Download Progressive Resistance to Apoptosis in a Cell

Progressive Resistance to
Apoptosis in a Cell Lineage
Model of Human
Proliferative Breast Disease
Susan L. Starcevic, Cornelis
Elferink, Raymond F. Novak
Background: Proliferative breast disease (PBD) may increase a woman’s
risk of developing breast cancer, perhaps by decreasing cellular sensitivity
to apoptosis. To determine whether resistance to apoptosis develops during
PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the
morphologic changes of PBD in nude/
beige mice. Methods: The series of cell
lines used was MCF-10A cells (parental
preneoplastic human breast epithelial
cells), MCF-10AT cells (transformed
with T24 Ha-ras), and MCF-10ATG3B
cells (derivative cells that progress to
carcinoma). Fas-mediated apoptosis,
induced when a Fas monoclonal antibody bound to and activated the Fas
receptor on these cells, was assessed
morphologically and by flow cytometry. Levels of proteins involved in Fasmediated apoptosis and cleavage of
poly(adenosine diphosphate-ribose)
polymerase (PARP), an end product of
caspase activation, were determined by
immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical
tests were two-sided. Results: Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential
of cells: MCF-10A cells were extremely
susceptible, MCF-10AT cells were less
susceptible, and MCF-10ATG3B cells
were resistant. The percentage of apoptotic cells declined, from 24% to 8% to
6%, respectively. All lines produced
Fas ligand (FasL) and had comparable
levels of Fas receptor, FasL, Fasassociated death-domain protein, and
caspases 3 and 6. Levels of caspase 8
were similar in MCF-10A and MCF10AT cells but about 30% lower in
MCF-10ATG3B cells (P>.01 but <.05).
Levels of caspase 10 were about 20%
lower in MCF-10AT cells (P>.005 but
<.01) and about 59% lower in MCF10ATG3B cells than in MCF-10A cells
(P>.01 but <.05). PARP cleavage was
776 REPORTS
detected in MCF-10A and MCF-10AT
cells but not in MCF-10ATG3B cells.
Levels of Bax, Bid, and Bak proteins
were similar in all lines, but levels
of Bcl-2 were lower in MCF-10AT
and MCF-10ATG3B cells than in
MCF-A cells, and Bcl-2–Bax heterodimerization progressively declined
in the series. Conclusion: Resistance to
Fas-mediated apoptosis appears to develop progressively in the MCF-10AT
cell series. [J Natl Cancer Inst 2001;93:
776–82]
Breast cancer development is a multistep process that involves a sequence of
changes affecting critical homeostatic
pathways, such as those leading to apoptotic cell death. Apoptosis is critical to
cellular homeostasis, and the ability of
tumor cells to undergo apoptosis in response to various physiologic and therapeutic agents is decreased. Apoptosis mediated by Fas [also known as APO-1 and
CD95 (1)] involves ligand-induced oligomerization of the receptor and formation
of the death-inducing signal complex
(2), which includes the Fas-associated
death domain (FADD) (also referred to as
MORT1, i.e., mediator of receptorinduced toxicity 1) (3,4) and caspase 8
(3,5). Other members of the caspase family are activated and then cleave various,
functionally important cytosolic and
nuclear substrates, including poly(adenosine diphosphate [ADP]-ribose) polymerase [PARP (6)], which results in apoptotic
cell death.
Resistance to apoptosis and alterations
in Fas signaling have been observed in
breast carcinoma cell lines (7). Whether
resistance to apoptosis is uniquely associated with tumor cells or evolves in cells
well before the development of breast carcinoma, however, is unknown. To examine this question, we have used a cell
model of human proliferative breast disease (PBD). PBD is a term used to describe a sequence of progressive morphologic changes, including hyperplasia, that
Affiliation of authors: Institute of Environmental
Health Sciences, Wayne State University, Detroit,
MI.
Correspondence to: Raymond F. Novak, Ph.D.,
Institute of Environmental Health Sciences
(formerly the Institute of Chemical Toxicology),
2727 Second Ave., Wayne State University, Detroit,
MI 48201 (e-mail: [email protected]).
See “Notes” following “References.”
