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Int. J. Cancer: 70, 291–296 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Nicholas R.C. WILCKEN1, Elizabeth A. MUSGROVE1 and Robert L. SUTHERLAND1*
1Cancer Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Darlinghurst, Sydney, Australia
Both retinoids and anti-estrogens inhibit breast cancer cell
proliferation with accumulation of cells in the G1 phase of the
cell cycle, but the effect of retinoids is delayed compared to
that of anti-estrogens. To determine whether this temporal
difference is due to a simple delay in the action of retinoids on
a common site or to different sites of action within the G1
phase, we studied the cell cycle effects of retinoic acid (RA)
and the anti-estrogen ICI 164384 (ICI) in T-47D cells partially
synchronized by mevalonic acid rescue of lovastatin-induced
cell cycle arrest. We found that cells entering the cell cycle
semi-synchronously after mevalonic acid rescue of lovastatin
treatment were immediately susceptible to ICI but not RA.
This suggests that RA may act at a point up-stream and ICI at
a point down-stream of lovastatin action. Consistent with
this, cells recommencing cell cycle progression after RA
treatment were susceptible to the effects of lovastatin, while
cells pre-treated with ICI then rescued with estradiol were
not. In addition, cells rescued from cell cycle arrest induced
by either RA, ICI or lovastatin entered S phase with the same
kinetics. Our findings suggest, first, that within G1, RA acts
before and ICI acts after the point of lovastatin action and,
second, that despite these differences in the initiation of cell
cycle arrest, the final nature of the cell cycle arrest is similar.
Hence, retinoids and anti-estrogens may be expected to
target different cell cycle–regulatory molecules to initiate cell
cycle arrest, while overcoming this arrest may be accomplished by the activation of a common molecular pathway.
Int. J. Cancer, 70:291–296, 1997.
r 1997 Wiley-Liss, Inc.
Tamoxifen has been in clinical use for the treatment of breast
cancer for 2 decades and is currently being evaluated as a
preventive agent. Retinoids have also shown promise as potential
therapeutic and preventive agents for breast and other cancers,
though strong evidence from animal studies has yet to be translated
into success in clinical trials in breast cancer. While the cell cycle
effects of anti-estrogens (both steroidal and non-steroidal) on ER1
breast cancer cells are well defined, less is known about retinoid
action in these cells, yet potential differences and similarities
between the 2 agents may be relevant to both their molecular
mechanisms of action and their clinical use.
The potential importance of preventive agents has increased
recently with the identification of genes (BRCA1 and BRCA2)
which, when mutated, cause inherited breast cancer. With the
almost inevitable introduction of genetic screening for selected
groups will come the identification of women at very high risk of
breast cancer, for whom at present very little can be done. In the
experimental setting, the combination of retinoids and antiestrogens is more effective than either agent alone in preventing
mammary tumors (McCormick and Moon, 1986; Ratko et al.,
1989; Anzano et al., 1994), suggesting an attractive strategy for
breast cancer prevention in women at high risk. Delineating
separate mechanisms of action for retinoids and anti-estrogens may
lead to the ability to predict differential response rates, thus refining
prevention strategies. As well, if common steps are required to
overcome the cell cycle effects of retinoids and anti-estrogens, the
molecular characterization of such steps may point to mechanisms
of resistance to these treatments.
Anti-estrogens were first shown to inhibit cell cycle progression
in the G1 phase, and subsequently a series of experiments using
mitotically selected cells showed that breast cancer cells were
susceptible to anti-estrogens from early to mid-G1 (Sutherland et
al., 1983; Taylor et al., 1983). Molecular studies have consequently
focused on G1 events and the way in which anti-estrogens interact
with the proteins that control cell cycle progression in G1 (Watts et
al., 1995).
While the growth-inhibitory effects of retinoids in cell culture
are well documented in a variety of cell types (e.g., Lotan, 1979), as
are chemopreventive effects in rodent models of mammary cancer
(e.g., Moon et al., 1992), there is less information and less
consensus about the precise cell cycle effects seen in culture. This
is partly due to the known cell type-specific actions of retinoids,
making it difficult to extrapolate from one study to another.
