[CANCER RESEARCH 50. 5845-5850. September 15. I990|
Opposite Effects of Tamoxifen on in Vitro Protein Kinase C Activity and
Endogenous Protein Phosphorylation in Intact MCF-7 Cells1
M. Issandou, C. Faucher, F. Bayard, and J. M. Darbon2
Inserm UI68, Department of Endocrinology. CHL' Rangueil, UniversitéPaul Sabotier, 3I054 Toulouse Cedex, France
Ca2+- and phospholipid-dependent
ABSTRACT
PKC (22-24), an enzyme
We investigated the effects of the antiestrogen tamoxifen on MCF-7
cell protein kinase C either by using the in vitro histone kinase assay or
by studying the phosphorylation of its endogenous M, 28,000 protein
substrate in intact cells.
In the in vitro assay, tamoxifen inhibited the enzyme competitively
with respect to phospholipid, whereas estradici and morpholinobenzyl
phenoxy ethanamine, a specific ligand for antiestrogen binding sites, were
considerably less efficient.
In contrast, tamoxifen did not affect phosphorylation of the M, 28,000
protein induced by the phorbol ester 12-O-tetradecanoylphorbol 13acetate in intact MCF-7 cells. Estradiol and morpholinobenzyl phenoxy
ethanamine also had no effect. At high concentration (100 MM),tamoxifen
itself stimulated specific phosphorylation of this M, 28,000 protein.
Estradiol and morpholinobenzyl phenoxy ethanamine neither mimicked
nor interfered with this effect.
Our data suggest that the effect of tamoxifen on protein kinase C
activity depends on the phospholipid environment of the enzyme, and
opposite effects may be observed in intact cells to those seen in disrupted
cells. The action of tamoxifen on endogenous protein phosphorylation
was thought to be due to direct interaction with the phospholipid binding
domain of the enzyme rather than by interaction with the estrogen
receptor or the antiestrogen binding site. Nevertheless, our results do not
rule out a possible activation by tamoxifen of specific protein kinase(s)
and phosphatase(s). In any case, the antiproliferative activity of tamoxi
fen on MCF-7 cells cannot be attributed to its effects on protein kinase
C.
which plays a key role in transmembrane signaling of a wide
variety of extracellular stimuli, including growth factors, hor
mones, and other biologically active substances (25-27). The
physiological activator of protein kinase C is DAG, which
accumulates transiently because of a receptor-mediated break
down of inositol phospholipid (25,27). Tumor-promoting phor
bol esters, such as TPA, can mimic the intracellular messenger
DAG by activating protein kinase C directly (28). In fact, the
high-affinity phorbol ester receptor in target cells is now
thought to be PKC (29, 30).
The question is whether or not tamoxifen exerts its antiproliferating action by inhibiting protein kinase C (22-24). Such
an hypothesis implies that PKC plays a positive modulatory
role on cell growth. However, in MCF-7 human breast cancer
cells, our group (31-33) along with others (34, 35) have dem
onstrated that activators of PKC, such as phorbol esters and
diacylglycerols, completely inhibit cell proliferation.
In previous work, we showed that the arrest of MCF-7 cell
growth was correlated with the phosphorylation of an endoge
nous M, 28,000 protein which appears to be a specific substrate
of PKC (33, 36, 37). In the present study, we investigated the
effects of tamoxifen and morpho-BPE, a phenoxy-ethanamine
derivative which binds to the antiestrogen binding site but not
to the estrogen receptor (19), on both in vitro PKC activity and
protein phosphorylation in MCF-7 cells.
INTRODUCTION
MATERIALS
The triphenylethylene derivative tamoxifen is a synthetic
nonsteroidal antiestrogen which is used in the treatment of
human breast cancer to inhibit tumor growth (1, 2). Its mech
anism of action is not yet clearly understood, although there is
evidence that tamoxifen competes with estrogen at the estrogen
receptor (3-6). However, several reports indicate that not all of
its biological effects are mediated by the estrogen receptor (69). Tamoxifen and other related antiestrogens also bind specif
ically to a microsomal high-affinity site which is distinct from
the estrogen receptor (10-16). The nature and significance of
this ABS' remain to be established. It has been suggested to
play a role in the growth-inhibitory effect of antiestrogens (1719), although this has not been confirmed in other studies (20,
21).
