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Department of Pathology [E. K. L., J. R. M.], Program in Cell and Molecular Biology [E. K. L., J. R. M.], and the Department of Surgery [G. H., J. D. I., J. R. M.], Duke University Medical Center, Durham, North Carolina 27710
Abstract
Estrogen acts to promote DNA synthesis in the MCF-7 human breast cancer cell line via its interaction with high levels of estrogen receptor. The primary mode of estrogen action has been considered to be through transcriptional activation of genes containing estrogen response elements, including the immediate early genes c-myc and fos. Recent reports have indicated that estrogen, acting through the estrogen receptor, is capable of inducing the mitogen-activated protein kinase (MAPK) cytoplasmic signaling cascade. In this study, specific small molecule inhibitors of MAPK and phosphatidylinositol 3-kinase activity were used to determine the influence of these cascades on estrogen-mediated mitogenesis. Phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, as well as inhibitors of MAPK kinase-1, PD098059 and U0126, decreased the fraction of cells entering DNA synthesis after treatment with 17ß-estradiol. These compounds did not inhibit expression of myc or fos. However, the drugs did prevent the accumulation of cyclin D1 and hyperphosphorylated retinoblastoma protein, indicating that the block occurred at, or prior to, this point in the cell cycle. Although these compounds were effective in preventing estrogen-mediated mitogenesis, the downstream kinases extracellular signal-regulated kinase 1, extracellular signal-regulated kinase 2, and protein kinase B were not activated over basal levels by estrogen treatment. These studies suggest that estrogen initiates mitogenesis by inducing the transcription of immediate early genes, but cytoplasmic signaling pathways play an important role in the control of subsequent events in the cell cycle.
Introduction
Estrogen can act as an effective mitogen in cells that express the ER3 both in cell culture and in vivo (reviewed in Ref. 1 ). E2, the most prevalent estrogen produced by the ovaries, bound to ER results in receptor translocation to the nucleus and dimerization. The ligand-bound ER dimer can interact with DNA at palindromic EREs, resulting in modulation of transcriptional activity. Antiestrogen treatment of cells that depend upon estrogen for their growth blocks this transcriptional activation and results in a G0-G1 cell cycle arrest (2) . A number of genes contain putative EREs including the immediate-early genes, c-myc and c-fos (3 , 4) . Therefore, it has been postulated that the mechanism by which estrogen mediates mitogenesis is via transcriptional activation of these and other key cell cycle regulatory genes.
After immediate-early gene synthesis, the cell cycle converges on the expression and activity of the cyclins and CDKs, particularly the D-type cyclins. Estrogen treatment of growth-arrested MCF-7 cells, a cancerous breast epithelial cell line that overexpresses ER, results in increased expression of cyclin D1 (5) . Concurrently, the activity of cyclin D1 binding partners, CDK4 and CDK6, increases (6 , 7) . Formation of active cyclin D1-CDK4 and cyclin D1-CDK6 holoenzymes serves two functions: (a) redistribution of CDK inhibitors (such as p27Kip1 and p21Cip1) away from cyclin E, facilitating the formation of active cyclin E-CDK2 complexes; and (b) phosphorylation of the Rb protein, which is also phosphorylated by cyclin E-CDK2 (8, 9, 10) .
The subcellular localization of ER and its role as a transcription factor led to the assumption that estrogen action occurs primarily in the nucleus. However, several recent reports have shown that elements of cytoplasmic signaling cascades may also be estrogen responsive. In cells expressing ER, the MAPK family members Erk1 and Erk2 are activated within 5 min in response to E2 treatment (11, 12, 13, 14) . Furthermore, cell membrane-impermeable estrogen (estradiol conjugated to BSA) stimulates MAPK activity (15) . A possible explanation for this phenomenon comes from several studies that have reported the existence of plasma membrane-associated ER (16, 17, 18, 19) . Activation of MAPK raises the possibility that some or all of the mitogenic activity of estrogen may be mediated through this pathway.
This theory was also supported by a study demonstrating that a MAPK inhibitor (PD098059) prevented estrogen-responsive proliferation in cardiac fibroblasts (20) . Furthermore, estrogen was able to induce mitogenesis in NIH-3T3 cells transiently transfected with transcriptionally inactive ER, presumably because of the ability of E2 to activate MAPK (21) . Cumulatively, these studies suggest that MAPK activation may be a requirement for estrogen-induced cell cycle progression.