© Oxford University Press
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001
are observed in the breast before breast
cancer development and are associated
with a fourfold to fivefold increase in risk
for developing breast cancer (8). We
tested the hypothesis that selective resistance to apoptosis occurs progressively in
the development of PBD by investigating
Fas-mediated apoptosis in a series of preneoplastic human breast epithelial cell
lines [the MCF-10AT series: MCF-10A
(parental preneoplastic human breast epithelial cells) (9), MCF-10AT (transformed with T24 Ha-ras) (10,11), and
MCF-10ATG3B (derivative cells that
progess to carcinoma) (12)] that recapitulates the morphologic sequence of
changes of PBD when implanted in nude/
beige mice (12). Parental MCF-10A cells
do not form tumors when implanted
subcutaneously in nude/beige mice and
thus are eliminated. The MCF-10AT cell
line was generated by the insertion of a
mutated Ha-ras gene, T24 Ha-ras, into
MCF-10A cells (10,11). Although Ras
mutations are rare in human breast cancer,
60%–70% of primary human breast carcinomas express higher levels of Ras than
normal breast tissue (13–16). MCF-10AT
cells form small nodules when implanted
in vivo that persist for 1 year and sporadically progress to carcinomas (17). By the
continual re-establishment of the cells in
tissue culture from carcinomas, a cell line
designated MCF-10ATG3B was derived.
These cells form focal cribiforming ducts
within a month and progress to atypical
hyperplasia and ductal carcinoma in situ,
at a frequency of 25%–30%, and ultimately to invasive carcinoma (12) when
implanted in nude/beige mice.
MATERIALS
AND
METHODS
Cell Lines
The MCF-10AT cell series was obtained from
Dr. F. Miller (Karmanos Cancer Institute, formerly
the Michigan Cancer Foundation, Detroit, MI).
MCF-10A cells, the progenitor line of this series, are
spontaneously immortalized breast epithelial cells
obtained from a woman with fibrocystic breast disease (9). MCF-10A cells were transfected with a
mutated T24 Ha-ras gene to generate MCF-10AT
cells (10,11). Unlike the MCF-10A cells, MCF10AT cells persist as xenografts in nude/beige mice
and will develop into carcinomas in about 25% of
the animals (17). A family of MCF-10AT cell lines
was generated by re-establishing cells isolated from
the carcinomas in culture and subsequently reinjecting these cells into nude/beige mice (12). With ascending serial passage, the onset of PBD and the
development of invasive cancer appeared more
quickly after implantation. The MCF-10ATG3B cell
line was generated from cells that have been through
this process of transplantation in nude/beige mice
and re-establishment in culture three times. These
cells form focal cribiforming ducts within 1 month
and progress to ductal carcinoma in situ, at a frequency of 25%–30%, and ultimately progress to invasive carcinoma when implanted in nude/beige
mice (12).
Cells were maintained in a humidified environment of 5% CO2/95% air at 37 °C and cultured
in Dulbecco’s modified Eagle medium/F-12 medium (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) supplemented with 10 ␮g/mL of human
insulin (Life Technologies, Inc.), 20 ng/mL of epidermal growth factor (Life Technologies, Inc.),
100 ng/mL of cholera toxin (Life Technologies,
Inc.), 0.5 ␮g/mL of hydrocortisone (Sigma Chemical Co., St. Louis, MO), 5% horse serum (Life
Technologies, Inc.), 100 U/mL of penicillin
(Life Technologies, Inc.), and 100 ␮g/mL of streptomycin (Life Technologies, Inc.).
Anti-Fas-Mediated Apoptosis
All cells were grown to 85% confluence and were
treated with an activating mouse monoclonal antibody (mAb) (immunoglobulin [Ig] M; 1 ␮g/mL)
against the human Fas receptor (Fas mAb clone
CH11; Upstate Biotechnology, Lake Placid, NY)
that binds to the receptor initiating the Fas pathway.
Fas-mediated apoptosis was assessed morphologically and quantitatively by flow cytometry.
Morphologic assessment of apoptosis. Cells
were grown on chamber slides, treated with Fas
mAb for 15 hours, and then stained with acridine
orange and ethidium bromide as described by
Gorman et al. (18). Briefly, cells were rinsed with
phosphate-buffered saline (PBS) (i.e., 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium
phosphate, and 150 mM NaCl [pH 7.4]) and then
stained with acridine orange and ethidium bromide
(each at 4 ␮g/mL) for 2 minutes. Cells were viewed
by epifluorescence and were photographed with a
Nikon MicroPhot-SA camera (Nikon, Mager Scientific Inc., Dexter, MI).
