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University of Colorado Health Sciences Center, Departments of Medicine and Pathology (G.I.O., C.A.L., K.B.H.), Department of Biochemistry, Biophysics and Genetics (I.E.S., R.A.S.), Department of Pharmacology (M.C.T., T.A.L.), The Molecular Biology Program (S.D.G., B.G., T.A.L., R.A.S., K.B.H.), Denver, Colorado 80262
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Opposing views that progesterone is a proliferative hormone in the breast are currently reflected in clinical practice (10, 11, 12). Progestins are added to estrogens for hormone replacement therapy at menopause because they block the proliferative and tumorigenic effects of unopposed estrogens in the uterus. However, women who have been hysterectomized are not given progestins, to spare their breasts from the presumed proliferative effects of these hormones (13, 14, 15). This is defended by the prevailing notion that progesterone is differentiative in the uterus but proliferative in the breast (1, 2).
It is now clear that control of proliferation and differentiation by many hormones and growth factors is linked by events that occur in G1 of the cell cycle (16, 17, 18, 19). Recent studies implicate up-regulation of the cyclin-dependent kinase (cdk) inhibitors, particularly p21 (p21Kin1,Waf1,Sdi1) (20), not only in inhibiting cell proliferation, but in promoting differentiation. In contrast, overexpression of cyclin D1 inhibits the differentiative program (21) and, in the breast, promotes cellular hyperplasia and tumor formation (9).
The molecular mechanisms underlying the proliferative and differentiative effects of progesterone at its target tissues have been difficult to assess for several reasons (2, 6). First, in most progesterone target cells the levels of PR are regulated by estradiol (22). Therefore, obligatory pretreatment with estradiol, itself a potent proliferative agent (23), confounds assessment of progesterone’s role on growth and other cellular processes. Second, as Musgrove et al. (24) have shown, short-term progestin treatments have dual effects on the cell cycle: they inhibit reentry of cells from mitosis into G1, but stimulate progression of cells through G1 (24). This complicates analysis of the role of progesterone in regulating proliferation acutely. The mechanisms underlying its sustained effects are unknown. Third, progesterone target tissues contain two isoforms of PR, the 120-kDa B receptors and the N-terminally truncated 94-kDa A receptors (25), that have unequal transcriptional activities (26, 27, 28). Additionally, the two isoforms are dissimilarly regulated and expressed during development, after hormone treatments, and in different target tissues and tumors (29, 30, 31, 32).
Our approach to reconciling these complexities, which limit studies of progesterone actions, has been to construct simpler model systems. To that end, we have used the T47DCO human breast cancer cell line, in which the two PR isoforms have escaped from estrogen controls and are constitutively expressed (33), to isolate the effects of progesterone and eliminate the confounding effects of estradiol. To define the role of each receptor isoform, we recently isolated and subcloned a PR-negative subline of T47DCO called T47D-Y and used it as the recipient into which either the B- or A isoform of PR was stably reintroduced to produce T47D-YB and T47D-YA cells (34).
In the present study, we used T47D-YB cells to analyze the effects of acute and sustained progesterone on the factors that regulate cell cycle progression and integrate growth-regulatory signals. This includes measurement of the protein levels of key cyclins (35, 36, 37, 38, 39); the levels and phosphorylation state of the retinoblastoma (Rb) tumor suppressor (40); the levels of cdk inhibitors (36, 41); and the kinase activity of cdk2 (17, 35, 37, 38, 39). Since T47D-YB cells are resistant to the proliferative effects of epidermal growth factor (EGF), we analyzed the role of cross-talk between the progesterone- and EGF-signaling pathways, on these proliferative events.
We find that after stimulating one round of cell division, a single pulse of progesterone arrests T47D-YB cells in late G1 of the second cycle, by sequentially raising the levels, first of p21 and then of p27Kip1. This is accompanied by an induction followed by inhibition of cdk2 activity. A second progesterone dose augments the growth arrest. However, during the arrested state, the cells become responsive to the proliferative effects of EGF, which can cause them to resume cycling. This occurs only after a single progesterone pulse; a second progesterone dose delays the p21 fall and blocks EGF responsiveness. We propose that progesterone is neither inherently proliferative nor antiproliferative, but that its effects on growth depend on whether treatment is transient or continuous; the former is stimulatory and the latter inhibitory. Additionally, cell cycle arrest in G1 may be accompanied by progesterone-induced cellular changes that can be permissive for growth-stimulatory effects by other factors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, panel A), A
receptor-containing YA cells (panels B and D), and B
receptor-containing YB cells (panels C and E), were grown in control
medium or in medium containing the following additions: the synthetic
progestin agonist R5020 alone; the antiprogestins RU486 or ZK98299; or
R5020 plus one of the antiprogestins. Sets of cells were harvested
every 4 h for 48 h, and the percent in S+G2/M for
each treatment group was determined by flow cytometry. Optimal
concentrations for each hormone were determined in preliminary studies.