Estrogen and its receptor may also interact with other cytoplasmic signaling components. In endometrial fibroblasts as well as selected malignant cell lines, E2 stimulates the activity of PKC (22) . Furthermore, in MCF-7 cells, synthesis of phosphatidylinositol and activation of phosphatidylinositol kinases can occur in response to E2 treatment (23) . ER itself can also act as a substrate for PKA. Serine-236 can be phosphorylated in vitro by PKA, which may be important in receptor dimerization (24) . Collectively, these reports suggest that cytoplasmic factors may be important intermediates in estrogen action.
In previous work, we found that MCF-7 cells grown in medium alone (no serum or added growth factors) proliferate in response to estrogen (25) . This system provides a model for studying the mitogenic effects of E2 in the absence of other exogenously added stimulatory molecules. In the current study, we have used a series of pharmacological compounds that specifically inhibit signaling intermediates that have been implicated in estrogenic action. Our results show that MAPK and PI3-K, but not PKA or PKC, activity are required for cell cycle progression in response to E2 stimulation. The cell cycle block caused by MAPK and PI3-K inhibitors occurs after the expression of immediate-early genes and prior to the induction of cyclin D1 expression, resulting in G1 arrest. Additional experiments show that E2 does not significantly activate MAPK or PKB, a downstream target of PI3-K, suggesting that either basal levels or delayed induction of these kinases are required for cell cycle progression in MCF-7 cells.
Results
Cytoplasmic Signaling Inhibitors Prevent Estrogen-induced
Mitogenesis.
The breast cancer epithelial cell line MCF-7 expresses high levels of
ER
. When these cells are serum-starved in phenol red-free media for
24 h, the largest fraction of cells accumulates and arrests early
in the G1 stage of the cell cycle. Treatment of
starved cells with estrogen alone (2 x
10-8 M E2)
stimulated cell cycle progression and resulted in a 3–4-fold increase
in the number of cells undergoing DNA synthesis, a rate that is
comparable or greater than the mitogenic effect of EGF (Fig. 1A)
.
|
). The antiestrogen, as expected,
effectively blocked E2- induced DNA synthesis,
reducing the percentage of cells in S phase to below the levels in
starved cells. Neither the PKA or PKC inhibitors (H-89 and BisI) had
any significant effect. However, the MEK1 inhibitor, PD098059, reduced
the rate of DNA synthesis by >50%, whereas the MEK1/MEK2 inhibitor,
U0126 (U), decreased the rate of proliferation to below the level
observed in starved cells. Inhibition of the PI3-K cascade with
LY294002 was almost as effective as the ICI antiestrogen in
blocking E2-induced S phase. However, wortmannin
was not as potent an inhibitor. These data suggest that the activities
of at least two cytoplasmic signaling molecules (MEK1 and PI3-K) are
critical for the mitogenic effect of estrogen.
To compare the effects of these inhibitors on another ligand that is
mitogenic in MCF-7 cells, we repeated the experiments on this breast
cancer cell line stimulated with EGF (30)
. As reported
previously (31)
, the ICI antiestrogen inhibited
EGF-induced mitogenesis. Furthermore, EGF-stimulated DNA synthesis was
inhibited by the same compounds to virtually the same degree as was
seen with E2 treatment (Fig. 1A)
.
To determine the efficacy of these drugs in inhibiting estrogen-induced
proliferation, we measured S-phase progression in the presence of a
range of inhibitor concentrations (1–50 µM). At the
lowest concentration for each drug, minimal effects, if any, were
observed (Fig. 1B)
. However, in the presence of 5
µM LY294002, the percentage of cells in S phase
was maintained at or near basal levels, a finding that is supported by
the published IC50 (1.4
µM) of LY294002 for abolishment of PI3-K
activity (26)
. Similar kinetics of inhibition were noted
with the MEK1/MEK2 inhibitor, U0126. PD098059 only weakly inhibits the
activation of MEK2 and has a much greater IC50
for the inhibition of MEK1 as compared with U0126 (29)
.
Therefore, it was not surprising to note that PD098059 did not have as
pronounced an inhibitory effect on estrogen-induced mitogenesis.
Effect of Estrogen on the Activation of the MAPK Cascade.
Inhibition of the PI3-K and MAPK cascades prevented efficient
estrogen-induced cell cycle entry in MCF-7 cells. Several recent
reports have demonstrated that the MAPK pathway is stimulated by
estrogen in an ER-dependent fashion (12
, 13
, 15
, 21
, 32)
.