Using in vitro assays, tamoxifen has been shown to inhibit
Chemicals. Histone HI, TPA, phosphatidylserine, 1,2-dioleoyl-glycerol, H7, and protease V8 (from Staphylococcus aureus) were obtained
from Sigma. Staurosporine was from Calbiochem. [7-"P]ATP (0.5 to
3 Ci/mmol), ['HJIeucine (146 Ci/mmol), and ["Pjphosphoric acid were
AND METHODS
purchased from Amersham. Acrylamide and bisacrylamide were from
Biorad. Tamoxifen was obtained from ICI.
Cell Culture. MCF-7 cells were adapted to grow in the absence of
serum (38) at 37°Cin RPMI 1640 without phenol red (Gibco), supple
mented with 2 g/liter sodium bicarbonate, pH 7.3, 2 mM L-glutamine.
1 Õ¿M
insulin (Novo Laboratories), and 0.1 ¿IM
transferrin.
RPh-4 variants were isolated from MCF-7 cells (39) from their
resistance to the inhibitory action of TPA on cell growth and were
routinely cultured in the same medium as MCF-7 cells but in the
presence of 50 ng/ml of TPA.
RTx-6 variants were isolated from MCF-7 cells grown in the presence
of 4% fetal calf serum stripped of endogenous hormones (13) and were
subcultured in RPMI 1640 medium without phenol red.
Protein Kinase C Assay. Subconfluent cells (40 x 10* cells) were
Received 7/26/89; accepted 6/4/90
The costs of publication of this article were defrayed in part by the payment
rapidly harvested in cold PBS and homogenized in 20 HIMTris-HCI,
of page charges. This article must therefore be hereby marked advertisement in
pH 7.5, containing 0.25 M sucrose, 2 mM EDTA, 2 mM EGTA, 100
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by the Institut National de la Santéet de la Recherche Médicale Aig/ml of leupeptin, and 5 mM 0-mercaptoethanol (Buffer A).
The cell lysate was centrifuged for l h at 105,000 x g. The super
and by the Association de la Recherche contre le Cancer.
* Present address: Inserm U133. Faculté
de Médecine.133 route de Narbonne.
natant was applied to a DEAE-cellulose column (DE52, 0.8 x 3 cm)
31062 Toulouse Cedex. France. To whom requests for reprints should be ad
equilibrated with 20 mM Tris-HCI, pH 7.5, containing 2 mM EDTA, 1
dressed.
mM EGTA, 50 Mg/ml of phenylmethylsulfonyl fluoride, and 5 mM ß'The abbreviations used are: ABS. antiestrogen binding site; PKC. protein
kinase C; EGTA. ethyleneglycol-bis(rf-aminoethylether)-A'.A"-tetraacetic
acid:
mercaptoethanol (Buffer B). Columns were washed with 10 ml of Buffer
ICso, 50% inhibitory concentration; TPA. 12-O-tetradecanoylphorbol 13-acetate;
B. PKC activity was eluted with 4 ml of Buffer B containing 0.13 M
DAG, diacylglycerol; morpho-BPE. morpholinoben/yl phenoxy-ethanamine:
NaCl.
DMSO, dimethyl sulfoxide; PBS. phosphate-buffered saline: TCA. trichloroacetic
PKC was assayed by measuring the incorporation of "P from [7-32P]
acid; SDS. sodium dodecyl sulfate: H7. I-(5-isoquinolinylsulfonyl)-2-methylpiperazine.
ATP into HI histone. The standard assay mixture (200 ni) contained
5845
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EFFECTS OF TAMOX1FEN ON PKC AND PROTEIN PHOSPHORYI.ATION
20 mM Tris-HCl, pH 7.4, 40 n% of histone, 10 ^M ATP (550 cpm/
pmol), 5 HIMMgCli, 40 ¿il
of sample, and either 0.5 HIMEGTA or 0.5
HIMCaCli and various amounts of phosphatidylserine and 1,2-dioleoylglycerol. After incubation for 5 min at 30°C.10% TCA was added to
stop the reaction. The protein precipitate was dissolved in N NaOH,
and the incorporation of "P was measured by scintillation counting in
Pico-fluor (Packard).
