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Advances in Brief |
Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Recently, much progress has been made toward identifying survival mechanisms counteracting apoptosis in mammalian cells subjected to stressful environments such as DNA-damaging agents, hypoxia, and growth factor deprivation (reviewed in Ref. 6 ). For example, activation of Akt, a downstream mediator of PI-3-K, has been demonstrated to inhibit apoptosis in many cell types including fibroblasts (7) , neurons (8) , epithelial cells (9) , and hematopoietic cells (10) . PI-3-K-dependent phosphorylation of Akt can be triggered by cell-specific survival factors such as insulin, IGF-I, platelet-derived growth factor, and interleukin 3; there is also some evidence in mammary cells that EGF (11) and the extracellular matrix (12) can initiate Akt-dependent survival signaling. However, the existence of an alternative survival pathway has been suggested by the recent discovery, in the LNCaP prostate cancer cell line, of a potent antiapoptotic signal that is triggered by androgen, EGF, or serum and yet is functionally independent of both PI-3-K and Akt activation (13) .
In this report, we have used serum-free media and specific growth
factor supplementation to study antiapoptotic pathways in MCF10A and
derivative cells overexpressing c-Myc (MCF10A-Myc). When plated at a
subconfluent density in serum-free conditions for 48 h, 
35% of
MCF10A cells and 45% of MCF10A-Myc cells undergo cell death,
characterized by nuclear condensation and pyknosis. By culturing MCF10A
cells in a defined media containing specific combinations of
hydrocortisone, EGF, and insulin, we observed that the presence of
hydrocortisone provides the most potent antiapoptotic signal.
Furthermore, our results demonstrate that the apoptosis induced by
hydrocortisone withdrawal is not associated with changes in FasL or
CD95/FasR expression levels, nor is it inhibited by an anti-CD95/FasR
blocking antibody. Finally, we show that glucocorticoid treatment
protects mammary cells from apoptosis independently of the
antiapoptotic PI-3-K and Akt/protein kinase B signaling pathways,
suggesting the existence of a novel survival pathway triggered by GR
activation.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Western Analysis.
Cells (4 x 105
) were cultured in 3-cm wells and
then washed twice with ice-cold PBS, followed by the immediate addition
of 500 µl of 2x Laemmli buffer. Cell lysates were then collected by
scraping cells in Laemmli buffer with a rubber spatula and further
homogenized by multiple passes through an 18-gauge needle. Lysates were
then boiled for 5 min and electrophoresed either in 8, 10, or 12%
SDS-PAGE gels. After electrophoresis, proteins were blotted onto
nitrocellulose membranes (Osmonics, Minnetonka, MN), and equal protein
loading was verified with Ponceau S staining. Nitrocellulose was then
rinsed with Tris buffered saline (TBS)/0.1% Tween and incubated with
one of the following antibodies: rabbit polyclonal anti-Akt or
anti-phospho-Akt (recognizing phosphorylated serine 473; New England
Biolabs, Beverly, MA); mouse monoclonal 124 anti-Bcl2 oncoprotein (Dako
Corp., Carpenteria, CA); mouse monoclonal 9E10 anti-c-Myc, rabbit
polyclonal N-18 anti-CD95/FasR, C-20 anti-CD95/FasR, N-20 anti-Fas-L,
C-178 anti-Fas-L (Santa Cruz Biotechnology, Santa Cruz, CA), or mouse
anti-FADD (Transduction Laboratories, Lexington, KY). After incubation
with the primary antibody and washing in TBS/0.1% Tween, the
appropriate secondary antibody, either antimouse IgG-horseradish
peroxidase or anti-rabbit IgG-horseradish peroxidase (Zymed, San
Francisco, CA) was added. The nitrocellulose was then rinsed
extensively with TBS/0.1% Tween, incubated in ECL substrate according
to the manufacturer’s directions (Amersham, Arlington Heights, IL),
exposed to film, and developed.
Growth Factor Deprivation-induced Apoptosis.
Cells were trypsinized and then plated overnight in MEBM containing
0.5% FCS (Atlanta Biologicals, Norcross, GA); 24 h later, the
medium was changed to serum-free MEBM with hydrocortisone (0.5
µg/ml), EGF (10 ng/ml), and insulin (5.0 µg/ml) added. For
apoptosis assays, cells were trypsinized and then seeded subconfluently
at 2.0 x 105
cells/3-cm-diameter
plate. Twenty-four h later, cells were rinsed twice in PBS and cultured
for 24, 48, or 72 h in the presence of either hydrocortisone,
dexamethasone, EGF, insulin, or combinations of these growth factors at
the concentrations listed above. RU486 (5 x 10-7 M), a potent GR antagonist, was
also used in selected experiments as indicated (Sigma Chemical Co., St.