Cytospin assessment of apoptosis. Medium
from Fas mAb-treated and untreated cells was
collected, fixed with an equal volume of neutral
buffered 4% formaldehyde, and centrifuged (200g
for 5 minutes at 4 °C) in a Sorvall Econospin cytocentrifuge (Du Pont, NEN, Wilmington, DE). Cells
were then stained with hematoxylin–eosin and
viewed by bright-field microscopy.
Flow cytometry. Cells were treated with Fas
mAb for 15 hours, harvested in 0.25% trypsin/0.1%
EDTA (in PBS), centrifuged for 5 minutes at 500g
at 4 °C, washed, and then resuspended in 1 mL
of propidium iodide staining solution (i.e., 50 ␮g/
mL of propidium iodide, 100 U/mL of ribonuclease
A, and 0.1% glucose) for 1 hour. Apoptotic cells
were quantitated on a FACScalibur (Becton Dickinson, San Jose, CA) flow cytometer. Red fluorescence (measured at 585/542 nm), indicative of propidium iodide uptake by damaged cells, was
measured by use of logarithmic amplification and
electronic compensation for spectral overlap.
Immunoblot Analysis and
Immunoprecipitation
bodies from Upstate Biotechnology) and Ras, Bcl-2,
and Bax (antibodies from Transduction Laboratories, Lexington, KY), Bid (antibody from R&D Systems, Inc., Minneapolis, MN), and PARP (antibody
from Clontech, Palo Alto, CA) in the three cell lines.
Cells were washed twice with PBS and then lysed in
50 mM HEPES (pH 7.2), 150 mM NaCl, 1.5 mM
MgCl2, 1.5 mM EGTA, 10% glycerol, 1% Triton
X-100, 1 mM MnCl2, 1 mM sodium orthovanadate,
leupeptin (10 ␮g/mL), 2 mM phenylmethylsulfonyl
fluoride, and 200 U of aprotonin. Cells were scraped
into lysis buffer, and the lysates were transferred
into Eppendorf tubes and passed through a 21-gauge
needle. Samples were incubated on ice for 1 hour,
and the lysates were clarified by centrifugation at
16 000g for 20 minutes at 4 °C. The supernatant is
termed the whole-cell lysate.
For immunoblot analysis, protein samples (20–
50 ␮g of protein per lane) from three dishes of
cells were resolved by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) on
a 7.5% or 15% gel, transferred to a nitrocellulose
membrane (Bio-Rad Laboratories, Inc., Hercules,
CA), and blocked for 2 hours in a solution containing Tris-buffered saline (TBS) (i.e., 20 mM Tris–
HCl and 500 mM NaCl [pH 7.5]), 5% milk powder,
and 0.05% Tween 20. For immunodetection, blots
were incubated with the appropriate primary antibody for 2 hours at room temperature, followed by
incubation with a secondary antibody conjugated to
horseradish peroxidase (diluted 1 : 10 000 in TBS
containing 1% milk powder and 0.05% Tween 20)
for 1 hour at room temperature.
Bax–Bcl-2 association was measured by coimmunoprecipitation with a Bcl-2 mAb (Santa Cruz Biotechnology, Santa Cruz, CA) and then by immunoblotting with Bax polyclonal antibody (Transduction
Laboratories). Whole-cell lysates (500 ␮g of protein) were precleared with 1 ␮g of mouse IgG and
20 ␮L of protein A–agarose (Santa Cruz Biotechnology) for 1 hour at 4 °C. Samples were transferred
to IMMUNOCATCHER spin filters (CytoSignal
Research, Irvine, CA) and were centrifuged at
16 000g for 1 minute at room temperature. Bcl-2
mAb at 1 ␮g/mL in lysis buffer was added to supernatants and was incubated for 1 hour at 4 °C.
Samples were then incubated with protein A–agarose overnight at 4 °C, centrifuged (16 000g for
1 minute at 4 °C) in spin filters, and washed three
times with lysis buffer. Antibody–protein complexes
were separated from the agarose beads by adding
40 ␮L of SDS–PAGE loading buffer (i.e., 62.5 mM
Tris–HCl [pH 6.8], 20% glycerol, 2% SDS, and 5%
2-mercaptoethanol), and immunoprecipitated proteins were separated by SDS–PAGE on 15% gels for
immunoblot analysis as described above. In the
reverse experiment, Bax–Bcl-2 complexes were immunoprecipitated with an antibody against Bax and
were immunoblotted for Bcl-2 as described above.