Protein Phosphorylation. Subconfluent cultures (1 x 10" cells/35mm dish) were washed twice in a phosphate-free Krebs-Ringer buffer
containing 20 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid.
pH 7.3, 0.1% bovine serum albumin, and 0.2% glucose and incubated
for 2 h at 37°Cin 1 ml of the same buffer containing 50 nCi of ["P|
phosphoric acid. Stimuli were then added for a further 30-min period.
Cells were washed twice with cold PBS, and 10% TCA was added.
TCA-precipitated proteins were dissolved in 150 pi of electrophoresis
sample buffer containing 0.06 M Tris-HCl. pH 6.7, 2% SDS, 8%
glycerol, 2% (i-mercaptoethanol, and 0.005% bromophenol blue. Sam
ples were then boiled for 5 min at 90°C.Proteins were fractionated by
electrophoresis on 4.5% and 12% (w/v) discontinuous SDS-polyacrylamide slab gel. After fixation of proteins with cold TCA and staining
with Coomassie blue, the gel was dried and then exposed to HyperfilmM P for 48 to 72 h.
Peptide Mapping by Limited Proteolysis. One-dimensional peptide
mapping was carried out according to the method of Cleveland et al.
(40) using protease V8 from S. aureus. Briefly, the M, 28.000 phosphorylated band was excised from the gel, directly loaded on a second
SDS-polyacrylamide (4.5 and 15%) slab gel. and then overlayed with
protease (20 and 200 ng). Digestion proceeded directly in the stacking
gel during the subsequent electrophoresis.
Protein Synthesis. Subconfluent cultures (1.5 to 3 x IO6cells per 60mm dish) were submitted to heat shock by incubation at 43°Cfor
IN MCF-7 TELLS
120r
20
SO
100
TAMOXIFEN
•
uM
200
500
MORPHO-BPE
Fig. 1. Effects of tamoxifen and morpho-BPE on MCF-7 PKC activity in
vitro. Protein kinase C activity was assayed on MCF-7 cell cytosol following
DEAE-cellulose chromatography. in the presence of calcium (10 ,-innh and
various concentrations of phosphatidylserine (PS) and 1,2-dioleoyl-glycerol (DG):
O and *. 20 Mg/ml of PS. 0.75 Mg/ml of DC: A. 40 ><g/mlof PS. 1.5 Mg/ml of
DG; O. 60 jig/ml of PS. 2.25 ¿ig/mlof DC: •and *. 80 fig/ml of PS. 3 i/g/ml
of DG. Tamoxifen was dissolved in DMSO and added at the indicated final
concentrations. All reaction mixtures (control included) contained 5% DMSO.
Morpho-BPE was dissolved in distilled water and added at the indicated final
concentrations. Data were expressed as the percentage of respective controls
(without addition of tamoxifen or morpho-BPE).
various times, or they were exposed to 100 MMtamoxifen for 30 min
at 37°C.After washing, cells were incubated for l h at 37°Cin 2 ml of
leucine-free medium containing 100 ^Ci of ('H)leucine. At the end of
NlCHjlj
incubation, cells were washed twice with 5 ml of cold PBS, and 10%
TCA was added. TCA-precipitated proteins were analyzed by SDSpolyacrylamide gel electrophoresis and autoradiography.
I am
RESULTS
Moroho-BPE
Fig. 2. Structures of tamoxifen (7am) and morpho-BPE.
Tamoxifen inhibited protein (histone) kinase C activity from
MCF-7 cells in a dose-dependent manner (Fig. 1). The effi
ciency of inhibition depended on the amount of phosphatidyl
serine (and diacylglycerol) used. The IC5us were around 50, 60,
85, and 250 »mo\for 20, 40, 60, and 80 Mg/ml of phosphati
dylserine, respectively (and 0.75, 1.5, 2.25, and 3 ng/m\ of 1,2dioleoyl-glycerol, respectively). Tamoxifen (20 jumol) inhibited
significantly (by 20%) PKC activity in the presence of 20 ßg/
ml of phosphatidylserine, whereas 200 ^M tamoxifen was re
quired to affect the enzyme activity in the presence of 80 ng/
ml of phosphatidylserine (i.e., the standard conditions for assay
of PKC). Similar results were obtained when the enzyme activity
was assayed with a constant concentration of 1,2-dioleoylglycerol (3 jug/ml) and various concentrations of phosphatidyl
serine (data not shown).