Louis, MO). After growth factor incubation, the cells were fixed with
37% formaldehyde and stained with DAPI as described previously
(7)
. Using a Zeiss Axioskop microscope with UV
illumination at x400, an average of 250 DAPI-stained cells/well was
scored to determine the percentage of apoptotic cells per experimental
population. Experiments were repeated at least three times to calculate
averages and SEs.
Anti-CD95/FasR Antibody-induced Apoptosis.
To investigate the role of the CD95/FasR in apoptosis, cells were
plated as described above. Mouse monoclonal anti-CD95/FasR stimulating
antibody CH11 (1 µg/ml; Upstate Biotechnology, Inc., Lake Placid, NY)
was cultured in the presence of all three growth factors with or
without pretreatment with an optimal blocking concentration (4 µg/ml)
of ZB4 Fas neutralizing antibody (Upstate Biotechnology, Inc., Lake
Placid, NY). In the experimental groups, mouse monoclonal ZB4 anti-Fas
neutralizing antibody was added to media lacking glucocorticoid or all
growth factors for 48 h to determine whether blockade of CD95/FasR
signaling inhibited MEC apoptosis. Control cells were treated with an
equivalent concentration of isotype-matched mouse IgG. All cells were
subsequently fixed, stained with DAPI, and scored for apoptosis.
Inhibition of PI-3-kinase Signaling.
The specific PI-3-kinase inhibitor wortmannin (Sigma Chemical Co., St.
Louis, MO) was used to investigate the role of PI-3-kinase in
glucocorticoid-induced protection from apoptosis. Cells were deprived
of all growth factors for 24 h and then stimulated with
hydrocortisone, EGF, or insulin after a 30-min pretreatment with
wortmannin (100 ng/ml in 0.01% DMSO/PBS) or vehicle alone (0.01%
DMSO/PBS). Cell lysates were harvested 2 h later, and
phosphorylation of Akt was assessed by Western analysis using a
phosphospecific anti-Akt serine 473 antibody; duplicate cultures were
scored for apoptosis at 8, 24, and 48 h.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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35% of
cells undergoing apoptosis, as characterized by nuclear blebbing and
chromatin condensation (Fig. 1A
, left panel). When added to serum-free media, neither
insulin nor EGF treatment inhibited apoptosis >15% (Fig. 1B)
; however, either hydrocortisone (Fig. 1B)
or
the synthetic glucocorticoid dexamethasone (Fig. 1A
, right
panel) provided potent inhibition of cell death. Cells were
observed for up to 1 week after treatment with glucocorticoid and
remained protected from apoptosis (data not shown). Thus,
glucocorticoids provide a potent survival signal to MCF10A cells grown
under serum-free conditions.
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, inset). Ectopic expression of c-Myc resulted in an
accelerated growth rate compared with the parent cell line (Fig. 1C)
. Overexpression of c-Myc also accelerated apoptosis
induced by hydrocortisone, insulin, and/or EGF withdrawal (Fig. 1D)
. However, only 10–20% of cells underwent apoptosis
after insulin or EGF withdrawal, whereas >40% of MCF10A-Myc cells
underwent programmed cell death after glucocorticoid deprivation. In
summary, overexpression of c-Myc accelerates both proliferation and
apoptosis in MCF10A cells, whereas glucocorticoid treatment provides a
potent survival signal in both c-Myc-overexpressing and the parent
MCF10A cells.
We next investigated the GR specificity of glucocorticoid action using
the GR antagonist RU486. Although the progesterone receptor can also be
antagonized by RU486, we could not detect any progesterone receptor
expression in MCF10A cells by Western analysis (data not shown). We
therefore treated MCF10A or MCF10A-Myc cells with glucocorticoid
(10-6 M) in the presence or absence
of RU486 (5 x 10-7
M). At this concentration, RU486 has been shown previously
to antagonize downstream transcriptional regulatory effects of the GR,
presumably through competition with glucocorticoid for GR binding
(19)
. As shown in Fig. 2A,
RU486 cotreatment effectively blocked the antiapoptotic
effects of glucocorticoid treatment. Furthermore, increasing
concentrations of dexamethasone (Fig. 2B)
could out compete
the proapoptotic effects of RU486. These results strongly suggest that
the glucocorticoid-induced survival pathway of MCF10A cells is mediated
specifically through activation of the GR.