Proteins were detected by enhanced chemiluminescence (Amersham Life Science Inc., Piscataway,
NJ) on Kodak X-OMAT film (Sigma Chemical
Co.) and quantitated by densitometry with a laser
scanning densitometer (Molecular Dynamics, Sunnyvale, CA) and the ImageQuant (Molecular
Dynamics) analysis program.
Statistical Analysis
Immunoblot analysis of whole-cell lysates was
used to examine the protein levels of Fas receptor,
FADD, Fas ligand (FasL), caspases, and Bak (anti-
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001
Statistically significant differences (P<.05) between groups were determined by analysis of vari-
REPORTS 777
ance, followed by a Tukey–Kramer multiple comparisons analysis (19). The data were normally
distributed, and all statistical tests were two-sided.
RESULTS
Apoptosis in the MCF-10A,
MCF-10AT, and MCF-10ATG3B Cell
Lines
We examined Fas-mediated apoptosis
in the MCF-10AT series of human breast
epithelial cells. To examine apoptosis
morphologically, we treated adherent
cells (about 85% confluent, cycling) for
15 hours with Fas mAb that can induce
apoptosis in Fas-sensitive cells and then
stained the cells with ethidium bromide
and acridine orange to visualize apoptotic
cells and debris or collected the medium
for cytospin preparations. As shown in
Fig. 1, A, we observed many apoptotic
MCF-10A cells, fewer apoptotic MCF-10AT
cells, and no apoptotic MCF-10ATG3B
cells, the most aggressive line examined.
Cell shrinkage and membrane blebbing of
MCF-10A cells were evident as early as
8 hours after treatment with Fas mAb. As
shown in cytospin preparations in Fig. 1,
B, we observed extensive cellular debris
and cells containing apoptotic bodies
from MCF-10A cultures, less cellular debris from MCF-10AT cultures, and very
little cellular debris and no apoptotic bodies from MCF-10ATG3B cultures. Flow
cytometry was used to measure the percent of propidium iodide-stained cells in
the sub-G1 phase (indicative of apoptotic
cells) after a 15-hour treatment with Fas
mAb. As shown in Fig. 1, C, 24% of the
MCF-10A cells, 8% of the MCF-10AT
cells, and 6% of the MCF-10ATG3B cells
were in the sub-G1 population. The subG1 population represented only 0.5% of
the untreated cells from all three lines. A
limitation of the flow cytometry procedure is that, during preparation, cells are
isolated from the dishes and centrifuged
(500g for 5 minutes at 4 °C) before staining with propidium iodide, which results
in a substantial loss of cells undergoing
apoptosis. Thus, while visualization of
cells with acridine orange and ethidium
bromide and cytospin preparations is
qualitative, it may provide a more accurate indication for differential sensitivity
to apoptosis because fewer cells are lost
during processing.
Expression of FasL, Fas, FADD, Ras,
and Caspases
Fas-mediated apoptosis is initiated
when the FasL binds to the Fas receptor;
the death-inducing signal complex, which
includes FADD and caspase 8 (2), is
formed; and the caspase cascade is activated. We used immunoblot analysis to
examine levels of these proteins in the
three cell lines studied. FasL, expressed
primarily in lymphoid tissue, was detected at comparable levels in all three
cell lines, as were Fas receptor and FADD
(Fig. 2, A). Consequently, differential expression of FasL or receptor or FADD
proteins cannot explain the different sensitivities of these cell lines to Fas mAb.
We also examined Ras protein levels because T24 Ha-ras genes were transfected
into the MCF-10AT cells. The level of
Ha-Ras protein was 2.2-fold higher in
MCF-10AT cells (P>.01 but <.05) and
2.4-fold higher in MCF-10ATG3B cells
(P>.01 but <.05) than in MCF-10A cells,
with the differences being statistically
significant (Fig. 2, B).
Activation of the caspase cascade begins when active caspases cleave and thus
activate inactive caspases downstream.
We used immunoblot analysis to examine
the expression of caspases 8 and 10,
proximal members of the caspase family,
and of caspases 3 and 6, effector caspases.
Levels of caspase 8 were comparable in
MCF-10A and MCF-10AT cells but were
about 30% lower in Fas-resistant MCF10ATG3B cells (P>.01 but <.05). Levels
Fig. 1. Response of MCF-10A
(10A), MCF-10AT (10AT), and
MCF-10ATG3B (10ATG3B) cells
to Fas monoclonal antibody (Fas
mAb) treatment. Panel A: After a
15-hour incubation with Fas mAb,
cells were stained with ethidium
bromide and acridine orange. The
number of MCF-10A and MCF10AT cells decreased, and membrane blebbing was observed (arrows). MCF-10ATG3B cells treated
with Fas mAb were comparable to
untreated cells. Scale bar ⳱ 30 ␮m.
