con

centrations above that of its binding site. However, due to thelarge number of phorbol ester binding sites in the brain particulate fraction and to the very high affinity of [3H]PMA for these

sites, different assay conditions were used to measure thedependence of [3H]PMA binding on free [3H]PMA concentration. Conditions were chosen such that at low free [3H]PMA

concentrations the amount of ligand bound would still be smallrelative to the total amount of ligand in the assay. The assayswere performed in 10-ml polycarbonate tubes with an 8-mlvolume of 50 mM Tris-CI (pH 7.4) containing brain particulatefraction (10 fig protein), bovine serum albumin (4 mg/ml),[3H]PMA, and in some cases excess nonradioactive PMA (0.8

/UM).After additions were made, the tubes were incubated at39Â°for 75 min and then centrifuged at 100,000 x g for 45 minat 4Â°in a 50Ti rotor in a Brinkman L5-50 ultracentrifuge. A 4-

ml aliquot of supernatant was withdrawn to determine free[3H]PMA concentration. The pellet was recovered from the tube

in 0.5 ml of 1% (w/v) sodium dodecyl sulfate.Quantitation of Binding. Radioactive samples (pellets and

supernatants) were counted in liquid scintillation fluid (Scinti-

verse; Fisher Scientific Co., Medford, Mass.). Efficiency ofcounting was determined by the channels-ratio method. Spe

cific binding to the particulate fraction pellet was the differencebetween total binding and nonspecific binding. Nonspecificbinding was determined as follows. Under the conditions ofeach binding assay, partitioning of [3H]PDBU and [3H]PMA

between pellet and supernatant was measured in the presenceof excess nonradioactive PDBU (30 fiM) and excess nonradioactive PMA (0.8 fiM), respectively. Nonspecific binding to theparticulate fraction in each experimental tube was calculatedfrom this partition coefficient and the measured amount ofradioactive ligand in the supernatant of that tube.

In competition studies, the large number of specific phorbolester binding sites in the brain particulate fraction necessitatedcorrection of the nominal free concentrations of nonradioactiveligands for stoichiometry of binding. The correction was >25%at the concentration of ligand giving half-maximal inhibition

only for ligands with high binding affinities (PMA, mezerein,and phorbol 12,13-didecanoate). For highly lipophilic competing ligands (PMA and phorbol 12,13-didecanoate), the freeconcentration would be expected to be decreased due tononspecific partitioning into the particulate fraction. In practice,we could determine partitioning only of PMA and PDBU, the 2derivatives we have available in labeled form.

K, values were calculated from the expression (8)

K, = /so/ti + Ã--/Kfl)

where Ã•.is the concentration of free radioactive ligand, Kd isthe dissociation constant of radioactive ligand, and /50 is theconcentration of free nonradioactive ligand yielding 50% inhibition of binding.

Heat and Enzyme Treatments. To assess sensitivity ofphorbol ester binding to heating, brain particulate fraction (100jug protein) was incubated in 0.15 ml of 50 HIMTris-CI (pH 7.4)at 100Â° for 5 min. After heating, binding of [3H]PDBU to the

boiled particulate fraction was assayed in a 0.25-ml volume as

described above. Treatment with papain was carried out asfollows. Brain particulate protein (0.725 mg/ml) was incubatedfor 90 min at 39Â°in 4 ml of 50 mM Tris-CI (pH 7.4) containing

papain (0.4 mg/ml) activated with 1 mM L-cysteine. Controlswere treated with 1 mw L-cysteine without papain. After incubation, the particulate fractions were centrifuged at 100,000x g for 45 min, and the resulting pellets were resuspended in1.2 ml of 50 mM Tris-CI (pH 7.4) containing bovine serum

3636 CANCER RESEARCH VOL. 40



Specific Binding of Tumor Promoters

albumin (4 mg/ml). Aliquote (0.1 ml) of the suspension wereassayed for [3H]PDBU binding in a volume of 0.25 ml as

described above.Treatment with phospholipase A2 was performed as follows.

