From the Program in Chemical Biology and Division of
Medical Oncology, Duke University, Durham, North Carolina 27710, the
§ Program in Electron Microscopy, Fred Hutchinson Cancer
Research Center, Seattle, Washington 98109, and the ¶ Institute
of Urology, University of Padova, Padova, Italy 35128
Received for publication, August 29, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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Despite the widespread clinical use of tamoxifen
as a breast cancer prevention agent, the molecular mechanism of
tamoxifen chemoprevention is poorly understood. Abnormal expression of
p53 is felt to be an early event in mammary carcinogenesis. We
developed an in vitro model of early breast cancer
prevention to investigate how tamoxifen and 4-hydroxytamoxifen may act
in normal human mammary epithelial cells (HMECs) that have acutely lost
p53 function. p53 function was suppressed by retrovirally mediated
expression of the human papillomavirus type 16 E6 protein. Tamoxifen,
but not 4-hydroxytamoxifen, rapidly induced apoptosis in p53( Apoptosis, or programmed cell death, is critical for embryogenesis
and for normal tissue homeostasis (1). Deregulated apoptotic signaling
is felt to contribute to human cancer and autoimmune disorders (2, 3).
Chemotherapeutic agents are also felt to exert many of their cytotoxic
effects by induction of apoptosis, and chemotherapy resistance
frequently correlates with resistance to apoptotic signaling (4).
Apoptosis is morphologically characterized by specific structural
changes including margination of chromatin, nuclear condensation, cell
shrinkage, and formation of apoptotic bodies (5). There is much
evidence that apoptotic signaling activates highly regulated and
specific proteolysis mediated by caspases (6). Caspases are a highly
conserved family of aspartic acid-specific proteases that are
synthesized as zymogens and are converted to active heterodimers by
proteolytic cleavage (7, 8). Activated caspases are thought to be
responsible, in part, for cellular changes that occur during the
execution phase of apoptosis such as DNA fragmentation, chromatin condensation, and formation of apoptotic bodies (9). A large body of
evidence supports a cascade model for effector caspase activation; a
proapoptotic signal culminates in release of mitochondrial cytochrome
c, resulting in activation of initiator caspases that, in
turn, activate effector caspases, resulting in cellular disassembly (9).
Recent evidence suggests that mitochondria play a central role in
apoptosis as integrators of cellular apoptotic signal transduction and
in amplification of the apoptotic response (10). Disruption of
mitochondrial electron transport and energy metabolism is recognized as
an early event in apoptosis and precedes the appearance of morphologic
changes characteristic of apoptosis (10). Mitochondrial dysfunction is
characterized by an increase in mitochondrial membrane permeability and
loss of membrane potential ( The estrogen agonist/antagonist tamoxifen is a triphenylethylene that
has been shown to act as both a chemotherapeutic agent for the
treatment of breast cancer and, more recently, as a breast cancer
chemoprevention agent. The Breast Cancer Prevention Trial demonstrated
a 45% reduction in breast cancer incidence among the participants who
took tamoxifen, 6 years after its inception (12). This was the first
study to demonstrate that a chemopreventive agent could reduce the
incidence of breast cancer. However, many questions surround the
results from the Breast Cancer Prevention Trial. 1) Did "true"
chemoprevention occur or were the benefits of tamoxifen due to ablation
of preclinical breast cancer? 2) Tamoxifen has been shown to induce
both growth arrest and apoptosis (13-15). Did tamoxifen act as a
cytostatic or cytotoxic agent in the Breast Cancer Prevention Trial?
p53 is a critical regulator of cell cycle control, and the high
frequency with which p53 is functionally inactivated in early human
breast cancer attests to its key role in preventing mammary carcinogenesis (16-18). Approximately 50% of all primary
node-negative breast cancers have deleted or mutated p53, and
individuals with germ line heterozygous mutations in p53, or
Li-Fraumeni's syndrome, demonstrate an increased risk of breast cancer
(19-21). Furthermore, aberrant expression of p53 in mammary epithelial
cells is a predictor of risk for the subsequent development of breast
cancer. 1) The accumulation of p53 protein (but not c-ErbB-2) in
benign breast lesions is a significant predictor for the subsequent
development of breast cancer (22, 23). 2) p53 protein is frequently
overexpressed (36%) in benign mammary epithelial cells obtained from
high risk women but not observed (0%) in low risk women (22). 3)
Aberrant expression of p53 in the setting of mammary hyperplasia is a
significant predictor of the subsequent development of breast cancer in
high risk women (23). These observations suggest that loss of p53 function may be an early event in breast carcinogenesis.