Panel B: Cytospin preparations of
culture medium from cells treated
with Fas mAb for 15 hours were
stained with hematoxylin–eosin.
MCF-10A and MCF-10AT culture
medium contained cellular debris
and apoptotic cells, demonstrating
nuclear fragmentation (arrows).
The medium from MCF-10ATG3B
cells contained very few cells and
little debris. The medium from untreated cells contained little debris.
Scale bar ⳱ 100 ␮m. Panel C: Flow
cytometry of propidium iodidestained untreated cells and cells treated with Fas mAb for 15 hours. Cells were stained with propidium iodide (50 ␮g/mL), and apoptotic cells were quantitated on
a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). Compared with untreated cells, MCF-10A cells treated with Fas mAb had an increased sub-G1
population, and MCF-10AT and MCF-10ATG3B cells treated with Fas mAb had an increased but smaller sub-G1 populations. The percentages of cells that are in
the sub-G1 population are indicated.
778 REPORTS
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001
Fig. 2. Levels of Fas ligand (FasL), Fas receptor (Fas), Fas-associated
death domain (FADD),
Ras, and caspase proteins. Proteins (50 ␮g
per lane) were separated
by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis on 7.5%
gels and transferred to
nitrocellulose membranes. Blots were
probed overnight with
the specific primary antibody, and the bound
antibody was detected
with the appropriate
horseradish peroxidaseconjugated secondary
antibody. Band density
was determined by use
of scanning laser densitometry and was quantified by use of the ImageQuant analysis program
(Molecular Dynamics,
Sunnyvale, CA). Statistical analyses were performed by an analysis of
variance followed by a
Tukey–Kramer analysis;
statistically significant P
values were less than
.05. All statistical tests
were two-sided. Panel
A: Levels of endogenous FasL, Fas receptor, and FADD in MCF-10A (10A), MCF-10AT (10AT), and
MCF-10ATG3B (10ATG3B) cell lines were determined by immunoblot analysis of whole-cell lysates. Panel
B: Ras protein levels were examined in the cell lines by immunoblot analysis. Ras protein levels increased
with progression through the cell series. Compared with levels in MCF-10A cells, levels of Ras protein were
2.2-fold higher in the MCF-10AT cells and 2.4-fold higher in the MCF-10ATG3B cells (P<.05). The Ras
blot was probed with an anti-␣-tubulin antibody as a loading control for equivalent protein content. The three
separate bands represent triplicate samples. Panel C: Levels of caspases 3, 6, 8, and 10 were examined in
the whole-cell lysates by immunoblot analysis. Levels of caspase 3 and 6 were comparable in all three lines.
Levels of caspase 8 were about 30% lower in the MCF-10ATG3B cells than in the MCF-10A and MCF10AT cells. Levels of caspase 10 were about 20% lower in the MCF-10AT cells and about 59% lower in the
MCF-10ATG3B cells than in the MCF-10A cells.
of caspase 10 were about 20% lower in
the MCF-10AT cells (P>.005 but <.01)
and about 59% lower in the MCF10ATG3B cells (P>.01 but <.05) than in
the parental MCF-10A cells. Levels of
downstream caspases 3 and 6 did not vary
appreciably in the three lines (Fig. 2, C).
Thus, caspase 8 and 10 levels are lower in
MCF-10ATG3B cells than in parental
MCF-10A cells and are associated with
the resistance of these cells to Fasmediated apoptosis.
PARP Cleavage as Evidence of
Caspase Activation
Essential cellular proteins cleaved and
inactivated by the caspase cascade include the nuclear protein PARP (116 kd),
which has been implicated in maintaining
genomic integrity and in participating in
the repair of DNA strand breaks (20). Because caspase activation initiates positive
feedback loops involving many caspases
that amplify the signal and also increases
the level of complexity, we used immunoblot analysis to examine whether PARP
cleavage, which produces an 85-kd cleavage product and is an end result of
caspase activation, occurred after treatment with Fas mAb in the three lines. After a 3.5-hour treatment with Fas mAb
(Fig. 3, A), the PARP cleavage product
was detected in the MCF-10A cells but
not in the MCF-10AT or MCF-10ATG3B
cells. After an 8-hour treatment, the
PARP cleavage product was detected in
MCF-10AT cells. However, even after a
25-hour treatment, the PARP cleavage
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001
product was not detected in MCF10ATG3B cells, but the level of parent
PARP was clearly decreased (Fig. 3, B).
