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W. Alton Jones Cell Science Center, Lake Placid, New York 12946
Address all correspondence and requests for reprints to: Dr. JoEllen Welsh, Senior Scientist, W. Alton Jones Cell Science Center, 10 Old Barn Road, Lake Placid, New York 12946. E-mail: jwelsh{at}cell-science.org
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Abstract |
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Introduction |
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To probe the mechanisms whereby vitamin D signaling modulates apoptosis, we have selected a subclone of MCF-7 cells (termed MCF7D3Res cells) that is resistant to the growth inhibitory effects of 1,25-(OH)2D3 (12). MCF-7D3Res cells do not exhibit cell cycle arrest or apoptosis following treatment with up to 100 nM 1,25-(OH)2D3, yet these cells retain sensitivity to antiestrogens such as tamoxifen. The mechanism of 1,25-(OH)2D3 resistance is unclear, although we have demonstrated that the MCF-7D3Res cells express the vitamin D receptor (VDR), which is able to bind ligand and several known vitamin D response elements (12). These data suggest that 1,25-(OH)2D3 resistance in these cells is not due to complete abrogation of VDR-mediated effects and may represent an uncoupling of growth regulation from VDR signaling. Because previous reports have implicated the cell cycle regulatory proteins p53, p21, and Rb in mediating the growth inhibitory effects of both 1,25-(OH)2D3 and TPA, the second objective of these studies was to compare expression of these proteins in MCF-7 cells and MCF-7D3Res cells after treatment with 1,25-(OH)2D3 and TPA.
Our results indicate that 1,25-(OH)2D3 and TPA exert similar but independent effects on growth and cell cycle regulatory proteins and disparate effects on MCF-7 cell apoptosis. These data suggest that 1,25-(OH)2D3, but not TPA, generates distinct signals that lead to induction of apoptosis in MCF-7 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MEM medium (Life Technologies, Inc.,
Gaithersburg, MD) containing 25 mM HEPES and 5% FBS (Life
Technologies). Cells were routinely plated at 2 x 104
cells/ml and passaged every 3–4 days. Stock cultures of
MCF-7D3Res cells were routinely grown in medium containing
100 nM 1,25-(OH)2D3 (Biomol,
Plymouth Meeting, PA). For experiments, MCF-7 and
MCF-7D3Res cells were plated in medium containing 5% FBS
without 1,25(OH)2D3, and treatments with
1,25(OH)2D3, TPA or ethanol vehicle were
initiated 2 days after plating. Doses of
1,25(OH)2D3 up to 100 nM were used
for these experiments to maximize the differences in growth kinetics
between MCF-7 and MCF-7D3Res cells. Although 100
nM 1,25(OH)2D3 is higher than the
physiologic concentration of this hormone, similar doses are routinely
used for in vitro studies of VDR target gene regulation.
Determination of total cell number and DNA fragmentation
For measurement of cell number or DNA fragmentation, cells were
plated in 24-well plates at a density of 2 x 103
cells/well. For quantitation of total cell number, cells were fixed
with 1% glutaraldehyde for 15 min, incubated with 0.1% crystal violet
(Fisher Scientific, Pittsburgh, PA) for 30 min, destained with
H2O, and solubilized with 0.2% Triton X-100. Absorbance at
562 nm (minus background absorbance at 630 nm) was determined on a
microplate reader. Quantitation of apoptotic cell death was achieved
with a commercially available cell death ELISA system (Boehringer
Mannheim, Indianapolis, IN) that detects DNA fragmentation, a
characteristic feature of apoptosis. During apoptosis, nuclear
disintegration results from activation of an endonuclease that cleaves
DNA at the internucleosomal linker regions, generating soluble
DNA-histone complexes. In the cell death ELISA assay, these DNA-histone
fragments are detected in cytosolic extracts using a sandwich ELISA
with monoclonal antibodies directed against DNA and histones. Thus, an
increase in soluble DNA-histone fragments indicates an increase in the
number of cells undergoing apoptosis. Cytosolic extracts derived from
control and treated cells were equalized on the basis of total cell
number (determined by crystal violet staining) and analyzed according
to the manufacturer’s protocol.
Morphology and in situ end labeling
Cells were plated on coverslips in six-well plates at a density
of 1 x 104 cells/ml and were treated with ethanol,
100 nM 1,25-(OH)2D3 or 1
nM TPA± 100 nM
1,25-(OH)2D3 2 days later. Cells were fixed
after 72 h, and DNA fragmentation was detected with the In
Situ Cell Death Detection kit (Boehringer-Mannheim) according to
manufacturer’s protocol. This assay labels individual cells undergoing
apoptosis by terminal transferase-mediated addition of fluorescein dUTP
at DNA strand breaks. Following washing and mounting, cells were viewed
on a Nikon Optiphot 2 microscope and photographed.