We also investigated the effect of the phenoxy-ethanamine
derivative, morpho-BPE (Fig. 2), another inhibitor of MCF-7
cell proliferation (19), on PKC activity. In contrast to tamoxi
fen, concentrations of morpho-BPE up to 50 ¿¿mol
did not
inhibit enzyme activity at any of the phospholipid concentra
tions tested. A dose as high as 500 MMof morpho-BPE only
led to a 20% inhibition of PKC activity in the presence of 80
Mg/ml of phosphatidylserine (Fig. 1). Estradici (100 ¿imol)did
not affect PKC activity under these conditions, and only led to
a 30% inhibition of enzyme activity in the presence of 20 ^g/
ml of phosphatidylserine (data not shown). None of the com
pounds tested affected basal histone kinase activity measured
in the absence of Ca2* and phospholipid (data not shown).
It can be seen from Fig. 3 that tamoxifen also inhibited PKC
activity when 1,2-dioleoyl-glycerol was replaced with TPA. As
in the previous experiments, inhibition kinetics was highlydependent on the concentration of phosphatidylserine (IC5U=
25, 75, 90, and 150 ^mol for 20, 40, 60, and 80 Mg/ml of
phosphatidylserine). lending further support to a competitive
inhibition of the enzyme by tamoxifen with respect to phospha
tidylserine.
Since the inhibitory action of tamoxifen on PKC appeared
to depend on the phospholipid environment, we wondered
whether tamoxifen would affect the enzyme activity in intact
MCF-7 cells. Fig. 4 shows that tamoxifen (up to 100 //mol) did
not affect the TPA-induced phosphorylation of the M, 28,000
protein. Morpho-BPE (100 ¿¿mol)
and estradiol (100 /¿mol,not
shown) were also without effect on this TPA-induced protein
phosphorylation.
Surprisingly, when added alone at high concentration (100
pinol), tamoxifen consistently stimulated phosphorylation of
the M, 28,000 protein. In contrast, at the same concentration
of 100 /jmol, neither morpho-BPE (Fig. 4) nor estradiol (see
below Fig. 7) stimulated this phosphorylation.
We have recently reported that the M, 28,000 PKC substrate
may be ascribed to the stress protein family.4 Since cytotoxic
concentrations of tamoxifen may induce stress of MCF-7 cells,
we wondered if the stimulation of the M, 28,000 protein phos-
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EFFECTS OF TAMOXIFEN ON PKC AND PROTEIN PHOSPHORVLATION IN MCF-7 CELLS
140
"•Il
28
— 28
50
HM
100
200
500
TAMOXIFEN
Fig. 3. Inhibition by tamoxifen of PKC activity in the presence of TPA and
various concentrations of phosphatidylserine. Protein kinase C activity was as
sayed on MCF-7 cell cytosol following DEAE-cellulose chromatography. in the
presence of calcium (IO .moli TPA (10 ng/ml). and various concentrations of
phosphatidylserine (20.40.60. and 80 jjg/ml). Tamoxifen was dissolved in DMSO
and added at the final indicated concentrations. Controls contained the same
concentration of solvent DMSO (5%). Data were expressed as the percentage of
controls.
0.5h 1h
O
O
2h
3h
4h
5h
6h
HS
Fig. 5. Effects of heat shock and tamoxifen treatment on M, 28,000 protein
synthesis in MCF-7 cells. MCF-7 cells were incubated at 37'C (CONT) or at
43'C for various times (HS). or they were exposed to 100 MMtamoxifen for 30
min at 37'C (TAM). After washing, cells were incubated for I h at 37'C in 2 ml
of leucine-free medium containing 100 ^Ci of |'H]leucine. At the end of incuba
tion, cells were washed twice with 5 ml of cold PBS, and 10% TCA was added.
TCA-precipitated proteins were analyzed by SDS-polyacrylamidc gel electrophoresis and autoradiography.
28-
phorylation caused by the antiestrogen could have been due to
an increase in the cellular content of this protein. Fig. 5 shows
that 100 ßM
tamoxifen was unable to induce synthesis of the
M, 28,000 protein, whereas a 30-min heat shock did.
Fig. 6A shows that the two PKC inhibitors H7 and staurosporine markedly inhibited the TPA-induced phosphorylation
of the M, 28,000 protein in MCF-7 cells, but were without
influence on the antiestrogen effect. However, peptide mapping
of the A/r 28,000 phosphoprotein revealed identical phosphopeptides upon phorbol ester and tamoxifen stimulation (Fig.