Thus, Fas mAb-induced PARP cleavage
occurs readily in the MCF-10A cells and
after a lag in the MCF-10AT cells but
fails to occur in the MCF-10ATG3B cells,
a result that parallels the progressive resistance of these cells to apoptosis and
their ability to progress to PBD when implanted subcutaneously in nude/beige
mice.
Analysis of Bcl-2, Bax, Bak, and Bid
Expression
Members of the Bcl-2 family may
positively and negatively modulate the
Fas-signaling cascade. Consequently, we
examined levels of the Bcl-2 family members Bax, Bak, Bid, and Bcl-2 proteins in
all three lines to determine whether they
were involved in the loss of Fas sensitivity. Levels of proapoptotic Bax protein
were generally higher in the MCF10ATG3B cells than in the MCF-10A
cells, although the differences were not
statistically significant (Fig. 4, A). Levels
of proapoptotic Bak and Bid proteins
were comparable in all three lines (Fig. 4,
A). Levels of the antiapoptotic Bcl-2 protein decreased statistically significantly,
with the Fas-susceptible parent MCF-10A
cells having 51% more Bcl-2 than the
MCF-10AT cells (P>.01 but <.05) and
58% more Bcl-2 than the MCF10ATG3B cells (P>.005 but <.01) (Fig. 4,
B). Bcl-2 levels, however, were not statistically significantly different between
the MCF-10AT and MCF-10ATG3B cells.
Association of Bcl-2 and Bax
In vivo, Bcl-2 forms homodimers and
heterodimers with other Bcl-2 family
members, including Bax (21). When Bax
predominates, apoptosis is accelerated
and the death-repressor activity of Bcl-2
is inhibited. Consequently, we examined
the Bcl-2–Bax association in our series by
immunoprecipitating Bcl-2–Bax complexes in whole-cell lysates with Bcl-2
antibodies, followed by immunoblot
analysis for Bax. We readily detected
Bcl-2–Bax complexes in the MCF-10A
and MCF-10AT cells but not in the MCF10ATG3B cells (Fig. 4, C), and fewer
complexes were detected in the MCF10AT cells than in the MCF-10A cells. To
confirm that coimmunoprecipitation with
Bcl-2 antibodies was effective, the blot
was stripped and reprobed for Bcl-2.
Bcl-2 was detected in all three cell lines
REPORTS 779
Fig. 3. Poly(adenosine
diphosphate-ribose)
polymerase (PARP)
cleavage after treatment
with Fas monoclonal antibody (mAb). Proteins
(30 ␮g per lane) were
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 7.5% gels and
transferred to nitrocellulose membranes. Blots
were probed overnight
with anti-PARP antibody, and bound antibodies were detected
with a horseradish peroxidase-conjugated secondary antibody. Band
density was determined
with scanning laser densitometry. Panel A: Immunoblot analysis of
MCF-10A (A), MCF10AT (AT), and MCF10ATG3B (3B) cells
treated with Fas mAb
for 3.5 hours. The 85-kd
PARP cleavage product
was detected only in the
MCF-10A (A) cells (arrow). Panel B: Immunoblot analysis of the
time course in hours of PARP cleavage in the MCF-10AT (10AT) and MCF-10ATG3B (10ATG3B) cells.
PARP cleavage was observed in the MCF-10AT cells at 11 and 15 hours but was not detected in the
MCF-10ATG3B cells. However, in the MCF-10ATG3B cells, PARP expression was decreased, even in the
absence of a cleavage product at 25 hours. Equal loading was demonstrated by probing the same cell lysates
for ␣-tubulin.
with the same pattern of expression as
described previously (Fig. 4, B), i.e., decreased Bcl-2 levels in the MCF-10AT
and MCF-10ATG3B cells relative to the
parental MCF-10A cells. In the reverse
experiment in which complexes were immunoprecipitated with an antibody
against Bax and immunoblotted for Bcl-2,
Bax–Bcl-2 complexes were evident in the
MCF-10ATG3B cells, but levels were
lower than in the MCF-10A and MCF10AT cells (Fig. 4, B). Thus, the levels of
Bcl-2–Bax complex detected in the three
lines reflect the response of these cells to
Fas-mediated apoptosis and their ability
to progress to PBD in nude/beige mice.