Steroid receptor ligand binding assays
Binding of 3H-1,25-(OH)2D3
or 3H-17ß-estradiol was measured in nuclear extracts
derived from MCF-7 and MCF-7D3Res cells treated with 100
nM 1,25-(OH)2D3, 100 nM
TPA, or vehicle for 48 h (13). Briefly, cells were homogenized in
high salt KTED buffer (300 mM KCl, 10 mM
Tris-HCl, 1 mM EDTA, 1 mM DTT, 10
mM molybdate, 25 µg/ml leupeptin, 25 µg/ml aprotinin,
100 mM PMSF, 1 mM NaVO3, 10
mM NaF, 10 mM benzamidine; pH 7.5) and
centrifuged (105,000 x g, 45 min) to yield a chromatin
extract that was incubated with 1 nM
3H-1,25-(OH)2D3 (Amersham Life
Science, Buckinghamshire, UK) or 2 nM
3H-17ß-estradiol for 24 h at 4 C. Bound and free
hormone were separated by addition of dextran coated charcoal,
incubation for 15 min, and centrifugation at 3500 x g
for 15 min. Bound 3H-1,25-(OH)2D3
or 3H-17ß-estradiol in the supernatant was counted in a
Beckman scintillation counter. Data are expressed as fmol
3H-1,25-(OH)2D3 or
3H-17ß-estradiol bound per mg protein after correction
for nonspecific binding, which was measured in parallel tubes incubated
with 250-fold excess unlabeled 1,25-(OH)2D3 or
diethylstilbestrol.
Western blotting of Rb, p21, p53, and VDR
For immunoblotting, cells were seeded at a density of 1.5
x 104 cells/ml and treated with
1,25-(OH)2D3, in the presence or absence of
TPA, or ethanol vehicle, 2 days after plating. At the indicated time
points, cell lysates were prepared and separated on SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with monoclonal
antibodies directed against human Rb (Pharmingen, SanDiego, CA), p21
(Oncogene Science, Cambridge, MA) or p53 (DAKO, Glostrup, Denmark).
Following incubation with horseradish peroxidase conjugated antimouse
Ig, specific bands were detected by enhanced chemiluminescence using
products from Amersham. For VDR, proteins were precipitated from high
salt nuclear extracts (prepared as described for ligand binding
studies), separated on SDS-PAGE, transferred to nitrocellulose, and
blotted with a monoclonal antibody directed against the VDR (clone
9A7
, Neomarkers, Fremont, CA) followed by horseradish peroxidase
conjugated antirat secondary antibody and chemiluminescent detection.
Blots were scanned by laser densitometry and data are given relative to
vehicle treated values. As indicated in individual figure legends, each
blot is representative of two or more independent experiments which
yielded similar results.
Statistical evaluation
Data are expressed as mean ± SE, with the
number of replicates indicated in the figure legends. When not shown,
SEMs were less than 5% of the mean. One-way ANOVA was used
to assess statistical significance between means. Differences between
means were considered significant if p values less than 0.05 were
obtained with the Bonferroni method using the GraphPad Instat computer
program (Intuitive Software for Science, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, both
1,25-(OH)2D3 and TPA significantly reduced
MCF-7 cell number, but distinct growth patterns were observed with each
agent. In MCF-7 cultures treated with TPA, total cell number continued
to increase, but at a slower rate than in vehicle treated cultures. In
MCF-7 cultures treated with 1,25-(OH)2D3, total
cell number did not increase after day 3. In cultures treated with both
1,25-(OH)2D3 and TPA, total cell number did not
increase after day 1. In the MCF-7D3Res cultures,
1,25-(OH)2D3 had minimal effects on cell
number, yet TPA significantly reduced cell numbers. Furthermore, TPA
was able to partially sensitize MCF-7D3Res cells to the
effects of 100 nM 1,25-(OH)2D3
because cell numbers were significantly lower in
MCF-7D3Res cultures treated with both
1,25-(OH)2D3 and TPA than in those treated with
TPA alone.