Although abnormal expression of p53 predicts a poor response to
tamoxifen chemotherapy, little is known about the fate of normal human
mammary epithelial cells
(HMECs)1 that acutely lose
p53 function during tamoxifen chemoprevention. We sought to model
tamoxifen chemoprevention in normal HMECs that have acutely lost p53
function. p53 function was suppressed utilizing retrovirally mediated
introduction of human papilloma type 16 (HPV-16) E6 protein (24). The
E6 protein of the cancer-associated HPV-16 binds to p53 and targets it
for degradation through the ubiquitin pathway (25, 26) and provides a
model for the isolated loss of p53 function.
Tamoxifen is extensively metabolized in vivo, producing
demethylated and hydroxylated derivatives that can be detected in patients receiving tamoxifen treatment (27-29). The major metabolites of tamoxifen are felt to be 4-hydroxytamoxifen and
N-demethyltamoxifen (27). Whereas certain metabolites are
biologically inactive, 4-hydroxytamoxifen has a higher affinity for the
estrogen receptor than tamoxifen in vitro (18, 30). The
ability of tamoxifen to inhibit proliferation has been extensively
studied. However, the molecular mechanism by which tamoxifen initiates
apoptosis is poorly understood. We sought to investigate whether
tamoxifen and its related metabolite, 4-hydroxytamoxifen, may induce
growth arrest and/or apoptosis in HMECs that have acutely lost p53
function as a model of anti-estrogen chemoprevention. Surprisingly, we observed that tamoxifen but not 4-hydroxytamoxifen induced
apoptosis in p53( Materials--
A 1.0 mM stock solution of tamoxifen
and 4-hydroxytamoxifen (Sigma) was prepared in 100% ethanol and stored
in opaque tubes at Cell Culture and Media--
Normal human mammary epithelial cell
(HMEC) strain AG11132 (M. Stampfer, 172R/AA7) was purchased from the
National Institute of Aging, Cell Culture Repository (Coriell
Institute) (32). HMEC strain AG11132 was established from normal tissue
obtained at reduction mammoplasty, has a limited life span in culture, and fails to divide after ~20-25 passages. AG11132 cells exhibit a
low level of estrogen receptor staining, characteristic of normal mammary cells. AG11132 was at passage 8 at the time of receipt. Cells
were grown in mammary epithelial cell basal medium (Clonetics, San
Diego, CA) supplemented with 4 µl/ml bovine pituitary extract (Clonetics), 5 µg/ml insulin (Upstate Biotechnology Inc., Lake Placid, NY), 10 ng/ml epidermal growth factor (Upstate Biotechnology Inc.), 0.5 µg/ml hydrocortisone (Sigma),
10 Cell Synchronization--
Approximately 2 × 106 p53(+) HMEC-LXSN or p53( Retroviral Transduction--
The LXSN16E6 retroviral vector
containing the HPV-16 E6 coding sequence (provided by D. Galloway) has
been described previously (24). AG11132 normal human mammary epithelial
cells (passage 9) were plated in four T-75 tissue culture flasks in
Standard Medium and grown to 50% confluency. Transducing virions from
either the PA317-LXSN16E6 or the control PA317-LXSN (without insert) retroviral producer line were added at a multiplicity of infection at
1:1 in the presence of 4 µg/ml Polybrene (Sigma) to log phase cells
grown in T-75 flasks. The two remaining T-75 flasks were not infected
with virus. After 48 h the 2 flasks containing transduced cells
and 1 flask with untransduced cells were selected with Standard Medium
containing 300 µg/ml G418. Cells were continued in G418 containing
medium for 1 week, until 100% of control untransduced cells were dead.
The 4th flask of unselected, untransduced parental control cells was
passaged in parallel with the selected, transduced experimental and
vector control cells. Parental AG11132 cells are designated HMEC-P, and
transduced cells expressing the HPV-16E6 construct are designated
p53( Western Blotting--
Preparation of cellular lysates and
immunoblotting were performed as described previously (34, 35). Equal
amounts of protein lysates (~100 µg of total protein) were loaded
on 10% polyacrylamide gels, and the gels were run and then
electroblotted (Hoeffer) at 80 mA for 45 min onto Hybond-ECL membrane
(Amersham Pharmacia Biotech). The membrane was blocked with 20% bovine
serum albumin (Sigma) in PBS overnight at RT and then incubated with a
1:100 dilution of mouse anti-human p53 (Oncogene Science Ab-2). The membrane was washed three to five times at RT with 250 ml of PBS containing 0.1% Tween and then incubated with either a horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) at a
1:35,000 dilution, or a 1:2000 dilution of horseradish
peroxidase-conjugated protein A (Sigma) for 1 h at RT. The blot
was washed again, and complexes were detected by ECL Western blotting
Detection Reagents (Amersham Pharmacia Biotech) as described by the manufacturer.