6Ä).
We have recently selected variants of the MCF-7 line (RPh4) which are resistant to the inhibitory effect of TPA on cell
proliferation (39). These variants are routinely cultured in the
presence of phorbol ester. We found that PKC content in RPh4 cells depended on the duration of subculture in the absence
of TPA. Recovery of enzyme activity was low (35%), interme
diate (70%), and total (100%) after 8 days (RPh-4: d8), 15 days
(RPh-4: dl5), and 3 mo (RPh-4: Rv), respectively, of subculture
in TPA-free medium.J
-28
TPA
Fig. 4. Effects of TPA. tamoxifen. and morpho-BPE on protein phosphor) 1ation in intact MCF-7 cells. Subconfluent cells were incubated for 30 min in
phosphate-free Krebs-Ringer buffer containing 50 ^Ci of ("P]phosphoric acid in
the absence (Com) or in the presence of 100 ng/ml of TPA. various concentrations
(I. 10. and 100 ¿jmol)of tamoxifen (Tant), lOO^M morpho-BPE. or combination
of TPA and tamoxifen or TPA and morpho-BPE (+TPA). Tamoxifen was
dissolved in DMSO. The control at the same final concentration (lr<) is also
shown (Coni. DMSO). Morpho-BPE was dissolved in distilled water. TCAprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography. The arrow indicates the position of the M, 28.000 protein.
We found that neither tamoxifen (100 ¿/mol)nor TPA stim
ulated phosphorylation of the M, 28,000 protein in RPh-4: d8
cells (Fig. 7). Surprisingly, tamoxifen was also ineffective in
stimulating the phosphorylation of the specific M, 28,000 PKC
substrate in RPh-4: dl5 and RPh-4: Rv cells, whereas it inhib
ited the TPA-induced phosphorylation of the M, 28,000 pro
tein.
Furthermore, tamoxifen reduced the "P labeling of a A/r
4 J. M. Darbon et al., manuscript submitted for publication.
5847
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EFFECTS OF TAMOXIFEN
ON PKC AND PROTEIN PHOSPHORYLAT1ON
IN MCF-7 CELLS
0
Fig. 6. Effects of H7 and staurosporine on
TPA- and camoxifen-induced M, 28.000 pro
tein phosphorylation. In A, MCF-7 cells were
incubated for 30 min in the absence (CONT)
or in the presence of 400 ¿IM
H7 (H7), 100 n.M
staurosporine (ST). 100 ng/ml of TP A ( TPA),
100 UMt;uiinviti'ii (TAM), or combinations of
TPA and H7, TPA and staurosporine. tamoxifen and H7. or tamoxifen and staurosporine.
Phosphorylated proteins were analyzed by
SDS polyacrylamide gel electrophoresis and
autoradiography. In B. the M, 28.000 protein
phosphorylated after TPA and tamoxifen stim
ulation was excised from the gel and submitted
to peptide mapping analysis by limited proteolysis with 20 ng (I) and 200 ng (2) of protease
V8. Arrows indicate the position of phosphopeptides revealed after autoradiography of the
second SDS-polyacrylamide gel.
Fig. 7. Effects of tamoxifen and estradiol
on protein phosphorylation in intact MCF-7
and RPh-4 cells. Subconfluent cells were in
cubated as described in the legend to Fig. 4 in
the absence (control, a) or in the presence of
100 ng/ml of TPA (ft). 100 ^M tamoxifen (c),
100 MMestradiol (d). 100 ß\itamoxifen plus
100 /¡Mestradiol (e), or 100 ng/ml of TPA
plus 100 MMtamoxifen (/). Estradiol and ta
moxifen were dissolved in DMSO (final con
centration, 1% as in the control). RPh-4 cells
were subcultured in TPA-free medium for 8
days (RPh-4: d8). 15 days (RPh-4: d 15) or 3
mo (RPh-4: Rv) before the experiment. Arrows
indicate the position of M, 28.000 and M,
20,000 proteins. The labeling of the M, 28.000
protein observed in RPh-4: d8 control was
probably due to small amounts of the highly
lipophilic phorbol ester remaining in the cell
culture despite the period of TPA starvation.