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. Furthermore, CH11-induced apoptosis was inhibited after
pretreatment with an anti-FasR blocking antibody, ZB4 (Fig. 3A)
. However, cells incubated with the ZB4 blocking antibody
and then subjected to hydrocortisone withdrawal were not inhibited from
cell death (Fig. 3A)
, suggesting that glucocorticoid
withdrawal triggers apoptosis through a CD95/FasR-independent
mechanism.
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B activity (20)
and FasL has been identified as a NF-B transcriptional target
(21)
, we next examined whether glucocorticoid withdrawal
increases either FasL or CD95/FasR expression levels. Although both
MCF10A and MCF10A-Myc cell lines expressed detectable baseline levels
of FasL (Fig. 3B)
and its receptor CD95/FasR (Fig. 3C)
, expression of these proteins did not change after
glucocorticoid withdrawal and induction of apoptosis. Therefore, cell
death triggered by glucocorticoid withdrawal does not appear to
correlate with either cell surface activation of the FasR or changes in
expression levels of receptor or ligand.
The CD95/FasR and the other tumor necrosis factor receptor homologues
that contain a death domain in their cytoplasmic region (DR3 and TRAIL
receptors DR4 and DR5) use the FADD protein for signaling. Because FADD
has previously been implicated in the pathway of Myc-accelerated
apoptosis in fibroblasts subjected to serum deprivation
(22)
, it remained possible that glucocorticoid withdrawal
activates apoptosis through activation of a FADD-dependent mechanism.
Expression of DN-FADD failed to inhibit MCF10A cell death from
glucocorticoid withdrawal (Fig. 2D)
; however, a consistent
inhibition of accelerated apoptosis was seen in MCF10A-Myc cells
overexpressing DN-FADD. Thus, growth factor deprivation-induced
apoptosis appears to be independent of CD95/FasR signaling in MCF10A
cells; however, c-Myc-mediated acceleration of apoptosis in these cells
is inhibited by DN-FADD.
GR Signaling Acts through an Akt- and PI-3-K-independent Pathway.
Because Akt activation has proven to be a common survival signaling end
point, we tested the hypothesis that expression of an activated Akt
would protect against glucocorticoid withdrawal-induced apoptosis.
Myristylated Akt (MyrAkt-pBabePuro), a constitutively active form
of membrane-bound Akt (16
, 23)
, was introduced into MCF10A
and MCF10A-Myc cells. Control cells were transduced with the empty
pBabePuro retrovirus and selected in puromycin. Alternatively, cells
were transduced with Bcl2-pMV12, and hygromycin-resistant cells were
selected. Overexpression of MyrAkt or Bcl2 was verified by Western
analyses (Fig. 4A)
. Cell lines were then evaluated for inhibition of
apoptosis induced by glucocorticoid withdrawal. Expression of
constitutively active MyrAkt in either MCF10A-Myc (Fig. 4B)
or MCF10A cells (data not shown) did not inhibit apoptosis (Fig. 4B)
. However, ectopic overexpression of Bcl2 dramatically
inhibited apoptosis in response to survival factor withdrawal (Fig. 4B)
. Endogenous protein levels of Bcl2, or the Bcl2 family
member Bcl-XL, did not change after
glucocorticoid or serum withdrawal (data not shown). Thus,
glucocorticoid-induced survival signaling appears to act through an
Akt-independent pathway that is distal or parallel to the site of the
antiapoptotic function of Bcl2.
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,
suggesting that PI-3-K activity could be inhibited effectively in these
cells. Glucocorticoid treatment alone did not induce Akt
phosphorylation. Furthermore, wortmannin pretreatment of
glucocorticoid, insulin, EGF, or IGF-I-treated cells did not inhibit
cell survival at 24 h (Fig. 4D)
or up to 72 h
(data not shown). These results suggest that the GR-mediated survival
pathway functions independently of both PI-3-K and Akt signaling. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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In our experiments with MCF10A cells, the relatively slight inhibition
of apoptosis (10%) observed with the addition of either insulin or
EGF is consistent with previous studies (3)
conducted with
confluent samples of MCF10A cells. Glucocorticoid treatment alone,
however, could potently block apoptosis. Glucocorticoids also blocked
the accelerated apoptosis seen in MCF10A cells overexpressing c-Myc.
These findings are consistent with an earlier report in which
overexpression of another breast cancer-associated oncogene,
ErbB-2, sensitized the immortalized MEC line HB4A to
apoptosis from serum withdrawal; apoptosis could also be inhibited by
glucocorticoids, although the GR specificity or the mechanism of action
was not investigated (24)
. Together, these observations
suggest that initially oncogene overexpression may sensitize MECs to
apoptosis and that glucocorticoids may inhibit cell death through a
central mechanism. In support of this hypothesis, we have observed
generally higher rates of apoptosis in immortalized MEC lines than in
primary
MECs,5
suggesting that the oncogenic changes favoring immortalization may also
lead to sensitization of cells to apoptosis induced by growth factor
withdrawal.