Nevertheless, in the majority of studies involving breast cancer
cells, DNA synthesis is reduced, and, as with anti-estrogen
treatment, cells accumulate in G1 phase (Ueda et al., 1980; Marth et
al., 1985; Rubin et al., 1994; Zhao et al., 1995). For both
anti-estrogens and retinoids, inhibition of cell cycle progression is
closely correlated with reduced cell proliferation, but the effects of
retinoids and anti-estrogens on G1 progression have different
kinetics (Wilcken et al., 1996).
We therefore sought to define more closely the relationship
between retinoids and anti-estrogens with respect to their cell cycle
effects by extending our previously published data on asynchronously proliferating T-47D breast cancer cells to populations of
synchronous cells. Cell synchronization was achieved with the
mevalonic acid rescue of lovastatin-treated cells (described in
Keyomarsi et al., 1991). Lovastatin and related drugs inhibit
HMG-CoA reductase, a rate-limiting enzyme in the cholesterol
synthesis pathway, an action which in cell culture results in cell
cycle arrest early in the G1 phase (Jakobisiak et al., 1991). This can
then be reversed by addition to the culture medium of mevalonic
acid, the immediate product of HMG-CoA reductase, and as a
result cells recommence cell cycle progression in a synchronous
Our data using this cell culture system demonstrate that the cell
cycle effects of retinoic acid (RA) and the pure steroidal antiestrogen ICI 164384 are different, despite the fact that both agents
cause cells to accumulate in G1 phase and that differences in the
molecular pathways by which they control cell proliferation,
therefore, are to be expected.
T-47D cells were supplied by E.G. and G. Mason Research
Institute (Worcester, MA) for the National Cancer Institute Breast
Cancer Program Cell Culture Bank, and MCF-7 cells were from the
Michigan Cancer Foundation (Detroit, MI). RPMI 1640 medium
containing HEPES (N-2-hydroxyethylpiperazine-N8-ethanesulfonic acid) (20 mM), sodium bicarbonate (14 mM), L-glutamine (6
mM) and 20 µg/ml gentamicin was used throughout. Stock cultures
were maintained in medium supplemented with 10 µg/ml human
Contract grant sponsor: The National Health and Medical Research
Council of Australia.
*Correspondence to: Garvan Institute of Medical Research, St. Vincent’s
Hospital, Darlinghurst, NSW 2010, Australia. Fax: 61 (2) 9295 8321.
Received 16 July 1996; revised 1 October 1996.
insulin (Actrapid; CSL-Novo, North Rocks, Australia) and 10%
FCS, and cell growth experiments were carried out in medium
supplemented with 5% FCS. To maintain maximal growth rates
and therefore observe maximal growth inhibition, serum was not
charcoal-stripped. RA was purchased from Sigma (St. Louis, MO)
and Ro 13-7410 (P-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8,8,-tetramethyl2-naphthalenyl)]-1- propenyl-benzoate) was obtained from Dr. M.
Klaus (F. Hoffman-La Roche, Basel, Switzerland). ICI 164384
(N-n-butyl-N-methyl-[3,17b-dihydroxyestra-1,3,5 (10)-triene-7ayl] undecanamide) was obtained from Dr. A. Wakeling (Zeneca,
Macclesfield, UK). The anti-estrogen and retinoids were stored as
1,000-fold concentrated stock solutions at 270°C in either absolute
ethanol (ICI 164384 and RA) or DMSO (Ro 13-7410), and the
retinoids were sealed with argon and protected from light. Retinoid
stock solutions were discarded after 1 week. Lovastatin was
purchased from Merck Sharp and Dohme (Rahway, NJ) and
converted from its lactone pro-drug form to the open acid form by
dissolving of the compound in 1 M NaOH, neutralizing with HCl to
a pH of 7.2 and making up to a volume with distilled water to
produce a 10 mM stock solution that was stored at 220°C.
Mevalonic acid lactone was purchased from Sigma.