The dual specificity kinases, MEK1 and MEK2, phosphorylate a threonine
and a tyrosine residue in the regulatory sites of Erk1 and Erk2,
resulting in the activation of these MAPKs (reviewed in Ref.
33
). We performed a series of experiments to determine
whether Erk1 and/or Erk2 were activated by E2
under our conditions (Fig. 2A)
. The dually phosphorylated forms of Erk1 and Erk2 were
specifically recognized on the immunoblot using a phosphospecific
monoclonal antibody, whereas total Erk1 and Erk2 was detected using a
separate antibody after stripping and reprobing the same blot. In this
case, we measured activation 5 min after treatment with either EGF, a
known inducer of MAPK activity, or E2. Compared
with the untreated control (starved cells), 10 ng/ml EGF induced a
dramatic increase in phosphorylated Erk1 and Erk2. Under the same
conditions, 2 x 10-8
M E2 had little or no
effect on the levels of activated Erk1 or Erk2 (Fig. 2A)
. A
number of repetitions of this experiment failed to show more than a
1.5-fold induction at any time point (from 1 to 20 min after treatment)
or at any cell density. Neither ICI 182,780 nor LY294002 had any effect
on active MAPK levels in E2-stimulated cells
(Fig. 2A)
. Although estrogen failed to activate MAPK, the
MAPK inhibitors (PD098059 and U0126) effectively reduced the basal
levels of phospho-Erk1 and -Erk2 (Fig. 2A)
.
|
. Estrogen had no effect on Erk1 or Erk2
levels, but increasing concentrations of these compounds inhibited
phosphorylation of these kinases. A clear reduction in phospho-Erk1 and
-Erk2 was noted with as little as 1 µM U0126
compared with starved or E2-treated cells. Peak
inhibition was achieved between 5 and 10 µM.
The PD098059 compound also reduced the levels of active
Erk1/Erk2 with maximum inhibition reached at 
10
µM. Even at the highest concentration tested
(50 µM), PD098059 was unable to completely
inhibit phospho-Erk1/Erk2 levels. These data are consistent with
relative S-phase inhibition observed with these two compounds and
suggest that maintenance of basal levels of phospho-Erk1/Erk2 may be
important in E2-mediated mitogenesis.
Effect of Estrogen on the Activation of the PI3-K Cascade.
PKB is a primary downstream target of PI3-K activation and has been
implicated in cyclin D1 protein stabilization (34)
.
Treatment with estrogen had little, if any, effect on phosphorylated
(active) PKB levels compared with stimulation with 30 ng/ml IGF-I, a
known activator of PI3-K in MCF-7 cells (Fig. 3A
; Ref. 35
). Neither PD098059, U0126, nor ICI
182,780 affected the phosphorylation of PKB; however, the PI3-K drug,
LY294002, was a potent inhibitor of PKB activation in response to IGF.
|
.
However, near-maximal inhibition of S-phase was achieved using 5
µM (Fig. 1B)
. As measured by
phosphorylation of PKB, inhibition of the basal activity of PI3-K does
not appear to correlate with suppression of
E2-induced growth.
Toxicity and Reversibility of PI3-K and MAPK Inhibitor Molecules.
We next examined toxicity and reversibility of the pharmacological
inhibitors of MAPK, PI3-K, and ER. Cell viability was measured by dye
exclusion after 24 h of drug treatment in the presence of 2 x 10-8 M E2
(Fig. 4)
. A slight decline in viability was observed in cultures treated with
the MAPK inhibitors PD098059 and U0126 and with the estrogen antagonist
ICI 182,780. We observed no increase in the apoptotic fraction with
these drugs, as measured by flow cytometry (data not shown). Next, we
measured the reversibility of the growth-inhibitory effects of each
drug by a washout experiment. MCF-7 cells were treated with the small
molecule inhibitors for 24 h in the absence of any mitogen prior
to washing three times with PBS and stimulating with E2
(Fig. 4)
. Cell cycle analysis showed that cells treated with LY294002
and PD098059 could respond mitogenically to estrogen stimulation after
the drugs had been removed. However, inhibition by U0126 and ICI
182,780 could not be reversed in this time frame. Because this did not
appear to be related to toxicity, it appears that these drugs exert a
long-lasting effect on their targets. The ICI antiestrogen acts by
inducing ER degradation; therefore, a long-lasting effect on ER
response is not surprising (36)
.
|
. Blocking PKA or PKC activation had no impact on
ER-mediated transcription, whereas the MEK1/2 inhibitor U0126 was as
effective as the ICI compound at preventing ER-mediated transcription.