As shown in Fig. 1, panel A, the percent of PR-negative Y cells in
S+G2/M during 50 h of treatment with 30 nM
R5020, 100 nM RU486, or 1 µM ZK98299 is no
different than untreated controls, supporting the idea that
proliferative responses to progestins and antiprogestins are dependent
on the presence of PR and ruling out any effects through glucocorticoid
receptors. These cells are also unresponsive to estradiol (not shown)
and EGF (see below). Compared with controls (set at 0), in either A
receptor (panels B and D) or B receptor (panels C and E) containing
cell lines, the percent of cells in S+G2/M starts to climb
approximately 12 h after R5020 is added to the medium, peaks at
20–24 h, and returns to basal levels approximately 36 h later, as
previously reported (24). During this transient increase in mitotic
activity, the percent of cells in S+G2/M rises from basal
levels of approximately 15–20% in the untreated controls, to 45–50%
in the R5020-treated sets. The two antiprogestins, RU486 or ZK98299,
when each is given alone, consistently have a transient
growth-suppressive effect that is more pronounced in the YA than in the
YB cells. However, in both cell lines, neither antagonist, when present
alone, has long-term growth-suppressive effects (data not shown). In
either cell line, the two antiprogestins completely block the
R5020-induced proliferative burst, suggesting again that growth effects
of progestins are PR-dependent. Since the two PR-containing cell lines
did not differ significantly, the remaining studies described here used
T47D-YB cells. Control of proliferation by antiprogestins in the
presence of cAMP does exhibit PR isoform specificity (K. Horwitz, in
preparation).
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was due to degradation of the
hormone during the 40 or more hours of cell culture. To determine
whether additional hormone would produce a sustained rise in
proliferation, T47D-YB cells were treated with R5020 at time 0, then
given a second pulse of R5020 at 48 h, and the percent of cells in
different phases of the cell cycle was measured for 96 h (Fig. 2A). Surprisingly, while the initial
proliferative rise at 20 h was clearly evident, the cells were
completely refractory to the second hormone challenge.
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), 10 h after the
start of R5020 treatment, B receptors are barely detectable. If the
cell culture medium is not changed, there is little recovery of
receptors for at least 50 h. However, as shown in Fig. 2B, after
progesterone treatment, B receptor down-regulation is transitory:
receptor levels are depressed 10 h after the hormone addition, but
they rapidly replenish to control levels because this natural ligand is
metabolized in T47DCO cells with a half-life of 2–4 h
(42). We therefore postulated that T47D-YB cells are refractory to a
second hormone challenge after initial treatment with R5020 (Fig. 2A)
because PR are still down-regulated at 40 h, and we reasoned that
if the cells were initially treated with progesterone, their progestin
sensitivity would be restored at 40 h. Figure 2A shows that this
is not the case. Like R5020, progesterone produces a transient increase
in cell proliferation. Nevertheless, 40 h after the initial
progesterone-induced growth, and despite adequate levels of receptors
(Fig. 2B), the cells are completely refractory to subsequent treatment
with R5020.
This result was surprising, and we wondered whether the replenished
receptors were somehow functionally incompetent (Fig. 3). To test this, we measured the ability
of replenished receptors to activate transcription of chloramphenicol
acetyl transferase (CAT) driven by a PR-responsive promoter. Parallel
sets of YB cells were pretreated with progesterone at zero time.
Twenty-four hours later a subset of cells was transfected with the
PRE2-TATAtk-CAT reporter, in which the proximal
promoter of the thymidine kinase gene is controlled by two upstream
progesterone response elements (PRE). The cells were glycerol shocked
at 46 h to complete the transfection and immediately treated with
a second dose of either progesterone or R5020 for an additional 24
(Fig. 3, inset) or 48 h (not shown) before they were
harvested, and CAT activity was measured in cell lysates. A control set
of transfected cells was left untreated at 40 h (Fig. 3, inset). Additionally, parallel sets of untransfected
cultures were treated with a second pulse of R5020 at 40 h, and
the cells were harvested periodically over the next 48 h for
analysis of cell cycle phases by flow cytometry (Fig. 3). As shown
above, after the initial progesterone-induced proliferative burst, the
cells enter a period of growth arrest, lasting at least 80 h, from
which they cannot be rescued by retreatment with R5020 at 40 h.