Cell growth experiments
For growth-inhibition assays, 105 cells were plated into 25 cm2
flasks. One to 2 days later, 5 µl aliquots of drug (ICI 164384, RA or
Ro 13-7410) were added directly to the culture medium. Cells were
then harvested and counted under phase contrast microscopy, and
DNA flow cytometry was performed as previously described
(Musgrove et al., 1989). Briefly, trypsinized cells were resuspended in medium at a concentration of 2 to 3 3 105 cells/ml.
Ethidium bromide (Sigma) and mithramycin (Pfizer, West Ryde,
Australia) were added at final concentrations of 40 µg/ml and 12.5
µg/ml, respectively, in the presence of 0.2% Triton X-100 (Sigma)
and, for mithramycin, at a final concentration of 7.5 mM MgCl2.
Stained samples were stored either at 4°C overnight or at room
temperature for 4 hr. DNA flow cytometry was performed on a
FACStar laser-based flow cytometer (Becton Dickinson, Mountain
View, CA), and data were acquired and analyzed using the
accompanying software (Becton Dickinson). DNA histograms
contained 30,000 events and had a coefficient of variation of 2–4%.
retinoids and anti-estrogens invariably leads to a delay in the
stereotypical cell cycle action of the retinoids compared with the
anti-estrogens (Wilcken et al., 1996). Figure 1 shows the mean
effect of equipotent concentrations of RA and the pure steroidal
anti-estrogen ICI 164384 on S phase entry in T-47D cells and
demonstrates that the same effect is also seen in MCF-7 breast
cancer cells. Since MCF-7 cells are both more sensitive to
anti-estrogens and less sensitive to retinoids than T-47D cells,
however, lower concentrations of anti-estrogen and higher concentrations of retinoid are required to achieve equipotency. As shown
in Figure 1, MCF-7 cells, therefore, have been treated with the
retinoid analogue Ro 13-7410 to match the added sensitivity of
MCF-7 cells to anti-estrogens. In both cell lines, the retinoids tested
had little effect on the percent of cells in S phase (%S phase) after
16 hr of treatment, while the anti-estrogen had a near maximal
effect at this time, yet by 40 hr the effects on %S phase were
broadly similar for both classes of agent. This delay in retinoid cell
cycle action is not seen in genomic actions of retinoids, such as the
transcriptional regulation of androgen and progesterone receptor
gene expression in T-47D cells (Clarke et al., 1991; Hall et al.,
1992; Wilcken et al., 1996), implying that access to receptors and
DNA is not a limiting factor in retinoid action.
Thus, in both breast cancer cell lines there is a difference
between retinoid and anti-estrogen effects which represents either
different mechanistic pathways or a simple delay in the onset of
retinoid action on the cell cycle. Subsequent experiments were
confined to T-47D cells and aimed at distinguishing between these
2 possibilities by comparing the effects of RA and ICI 164384 in
synchronized cells.
Inducing cell synchrony with lovastatin
Initial experiments were carried out to determine the concentration and duration of lovastatin treatment that gave the greatest
extent of G1 arrest and subsequent synchronous progression
through the cell cycle after mevalonic acid rescue in T-47D cells
(data not shown). The optimized procedure was as follows:
Cell synchronization
Cells (105) were plated into 25 cm2 flasks, and after 1–2 days
aliquots of 10 mM lovastatin were added to make a final concentration of 40 µM. Twenty-four hours later, medium was removed
and fresh medium added together with 2 M mevalonic acid to yield
a final concentration of 5 mM. In some experiments, RA, ICI
164384 or vehicle (0.1% ethanol) were also added at this time.
Reversal experiments
Two types of experiment were carried out. First, 105 cells were
plated into 25 cm2 flasks; after 1 day, RA was added for 48 hr, or
ICI 164384 or lovastatin for 24 hr, at which time the medium was
replaced in all flasks. Estradiol was then added to the cells
previously treated with RA or ICI 164384 and mevalonic acid to
the cells previously treated with lovastatin, and cells were then
harvested at intervals for DNA flow cytometry. Second, cells were
treated with either RA for 48 hr or ICI 164384 for 24 hr, after which
medium was replaced and estradiol added to all flasks. Then, either
lovastatin or H2O was added, and cells were harvested at intervals
for DNA flow cytometry.