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, vehicle treated MCF-7 cells exhibited
one band, representing the phosphorylated form of the Rb protein. In
contrast, an additional, faster migrating Rb band was detectable in
cells treated for 48 h with either 100 nM
1,25-(OH)2D3 or 1 nM TPA. This
lower band represents the hypophosphorylated form of the Rb protein,
which is associated with growth arrest in
G0/G1. In MCF-7 cells treated simultaneously
with both agents, the expression of the hypophosphorylated form of the
Rb protein was more prominent than with either agent alone at doses as
low as 1 nM 1,25-(OH)2D3. A clear
down-regulation of both hypo and hyper phosphorylated forms of the Rb
protein was evident in cells coincubated with 1 nM TPA and
100 nM 1,25-(OH)2D3. No changes in
Rb amount or phosphorylation state were detected after treatment of
MCF-7D3Res cells with up to 100 nM
1,25-(OH)2D3. However, there was
down-regulation of Rb protein after treament with 1 nM TPA,
and this effect was further enhanced when MCF-7D3Res cells
were coincubated with 1 nM TPA and 10 nM or
more 1,25-(OH)2D3.
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, p21 expression was initially low in
vehicle treated MCF-7 cells and increased with time in culture. MCF-7
cells treated with 1,25-(OH)2D3 exhibited an
early (24–48 h) increase in p21 protein compared with time matched
controls. However, after 72 h, the level of p21 in
1,25-(OH)2D3 treated MCF-7 cells plateaued and
was no longer different from vehicle treated cultures. In contrast, TPA
treated MCF-7 cells exhibited a more intense elevation of p21 protein
expression that persisted over the 96-h time course. When MCF-7 cells
were treated with both 1,25-(OH)2D3 and TPA,
p21 was up-regulated at 24 and 48 h and thereafter declined to
levels similar to that observed in time-matched vehicle treated cells.
In MCF-7D3Res cells, no significant induction of p21 by
1,25-(OH)2D3 was observed. However,
MCF-7D3Res cells treated with TPA exhibited a significant
increase in p21 protein expression with a pattern similar to that
observed in the parental MCF-7 cells. Expression of p21 in
MCF-7D3Res cells treated with both
1,25-(OH)2D3 and TPA was similar to that of
cells treated with TPA alone.
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). A similar pattern of transient
down-regulation was observed following TPA treatment of MCF-7 cells. In
contrast, when MCF-7 cells were treated with both
1,25-(OH)2D3 and TPA, p53 was dramatically and
persistently down-regulated begining at 24h. In MCF-7D3Res
cells, 1,25-(OH)2D3 treatment for up to
96 h did not alter p53 protein expression relative to time matched
vehicle treated controls. In contrast, TPA transiently down-regulated
p53 in MCF-7D3Res cells after 24 h, with a subsequent
up-regulation observed at 72h. When MCF-7D3Res cells were
treated with both compounds, p53 remained down-modulated throughout the
96-h time course, mimicking the pattern observed in parental MCF-7
cells.
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). Our previous studies have indicated
that no DNA fragmentation is induced by
1,25-(OH)2D3 in MCF-7D3Res cells
(12).
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(bottom
panels), cultures treated with ethanol vehicle exhibited typical
epithelial morphology on phase contrast, and mitotic cells were
frequent (panels a and e). In contrast, many cells in
1,25(OH)2D3 treated cultures were shrunken and
lay above the adherent cells of the monolayer and the nuclei in these
cells were positive for DNA strand breaks (panels b and f). TPA treated
MCF-7 cultures contained many giant cells with large, round nuclei and
few DNA strand breaks (panels c and g). However, in cultures treated
with both TPA and 1,25-(OH)2D3, most of the
MCF-7 cells were apoptotic (many with DNA strand breaks), and cells
with normal morphology were infrequent. As indicated in Fig. 6
(top panels), neither 1,25-(OH)2D3
or TPA treatment induced DNA strand breaks in MCF-7D3Res
cultures. When MCF-7D3Res cells were treated with both TPA
and 1,25-(OH)2D3, the cultures contained a
mixture of normal cells, apoptotic cells, and giant cells with large
secretory granules.
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, and Western blotting is presented in
Fig. 7. In the absence of ligand,
1,25-(OH)2D3 binding was lower in
MCF-7D3Res cells than in parental MCF-7 cells. It is
important to note that these data represent VDR expression after 4 days
in the absence of ligand because cells were treated 48 h after
plating and then exposed to ethanol for 48 h. Because the
MCF-7D3Res cells are normally continuously cultured in 100
nM 1,25-(OH)2D3, the lower levels
of VDR after 4 days in the absence of ligand may indicate a difference
in stability of the unoccupied VDR (14, 15) in these cells. On Western
blots of the same nuclear extracts, the 9A7 antibody recognized a
doublet at approximately 50 kDa. MCF-7 cells exhibited approximately
equal amounts of the two immunoreactive bands, whereas
MCF-7D3Res cells predominantly expressed the upper band.
