(Received for publication, September 1, 1995; and in revised form, October 13, 1995)
From the
Under normal culturing conditions, the T47D human breast cancer
cell line expresses progesterone receptor constitutively and is
responsive to estrogen. Because the tumor suppressor protein p53 plays
a central role in determining genetic stability and cell proliferation,
we have examined the effects of 17-estradiol, the synthetic
progestin R5020, and the antiprogestin RU486 on the levels of this
protein in T47D cells. Western blot analysis of cellular extracts,
performed with a monoclonal antibody capable of quantitatively
supershifting a specific p53-p53 response element complex in a gel
mobility shift assay, detected a single immunoreactive band
representing p53. When cells were grown for 4-5 days in culture
medium containing charcoal-treated fetal calf serum, p53 levels
declined to 10% of the level seen in the control (no charcoal
treatment) group. Supplementation of culture medium containing
charcoal-treated calf serum with 0.1-1 nM 17
-estradiol restored p53 to its normal levels. A 4-day
treatment of cells with R5020 or RU486 lowered the p53 levels in cells
grown in normal culturing conditions to 15 and 30% of control levels,
respectively. R5020 and RU486 treatments also caused down-regulation
and/or hyperphosphorylation of the progesterone receptor, which
correlated with the down-regulation of p53. These observations indicate
that in T47D cells, p53 is up-regulated by estradiol while R5020
down-regulates this protein. Since estradiol is known to promote cell
proliferation, the induction of p53 observed in this study leads us to
propose that estradiol stimulates p53 to regulate proliferation of T47D
cells in culture.
The development, growth, and differentiation of human breast is
under the influence of a number of hormones including the sex steroids,
estradiol (E) (
)and progesterone. In cases where
the breast tissue transforms into a tumorous entity, it often continues
to respond to circulating levels of these hormones provided it
expresses receptors for the corresponding hormone. While
hormone-insensitive malignancies do not appear to express functional
receptors, hormone-sensitive cancers may overexpress progesterone
receptor (PR) and estradiol receptor (McGuire, 1978). Treatment of
breast cancers with hormones or antihormones presumes the presence of
functional receptors, which via activation or inactivation of receptors
mediate regression of cancerous tissues. Although breast cancers may
initially respond to endocrine therapy, the tissue can transform into a
hormone-insensitive entity. The mechanisms underlying the evolution of
hormone-sensitive tumors to hormone-insensitive states are not known
(Horwitz, 1994). It is, therefore, crucial to determine whether
treatment with hormones and/or antihormones might affect a shift toward
a progressively more malignant state of breast cancer.
The
biological activity of the tumor suppressor protein p53 is associated
with suppression of cell growth. It is now widely recognized that p53
may be the most frequently mutated protein in human cancers (Oren,
1992). Because of the crucial role that p53 plays in tumor suppression,
we explored the role of E and progestin in regulating p53
in the T47D cell line, which is responsive to both of these steroid
hormones.
We have observed that in T47D cells, E, R5020,
and RU486 are able to induce a proliferative state, which is detected
as an increase in total cell number when cells are cultured in
charcoal-treated serum (Iwasaki et al., 1994). Effects of
progestins and antiprogestins on the expression of proto-oncogenes have
been explored (Schuchard et al., 1993), but there is a paucity
of information regarding the influence of steroid hormones on tumor
suppressor function in hormone-responsive breast cancer cells. We
present evidence here that in T47D cells, p53 is significantly
increased upon E
treatment while it is down-regulated by
both R5020 and RU486.