Differential effects of retinoids and anti-estrogens
on asynchronous cells
We have previously shown that, while the final effect on cell
cycle phase distribution is the same for several retinoids and
anti-estrogens in T-47D cells (an accumulation in G1 phase and a
decline in S phase), the time course of these effects is different.
Thus, treatment of T-47D cells with a series of structurally different
FIGURE 1 – Effects of retinoids and anti-estrogens on %S phase in
asynchronous T-47D and MCF-7 cells. T-47D or MCF-7 cells proliferating in medium supplemented with 5% FCS were treated with
retinoid, anti-estrogen or vehicle (0.1% ethanol) and harvested after 16
and 40 hr for determination of cell cycle phase distribution by flow
cytometry. Data are presented relative to the %S phase of vehicletreated cells. T-47D cells were treated with RA (1026 M, filled
histograms: mean 6 SEM) or the anti-estrogen ICI 164384 (1027 M,
striped histograms: mean 6 range). MCF-7 cells were treated with the
retinoid Ro 13-7410 (1025 M, filled histograms: mean 6 range) or the
anti-estrogen ICI 164384 (3 3 1029 M, striped histograms:
mean 6 range).
exponentially growing cells were incubated with 40 µM lovastatin
for 24 hr, at which time the medium was replaced and 5 mM
mevalonic acid added. Cells were then harvested at intervals over
the subsequent 56 hr and cell cycle phase distribution measured.
Figure 2 shows that cell synchrony was achieved, with a synchronous cohort of cells entering S phase 8 hr after treatment with
mevalonic acid. A maximal 4-fold increase in %S phase to
approximately 40% was observed between 20 and 24 hr. This initial
increase in %S phase was followed 8–10 hr later by an increase in
the number of cells in G2 1 M phase of a similar proportional
magnitude to the increase in %S phase (i.e., about 4-fold). In
addition, there was a second, smaller peak of cells in S phase at
about 40 hr, indicating that the next generation of cells had entered
a second cell cycle in partial synchrony. Therefore, the results are
consistent with studies in other cell types (Keyomarsi et al., 1991):
a synchronous population of cells entered the cell cycle after rescue
from lovastatin arrest, progressed through S phase and on to
mitosis and thence to a second partially synchronous cell cycle.
Although this method does not yield a perfectly synchronous
population, the degree of enrichment of cells in S phase seen was
sufficient to allow meaningful interpretation of the effects of
anti-estrogens and retinoids on cell cycle progression. The technique also represents a good compromise between the desirable
effect of achieving complete synchrony and the undesirable effect
of using synchronizing agents that may themselves have confounding cell cycle or metabolic effects. For example, the standard
method for obtaining a population of synchronized cells is mitotic
selection since this technique selects cells that are at the beginning
of the cell cycle but does not affect underlying RNA and protein
synthesis. However, there is cell line-dependent variability in the
purity of the selected population, and T-47D cells produce a smaller
proportion of synchronized cells than do MCF-7 cells. Nevertheless, mitotic selection has been performed on T-47D cells (Musgrove, 1992), and the resultant progression of cells through G1
phase and into S phase is very similar to that shown in Figure 2,
with little change seen in %S phase over the first 9 hr and a steep
rise then occurring to a peak of about 50% after about 21 hr. In
addition, lovastatin treatment does not affect overall RNA and
protein synthesis (Jakobisiak et al., 1991; Keyomarsi et al., 1991)
and, therefore, is likely to be superior to other methods of
synchronization, such as growth factor withdrawal or treatment
FIGURE 2 – Synchronization of T-47D cells. T-47D cells proliferating in medium supplemented with 5% FCS were treated with 40 µM
lovastatin for 24 hr, after which medium was replaced and 5 mM
mevalonic acid added. Cells were then harvested at intervals over 56 hr
for determination of cell cycle phase distribution by flow cytometry. S
phase data are the mean 6 SEM of 3 independent experiments. G2 1 M
data are from a representative experiment.
with hydroxyurea, but similar in efficacy to mitotic selection in
T-47D cells.