However, treatment with 1,25-(OH)2D3 for
48 h up-regulated 1,25-(OH)2D3 binding and
the expression of the lower band on Western blots in both MCF-7 and
MCF-7D3Res cells. The extent of up-regulation of ligand
binding was higher in the MCF-7D3Res cells (2.5-fold
increase) compared with the parental MCF-7 cells (1.6-fold increase),
and the level of VDR in 1,25-(OH)2D3 treated
MCF-7D3Res cells approached that of the untreated MCF-7
cells. Following exposure to TPA, both ligand binding and the
expression of the lower mol wt band of the doublet were up-regulated in
the MCF-7 cells, whereas no significant increase was observed in
the MCF-7D3Res cells.
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) and TPA, but not 1,25-(OH)2D3,
down-regulated estradiol binding in these cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies demonstrated that the effect of 1,25-(OH)2D3 on MCF-7 cell growth arrest, previously shown to be at the G0/G1 boundary (6) was preceded by induction of p21 and dephosphorylation of Rb protein. A similar correlation between 1,25-(OH)2D3 mediated growth arrest, p21 induction and Rb phosphorylation has been reported in human leukemic cell lines (20, 21). Although others have reported that 1,25-(OH)2D3 and its analogs KH1060 and EB1089 up-regulated p53 in MCF-7 cells (17, 22), we observed a transient down-regulation of p53 after treatment with 1,25-(OH)2D3, and the expression of p53 in 1,25-(OH)2D3 treated cells never exceded that of time matched vehicle control cultures. Our data are consistent with those of Fan and Yu (18), who reported down-regulation of p53 by 1,25-(OH)2D3 in CAMA-IEe breast cancer cells. Further comparative studies are necessary to determine whether discrepancies in the effect of 1,25-(OH)2D3 on p53 expression reflect culture conditions, cell characteristics, or timing and dose of treatments. In any case, our results demonstrated that 1,25-(OH)2D3 induced p21 expression in the absence of p53 up-regulation in MCF-7 cells, consistent with reports of p53 independent induction of p21 in mammary cells (23). The recent identification of a vitamin D3 response element in the human p21 gene that is transcriptionally activated by 1,25-(OH)2D3 in a p53-independent manner (21) lends further credence to the hypothesis that 1,25-(OH)2D3 directly induces p21 in association with growth arrest of MCF-7 cells.
Additional insight into the mechanisms of vitamin D-mediated growth regulation was derived from the MCF-7D3Res cell line, whose growth is unaffected by 1,25-(OH)2D3 (12). In contrast to the parental MCF-7 cells, the MCF-7D3Res cells did not exhibit any changes in p21 or p53 expression, or Rb phosphorylation, after treatment for up to 96 h with 100 nM 1,25-(OH)2D3. However, TPA did induce growth arrest in the MCF-7D3Res cells, which was associated with up-regulation of p21, transient down-regulation of p53 and dephosphorylation of Rb. Because MCF-7 and MCF-7D3Res cells exhibited comparable responses to TPA mediated growth inhibition, it is clear that the growth regulatory effects of TPA and 1,25-(OH)2D3 in breast cancer cells can be dissociated, suggesting independent mechanisms of action. In addition, these data suggest that there are no inherent defects in p21 or Rb function in MCF-7D3Res cells and indicate that a specific defect in 1,25-(OH)2D3 regulation of these proteins may be linked to vitamin D3 resistance. Further studies to compare the transcriptional activation of the p21 promoter by 1,25-(OH)2D3 in MCF-7 and MCF-7D3Res cells are in progress.
Although 1,25-(OH)2D3 and TPA exerted similar effects on p53 and Rb in MCF-7 cells, there were distinct differences in the kinetics and magnitude of changes in p21 expression after treatment with the two agents. 1,25-(OH)2D3 treatment induced a smaller and transient up-regulation of p21, whereas TPA induced a more dramatic and sustained up-regulation of p21 in MCF-7 cells. This was unexpected because, at the doses used in this study, 1,25-(OH)2D3 exerted a more dramatic effect on MCF-7 cell number than did TPA. The lack of correlation between cell number and p21 expression may reflect the differential effects of TPA and 1,25-(OH)2D3 on growth arrest vs. apoptosis because 1,25-(OH)2D3 induced apoptosis after 72 h, whereas TPA did not. A detailed analysis of MCF-7 cell cycle kinetics after treatment with TPA and 1,25-(OH)2D3 will be necessary to resolve this issue.