Transcription declined 50% in the presence of the MEK1 inhibitor
PD098059 and with the PI3-K inhibitor LY294002.
|
The specificity of drug effects on ER-mediated transcription was
examined by studying their impact on the transcriptional activity of
another steroid hormone, progesterone. MCF-7 cells were transiently
transfected with a human progesterone receptor expression vector and a
luciferase gene with a progesterone-responsive promoter. Addition of
10-7 M progesterone for 24 h
stimulated a 5-fold increase in luciferase activity, an activity that
was not inhibited in the presence of LY294002 and ICI 182,780 (Fig. 5B)
. Both MAPK inhibitors prevented maximal induction by
50%. Therefore, these cytoplasmic inhibitors have pleiotropic
effects on nuclear hormone-mediated transcription.
The level of the ER itself may be decreasing in response to the
inhibitory compounds, which could account for the decline in
transcriptional activity. Therefore, we measured the levels of ER by
immunoblotting over the course of a 24-h treatment regimen (Fig. 5C)
. ER is known to decline in response to
E2 treatment (37
, 38)
; we observed
this phenomenon with ER protein levels reduced 3-fold after 9 h of
estrogen stimulation compared with basal levels in starved cells.
Estrogen stimulation in the presence of LY294002, wortmannin, or
PD098059 did not affect ER protein levels compared with
E2 alone. Furthermore, in the absence of
estrogen, these drugs caused no change in the steady-state levels of ER
found in serum starved cells. Consistent with published data, ICI
182,780 reduced ER levels in the presence or absence of steroid hormone
(36)
. Unlike PD098059, treatment with U0126 decreased ER
levels with kinetics similar to those observed with ICI 182,780. The
diminishment of ER in the presence of U0126 and with ICI 182,780 is
consistent with the dramatic inhibition of ER-mediated transcription at
later time points.
Inhibitor Effects on Estrogen-induced Gene Transcription.
The kinase inhibitors did exert a suppressive effect on
estrogen-mediated transcription measured by reporter gene assay. These
assays were performed between 6 and 24 h of drug treatment, long
after the signaling cascades or the ER may transduce any immediate
effects on the cell cycle. Decreased ER transcription may be a
secondary effect of the arrest rather than a direct effect of kinase
inhibitors. Expression of the immediate-early genes occurs rapidly
after E2 treatment; therefore, we measured the
expression of these genes directly by Northern blotting. We observed
maximal expression of the two putative estrogen-responsive
immediate-early genes, c-myc and c-fos, within
1 h of E2 treatment (Fig. 6)
. None of the signal transduction inhibitors (bisindolylmaleimide I,
H-89, LY294002, and PD098059) had an effect on the kinetics or
magnitude of estrogen-induced myc and fos expression (Fig. 6
and data
not shown). Estrogen induction of myc and fos protein was also not
inhibited by these compounds (data not shown). The ICI antiestrogen
completely inhibited the induction of both c-myc and
c-fos mRNA (and protein) as expected (Fig. 6
and data not
shown). Despite decreasing the steady-state levels of ER, U0126 had no
effect on the induction of myc and fos in response to estrogen
treatment. Therefore, although the compounds did affect estrogen
transcription measured in the transient assays, they had no effect on
immediate-early gene synthesis. These results suggest that induction of
these genes is directly dependent upon the hormone receptor’s nuclear
action. Furthermore, inhibition of estrogen transcription in the
reporter gene assay at later times is not correlated with effects on
the transcription of these endogenous genes.
|
12 h after the addition
of estrogen, with peak S phase values reached at 24 h
(25)
. Effective inhibition of cell cycle progression was
observed when LY294002 was added up to 6 h after exposure to
estrogen (Fig. 7)
. By 12 h, the LY compound was no longer an effective inhibitor.
In parallel experiments using the MEK1 inhibitor (PD098059), we
observed a gradual increase in S-phase progression with delayed
addition of the compound after E2 stimulation.
Although the demarcation in PD098059 activity was not as clearly
defined, the profile from both compounds suggested that inhibition of
events early in G1 is important for the activity
of these drugs in preventing S-phase progression.
|
. This modest induction is consistent with other reports
and with the 3–4-fold increase in S phase that we routinely observed
under these conditions (41)
. As expected, cyclin D1
accumulation resulted in the activation of CDK2 and Rb
hyperphosphorylation (Fig. 8, B and C
; Ref.