DISCUSSION
Women with PBD, particularly those
with atypical hyperplasia, have a fourfold
to fivefold increased relative risk for developing breast cancer (8). We have used
the MCF-10AT series of human breast
epithelial cell lines, which reproduce the
sequence of pathologic changes observed
780 REPORTS
in human PBD when implanted in vivo in
nude/beige mice, to investigate the possibility that Fas-mediated apoptosis evolves
during PBD. With increased numbers of
serial passages in mice, the onset of PBD
and the development of invasive carcinoma appear earlier with these cells (12).
Because these cells were established in
culture from lesions representing successive transplant generations, alterations in
apoptosis and the signaling components
regulating apoptosis may reflect the
changes that occur in vivo during PBD.
The ability to study the apoptotic response of these genotypically related
cells, compared with unrelated cell lines,
is important because progression from
normal epithelia to preneoplastic epithelia
represents an early stage in the development of neoplastic disease. Our results
with the MCF-10AT series demonstrate
that, although protein levels of Fas receptor, FADD, FasL, Bak, Bax, Bid, and
caspases 3 and 6 are generally comparable in all three cell lines, the parent
MCF-10A cells rapidly undergo apoptosis
when exposed to an activating Fas mAb,
MCF-10AT cells undergo apoptosis after
delay or lag but are clearly susceptible to
Fas mAb, but the third-generation MCF10ATG3B cells were predominantly resistant to Fas-mediated apoptosis.
Fas-mediated apoptosis is initiated
when the FasL binds to the receptor. Although the FasL is expressed predominantly on cytolytic T cells (22), FasL
has also been identified in hepatoma and
lung cancer cells (23,24). We show that
these breast epithelial cells produce FasL
and that MCF-10A, MCF-10AT, and
MCF-10ATG3B cells have comparable
amounts of FasL. The observations that
breast epithelial cells express FasL and
the Fas receptor suggest that breast epithelial cells, like T cells, may use this
apoptotic pathway to regulate cell turnover. This observation is not entirely surprising, given that, physiologically, breast
epithelial cell proliferation increases
markedly during pregnancy and lactation,
followed by a dramatic reduction after
weaning (involution). This reduction in
cell numbers presumably involves apoptosis.
Although the cells in this lineage exhibit comparable levels of the Fas receptor, their responses to Fas mAb differ. It
has been reported (25,26) that the sensitivity of cells to Fas-induced apoptosis is
not always associated with the level of
expression of Fas at the cell surface. Because MCF-10ATG3B cells contain mutated T24 Ha-ras, it could be argued that
such a transformation caused the decreased Fas sensitivity in these cells. Immunoblot results, where Ha-Ras protein
levels were observed to increase progressively in this lineage, clearly support this
possibility. Fibroblasts containing oncogenic K-Ras are resistant to Fas-mediated
apoptosis through activation of the mitogen-activated protein kinase (27). Likewise, Fenton et al. (28) demonstrated resistance to Fas-mediated apoptosis in a
series of Ras-transformed cell lines.
Fas-receptor activation recruits FADD
and then caspase 8, which initiates the
caspase cascade that leads to proteolytic
cleavage of functionally important cellular enzymes, including PARP. PARP is
arguably the best characterized proteolytic substrate of the caspase cascade, being
cleaved in the final execution phase of
apoptosis. We readily detected PARP
cleavage in the MCF-10A cells 3.5 hours
after a Fas mAb treatment and in the
MCF-10AT cells after 8 hours, but we did
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001
Fig. 4. Levels of Bax, Bak, Bid, and
Bcl-2 proteins. Proteins (50 ␮g per
lane) were separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis on 15% gels and were
transferred to nitrocellulose membranes. Blots were probed overnight
with the corresponding primary antibody, and bound antibodies were detected with horseradish peroxidaseconjugated secondary antibodies.