Brain particulate protein (100 fig) was incubated for 60 min at39Â°in 0.15 ml of 50 rnw Tris-CI (pH 7.4) containing phospho

lipase A2 (20 fig/ml). CaCI2 (10 mw) was included during theincubation, since phospholipase A2 requires Ca2+ for activity

(33). Controls were treated with 10 HIMCaCI2 without enzyme.At the end of the incubation, EDTA was added to both test andcontrol tubes at a final concentration of 50 mw to chelate Ca2+and inhibit phospholipase activity. Specific binding of [3H]PDBU

was then assayed in a volume of 0.25 ml as described above.Sensitivity of [3H]PDBU binding to neuraminidase was tested

as follows. Brain particulate protein (100 jug) was incubated for15 min at 39Â°in 0.15 ml of 50 HIM Tris-CI (pH 7.4) containing

neuraminidase (20 /ig/ml). Controls were incubated withoutenzyme. Following the incubation, [3H]PDBU binding was as

sayed in a volume of 0.25 ml as described above.

RESULTS

Mouse brain was examined as part of an ongoing survey ofthe tissue distribution of phorbol ester binding activity. Thewhole particulate fraction from brain was used in binding assays for estimation of total binding activity in the tissue. Nobinding to the cytosol fraction was detectable.5 More extensive

fractionation of the particulate fraction was not carried outbecause of concern over potential loss of binding activity. Highlevels of specific [3H]PDBU binding to the particulate fraction

were observed (Chart 1). At saturation, 28.4 Â±1.0 (S.E.; n =3 experiments) pmol of [3H]PDBU were bound per mg of

protein. If binding were to a protein with a molecular weight of100,000, this protein would constitute 0.3% of the total particulate protein (0.2% of the total cellular protein). The mean Kddetermined from 3 experiments was 7.4 Â± 0.5 nM. As described in "Materials and Methods," specific binding wasdetermined as the difference in total [3H]PDBU binding in the

absence and presence of a large excess of nonradioactivePDBU. Nonspecific binding per mg of protein (also indicated inChart 1) was within 15% of that observed previously for preparations from chick embryo fibroblasts (16) and mouse skin(10). Because of the high level of specific binding, however,specific binding accounted for a much greater proportion oftotal binding than in these other systems. Specific bindingranged from 98% of total binding at the lowest [3H]PDBU

concentration examined to 88% at the highest. At the Kd forPDBU, specific binding was 96% of the total.

Specific [3H]PDBU binding was measured in the presence of

bovine serum albumin. Control experiments indicated that bovine serum albumin had no effect on either the Ka for [3H]PDBU

binding or the amount of specific binding at saturation. Sincebovine serum albumin minimized fluctuations in free [3H]PDBU

concentrations, however, it was routinely included in the binding assays.

The binding activity of the particulate preparation was sensitive to heating (Table 1). All activity was destroyed by boilingfor 5 min. Binding was likewise sensitive to papain providedthat high concentrations of the protease were used. The bind-

10 20 30 40

FREE [3H] PDBu, nM50

Â¡J

10 15 25

BOUND[3H] PDBu,pmol/mg

Chart 1. Binding of [ 'H)PDBU to mouse brain. A, binding as a function of freeligand concentration. Specific binding (â€¢)and nonspecific binding (O) of [ 'Hj-

PDBU to brain particulate fraction (100 ^g protein) were determined in a 0.25-mlincubation volume as described in "Materials and Methods.' The curve was

calculated with the dissociation constant and the amount of specific binding atsaturation, both obtained from the Scatchard plot of the data. Points, mean of 3determinations within one experiment; oars, S.E. Two additional experimentsgave similar results. B, Scatchard plot of the specific binding data in A. Parameters for the line were calculated by linear regression analysis (r = 0.99).

Table 1Effects of heat and enzyme treatments on specific 1JHÂ¡PDBUbinding

Binding assays and the various treatments were performed as described in'Materials and Methods."