Coincubation of MCF-7 cells with both 1,25-(OH)2D3 and TPA exerted complementary effects on growth arrest and apoptosis. Although TPA alone did not induce apoptosis in MCF-7 cells, 1,25-(OH)2D3 mediated DNA fragmentation was potentiated in the presence of TPA. Despite the maximal effect on growth arrest and apoptosis under cotreatment conditions, p21 was only transiently up regulated after treatment with both 1,25-(OH)2D3 and TPA. Because of the extensive apoptosis observed in MCF-7 cells treated with both 1,25-(OH)2D3 and TPA, it is possible that cells that exhibited high levels of p21 (at 24–48 h) were eliminated by apoptosis, and the lower levels of these protein (which were similar to levels observed in untreated cells) reflects expression in surviving cells. In contrast to p21, a rapid and sustained down-regulation of p53 was observed in MCF-7 cells treated with both 1,25-(OH)2D3 and TPA. This was also unexpected because neither agent alone induced persistent down-regulation of p53, but again this may reflect the high rate of apoptosis in these cultures. Further studies are therefore needed to determine whether the changes in p21 and p53 occur in cells actively undergoing apoptosis or cells that remain viable.
In the MCF-7D3Res cells, simultaneous treatment with TPA and 1,25-(OH)2D3 exerted more dramatic effects on growth, down-regulation of p53 and expression/phosphorylation of Rb than did TPA alone. However, co-incubation of MCF-7D3Res cells with TPA and 1,25-(OH)2D3 did not potentiate the effect of TPA on p21 expression. This could reflect the lesser degree of growth arrest/apoptosis in MCF-7D3Res cells treated with 1,25-(OH)2D3 and TPA (as compared with the parental MCF-7 cells) or may indicate dysregulation of p21 in the MCF-7D3Res cell line.
Data from both ligand binding and Western blots indicated that TPA up-regulated VDR expression in MCF-7 cells, suggesting that enhanced sensitivity to 1,25-(OH)2D3 may contribute to the synergistic effects of these two agents on growth. Our data are compatible with TPA mediated up-regulation of the VDR in rat osteosarcoma (ROS 17/2.8) cells (24) but contrast with the down-regulation of the VDR in TPA treated renal and 3T3 cells (25, 26). These discrepancies, which may reflect cell specificity in PKC isozyme expression, may be important determinants of tissue-specific vitamin D3 mediated gene expression. Because PKCß mediated phosphorylation of the VDR may alter the receptor’s ability to bind to the vitamin D3 response element (27), TPA may also influence VDR regulated events downstream of ligand binding. Of note, no increase in 1,25-(OH)2D3 binding or VDR protein expression was observed in the MCF-7D3Res cells following TPA treatment, suggesting a possible defect in VDR regulation in these cells.
In agreement with previous data (6, 28, 29), 1,25-(OH)2D3 and TPA independently down-regulated estradiol binding in MCF-7 cells within 48 h. In MCF-7D3Res cells, estradiol binding was reduced relative to MCF-7 cells, and treatment with 1,25-(OH)2D3 had no effect on estradiol binding. However, TPA mediated down-regulation of ER was comparable in MCF-7 and MCF-7D3Res cells. These results suggest that a specific defect in vitamin D regulation of estrogen signaling may contribute to the 1,25-(OH)2D3 resistance of these cells. Further studies are necessary to compare the expression of estrogen-regulated proliferation and/or survival factors, in relation to growth arrest or apoptosis in MCF-7 and MCF-7D3Res cells.
In summary, these studies indicate that 1,25-(OH)2D3 and TPA exerted independent growth inhibitory effects on MCF-7 cells, but only 1,25-(OH)2D3 induced characteristic apoptotic morphology and DNA fragmentation. Although 1,25-(OH)2D3 and TPA exerted similar effects on the cell cycle regulatory proteins, Rb and p53, distinct differences in the kinetics and magnitude of p21 protein regulation were observed in response to these two agents. Induction of apoptosis in MCF-7 cells following treatment with 1,25-(OH)2D3 did not correlate with an increase in p21 or p53. In MCF-7D3Res cells, 1,25-(OH)2D3 had no effect on growth or expression of Rb, p53 or p21, yet TPA induced growth arrest of MCF-7D3Res cells, which was associated with up-regulation of p21 and down-regulation of both Rb and p53. Furthermore, TPA partially sensitized the MCF-7D3Res cells to 1,25-(OH)2D3, suggesting that phosphorylation may play a role in dictating MCF-7 cell sensitivity to vitamin D3 mediated growth regulation. These data also suggest that cell cycle arrest is not sufficient to trigger apoptosis in MCF-7 cells, indicating that 1,25-(OH)2D3 generates additional signals which are necessary for the induction of cell death.
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Footnotes |
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Received June 20, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in renal epithelial cells. J Biol Chem 269:12562–12569
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