However as the inset (Fig. 3) shows, this inhibition is not
due to incompetent receptors, since they can induce CAT transcription
in the same endocrine setting. Thus, despite progesterone pretreatment
at time zero, at 40 h R5020 and progesterone strongly induce
transcription from the PRE-TATAtk-CAT reporter. The control
cells that received no second hormone dose confirm that 40 h after
the initial progesterone dose, insufficient hormone remains in either
the cells or the medium from the primary treatment to transactivate the
promoter, so that the high levels of CAT activity seen in the 40-h
R5020- and progesterone treated sets must be due to the second dose. We
conclude that the replenished receptors are fully functional: they are
capable of binding ligand, of binding DNA at cognate PREs, and of
interacting with the requisite factors on a promoter to activate
transcription (43).
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shows the changes in
protein levels over a 70-h period, of cyclins D1, D3, and E, of the
inhibitors p21 and p27Kip1, and of Rb. These proteins
regulate progression of cells through G1 (36, 39). The cells were
treated with progesterone at time zero, followed 40 h later by no
treatment or by R5020. The cells were harvested periodically, and
aliquots of cell lysates were assayed for the cell cycle proteins by
immunoblotting, and for the percent of cells in S+G2/M by
flow cytometry. In Fig. 4, the flow cytometric data are shown by the
dashed line, since they represent the same data points that
are shown in detail in Fig. 3. After primary treatment with
progesterone, the levels of cyclin D1 rise transiently and then fall as
previously reported (24), coincident with the increased proliferative
activity observed in the first 24 h. D1 expression increases
within 2 h of progesterone addition (not shown). A second abortive
peak is observed between 25–40 h, and levels then fall again after
40 h. This second peak is not always observed; it is markedly
attenuated if the basal cell proliferation rate is relatively low. Note
that the study shown in Fig. 4 involves cells that have a rapid basal
proliferation rate, characteristically seen in late passages. The
changes in D3 levels resemble those of D1, in which an initial rise is
followed by a persistent fall, and retreatment with R5020 at 40 h
produces an abortive rise. Cyclin E levels fall to low levels as the
cells transit the cell cycle in the first 24 h after progesterone,
then rise spontaneously to very high levels and remain high, in cells
that receive no further treatment. This lack of cyclin E degradation
suggests that the cells are blocked in late G1 and cannot enter S phase
(44). Retreatment of the cells with R5020 at 40 h lowers cyclin E
levels only minimally, and the flow cytometry data confirm that these
cells do not resume cycling.
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We find that T47DCO cells and their descendents do not
express p16 (p16INK4,MTS1) (45), but they express p21
(p21Cip1,Waf1,Sdi1) (20) and p27Kip1
(p28lck1) (46). A rise in protein levels of the inhibitors
begins as cyclin levels are declining. The levels of p21 increase
first, peak approximately 36 h after the initial progesterone
pulse, and then fall. This decline can be delayed approximately 18
h by retreatment of the cells with progesterone at 40 h. Thus
elevated p21 levels are transiently stabilized by the second
progesterone pulse. The levels of p27Kip1 remain relatively
unchanged for 36–40 h after the initial progesterone treatment and
then start to climb as the cells arrest. Its levels eventually fall
(not shown here, but see Fig. 7D) in the absence of a second hormone
pulse, as the cells spontaneously recover their ability to proliferate.
This recovery rate varies somewhat among experiments and appears to be
related to the fall in p27. However, a second hormone dose produces
even higher and sustained levels of both p21 and p27Kip1
resulting in failure to recover and persistent growth suppression (see
Fig. 6B).
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). In
the first 6–12 h, levels of Rb are high but the protein is
predominantly in its inactive hyperphosphorylated state. In this
period, lacking sufficient amounts of inhibitory Rb, the cells undergo
one round of cell division. However, coincident with the G1 arrest in
the second cycle, 18–24 h after progesterone treatment, significant
levels of the hypophosphorylated active repressor form of Rb are
present. Thereafter, total Rb protein levels down-regulate by more than
90%, consistent with G1 cell cycle arrest in many systems (47). This
decline occurs in the presence (not shown) or absence (Fig. 4B) of a
second hormone pulse at 40 h.