Effects of ICI 164384 and RA on synchronized cells
The next experiment was designed to test whether cells synchronized by this method are susceptible to the growth-inhibitory effect
of the pure steroidal anti-estrogen ICI 164384. A model placing
lovastatin action early in G1 phase and ICI 164384 action somewhat later predicts that ICI 164384 would suppress some or all of
the S phase entry seen with lovastatin–mevalonic acid synchronisation, and this was observed. As Figure 3 demonstrates, cells
rescued with mevalonic acid reached a peak %S phase of 45% at 24
hr, while cells treated with 1027 M ICI 164384 at the same time as
mevalonic acid was added reached a peak of only 30%. This
suppression of S phase entry was followed by a decline in entry into
G2 1 M, demonstrating that fewer cells entered and completed the
cell cycle in the presence of ICI 164384. Furthermore, the second S
phase peak seen in the synchronized cells was completely ablated
by ICI 164384, showing that ICI 164384 was active during the
second round of replication. Therefore, sensitivity to the cell cycle
effects of ICI 164384 lies down-stream of the point at which
lovastatin arrests cells.
To test whether the relationship between RA and lovastatin
effects was the same as that for ICI 164384, the same experiment
was undertaken with RA. In this case (Fig. 4), RA did not
appreciably affect the synchronous entry of cells into S phase at 24
hr but did reduce the second, smaller wave of S phase entry, as had
ICI 164384 in the previous experiment. The proportion of cells
entering G2 1 M phase was also unaffected at 24 hr and reduced at
48 hr, consistent with the S phase data.
The results of these experiments demonstrate that the cell cycle
actions of RA and ICI 164384 are different since cells rescued from
FIGURE 3 – Effect of ICI 164384 on cells synchronized by lovastatin–
mevalonic acid. T-47D cells proliferating in medium supplemented
with 5% FCS were treated with 40 µM lovastatin for 24 hr, after which
medium was replaced and 5 mM mevalonic acid added together with
either 1027 M ICI 164384 or vehicle (0.1% ethanol). Cells were then
harvested at intervals over 56 hr for determination of cell cycle phase
distribution by DNA flow cytometry. Similar data were obtained in a
second independent experiment.
FIGURE 4 – Effect of RA on cells synchronized by lovastatin–
mevalonic acid. T-47D cells proliferating in medium supplemented
with 5% FCS were treated with 40 µM lovastatin for 24 hr, after which
medium was replaced and 5 mM mevalonic acid added together with
either 1026 M RA or vehicle (0.1% ethanol). Cells were then harvested
at intervals over 56 hr for determination of cell cycle phase distribution
by flow cytometry. Similar data were obtained in a second independent
RA-treated cells, however, no specific agonist was available;
therefore, the medium was changed to remove as much retinoid as
possible. Estradiol was also added as a control, though it has
previously been demonstrated that estradiol in this setting does not
have an effect on RA-treated cells (Wilcken et al., 1996). Thus, the
effects of both ICI 164384 and lovastatin were specifically reversed
(by estradiol and mevalonic acid, respectively), while RA was
simply removed from the medium.
Figure 5 shows a pooled time course of lovastatin-rescued cells
synchronously entering S phase, as seen in Figures 2–4, with a peak
value of 40–45% at 24 hr. Estradiol reversal of ICI 164384
treatment also led to synchronous re-entry into S phase but with
35% of cells in S phase at 24 hr, while removal of RA resulted in a
more modest reversal, with a peak S phase value of 28–30% at 24
hr. Therefore, the quantitative effect of ‘‘reversing’’ the 3 treatments differed, and this effect was independent of the starting %S
phases, all of which were similar. However, this is most likely a
reflection of the varying degrees with which the respective
treatments were reversed. Both lovastatin and ICI 164384 treatments were specifically reversed (with mevalonic acid and estradiol, respectively), while the effect of RA was ‘‘reversed’’ simply
by the addition of fresh medium, there being no direct way to
antagonize RA. Therefore, it might be expected that there would be
less S phase entry with RA ‘‘reversal’’. The same could apply to ICI
164384, where a ligand-receptor-DNA binding process must be
reversed by estradiol, while mevalonic acid addition restores a
single biochemical pathway. However, despite the difference in the
magnitude of S phase entry, the timing of entry into S phase after
reversal of cell cycle arrest was very similar for all 3 treatments.