Specific binding remain-Treatment ing (treatedxontrol ratio)

Heat (100Â°; 5 min)

Papain (0.4 mg/ml; 90 min)Phospholipase A2 (20 /ig/ml; 60 min)Neuraminidase (10 jig/ml; 15 min)

0.03 Â±0.0010.23 Â±0.030.18 Â±0.061.05 Â±0.02

bK. B. Delclos. and P. M. Blumberg, unpublished results.

Mean Â±S.E. for 2 experiments, each carried out in triplicate.

ing activity was relatively insensitive to trypsin treatment (notshown). Similar behavior has been reported for the aniÃ³ntransport system in RBC (7). Vesicles prepared from a totallipid extract of calf brain did not bind [3H]PDBU specifically,

and purified sulfatides, cerebrosides, and gangliosides had noeffect on [3H]PDBU binding to the mouse brain particulate

fraction (19). On the other hand, binding to the particulatefraction was inhibited by phospholipase A2 (Table 1). Lipids orthe proper lipid environment may therefore be necessary forthe phorbol esters to bind to their target. Binding was notaffected by neuraminidase.

In order to define the structure-activity requirements of the[3H]PDBU binding sites in brain, the potencies of nonradioactive

phorbol esters and related diterpenes for inhibiting the binding

OCTOBER 1980 3637



IV. G. Dunphy et al.

of [3H]PDBU were determined (Chart 2). The 6 biologically

active phorbol derivatives, PMA, phorbol 12,13-didecanoate,phorbol 12,13-dibenzoate, phorbol 12,13-diacetate, phorbol13-acetate, and phorbol 12,13,20-triacetate, all inhibited bind

ing; the slopes of the inhibition curves were similar. Bindingwas also inhibited by the phorbol-related diterpene ester, mez-

erein. The inhibition curves can be compared with that fornonradioactive PDBU, which was included as a control. Noinhibition was observed for the biologically inactive parentalcohol phorbol or for 4a-phorbol 12,13-didecanoate, the biologically inactive epimer of phorbol 12,13-didecanoate (Table

2).In order to measure accurately the potencies of the more

active derivatives for inhibiting [3H]PDBU binding, it was desir

able to limit the proportion of bound nonradioactive ligand tototal nonradioactive ligand. For several derivatives, therefore,the binding site concentration in the competition assays wasdecreased from that used for the majority of the derivatives

12 11 10 9 8 76 54 3

-/Off [COMPETING UGAND], M

Chart 2. Inhibition of specific ['HJPDBU binding by nonradioactive phorbol-related diterpenes. Specific binding of [ 'HjPDBU was measured as described in'Materials and Methods." In each experiment, specific binding of ['HJPDBU in

the presence of nonradioactive phorbol-related diterpenes was expressed as afraction of the total specific binding in the absence of competing ligand. In thecase of PMA, mezerein, and PDBU, competition assays were done using 40 jigof paniculate fraction protein in a total volume of 1.0 ml. For the remainingderivatives, 125 fig of particulate protein were used in a volume of 0.25 ml.Dilutions of nonradioactive phorbol-related diterpenes were made in a smallvolume of dimethyl sulfoxide The final concentration of dimethyl sulfoxide in alltubes was 0.95%. For each derivative except PMA and PDBU, the curvesrepresent the average of 2 experiments with triplicate determinations in eachexperiment. The curves for PMA and PDBU depict one of 2 experiments, each ofwhich gave similar results. The nominal concentrations of the nonradioactiveligands were corrected for decrease in concentration due to binding to theparticulate fraction and for competition with [3H]PDBU as described in "Materialsand Methods." In addition, concentrations of PMA were corrected for nonspecificpartitioning into the particulate fraction (see "Materials and Methods" and the

text) PMA (K,, 30 PM); Mz, mezerein (Kâ€ž1.4 nM); FDD. phorbol 12.13-dideca-noate (K,.