Because progesterone regulates the levels of the cdk inhibitors,
p21 and p27, in a complex manner, we examined the activity of the
cyclin-dependent protein kinase, cdk2, by its ability to phosphorylate
purified recombinant pRb (Fig. 4C). cdk2 is regulated both by p21 and
p27kip1 (20, 40, 41, 46). In untreated control cells at
early time points after cell plating, cdk2 activity is low and minimal
changes in cell proliferation are observed (Figs. 6B and 7B). cdk2
activity rises as untreated cells begin to cycle (32 h) and then falls
as cells reach confluence and become contact inhibited (72 h).
Interestingly, progesterone up-regulates cdk2 activity early (12 h),
then suppresses it at later time points (48–72 h) relative to
untreated controls. Thus, progesterone exerts biphasic effects on cdk
activity, consistent with its biphasic effects on cell
proliferation.
Indeed, the profound quality of the arrest in G1 is further confirmed
by analysis of cyclin A and B levels as shown in Fig. 5. These cyclins are produced in S- and
G2-phase, respectively, in preparation for mitosis (35, 37, 38). In
progesterone-treated T47D-YB cells, the levels of both cyclins reach a
peak during the initial proliferative burst, but then fall
precipitously to almost undetectable levels for at least 72 h
(Fig. 5, A and B), and they cannot be rescued by a second hormone pulse
at 41 h (Fig. 5C).
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), parallel sets of cells were
treated with progesterone at time zero, then exposed to R5020 starting
48 h later, or every 6 h thereafter. For each set of time
points, the ability of R5020 to induce proliferation was monitored for
the subsequent 36 h. Each hormone-treated set was compared with
control cells that had not received the second R5020 dose to monitor
spontaneous recovery. Figure 6 shows that resistance to R5020 persists
until approximately 72 h after the initial progesterone dose, at
which point R5020 produces a brisk proliferative response (Fig. 6A),
coincident with spontaneous recovery (not shown, but see Fig. 7C).
Thus, only when they are poised to resume growing spontaneously, do the
cells regain sensitivity to progestins. The extent and duration of this
second proliferative burst resemble that of the initial response seen
in naive, hormone-untreated cells (Figs. 1 and 2). The recovery time
varies somewhat among experiments, depending on the basal cell
proliferation rate at time zero; cells with a high basal rate, usually
late passage cells, recover more quickly. In general, T47D-YB breast
cancer cells remain in stasis for approximately 3 days after a brief
pulse of progesterone (recall the t1/2 is 2–4 h),
but then resume growing at the same rate as controls (Fig. 6B). On the
other hand, repeated exposure to progesterone or R5020 every 48 h
produces permanent growth arrest (Fig. 6B). This arrest is associated
with a 12- to 15-fold increase in the levels of p21 and a 6- to 7-fold
increase in the levels of p27Kip1 (Fig. 6C).
EGF Induces a Proliferative Response in Progesterone-Resistant
Cells
To determine whether, during the
progesterone-resistant state, T47D-YB cells are also resistant to other
mitogenic signals, we tested the effects of EGF. This mitogen is an
important growth factor in breast cancers, and clinically, an inverse
relationship exists between steroid receptor loss (with concomitant
hormone resistance) and the expression of EGF receptors (48, 49, 50). We
therefore tested the relationship, if any, between progesterone
resistance and EGF growth sensitivity (Fig. 7, A-E). Control T47D-YB cells express
low levels of EGF receptors, which are functionally competent in their
ability to signal to downstream cytoplasmic effectors, since a 10
nM pulse of EGF strongly induces mitogen-activated protein
(MAP) kinase activity (not shown). However, the naive cells are
resistant to the growth-stimulatory effects of EGF (Fig. 7A). In this
study, cells that had received no prior treatment were incubated with
10 nM recombinant human EGF or left untreated, and the
percent of cells in S+G2/M was measured every 6 h for
30 h. As shown, the proliferation of cells treated acutely with
EGF did not differ significantly from controls.
To determine whether chronic EGF treatment affects growth, cells
received EGF continuously for 6 days in the presence or absence of
continuous R5020, and their proliferation rate was compared with that
of untreated or R5020-treated cells (Fig. 7B). As shown, EGF alone does
not accelerate growth above the control rate, and it cannot relieve the
growth suppression produced by continuous R5020.
Surprisingly therefore, T47D-YB cells can be sensitized to the
proliferative effects of EGF by a brief progesterone pulse (Fig. 7C).