Figure 1:
Characterization of the specific DNA
binding activity and evidence that anti-p53 antibody reacts with p53
from T47D extracts: gel mobility shift assay. A high salt extract (0.5 M ionic strength) was prepared from confluent T47D cells
cultured in whole serum. All DNA binding reactions contained 5 µl
of T47D extract (total protein, 15 µg), 100,000 cpm of P end-labeled specific p53RE probe, and 5 µl of 4
gel mobility shift assay buffer (80 mM Hepes, 40%
glycerol) in a total volume of 20 µl. Individual lanes also
contained: lane 1, 0.6 µg of salmon sperm DNA; lane
2, no salmon sperm DNA; lane 3, 0.6 µg of salmon
sperm DNA; lane 4, 1.7 µg of salmon sperm DNA; lane
5, 0.6 µg of salmon sperm DNA plus 0.7 µg (36 pmol) of
non-radiolabeled probe; lane 6, 0.6 µg of salmon sperm DNA
plus 0.25 µg of anti-p53 antibody; lane 7, 0.6 µg of
salmon sperm DNA plus 1.25 µg of anti-p53 antibody; lane
8, 0.6 µg of salmon sperm DNA plus 0.1 µg of PAb240; lane 9, 0.6 µg of salmon sperm DNA plus 0.5 µg of
PAb240. Cell extract, salmon sperm DNA, non-radiolabeled probe, and
antibodies were incubated for 15 min at 4 °C prior to addition of
P-labeled probe. Note that lanes 1 and 2 were run on a separate gel from lanes 3-9. p53REc, p53 response element
complex.
Figure 2: p53 levels are dependent on serum factor(s) removed by charcoal treatment: Western blot analysis. T47D cells were cultured for various times in medium containing whole or charcoal-treated serum. All the cells were initially plated in whole serum for 2 days. The numbers across the top of the figure represent time (days) in medium containing charcoal-treated serum. The lane marked C represents the control, which was cells cultured in whole serum only. All cells were harvested at the same time after 10 days of culturing. Cells were extracted as described under ``Experimental Procedures.'' Extracts were analyzed for protein concentration, and a total of 100 µg of protein/lane for each condition was subjected to SDS-PAGE and Western blot analysis as described under ``Experimental Procedures.''
Figure 3:
Estradiol reverses the effect of charcoal
treatment of serum on p53 levels: Western blot analysis. T47D cells
were plated for 2 days in medium containing whole serum and then for
another 6 days in charcoal-treated serum plus various concentrations of
E as indicated. The lane marked C represents cells cultured in whole serum for the entire time with
no exogenous E
treatment. The lane marked 0 contained no E
. All cells were harvested at the same
time after 8 days of culturing, and cellular extracts were prepared as
outlined under ``Experimental Procedures.'' The extracts were
analyzed for protein concentration, and a total of 100 µg of
protein/lane for each condition was subjected to SDS-PAGE and
Western blot analysis as described under ``Experimental
Procedures.''
Figure 4: The progestin, R5020, and the PR antagonist, RU486, decrease the levels of p53 in cells cultured in whole serum: Western blot analysis. Cells were first cultured for 6 days in whole serum prior to addition of ligand and then in whole serum plus a 10 nM concentration of the ligands as indicated for 4 days. All cells were harvested at the same time after 10 days of culturing. Cells were extracted as described under ``Experimental Procedures.'' Extracts were analyzed for protein concentration, and a total of 100 µg of protein/lane for each condition was subjected to SDS-PAGE and Western blot analysis. Membranes were probed for p53 and PR. PR was probed by using monoclonal anti-PR antibody AB52 (Estes et al., 1987). P, progesterone; TA, triamcinolone acetonide; F, cortisol.
The wild type protein product of the tumor suppressor gene,
p53, is a nuclear phosphoprotein, which functions as a transcription
factor directly regulating the expression of factors involved in cell
cycle control (Kastan et al., 1992) and programmed cell death
(Miyashita and Reed, 1995; Miyashita et al., 1994; Oltvai et al., 1993). The ability of p53 to act as a transcription
factor is determined by its sequence-specific DNA binding activity
(Kern et al., 1991; Hupp et al., 1992; Cho et
al., 1994). The occupancy of response elements by p53 and its
ability to trans-activate responsive genes would at least partially
depend on its intranuclear concentration. Thus, measured levels of this
protein are an important determinant of its activity. Since we examined
the effects of steroids (E, R5020, and RU486) on the levels
of p53 using Western blot analysis, it was important to demonstrate the
specific reactivity of the anti-p53 antibody. For this, we employed the
gel mobility shift assay. The observation that a specific p53-REc could
be detected in whole cell extracts indicates that at least with respect
to its specific DNA binding activity as shown in Fig. 1, p53 in
T47D cells is normal. It is not surprising that PAb240, an antibody
that recognizes mutant forms of p53, neither blocked the formation nor
supershifts the p53-REc. Our results are in agreement with a similar
observation reported in the literature on the inability of PAb240 to
supershift the p53-REc (Halazonetis et al., 1993). The
anti-p53 antibody appeared to increase the affinity of p53 for its
response element as well as supershift the p53-REc (Fig. 1).