.24 .36 .48 ISO

1/FREE [3H]PDBu, (nM) -f

Chart 3. Analysis of inhibition of specific f'HJPDBU binding by nonradioactivephorbol 12,13-diacetate (PDA). Specific binding of [3H]PDBU to brain participatefraction as a function of the free concentration of [3H]PDBU was determined as

described for Chart 1 in the presence of the indicated concentration of nonradioactive phorbol 12,13-diacetate. Points, average of 4 determinations in a singleexperiment. Two additional experiments gave similar results. The lines werecalculated by linear regression analysis.

spite of the high lipophilicity of this compound (30). Scatchardanalysis of the binding data showed that [3H]PMA bound to a

single class of sites with a Kd of 66 Â±3 PM(n = 3 experiments).At saturation, 21.4 Â±0.8 pmol (n = 3 experiments) of [3H]PMAwere bound per mg of protein. Nonspecific binding of [3H]PMA

was measured in the presence of excess nonradioactive PMAas described in "Materials and Methods." As expected, nonspecific binding was linear with free [3H]PMA concentration.From the lowest to the highest [3H]PMA concentrations exam

ined, specific binding varied from 95% to 82% of total binding.At the Kd for PMA, specific binding was 91 % of the total.

The conditions of the [3H]PMA binding assay were modifiedfrom those of the [3H]PDBU assay in order to ensure that a low

proportion of total ligand would be bound. Under the conditionsof the [3H]PDBU assay (100 /Â¿gparticulate protein in a 0.25-mlincubation volume), 99% of the [3H]PMA would be expected to

be specifically bound at its Kd. In this situation, small amountsof impurities either preexisting in the [3H]PMA stock or arising

by degradation during the binding assay could hamper accurate measurement of free [3H]PMA concentration. To avoid this

problem, we decreased the concentration of phorbol esterbinding sites (10 jug particulate protein in an 8-ml incubationvolume) in the [3H]PMA binding assay to roughly one-half theKa for [3H]PMA, the lowest concentration conveniently attain

able. At this binding site concentration, less than 20% of thetotal [3H]PMA was bound at half-maximal specific binding.Under these assay conditions, [3H]PMA binding reached equi

librium more slowly (Chart 5) than had previously been observed for [3H]PDBU. Time of incubation was accordingly

lengthened to 75 min from 30 min. Another consideration inchoosing the [3H]PMA assay conditions was that, due to thefairly low specific activity of the [3H]PMA available, it was

necessary to have a large volume of supernatant with which tomeasure free [3H]PMA concentrations; 66 pw [3H]PMA repre

sented 1300 dpm/ml.Scatchard analysis showed a slightly lower number of sites

for [3H]PMA than for [3H]PDBU. The difference probably re

flects the necessarily different procedures used in the determination of Kd's for the 2 ligands. When [3H]PMA was bound

under the conditions of the PDBU assay, specific binding atsaturation (30 Â± 2 pmol/mg; n = 4) was similar to that forPDBU.

10

0.1 0.2 0,3

FREE [3H] PMA, nM

BOUND [3H] PMA, pmol/mg

Chart 4. Binding of [3H)PMA to mouse brain. A. binding as a function of freeligand concentration. Specific binding (â€¢)and nonspecific binding (O) of [JH]-

PMA to brain particulate fraction (10 fig protein) were determined using an 8-mlincubation volume as described in 'Materials and Methods. The curve was

calculated with the dissociation constant and the amount of specific binding atsaturation, both obtained from the Scatchard plot of the data. Points, mean of 3determinations in a single experiment; bars, S.E. The results of 2 additionalexperiments were similar. B. Scatchard plot of the specific binding data in A.Parameters for the line were calculated by linear regression analysis (r = 0.98).

150-

. 100

50-

30 90 120

TIME, minuits

Chart 5. Time course of specific [3H]PMA binding. Specific binding of [3H]-

PMA to brain particulate fraction (10 ng) was determined using an 8-ml incubationvolume as described in "Materials and Methods," except that time of incubationat 39Â°was varied as indicated. The free concentration of [3H]PMA in the assay

was approximately 90 PM. Points, mean of 3 determinations; bars. S.E.