In this study, sets of T47D-YB cells that had been pretreated with
progesterone at time 0 were challenged with EGF at various time points,
starting at 36 h. Control sets received no second progestin
treatment to monitor spontaneous recovery from the progesterone-induced
arrest. While EGF given 36 h after progesterone was ineffective,
the cells acquire sensitivity to EGF starting approximately 46 h
after progesterone pretreatment and exhibit an extensive proliferative
burst after a 6-h lag. Cells treated with EGF 52 h after
progesterone respond even faster. Recall that at these time points the
cells are insensitive to progesterone (Fig. 6) and have not recovered
spontaneously (Fig. 7C, dashed line). These and other
studies (not shown) suggest that there is a critical period during
progesterone-induced growth arrest, in which T47D-YB cells acquire
sensitivity to the proliferative effects of EGF, which accelerates
their reentry into the cell cycle.
What accounts for this brief sensitivity to EGF? Figure 7D shows that
after the progesterone pulse, p21 levels rise, peak at approximately
30 h, and then fall by 36 h, at a time preceding the rise in
p27Kip1 levels. Figure 7E shows a similar pattern from
another experiment, in which it is evident that approximately 48 h
after a single progesterone pulse, there is a brief period of time
characterized by relatively low levels of both inhibitors, during which
EGF can influence cell proliferation. This pattern occurs only after
the initial signal produced by the short half-life of one progesterone
pulse. More prolonged progestin exposure, produced either by repeated
treatment with progesterone or by continuous treatment with a poorly
metabolizable synthetic progestin such as R5020, prevents or slows the
fall in p21 levels (Fig. 4) so that this event overlaps with, rather
than precedes, the rise in p27Kip1. We conclude that EGF
cannot overcome the continuous inhibitor levels produced by a sustained
progestin signal, as shown in Fig. 7B.
However, the brief window, characterized by low inhibitor levels after
one pulse of progesterone, differs from the hormone-untreated condition
in one respect. Namely, that EGF receptors are strongly up-regulated by
the progesterone pulse (Fig. 8). These
data, derived from immunoblots, show that progesterone treatment
increases the number of EGF receptors by 3- to 5-fold between 24 and
48 h. Similar effects of progestins have been previously described
(51, 52).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Continuous Progesterone Is Autoinhibitory
The immediate response of proliferating breast cancer cells to
progesterone is an acceleration of the cell cycle driven by increased
levels of cyclins D1 (8), D3, and E and by accumulation of inactive
forms of Rb in the face of persistent low steady state levels of p21
and p27Kip1. Under these conditions cyclin D-cdk4 complexes
accumulate above the inhibitory threshold of p27Kip1 (53),
allowing the cells to progress past the G1 restriction point and enter
mitosis. Because the breast tumor cells used in these studies are
proliferating rapidly initially (at a "low" proliferation rate,
15–25% of cells are in S+G2/M at any time), and
progesterone more than doubles this rate (leading to more than 50% in
S+G2/M), the cells proceed through mitosis and reenter G1
of the second cycle in a partially synchronized state. At this point
however, the cells are unable to adequately replenish the depleted
stores of cyclin Ds, Rb protein levels are extensively down-regulated,
cyclin E levels rise sharply, followed first by rising p21, and later
by rising p27Kip1 levels, while cdk2 activity declines.
Cyclin A, required for progression through S phase and cyclin B, the
primary mitotic cyclin, are completely down-regulated. Taken together
these data indicate that the cells are arrested late in G1 (44).
One surprising finding is that additional progesterone cannot override the growth suppression produced by the first progesterone dose, despite the presence of adequate levels of transcriptionally competent PR. Thus, the proliferative block is not at the level of the PR-signaling system. In fact, we propose that the growth arrest may actually require the presence of functional PR, since it appears to be due to sustained up-regulation of p21 and p27Kip1 produced by a positive feedback loop initiated by at least two progestin treatments, given before the time at which the cells would spontaneously recover. Thus, we believe that sustained progesterone is autoinhibitory, in contrast to transient progesterone, which is stimulatory. This model has important implications for the scheduling of progestin treatments in clinical settings, since it predicts that the effects of continuously administered progestins (54) differ significantly from those of episodically or cyclically administered progestins (13, 14); the former would be growth inhibitory and the latter stimulatory. These data also suggest that the cyclical progesterone of the menstrual cycle can have different physiological consequences than the continuous progesterone of pregnancy. A model in which the rate and duration of progesterone treatment control the type of response observed would reconcile contradictory views that this hormone is either proliferative or differentiative. A similar model, in which a proliferative vs. differentiative end point is controlled by the duration of MAP kinase signaling was recently described (55).