Since the data presented above suggest that the 3 agents tested have
different points of action, this result implies that the final growth-
lovastatin arrest were immediately susceptible to the effects of ICI
164384 but not RA. An explanation compatible with these observations is that ICI 164384 acts at a point in G1 phase after the action of
lovastatin, while cells are susceptible to RA action only before the
point at which lovastatin acts since cells released from lovastatin
were not susceptible to RA until the next cell cycle.
However, a major factor confounding such an interpretation is
the delay in the onset of RA action on the cell cycle, as
demonstrated in Figure 1. Thus, these results are also compatible
with RA and ICI 164384 acting at the same point in G1 phase but
with the delay in RA action explaining the differential effects on
synchronized cells.
Effects of lovastatin on cells recovering from ICI 164384
or RA treatment
Two further sets of experiments were carried out to address this
question. First, it could be postulated that if drugs which arrested
cell cycle progression at different points within G1 phase were
removed or their effects reversed, cells might enter S phase at
different times. Therefore, the time taken to recover from cell cycle
arrest could provide some indication as to where in the cell cycle
arrest had occurred. This question was examined by treating cells
with RA (48 hr), ICI 164384 (24 hr) or lovastatin (24 hr) and then
reversing the effects of these treatments as much as possible, to
allow the recommencement of cell cycle progression.
In the case of lovastatin-treated cells, this was straightforward,
and Figures 2–4 demonstrate how effectively fresh medium with
added mevalonic acid overcomes the effect of lovastatin. Maximal
reversal of anti-estrogen was achieved by changing the medium
and adding estradiol to the ICI 164384-treated cells. For the
FIGURE 5 – Effect of reversing the cell cycle arrest caused by
lovastatin, ICI 164384 or RA. T-47D cells proliferating in medium
supplemented with 5% FCS were treated with 40 µM lovastatin for 24
hr (L, h) or 1027 M ICI 164384 for 24 hr (ICI, n) or 1026 M RA for 48
hr (RA, s), and at time zero these treatments were reversed. The
medium in all flasks was changed; 5 mM mevalonic acid was added to
lovastatin-treated cells and 1027 M estradiol was added to RA- and ICI
164384-treated cells. Cells were then harvested at intervals over 28 hr
for determination of cell cycle phase distribution by flow cytometry.
Data points represent the mean 6 SEM of 3 independent experiments.
arrested state is similar for all 3 agents. An alternative explanation
is that rescued cells, having been arrested at different points in the
cell cycle, proceed through G1 phase at different rates, thus arriving
at S phase at the same time.
Finally, if the point of action of RA is indeed earlier in G1 phase
than the point of lovastatin action, one would predict that cells
released from RA-induced arrest would be immediately susceptible
to lovastatin, while cells released from ICI-induced arrest would
not. If RA acted after lovastatin, such cells would need to progress
through the cell cycle to a round of replication before being
affected by lovastatin. Therefore, exponentially growing cells were
treated with either RA for 48 hr or ICI 164384 for 24 hr, then, the
resulting block in cell cycle progression induced by each drug was
relieved by changing to drug-free medium with estradiol added, as
in the previous experiment. At the same time, lovastatin (or H2O as
a control) was added, and cells were harvested at intervals over a
period of 28 hr.
The results of this experiment are shown in Figure 6. Cells
rescued from RA-induced arrest re-initiated cell cycle progression,
with %S phase more than doubling from 12% after 48 hr of RA
treatment to 28% 24 hr after this treatment was reversed, as seen in
Figure 5. However, in the presence of lovastatin, cells remained in
an arrested state (Fig. 6). In contrast, while cells rescued from ICI
164384-induced arrest re-entered S phase (Figs. 5, 6), lovastatin
added at the time of reversal had no major effect on this cell cycle
progression (Fig. 6). Consistent with the lack of effect lovastatin
had on recovery from anti-estrogen treatment, shown in Figure 6,
Bonapace et al. (1996) have reported that estradiol can induce cell
cycle progression in MCF-7 cells treated with the closely related
simvastatin, demonstrating that estradiol-mediated pathways can
override the effects of HMG-CoA reductase inhibitors.