43
). The pharmacological compounds demonstrated a good
correlation between antimitogenic activity and inhibition of the Rb
pathway. The ICI antiestrogen, as reported previously
(44)
, and the MAPK inhibitor U0126 both prevented cyclin
D1 induction and activation of CDK2 and led to decreased levels of
hyperphosphorylated retinoblastoma protein. These findings are
consistent with the effects of ICI and U0126 on mitogenesis. Of the
PI3-K inhibitors, wortmannin was not as potent at preventing the
accumulation of cyclin D1 protein as LY294002, a finding that is
consistent with the relative S-phase inhibition observed with these
compounds. The MEK1 inhibitor PD098059 also inhibited cyclin D1 levels;
however, the effect appeared to be more transitory as cyclin D1 began
to accumulate at later time points (Fig. 8A
and data not
shown). This was also reflected by the incomplete inhibition of CDK2
activation, Rb hyperphosphorylation, and cell cycle progression. From
these experiments, we place the critical antimitogenic effects of the
PI3-K and MAPK inhibitors at or before the level of cyclin D1
induction.
|
Estrogen, a nuclear hormone, exerts a mitogenic effect on MCF-7 cells without the addition of any other growth factors, which facilitates the study of this pathway in isolation. A great deal of work on estrogen-induced growth has led to several conclusions: (a) the response depends upon the presence of functional estrogen receptor; (b) immediate-early genes are transcriptionally activated; and (c) cyclin D1 is an important intermediate in inducing this cell cycle cascade (reviewed in Ref. 45 ). If one assumes that the primary mode of estrogen action is via its receptor acting as a ligand-inducible transcription factor, mitogenesis could be explained by the direct transcriptional activation of immediate-early genes that contain functional EREs. The recent reports of estrogen activation of cytoplasmic signaling components, particularly the MAPK pathway, complicate this relatively simple paradigm (11 , 13 , 15 , 22, 23, 24 , 46 , 47) . It is in this context that the current studies were performed.
We found that disruption of the PI3-K and MAPK cascades by specific inhibitors (PD098059, which inhibits MEK1; U0126, which inhibits MEK1 and MEK2; and LY294002 and wortmannin, which inhibit PI3-K) prevented or reduced the ability of MCF-7 cells to enter S phase in response to estrogen. These drugs arrested cells in G1 and did not induce any significant cytotoxicity. We also demonstrated that E2 does not measurably activate either PKB or MAPK under conditions that result in a mitogenic response. We did observe variable effects on estrogen-mediated transcription assayed on artificial EREs; however, the immediate-early genes c-myc and c-fos were both induced in the presence of these drugs. Both the PI3-K and MAPK inhibitors prevented cyclin D1 accumulation, which correlated with an absence of CDK2 activity and Rb phosphorylation. We concluded that inhibition of estrogen-mediated mitogenesis by these drugs was likely attributable to this repression of cyclin D1 expression, CDK2 activation, and Rb phosphorylation.
This study raises several issues regarding the mechanism of estrogen action and mitogenesis in general. The lack of any reproducible induction of MAPK activity, as measured by Erk1 and Erk2 phosphorylation, contradicts several published reports (13 , 15 , 21 , 32 , 47) . In light of these reports, we examined this parameter many times using different time points, cell densities, and variable concentrations of charcoal-stripped serum. On occasion, we did observe a small increase (<1.5 fold) in Erk1 and Erk2 phosphorylation between 2 and 5 min after estrogen stimulation. Published accounts of this activation have also shown a relatively modest induction (between 1.5- and 3-fold) over control values (13 , 47) . In all reported cases and in our experiments, the magnitude of activated MAPK resulting from a cell surface signal such as EGF far exceeds any induction attributable to estrogen (32) . Furthermore, estrogen can overcome a cell cycle block induced by Simvastatin, an HMG-CoA reductase inhibitor, without detectable MAPK activation (48) . It is also clear from our experiments that estrogen is mitogenic in the absence of measurable MAPK activation, which raises the issue of the physiological relevance of the response when it is observed.
MAPK and PI3-K inhibitors were shown to inhibit ER-mediated transcription, assayed using a gene reporter system. However, these inhibitors do not prevent the expression of immediate-early genes in response to estrogen. At least one plausible explanation can reconcile this apparent discrepancy. Maximal expression of c-myc and c-fos occurs after 1 h of stimulation, whereas luciferase activity required 6 h for a measurable response. Therefore, effects on ER transcription may not be manifest until after the rapid immediate-early response. How critical this delayed inhibition of ER activity is in mediating the growth arrest remains to be determined.