The mechanism of progesterone-mediated induction of p21 and p27 is unknown. However, aside from its p53-dependent regulation in response to DNA damage, p53-independent transcriptional activation of p21 has recently been shown to be regulated by the MAP kinase pathway after stimulation of cells with growth factors (56). We are currently investigating the mechanism of p21 and p27 regulation by progesterone. Interestingly, in addition to its action as a cyclin-dependent protein kinase inhibitor and cell-cycle inhibitor, p21, at low concentrations, promotes the assembly of active cyclin/cdk/proliferating cell nuclear antigen complexes and exerts a positive influence on cell growth; kinase activity increases 3-fold upon the addition of low concentrations of p21 to lysates containing cyclin A and cdk2 (57). Thus, quaternary complexes containing one p21 molecule are fully active, while inhibition of cyclin kinases requires association of more than one p21 subunit. This may explain the biphasic effects of progesterone on breast cancer cell growth. In this model, the initial proliferative burst is supported by the assembly and activation of cdk/cyclin/proliferating cell nuclear antigen/p21 complexes. However, as p21 and p27 expression increases, these quaternary complexes become inactive due to the addition of multiple inhibitor subunits, and the cells are growth inhibited. A second exposure to progesterone produces sustained elevation of p21 and prolongs growth inhibition. Consistent with this hypothesis, Matsushine et al. (58) showed that cyclin D and cdk4 do not associate in serum-starved cells, but undergo association and activation upon serum stimulation, a condition that increases p21 levels in a p53-independent manner. Similarly, LaBaer et al. (59) found that the addition of low concentrations of p21 and p27 lead to a 35- and 80-fold increase in the assembly and activity of cyclin D-cdk4 complexes, respectively, while high concentrations inhibited activity, suggesting new roles for these inhibitors as adaptor proteins that assemble and program kinase complexes (59).
Progesterone Enhances Sensitivity to EGF and Up-Regulates EGF
Receptors
Failure of progestins to reinitiate proliferation after a single
progesterone pulse is not due to insensitivity of the cell-cycling
machinery, since an alternate mitogenic signal emanating from the cell
surface can transiently reactivate proliferation. The mechanisms by
which one pulse of progesterone sensitizes the cells to the
proliferative effects of EGF appears to be related to its ability to
up-regulate the levels of EGF receptors, as previously described (51, 52). Untreated T47D-YB cells do not respond to proliferative signals by
EGF (Fig. 7A), although the cells express low levels of immunoreactive
EGF receptors (Fig. 8), and they respond to EGF by activating the
downstream effectors, p42 and p44 MAP kinases (not shown). Thus, the
EGF receptor-mediated signal transduction pathway leading to activation
of cytoplasmic kinase cascades known to regulate cellular processes
including growth (60), is functionally intact in these cells. At high
EGF receptor levels, however, EGF appears to be able to induce the
arrested cells to reenter the cell cycle, as long as the levels of the
cdk inhibitors are relatively low. It is possible that at high levels,
EGF receptors can engage novel signaling proteins (61) or be capable of
associating with novel cell-surface partners (62) that are unavailable
at low receptor concentrations. Similar, phenotypically different
response, dependent on the number of active cell surface receptors,
have been described (63, 64, 65).
On the other hand, our data suggest that the high levels of EGF
receptors cannot overcome the inhibitory effects of high levels of p21
or p27Kip1 produced by a second dose of progesterone. This
may explain EGF-dependent proliferation after a single pulse of
progesterone but resistance after a second pulse or during sustained
progesterone treatment. We have shown that one progesterone pulse leads
to a transient rise in p21 levels and that p27Kip1 levels
start to rise as p21 levels are falling. Therefore we postulate that
after a single progesterone pulse, there is a transient window of EGF
responsiveness generated by increased EGF receptor levels and falling
p21 levels, before the rise in p27Kip1 levels. A second
dose of progesterone delays the fall in p21 and increases the levels of
p27Kip1 (Fig. 4), eliminating the window to EGF
responsiveness. This suggests that in breast cancer cells,
proliferative sensitivity to EGF is dependent on the cell cycle state
and progestational history of the cells.
There is considerable evidence linking the EGF and progesterone
signaling pathways in breast cancer. This includes attenuation of
progestin responsiveness and decreases in PR levels in cells treated
with EGF (66); augmentation of the proliferative, differentiative, and
transcriptional effects of progestins by cotreatment with EGF (67, 68, 69);
and progestin-specific regulation of EGF and EGF receptor levels (Fig. 8 and Refs. 49, 51, and 66). There are also provocative clinical data
linking enhanced expression of EGF receptors to acquisition of steroid
hormone resistance in breast cancer (48, 50).