The "P labeling of the M, 20.000 protein in
TPA plus tamoxifen-treated RPh-4: d8 cells
was most probably artifactual as it was not
observed in two other similar experiments.
-«27
-«21
-«17
-«14
28»-
-«28
«
RPh-4:d8
RPh. 4 : Rv
RPh-4
: d15
20 ..
bcdefabcdef
20,000 protein. Preliminary data indicate that Ca2+ ionophore
and ionomycine increase slightly the degree of phosphorylation
of this protein in intact MCF-7 cells, but do not affect the M,
28,000 protein (not shown). The M, 20,000 protein may thus
be a substrate of calmodulin kinase. It is of interest in this
respect that tamoxifen has been reported to be a possible
antagonist of calmodulin (41). Alternatively, the decrease in f2P
incorporation into the M, 20,000 protein in MCF-7 and RPh4 cells as well as the lack of "P labeling of the M, 28,000
protein in RPh-4 cells may be due to antiestrogen stimulation
of specific phosphatases.
Fig. 7 also shows that estradiol did not compete with tamox
ifen in stimulating phosphorylation of the M, 28,000 protein
in MCF-7 cells. We have recently selected other MCF-7 cell
variants (RTx-6) which are resistant to the inhibitory effect of
tamoxifen on cell proliferation, and which lack antiestrogen
binding sites (13). Fig. 8 shows that tamoxifen induced phos
phorylation of the Mr 28,000 protein in the RTx-6 as in the
MCF-7 cells. Estradiol was also unable to mimic the antiestro
gen effect in these cells (data not shown).
DISCUSSION
We report here opposite effects of tamoxifen on protein
kinase C activity measured in vitro and protein phosphorylation
observed in intact MCF-7 cells. While the triphenylethylene
antiestrogen inhibited the enzyme activity competitively with
respect to phospholipid concentration in the in vitro assay, it
did not reduce significantly TPA-induced phosphorylation of
the M, 28,000 PKC substrate in intact MCF-7 cells. In contrast,
tamoxifen at high concentration (100 ^mol) induced phosphor
ylation of the M, 28,000 protein, although it did not increase
5848
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EFFECTS OF TAMOXIFEN
MCF
- 7
RTx-
ON PKC AND PROTEIN PHOSPIIORYLATION
6
28-
-
20 .-
/
20
O
o
Fig. 8. Effect of tamoxifen on protein phosphorylalion in intact MCF-7 and
RTx-6 cells. MCF-7 cells and RTx-6 variants were grown in the presence of 4cc
fetal calf scrum stripped of endogenous steroid hormones. After washing in
phosphate-free Krebs-Ringer buffer, subconfluenl cells »ere incubated in the
absence (COAT) or in the presence of 100 ng/ml of TPA (TPA) or 100 /jM
lamoxifen (Tarn) as indicated in the legend to Fig. 4. Phosphorylated proteins
were analyzed in duplicate by SDS-polyacrylamide gel electrophoresis and autoradiography.
the basal protein kinase activity measured in vitro in the absence
of calcium and phospholipid.
The action of this triphenylethylene antiestrogen on protein
phosphorylation is unlikely to be mediated by either the estro
gen receptor or the ABS for the following reasons, (a) The
specific ligands estrogen and morpho-BPE did not mimic or
interfere with the effect of tamoxifen on M, 28,000 protein
phosphorylation. (b) Tamoxifen stimulated M, 28.000 protein
phosphorylation in RTx-6 cells which lack ABS. (c) MorphoBPE which has the same affinity for ABS as tamoxifen (19)
was 6 times less potent than tamoxifen in inhibiting the enzyme
activity when assayed under the same conditions (20 pg/ml of
phosphatidylserine). Estradiol also appears to have little action
on PKC activity in vitro.
The inhibitory effect of tamoxifen appears to be competitive
with respect to phosphatidylserine and probably reflects a direct
interaction with the phospholipid binding domain of the en
zyme, which has been observed in studies on PKC from rat (22,
23, 42) or pig (24) brain. However, tamoxifen may interact with
the catalytic domain of the enzyme (43). In our study, the lower
efficiency of morpho-BPE in inhibiting PKC is in line with the
weak hydrophobic nature of this amphiphilic ligand compared
with tamoxifen.