Estrogen stimulates the production and secretion of growth factors that can function in an autocrine fashion (49) . For example, protein levels of fibroblast growth factor-1 increase in response to E2 treatment in MCF-7 cells and may act synergistically with the hormone to generate a greater proliferative response (50 , 51) . Fibroblast growth factor-2 is also up-regulated in response to E2 stimulation of human endothelial cells and is required for the MAPK activation that is observed 3 h after exposure to estrogen (52) . Continuous incubation in the presence of MEK inhibitors would prevent the activation of MAPK by growth factors acting in an autocrine loop. Therefore, the cell cycle block observed with PD098059 and U0126 may be a result of inhibiting autocrine cascades necessary to realize the mitogenic potential of estrogen. Our finding that an antiproliferative response can still be achieved when adding the inhibitory drugs up to 6 h after hormone stimulation further substantiates this theory.
Our results strongly suggest that cyclin D1 induction is a key intermediate in estrogen mitogenesis. Several reports have focused on the role of cyclin D1 activation in estrogen-mediated mitogenesis. MCF-7 cells overexpressing cyclin D1 are unable to proliferate in the presence of antiestrogens (53) . Planas-Silva and Weinberg (41) demonstrated that estrogen relieves a cell cycle block in tamoxifen arrested MCF-7 cells by increasing levels of cyclin D1 protein, which causes the redistribution of p21cip1/waf1 away from the cyclin E/CDK2 complexes, allowing Rb phosphorylation and cell cycle progression. Prall et al. (42) derived MCF-7 cell lines with inducible expression of c-myc or cyclin D1. Cell cycle arrest of these lines by the estrogen antagonist ICI 182,780 was reversed by expression of either myc or cyclin D1. In both cases, loss of the CDK inhibitor p21 from cyclin E/CDK2 complexes was noted. Interestingly, c-myc overexpression did not result in elevated cyclin D1 levels, and when cyclin D1 was induced, myc levels remained constant, implicating the existence of multiple pathways to remove p21. Our data indicate that c-myc induction is not sufficient to allow estrogen-mediated cell cycle progression in the face of diminished cyclin D1 expression.
Functional activity of ectopically expressed ER is not sufficient for the expression of cyclin D1 and subsequent proliferation in response to estrogen (54) . However, Castoria et al. (21) reported recently that fibroblasts transiently transfected with a transcriptionally inactive ER (lacking the DNA binding domain) exhibited E2-dependent cell cycle progression, presumably attributable to the ability of the receptor to activate the MAPK cascade. This report suggests that proliferation in response to estrogen may be a result of nonnuclear events rather than ER-mediated transcription. In contrast, in a host of other studies, ectopic expression of active ER (measured by transcriptional activity) is not sufficient to convert cells into E2 responders (55, 56, 57, 58, 59, 60, 61) . Our data indicate that E2-induced proliferation can occur in the absence of detectable MAPK activation. Although other studies have shown E2-induced MAPK activity, the link between MAPK and mitogenesis after E2 stimulation has not been established. Specific culture conditions, experimental conditions, and divergence of cells in long-term culture may explain the discrepant results.
The question raised by our data is how the inhibitors of cytoplasmic signaling prevent S phase although the targets of these inhibitors are apparently not activated. Repression of cyclin D1 may be the key to understanding these observations. The molecular mechanism by which the PI3-K cascade functions in cyclin D1 accumulation has begun to be characterized as a posttranscriptional phenomenon. In the absence of mitogen, cyclin D1 is targeted for ubiquitin-dependent degradation because of the presence of a phosphate group at threonine 286, a substrate for GSK-3ß (62 , 63) . Inactivation of GSK-3ß occurs as a result of PKB activation, a downstream target of the PI3-K cascade (Ref. 44 and reviewed in Ref. 64 ). Thus, the cell cycle block induced by LY294002 in the presence of E2 may be attributable to the inability of GSK-3ß to be inactivated by PKB, thereby preventing the accumulation of cyclin D1.
The role of MAPK in cyclin D1 accumulation is less well defined. Typically, activation of MAPK results in nuclear translocation and activation of immediate-early gene expression. Several studies, however, do suggest a more direct effect. Using a Chinese hamster fibroblast cell line, CCL39, Lavoie et al. (65) demonstrated that not only was MAPK activation required for expression of endogenous cyclin D1, but also sufficient for full induction of this protein in the absence of growth factors. One theory to explain this phenomenon is that activated MAPK phosphorylates PHAS-1, which in turn impacts mRNA cap recognition and translation via eIF-4E (66) . Therefore, inhibition of these cascades may prevent cyclin D1 accumulation at the level of mRNA utilization.