Is Progesterone Proliferative or Differentiative?
Proliferation and differentiation are complex processes governed
by the concerted activity of multiple regulatory factors. Both
processes appear to have the common requirement that cells stop in G1
to await appropriate directional signals (16, 18, 19). Our
demonstration that progesterone can advance cells to this checkpoint,
while it sensitizes the cells to the actions of a growth factor,
provides a model that may reconcile opposing views that progesterone is
either a proliferative or a differentiative hormone. We suggest that
progesterone is neither, but that it is a competency factor necessary
to drive cells into either pathway, and that breast cell proliferation
and differentiation are intricately connected. In this model,
progesterone accelerates cells to the G1 checkpoint in the second
cycle, whereupon other, possibly tissue-specific, factors determine the
fate of the cell. Therefore the final state of progesterone target
tissues is determined by cross-talk between progesterone and growth or
differentiative factors that remain to be defined. However, the
progesterone treatment regimen may be a key factor in the ultimate
response produced, with transitory progesterone being permissive of
such cross-talk while sustained progesterone is inhibitory.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies were obtained from the following sources: anti-cyclin A, -cyclin B1, and -cyclin D1 were from Upstate Biotechnology (Lake Placid, NY); anti-cyclin D3 and E were from Pharmingen (San Diego, CA); anti-p21 and anti-p27Kip1 were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-pRb was a gift from Wen-Hwa Lee (University of Texas Health Science Center, San Antonio, TX); anti-EGF receptor 20.3.6 was a gift from Roger Davis (University of Massachusetts Medical School, Worcester, MA); anti-PR AB52 and B30 were produced in our laboratories (49); and horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad Laboratories (Hercules, CA).
Flow Cytometry
Cells (2 x 105) were plated into duplicate
wells of six-well plastic dishes with 3 ml of serum-containing medium.
After 24 h, progesterone, or the synthetic agonist R5020
(Roussel-Uclaf, Romainville, France) or the antiprogestins RU486
(Roussel-Uclaf) or ZK98299 (Schering AG, Berlin, Germany) were added in
ethanol, at final concentrations of 30 nM, 100
nM, or 1 µM, respectively. Control medium
contained only ethanol. Some cells received 60 ng/ml (10
nM) human recombinant EGF (Upstate Biotechnology,
Inc.).
Cells were harvested at the start of treatment (control, zero time) and every 4 or 6 h after hormone addition, into 1 ml of Hank’s EDTA and vigorously pipetted. The cell suspension was pelleted, resuspended into 1 ml of Krishan’s stain (70) containing propidium iodide and ribonuclease (RNase), and again vigorously pipetted. Samples were cooled to 4 C, and 10,000 cells were analyzed on an Epics 752 flow cytometer (Coulter Electronics, Hialeah, FL), using an incident beam from an argon laser at 488 nm, 500 mW. The cells were gated on forward angle vs. 90° light scatter to eliminate cellular debris and doublets. Red fluorescence, corresponding to DNA, was collected through a 590-nm longpass filter, and histograms of DNA content vs. cell number were constructed. Cell cycle analyses of the DNA histograms were performed using the ModFit Analysis program (Veritey Software House, Topsham, ME), which provides fits for the GO/G1, S and G2/M fractions of the population. The S- and G2/M-phase fractions were combined into a single growth fraction. In some figures, the percent of cells in S+G2/M in the hormone-treated sets were compared with the percent of cells in S+G2/M in the untreated controls, whose levels were set at 0. For long-term growth studies, cells were harvested into 1 ml of Hank’s EDTA and pipetted vigorously to obtain single-cell suspensions, and aliquots were counted using a hemocytometer.