These data are again consistent with the hypothesis that ICI
164384 and RA act at different points in the cell cycle. While cells
recovering from treatment with lovastatin, ICI 164384 or RA enter
S phase at the same time (albeit with some differences in
magnitude), cells recovering from treatment with ICI 164384 or
RA do not respond to treatment with lovastatin in the same way.
Thus, the hypothesis generated from the data presented in Figures 3
and 4 (that within the cell cycle RA acts before and ICI 164384
after lovastatin) is supported.
Since in breast cancer cells both anti-estrogens and retinoids
inhibit cell cycle progression in the G1 phase of the cell cycle, both
agents must act, either directly or indirectly, on molecules that are
necessary for transit through G1. Progression through G1 phase is
regulated by the sequential activation of cyclin-CDK complexes,
which in turn phosphorylate down-stream targets, the best defined
of which is pRB, the gene product of the tumor-suppressor gene
RB-1. Hyperphosphorylation of pRB is necessary for entry into S
phase, and there is, thus, a strong link between G1 cyclin induction,
CDK activation, pRB phosphorylation and transit through G1 phase
(Sherr, 1994). While the major event that activates CDKs is the
binding of cyclin, both phosphorylation and dephosphorylation of
certain residues on the CDK itself are also required.
Anti-estrogen treatment of breast cancer cells is accompanied by
decreases in cyclin D1 mRNA and protein levels, CDK4 activity
and pRB phosphorylation, followed by G1 cell cycle arrest (Watts
et al., 1995; Wilcken et al., 1996). The time sequence of these
events strongly suggests a causative relationship. Conversely, RA
caused a fall in CDK4 activity and pRB phosphorylation without
affecting cyclin D1 levels (Wilcken et al., 1996). While such
findings suggest that RA may act by inducing an inhibitor of CDK4
activity, this remains to be demonstrated. Moreover, the need to
induce the synthesis of such a molecule to levels where CDK
activity is inhibited could explain the delay in RA action on
asynchronous cells seen in Figure 1. Another untested possibility is
that RA indirectly affects the necessary phosphorylation or dephosphorylation of CDK4.
FIGURE 6 – Effect of lovastatin on cells re-entering the cell cycle
after reversal of RA or ICI 164384 treatment. T-47D cells proliferating
in medium supplemented with 5% FCS were treated with 1026 M RA
for 48 hr or 1027 M ICI 164384 for 24 hr, and at time zero these
treatments were reversed. The medium was changed and 1027 M
estradiol added to all flasks plus either 40 µM lovastatin (open
symbols) or H2O (closed symbols). Cells were then harvested at
intervals over 28 hr for determination of cell cycle phase distribution
by flow cytometry. Data points represent the mean 6 SEM of 3
independent experiments.
The cell cycle studies reported here thus support the notion that
the molecular targets of anti-estrogens and retinoids are likely to
differ and that initial targets of RA action might be expected to
regulate progression through an early part of G1 phase. However,
we have also shown that reversal of cell cycle arrest induced by
either RA or ICI 164384 (or lovastatin) resulted in entry into S
phase at the same time, suggesting that the maintenance of a
growth-arrested state in both cases may be overcome by the
activation of common steps. Molecular studies point to the
importance of CDK4 function, and the regulation of this molecule
(albeit by different mechanisms) may be a critical factor in the
action of both retinoids and anti-estrogens.
Finally, the demonstration of a difference in cell cycle action and
the implication of different molecular mechanisms of action lend
weight to animal data that consistently show synergism between
anti-estrogens and retinoids when these are used as anticarcinogenic agents. Collectively, these observations strongly
suggest that a combination of these agents may be a more effective
mode of cancer protection in those at high risk than the use of one
or other agent alone.
This study was supported by the National Health and Medical
Research Council of Australia (NHMRC). N.R.C.W. is the recipient of an NHMRC Medical Postgraduate Research Scholarship.
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