We propose that whereas estrogen may directly impact some components of cytoplasmic signaling cascades, the primary mode of action is within the nucleus. The finding that MAPK and PI3-K inhibitors prevent estrogen-induced mitogenesis indicates that cytoplasmic events are still required in this process. This underscores the importance of determining the molecular events required for the accumulation of cyclin D1 protein and the role of estrogen in autocrine loops, which may stimulate signaling cascades.
Materials and Methods
Materials.
ICI 182,780 was purchased from Tocris. E2, EGF,
IGF-I, LY294002, wortmannin, bisindolylmaleimide I, and H-89
(dihydrochloride) were obtained from Calbiochem. PD098059 was obtained
from New England Biolabs. A monoclonal antibody against
diphosphorylated Erk1 and Erk2 was purchased from Sigma Chemical Co.;
polyclonal antibodies against Erk2 (clone K-23), ER (G-20), CDK2
(M-2), the monoclonal antibody recognizing cyclin D1 (HD11), and normal
goat IgG were obtained from Santa Cruz Biotechnology. Actin (C4)
monoclonal antibody and protein G-agarose beads were from Boehringer
Mannheim, and the monoclonal antibody against Rb protein (G3–245) was
purchased from PharMingen. Polyclonal antibodies against PKB and
Ser-473-phosphorylated PKB were obtained from New England Biolabs.
Donald P. McDonnell kindly provided the estrogen-responsive luciferase
constructs (3X-ERE and TK-ERE), the progesterone-responsive luciferase
construct (3X-PRE), and the human progesterone receptor expression
plasmid.
Cell Culture.
MCF-7 cells were obtained from American Type Culture Collection and
propagated in RPMI 1640 containing 10% (v/v) FBS (Life Technologies,
Inc.). ER content was measured using the ERICA kit (Abbot Labs, Inc.)
and quantitated using the CAS 200 Image Analyzer (Cell Analysis
Systems, Inc.). MCF-7 cells express 170 fmol/mg of protein as compared
with <10 fmol/mg of protein found in normal breast epithelial cells.
For experimental purposes, cells were seeded in six-well format at a density of 3 x 105 cells/well or in a 60-mm dish at a density of 5 x 105 cells. After 24 h, cells were washed one time with phenol red-free RPMI without FBS. Cells were then incubated for an additional 24 h in this medium to effect growth factor deprivation (67) .
Cell Cycle Analysis/Viability.
After 24 h of stimulation with 2 x
10-8 M E2 in
the presence or absence of inhibitory drugs, cells were trypsinized and
pelleted prior to washing once in PBS. Cell pellets were fixed in 70%
ethanol (in PBS) overnight on ice. Cells were washed with PBS and
resuspended in 500 µl of PBS supplemented with 100 µg of RNase A
and 50 µg of propidium iodide. DNA content was determined on a per
cell basis using flow cytometry. Cell viability was assessed on
trypsinized cells by staining with erythrosin B and determining the
percentage of viable cells (500 cells counted) by microscopic
examination in three separate experiments.
Northern Blotting.
Total RNA was extracted from 5 x 105 cells
using the Trizol reagent according to the manufacturer’s instructions
(Life Technologies). Five µg of RNA were electrophoresed on a 1%
agarose-2.2 M formaldehyde gel and then transferred onto a
nylon membrane (ICN). Blots were cross-linked using UV irradiation and
hybridized with a 32-P-labeled probe prepared by
random priming (cDNA plasmid clones were purchased from American Type
Culture Collection). Blots were washed for 10 min at 65°C with 2x
SSC (150 mM NaCl and 15 mM
Na3C6H3O7,
pH 7.0) containing 1% SDS and then with 0.1x SSC/0.1% SDS prior to
autoradiography at -80°C with intensifying screens. IMAGEQuant
(Molecular Dynamics) was used to quantify the expression levels of the
gene of interest, and loading inconsistencies were corrected using
detected levels of the housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase.
Transient Transfections/Luciferase Assay.