Immunoblotting
For measurements of PR, whole cell extracts were prepared in 0.4
M KCl as previously described (27, 34). Receptors were
resolved by electrophoresis on an 11% polyacrylamide gel containing
SDS and then transferred to nitrocellulose. After incubation with
anti-PR monoclonal antibodies AB-52 and B-30 (71), the receptor bands
were detected by enhanced chemiluminescence (Amersham, Arlington
Heights, IL). For cell cycle proteins, cells were harvested at 50–80%
confluence and washed in PBS. Aliquots were removed for analysis by
flow cytometry to simultaneously determine cell cycle distribution. The
remaining cells were resuspended in Laemmli sample buffer (72) at
1–4 x 107 cells per ml, immediately boiled for 5
min, sheared through a syringe needle to reduce viscosity, aliquoted,
and stored at -80 C. Volumes of cell extracts normalized to
approximately 50 µg total protein, as measured by Ponceau S, were
subjected to gel electrophoresis. For measurement of EGF receptors,
cells were resuspended in RIPA buffer (0.1 M NaCl, 6
mM Na2HPO4, 4 mM
NaH2PO4, 1% deoxycholic acid, 1% NP-40, and
0.1% SDS) (61) for 10 min at 4 C, and centrifuged at 10,000 rpm to
produce a membrane-containing pellet. The pellet was reextracted with
RIPA and repelleted, and membranes (60 µg) were resuspended in four
volumes of water, sheared through a 28-gauge needle, boiled in Laemmli
sample buffer, and resolved by electrophoresis on an 8% polyacrylamide
gel. Proteins were transferred for 45 min at 0.5 A to Immobilon P
membranes (Millipore, Bedford, MA) using a Genie Electroblotter (Idea
Scientific, Minneapolis, MN). After incubation with the appropriate
antibodies, protein bands were detected by enhanced chemiluminescence
(Amersham). Film exposures ranged from 2 sec to 1 h depending on
the primary antibody. Bands were quantitated using a digital scanner
and the Image program (NIH) and, where appropriate, normalized to
levels of pSTAIRE sequence-containing cdks.
Transfection and Transcription
T47D-YB cells were plated and grown in 100-mm2 cell
culture plates in MEM supplemented with 5% FCS. Progesterone (30
nM) was added 40 h prior to completion of
transfection. Transfection of plasmid DNA into cells was performed
24 h after the start of progesterone treatment by calcium
phosphate coprecipitation using 1 µg of the
PRE2-TATAtk--CAT reporter (27), 3 µg of the
ß-galactosidase expression plasmid PCH110 (Pharmacia, Piscataway, NJ)
to correct for transfection efficiency, and 15 or 16 µg Bluescribe
carrier plasmid (Stratagene, La Jolla, CA) for a total of 20 µg DNA,
as previously described (27). Sixteen hours later transfection was
completed when the medium was aspirated, and the cells were shocked at
room temperature for 4 min with 5 ml HBSS containing 20% glycerol.
After the cells were washed twice with 10 ml serum-free MEM to remove
the glycerol, 10 ml MEM containing 5% FCS were added per dish, either
without or with 30 nM progesterone or R5020. Cells were
harvested after an additional 24 or 48 h. Cells in duplicate
plates were lysed by freeze-thawing in 200 µl of 0.25 M
Tris, pH 7.8. Lysates (50 µl) were assayed for ß-galactosidase
activity, and normalized aliquots were assayed for CAT activity by TLC
as described (27).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by NIH Grants CA-26869 and DK-48238, by Grant DAMD17–94-5–4026 from the U.S. Army Medical Research and Development Command, and by the National Foundation for Cancer Research (to K.B.H.); CA-58187 (to R.A.S.); U.S. Army AIBF1563 (to T.A.L.); Colorado Cancer League (to C.A.L.); and by the Flow Cytometry and Tissue Culture Core Laboratories of the University of Colorado Cancer Center. S.D.G. was supported by a graduate student stipend from the Lucille P. Markey Charitable Trust.
Received for publication February 17, 1997. Revision received June 24, 1997. Accepted for publication July 15, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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J. M. Kokontis, N. Hay, and S. Liao Progression of LNCaP Prostate Tumor Cells during Androgen Deprivation: Hormone-Independent Growth, Repression of Proliferation by Androgen, and Role for p27Kip1 in Androgen-Induced Cell Cycle Arrest Mol. Endocrinol., July 1, 1998; 12(7): 941 - 953. [Abstract] [Full Text]
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G. I. Owen, J. K. Richer, L. Tung, G. Takimoto, and K. B. Horwitz Progesterone Regulates Transcription of the p21WAF1 Cyclindependent Kinase Inhibitor Gene through Sp1 and CBP/p300 J. Biol. Chem., April 24, 1998; 273(17): 10696 - 10701. [Abstract] [Full Text] [PDF]
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)
or the antiprogestins RU486 (•) or ZK98299 (
) for 48 h. Cells
were harvested every 6 h, and the percent of cells in
S+G2/M was measured by flow cytometry. B, T47D-Y cells
stably expressing A receptors (YA) were treated with R5020 (
), or left untreated (