Tamoxifen significantly reduced PKC activity at /IM concen
trations which have been reported to inhibit MCF-7 cell growth.
However, in this cell type, there is increasing evidence that
PKC plays a negative modulatory role on cell proliferation.
Inhibition of the enzyme should, therefore, stimulate cell
growth. Moreover, the inhibition at MMconcentrations in vitro
was only observed at low concentrations of phospholipid, which
IN MCF-7 CELLS
do not necessarily correspond to the true environment of the
enzyme in the intact cell.
We also showed that concentrations of tamoxifen up to 100
ßtnoidid not reduce the phosphorylation of the specific M,
28,000 PKC substrate which is induced by TPA in MCF-7 cells.
This result is in disagreement with a recent report showing that
tamoxifen inhibits the calcium- and phosphatidylserine-induced
phosphorylation of various endogenous proteins from rat brain
and ovary homogenates (24). However, disruption of cell integ
rity may dramatically modify the phospholipid environment of
PKC.
On the other hand, the lack of effect of morpho-BPE on
phosphorylation of the M, 28,000 protein is in agreement with
the inefficiency of yV,/V-diethyl-2-[4-(phenyl methyl)phenoxy]ethanamine-HCl, another phenoxy-ethanamine derivative, in
blocking phosphorylation of the M, 47,000 protein which is
induced by TPA in platelets (44).
The fact that tamoxifen itself increases phosphorylation of
the M, 28,000 protein is intriguing, although it is probably not
linked to its cytostatic antiproliferative action as it was only
observed at 100 pmo\ which is cytotoxic for MCF-7 cells. More
surprising was the inability of tamoxifen to induce phosphor
ylation of the M, 28,000 protein in RPh-4 cells which contain
the same amount of functional PKC as MCF-7 cells. Although
MCF-7 and RPh-4 cells may contain distinct PKC isoforms
with different sensitivities to tamoxifen, this result might also
indicate that the M, 28,000 protein is phosphorylated by an
other type of protein kinase in tamoxifen-treated MCF-7 cells.
The inability of PKC inhibitors H7 and staurosporine to block
the M, 28.000 protein phosphorylation induced by the anties
trogen lends support to this idea. However, this latter finding
may suggest that tamoxifen prevents the inhibitory action of
both these compounds on PKC. Indeed, preliminary experi
ments indicate that tamoxifen, at a concentration which does
not affect PKC activity, obviates the inhibitory effect of H7
under the in vitro assay.
In any case, previous work in our laboratory demonstrated
that the M, 28,000 was a specific substrate for PKC. Only serine
residues were found to be phosphorylated on this M, 28,000
protein (33), which was not induced by activators of either the
cyclic AMP-dependent (36) or the calmodulin kinases.5 More
over, permeant diacylglycerols and exogenous phospholipase C
mimicked the action of phorbol esters on phosphorylation of
the M, 28,000 protein (33, 36). Finally, peptide mapping of the
phosphorylated M, 28,000 protein after TPA and tamoxifen
stimulation suggested that these two compounds may activate
the same kinase. The differences observed in the behavior of
PKC in RPh-4 and MCF-7 cells after stimulation with tamox
ifen may thus be due to differences in the microenvironments
of the enzyme in these two sublines. This may explain why the
antiestrogen inhibited the TPA-induced phosphorylation of the
M, 28,000 protein in the RPh-4 but not in the MCF-7 cells.
Activation of specific phosphatases in RPh-4 cells by tamoxifen
cannot, however, be ruled out.
Taken together, our results indicate the importance of the
phospholipid environment on the regulation of PKC activity.
This may account for the differences in the effects of tamoxifen
on PKC activity observed in the in vitro assay and in intact
MCF-7 cells.
ACKNOWLEDGMENTS
The authors are indebted to Dr. M. Poirot and J. C. Faye for helpful
discussion and for the gift of morpho-BPE. We also thank R. Baritaud
for his technical assistance.
5 Unpublished results.
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EFFECTS OF TAMOXIFEN
ON PKC AND PROTEIN PHOSPHORYLATION
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5850
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research.
Opposite Effects of Tamoxifen on in Vitro Protein Kinase C
Activity and Endogenous Protein Phosphorylation in Intact
MCF-7 Cells
M. Issandou, C. Faucher, F. Bayard, et al.
Cancer Res 1990;50:5845-5850.
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