OCTOBER 1980 3639



W. G. Dunphy et al.

1.0

0.5

I

Table 3Comparison ol the tumor-promoting and binding activities of the phorbol-related

diterpene esters in the mouse

12 11 10 9 8 7 6

-log [COMPETING LIGANDI, M

Chart 6. Inhibition of specific [3H]PMA binding by nonradioactive phorbolesters. Specific binding of [3H]PMA to brain paniculate fraction (40 pig protein)was measured in a 1.0-ml reaction volume as described in "Materials andMethods.' In each experiment, specific binding of [;1H]PMA in the presence of

nonradioactive phorbol esters was expressed as a fraction of the total specificbinding in the absence of competing ligand. Dilutions of nonradioactive phorbolesters were carried out in a small volume of dimethyl sulfoxide. The finalconcentration of dimethyl sulfoxide in all tubes was 0 95%. The nominal concentrations of the nonradioactive phorbol esters were corrected for the decrease inconcentration due to binding to the particulate fraction and for competition with( 'H]PMA as described in Materials and Methods. Concentrations of PMA were

also corrected for nonspecific partitioning into the particulate fraction (see'Materials and Methods and the text). The nonradioactive ligands were PMA

(K,. 70 PM) and PDBU (K,. 14 nM). Curves from Chart 2 showing inhibition ofspecific ( 'H]PDBU binding by PMA ( ) and by PDBU ( ) are presented

for comparison.

Nonradioactive PDBU inhibited [3H]PMA binding at least 95%

(Chart 6). Moreover, the competition curves for inhibition of[3H]PMA binding by nonradioactive PMA or PDBU (Chart 6,solid curves') showed quantitative agreement with those obtained for inhibition of [3H]PDBU binding (Chart 6, dotted anddashed curves'). We conclude that the major high-affinity bind

ing sites in brain for PMA and PDBU are identical.

DISCUSSION

PMA has occupied a unique position as the most potenttumor promoter among the phorbol-related diterpene esters.On the other hand, our structure-activity studies characterizingthe loss of fibronectin and the stimulation of 2-deoxyglucose

transport induced in chicken embryo fibroblasts by the phorbolesters indicated that the different phorbol derivatives, althoughranging greatly in potency, induced similar maximal responses(14, 15). This same observation was made for induction ofplasminogen activator in chicken embryo fibroblasts (48). Likewise, the in vitro [3H]PDBU binding assays on preparations

from chicken embryo fibroblasts, mouse skin, and mouse brainshowed the different biologically active phorbol-related diter-penes all to inhibit [3H]PDBU binding. The present studies withmouse brain, demonstrating that [3H]PMA binding can be

blocked >95% by PDBU, strongly argue that PMA, PDBU, andthe other phorbol derivatives in this series which were examined all act by binding to the same site.

The available data on tumor-promoting activity are consistentwith this conclusion (see Table 3). Most of the in vivo data citedare from comparisons made at suboptimal concentrations ofderivatives. For rigorous analysis of the tumor-promoting activities of the phorbol-related diterpene esters, complete dose-

response studies need to be performed for each derivative. In

DerivativePMA

Phorbol 12,13-didecanoateMezereinPhorbol 12,13-dibutyratePhorbol 12,13-dibenzoatePhorbol 12,13-diacetatePhorbol 12.13.20-lriacetatePhorbol 13-acetateRelative

tumor-promotingactivity1

1.549

180 or 7220-100

>50>200

NT0(44)

(34)(39, 44)

(1)(36)(25)Potency

in bindingassay (K, relative to

PMA)Brain


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OCTOBER 1980 3641



1980;40:3635-3641. Cancer Res William G. Dunphy, K. Barry Delclos and Peter M. Blumberg Mouse Brain

H]Phorbol 12-Myristate 13-Acetate to312,13-Dibutyrate and [H]Phorbol3Characterization of Specific Binding of [

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characterization of specific binding of [3h]phorbol 12,13 ......and has made possible experiments...

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