Band densities were determined with
scanning laser densitometry and were
quantified with the ImageQuant
analysis program (Molecular Dynamics, Sunnyvale, CA). Statistical analyses were performed with an analysis
of variance followed by a Tukey–
Kramer analysis; P less than .05 was
considered to be statistically significant. All statistical tests were twosided. Panel A: Levels of Bax, Bak,
and Bid proteins were determined
from three dishes. Panel B: Bcl-2
immunoblot and densitometric quantitation in all three cell lines. Data are
the mean ± 95% confidence interval
(n ⳱ five dishes). The asterisk (*)
indicates statistically significantly
different from MCF-10A (10A) cells
(P<.05). Panel C: Coimmunoprecipitation analysis of Bcl-2–Bax association. Cell lysates (500 ␮g) were
immunoprecipitated (IP) with Bcl-2
or Bax antibodies; immunoprecipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 15% gels and were transferred to nitrocellulose
membranes. The resulting western blot (WB) was probed with antibodies against Bax or Bcl-2, as indicated.
Note the decreased Bcl-2–Bax heterodimerization in the MCF-10AT cells (10AT) compared with the
MCF-10A cells (10A) and the absence of the complex in the MCF-10ATG3B cells (10ATG3B) (top panel).
In the reverse experiment, the Bax–Bcl-2 complexes were evident in the MCF-10ATG3B cells, but levels
were lower than in the MCF-10A and MCF-10AT cells (bottom panel).
not detect it in MCF-10ATG3B cells after
25 hours of treatment. However, decreased levels of PARP were clearly observed at that time, which may indicate
a subsequent mechanism for continued resistance to apoptosis. Decreased levels
of PARP and a marked resistance to inducers of apoptosis have been demonstrated in HL-60 cells after treatment
with retinoic acid or dimethyl sulfoxide
(29). PARP levels are also decreased
during monocyte/macrophage and neutrophilic differentiation (30). Thus, these results suggest that, although the precise
role of PARP has yet to be defined,
decreased levels of PARP may be critical
for the development of resistance to apoptosis and for differentiation/dedifferentiation.
In addition to Fas, several other proteins play a major role in regulating apoptosis. For example, the Bcl-2 gene family contains many related and interacting
molecules with antiapoptotic (e.g., Bcl-2)
and proapoptotic (e.g., Bax, Bid, and Bak)
activities. In these cells, levels of Bax,
Bak, and Bid proteins were comparable in
all three lines. Levels of antiapoptotic
Bcl-2 protein were higher in Fas-sensitive
MCF-10A cells than in Fas-susceptible
MCF-10AT cells and Fas-resistant MCF10ATG3B cells. Although most reports
indicate that Bcl-2 levels are increased in
breast cancer cells, Leek et al. (31) and
Silvestrini et al. (32) have reported an association between loss of Bcl-2 and poor
clinical outcome in women with breast
cancer. An approximately equal amount
of Bcl-2–Bax dimerization was observed
in Fas-susceptible MCF-10A and MCF10AT cells, but less was observed in
Fas-resistant MCF-10ATG3B cells. Heterodimerization of Bcl-2 and Bax decreases the antiapoptotic capacity of
Bcl-2 (33,34). The progressive decline in
levels of Bcl-2–Bax heterodimers in the
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001
three lines tested is, therefore, consistent
with the cell’s acquired resistance to apoptosis, but other antiapoptotic members
of the Bcl-2 family such as Bcl-xL may
also participate.
In conclusion, we have shown that
MCF-10A cells are much more susceptible to Fas-mediated apoptosis than
MCF-10AT cells and that MF-10ATG3B
cells, which exhibit the most rapid development of atypical hyperplasia and carcinoma in situ when implanted in vivo, are
resistant to Fas-mediated apoptosis. This
resistance appears to be associated with
an increase in Ras expression, a progressive loss of Bcl-2–Bax heterodimer formation, decreased levels of caspases 8 and
10, and subsequently decreased levels of
PARP in MCF-10ATG3B cells.
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NOTES
Supported in part by Public Health Service grants
ES02521 (to R. F. Novak) and ES07800 (to C. Elferink) and by the Cell Culture Core and Imaging and
Cytometry Core from Center grant P30ES06639
from the National Institute of Environmental Health
Sciences, National Institutes of Health, Department
of Health and Human Services.
We thank Drs. Susan Bortolin, Susan Land, Kimberley Woodcroft, and Michael McCabe for their
helpful suggestions. We also thank Mr. Mark Cameron and Ms. Sarah Khodadadeh for their assistance
with the Flow Cytometry data and Ms. Jennifer
Ortwine for her assistance with graphics.
Manuscript received July 7, 2000; revised February 21, 2001; accepted March 2, 2001.
Journal of the National Cancer Institute, Vol. 93, No. 10, May 16, 2001