The antiapoptotic effect of glucocorticoids on MECs is also consistent with earlier reports in an in vivo murine model of mammary gland remodeling after lactation in which glucocorticoid treatment inhibited involution and apoptosis (25 , 26) . In these studies, the major effect of glucocorticoid treatment was hypothesized to be transcriptional repression of genes such as stromelysin-1, gelatinase A, and urokinase-type plasminogen activator that are normally expressed in stromal cells during mammary gland involution. However, our data suggest that a direct effect of glucocorticoids on epithelial cell survival pathways may also contribute to the potent antiapoptotic and anti-involutional effects observed in in vivo models of mammary gland remodeling.
MCF10A cells and human breast cancer cell lines have been shown to
express the CD95/FasR and to die after CD95/FasR activation (27
, 28)
. In addition, the CD95/FasR pathway has been implicated in
the mechanism of c-Myc-accelerated apoptosis in fibroblasts (22
, 29)
. Interestingly, FasL expression has been shown recently to
be up-regulated by NF-B activity (21)
. NF-B
activity, in turn, can be repressed by glucocorticoid treatment
(20)
, suggesting that glucocorticoid withdrawal might
increase the expression of Fas, promoting apoptosis. Although the
CD95/FasR pathway is intact in MCF10A cells, apoptosis because of
growth factor withdrawal is independent of FasL or CD95/FasR expression
and signaling. However, acceleration of apoptosis by c-Myc
overexpression was inhibited by DN-FADD expression. This observation is
consistent with the results of c-Myc-induced apoptosis reported
previously in fibroblasts, in which it was shown that DN-FADD inhibits
c-Myc-accelerated apoptosis in response to serum withdrawal
(22)
.
Despite the previously described role of Akt activation in EGF and extracellular matrix-mediated survival signaling in breast cancer cell lines, we found here that the GR-mediated survival pathway in MECs is independent of both the PI-3-K and Akt/protein kinase B pathways. Wortmannin, a potent PI-3-K inhibitor, did not diminish the survival resulting from glucocorticoid treatment; furthermore, an activated Akt could not rescue cells from glucocorticoid withdrawal-induced apoptosis. However, PI-3-K-mediated signaling, resulting in phosphorylation of Akt by serum, insulin, and EGF, remained intact in MCF10A cells, implying a possible role for the PI-3-K-Akt pathway in mitogenesis of MCF10A cells.
In summary, we have identified a novel GR-mediated survival pathway in mammary epithelial cells operating independently of the well-described Fas and PI-3-K mechanisms. The critical downstream targets of glucocorticoid activation involved in GR-mediated signaling remain to be determined. Because RU486 efficiently inhibits the antiapoptotic effect of glucocorticoids, we hypothesize that transcriptional targets of the activated GR may execute the survival signal. The protection from apoptosis afforded by Bcl2 overexpression in cells deprived of glucocorticoid implies that Bcl2 can function proximal or parallel to the critical site of action of glucocorticoid withdrawal. Future efforts will be directed toward identifying the precise molecular mechanisms of glucocorticoid-mediated survival signaling in MECs as well as the role this pathway might play in normal and abnormal growth of breast epithelium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant R21 CA66132 and
the National Women’s Cancer Research Alliance. 
2 To whom requests for reprints should be
addressed, at Department of Medicine, Section of Hematology/Oncology,
MC 2115, University of Chicago, Chicago, IL 60637. Phone:
(773) 834-2604; Fax: (773) 834-2650; E-mail: sconzen{at}medicine.bsd.uchicago.edu
3 The abbreviations used are: MEC, mammary
epithelial cell; EGF, epidermal growth factor; IGF, insulin-like growth
factor; PI-3-K, phosphatidylinositol 3-kinase; FasL, Fas ligand; FasR,
Fas receptor; GR, glucocorticoid receptor; MEBM, mammary epithelial
basal medium; DAPI, 4',6-diamidino-2-phenylindole; NF, nuclear factor;
FADD, Fas-associated death domain; DN, dominant negative.
4 C. A. Mikosz and S. D. Conzen, manuscript in
preparation.
5 C. A. Mikosz and S. D. Conzen, unpublished
observations.
Received 10/27/99. Accepted 1/ 4/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B activity by glucocorticoids. Mol. Endocrinol., 12: 355-363, 1998.B. J. Biol. Chem., 274: 987-992, 1999.This article has been cited by other articles:
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