Antibody-induced increases in the specific DNA binding activity of p53
have been reported previously (Halazonetis et al., 1993, Hupp et al., 1992). The same antibody we used to supershift the
p53-REc was also capable of detecting a single protein band in a
Western blot analysis of T47D extracts, which migrated at the expected
molecular weight.
Treatment of cells with R5020 or RU486 altered the
levels of p53 when cells were cultured in charcoal-treated serum, but
the effects were not dramatic (data not shown). Charcoal treatment of
serum appears to remove, among other components, a hormone or a factor
that induces PR. Accordingly, lowering the levels of PR by charcoal
treatment of serum limits progestin effects. We believe that among a
number of possible heterocyclic compounds susceptible to removal by
charcoal treatment, E may be a prime candidate. We have
demonstrated in this report that E
is sufficient to restore
the levels of p53 and recently have determined it is also sufficient to
restore PR levels. (
)This may explain how PR-mediated
down-regulation of p53 might occur with R5020 and RU486 treatment. For
optimal detection of the effects of R5020 and RU486 on p53 levels, the
cells require priming by E
to raise the PR and p53 to
effective levels. The E
-induced increase in p53 levels in
the absence of PR stimulation raises p53 levels as a check on the
E
-induced proliferative state of T47D cells. However, when
PR is activated by R5020 or RU486 under estrogenized conditions, PR
action predominates and p53 levels are decreased. A PR-mediated
mechanism for reducing p53 in T47D cells would thus require estrogenic
stimuli. However, demonstration of a direct link between PR and p53
levels must await future investigations.
RU486 appears to be acting
as a PR agonist in this system. The agonist effects of RU486 could be
explained on the basis that the activation function (AF1) in the
N-terminal transcriptional activation domain of PR is sufficient to
mediate down-regulation of p53 and that the ligand-dependent activation
function (AF2) in the hormone binding domain, which would be inhibited
by RU486, is not required (Gronemeyer et al., 1992).
Alternatively, RU486 has been shown to act as an agonist when cAMP
levels are increased in T47D cells (Beck et al., 1993).
Because E has been shown to increase intracellular cAMP
levels in cultured breast cancer cells (Aronica et al., 1994),
it is possible that the levels of E
in the whole serum or
upon addition to charcoal-stripped serum were sufficient to convert
RU486 to an agonist.
Because triamcinolone acetonide, but not cortisol, down-regulated p53 and caused an upshift and down-regulation of PR, it would appear that the former binds PR and activates it in the T47D cells. The failure of the natural PR ligand progesterone to mimic the effects of the potent progestin R5020 is probably due to its rapid metabolism in T47D cells whereas R5020 is more stable (Horwitz et al., 1986). The latter possibility is supported by the observation that progesterone treatment neither induces upshift of PR nor its down-regulation (Fig. 4).
Although studies on the effects of steroids on p53 function are limited, recent reports indicate a precedence of steroid hormone regulation of p53. Withdrawal of androgen appears to increase the levels of p53 in the epithelial and stromal cells of the rat ventral prostate resulting in increased levels of apoptotic cells (Banerjee et al., 1995). Increased levels of p53 in this case might be due to inactivation of androgen receptor as a result of DHT withdrawal. This effect may be analogous to our observations of PR-mediated alterations in p53 levels in T47D cells and is consistent with the results that show PR binding ligands specifically reduce the p53 level in T47D cells.
Whether androgen, estrogen, or progesterone receptors directly regulate gene expression related to p53 transcription or whether the mechanism is indirect or post-transcriptional remains to be determined.