For liposome delivery, cells were washed with serum-free medium
(Opti-Mem I; Life Technologies). The firefly luciferase gene
(Photinus pyralis), under the control of estrogen- or
progesterone-responsive promoters (artificial or from the human
vitellogenin gene) as well as a cytomegalovirus-driven luciferase
construct (Renilla reniformis) were combined (2.5 µg/ml
and 5 ng/ml, respectively) prior to transfection, following the
standard protocol for Lipofectin (Life Technologies). (Because of low
endogenous expression levels of progesterone receptor in MCF-7 cells, a
human progesterone receptor B expression construct was also included in
the transfection mix for experiments using progesterone). Cells were
incubated with the liposome solution for 5 h, at which time the
medium was aspirated and replaced with RPMI 1640 without phenol red,
supplemented with 10% (v/v) charcoal-stripped FBS. Twenty-four h
later, cells were treated with 2 x 10-8
M estrogen in fresh medium, either in the
presence or absence of inhibitory drugs. Cells were lysed after 24 h of stimulation, and luciferase activity was determined using the
Dual-Luciferase Reporter Assay System (Promega) and a 20/20
dual-channel luminometer (Turner Designs).
Immunoblotting.
Cells were lysed in 2x SDS sample buffer [25 mM Tris-HCl
(pH 6.8), 12.5% glycerol, 6% SDS, 0.045% bromphenol blue, 4%
ß-mercaptoethanol, and 1 mM sodium orthovanadate].
Protein separation was achieved using 10% SDS-PAGE and
electrotransferred to nitrocellulose membranes (Schleicher & Schuell).
To prevent nonspecific interactions of the antibody, the blots were
blocked for 1 h at room temperature with PBS containing 0.01%
Tween 20 (PBS-T) and 5% nonfat dry milk. Membranes were incubated with
primary antibodies in PBS-T milk, at recommended dilutions, overnight
at 4°C. Antigen-antibody complexes were visualized by incubation with
horseradish peroxidase-conjugated goat antirabbit or antimouse IgG
(Jackson Laboratories) for 1 h at room temperature, followed by
ECL detection (DuPont NEN Life Science). Quantitation of protein levels
was performed as detailed above, with actin levels being used to
correct for variable loading.
CDK2 Kinase Assay.
After 24 h of estrogen stimulation in the presence or absence of
inhibitory compounds, cells were lysed in NP40 lysis buffer [50
mM Tris (pH 8), 5 mM EDTA, 150 mM
NaCl, and 0.5% NP40] on ice for 30 min. Insoluble proteins were
removed by centrifugation, and 500 µg of the remaining lysate were
precleared with 1 µg of normal goat IgG and 20 µl of protein
G-agarose beads. Precleared lysate was immunoprecipitated with an
anti-CDK2 antibody and immunocomplexes were washed twice with lysis
buffer and once with kinase buffer [50 mM Tris (pH 7.5),
10 mM MgCl2, and 1 mM
DTT]. Five µg of histone H1, 1 µM dATP, and 5 µCi
[
32P]dATP in 50 µl of kinase buffer were
added to the beads prior to a 30-min incubation at 30°C. The reaction
was terminated with the addition of 10x SDS sample buffer, and the
samples were separated using 15% SDS-PAGE. The gel was dried and
exposed to X-ray film.
Acknowledgments
We thank Donald P. McDonnell for the generous gift of the 3X-ERE, TK-ERE, 3X-PRE, and hPR-B constructs; Lenora Q. Blount for invaluable assistance in cell culturing; Carey J. Oliver for helpful discussions and comments; and Rita Nahta for critical review of the manuscript.
Footnotes
1 This work was supported by Predoctoral Training
Grant DAMD17-97-1-7224 (to E. K. L.) from the Department of Defense
and the Duke University Medical Center Specialized Programs of Research
Excellence in Breast Cancer Grants PHS CA68438 and PHS CA73802. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Duke University Medical Center,
Box 3873, Durham, NC 27710. Phone: (919) 681-6133; Fax:
(919) 681-6291; E-mail: marks003{at}mc.duke.edu
3 The abbreviations used are: ER, estrogen
receptor; E2, 17ß-estradiol; ERE, estrogen response
element; CDK, cyclin-dependent kinase; Rb, retinoblastoma; MAPK,
mitogen-activated protein kinase; Erk, extracellular signal-regulated
kinase; MEK, MAPK kinase; PKA, protein kinase A; PKB, protein kinase B;
PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase; EGF,
epidermal growth factor; IGF, insulin-like growth factor; GSK, glycogen
synthase kinase; FBS, fetal bovine serum.
Received for publication 6/17/99. Revision received 12/20/99. Accepted for publication 12/21/99.
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