High Performance Liquid Chromatography (HPLC) Analysis of
Tamoxifen Metabolism--
p53(+) HMEC-P parental cells (passage 10),
p53(+) HMEC-LXSN vector controls (passage 10), p53( Growth Curves--
p53(+) HMEC-LXSN vector controls and p53( Detection of Apoptosis-annexin V Staining--
Annexin V-FITC/a
(Boehringer Ingelheim, Heidelberg, Germany) was used as per
manufacturer's recommendation with some modification. Approximately
5 × 105 p53(+) HMEC-LXSN or p53( Diphenylamine Assay--
2.5-5.0 × 105 cells
were plated per T-25 flask and were grown in Standard Medium. Tamoxifen
or 4-hydroxytamoxifen stock was added directly to the media on Day 0 to
bring the final concentration to 1.0 µM. Confluency did
not exceed 70%. Cells were trypsinized 0, 6, 12, 18, or 24 h
after treatment, washed in cold PBS, pelleted, and then resuspended in
100 µl of lysis buffer (5 mM Tris, pH 8.0, 20 mM EDTA, 0.5% Triton X-100) on ice for 15 min. The lysate was spun at 12,000 rpm for 30 min in a refrigerated
microcentrifuge. The supernatant was removed and transferred to
a second microcentrifuge tube. Both tubes were placed on ice,
and 1.0 ml of 0.5 N perchloric acid was added to the
nuclear pellet in the first tube and vortexed. Five hundred µl of 1 N perchloric acid was added to the cytoplasmic fraction in
the second tube and vortexed. Both tubes were spun at 12,000 rpm for 15 min. The supernatants were then discarded, and 1.0 ml of 0.5 N perchloric acid was added to the pellets. The tubes were
heated to 70 °C for 20 min to hydrolyze the DNA, then cooled to RT,
and 1.0 ml of diphenylamine solution (1.5 g of diphenylamine (Aldrich)
in 100 ml of glacial acetic acid, to which was added 1.5 ml of sulfuric
acid and 0.1 ml of 1.6% acetaldehyde on the day of use) was added. The
tubes were incubated 16-20 h at 30 °C. Absorbance was read
at 600 nm (36).
Transmission Electron Microscopy--
p53( Caspase Assays--
Activated caspase-3 and -9 were detected
utilizing the ApoAlertTM Caspase Fluorescent Assay Kit
(CLONTECH). p53( Assessment of Mitochondrial Changes--
Mitochondrial
transmembrane potential was measured by rhodamine 123 (Molecular
Probes) (38) and JC-1 red fluorescence (CLONTECH) (39). JC-1 mitochondrial aggregate formation was measured by JC-1 green
fluorescence (CLONTECH) (39). Relative
mitochondrial mass was measured by flow cytometry using 1 n-nonyl acridine orange (NAO; Molecular Probes) (40).
For rhodamine 123 staining, 1 × 106 cells/ml were
incubated at 37 °C in 0.5 mg/ml rhodamine 123. For JC-1 staining,
1 × 106 cells incubated with 10 µg/ml JC-1 for 10 min at 37 °C and were analyzed for red and green fluorescence. For
NAO staining, 1 × 106 cells were resuspended in 1.0 ml of 1.0 µM NAO in PBS. Fluorescence of individual
nuclei and whole cells was performed using a FACScan flow cytometer
equipped with an argon-ion laser at 488 nm and 250 milliwatts light
output and Lysis II software (Becton Dickinson Immunocytometry
Systems). Forward and side scatter were used to establish size gates
and exclude cellular debris. The excitation wavelength was 488 nm. The
observation wavelengths were 530 nm for green fluorescence and 585 nm
for red fluorescence. The red and green JC-1 fluorescence emissions
from each cell were separated and measured using the standard optics of
the FACScan. Ten thousand events were collected in list mode fashion,
stored, and analyzed on Multicycle AV software (Phoenix Flow Systems).
p53 Protein Suppression in HMECs--
Retrovirally mediated
expression of the HPV-16 E6 protein was utilized to suppress normal
intracellular p53 protein levels in HMECs. Western blots were performed
on p53(+) HMEC-LXSN vector controls (passages 10 and 18) and p53( HPLC Analysis of Tamoxifen Metabolism in Tamoxifen-sensitive and
-resistant HMECs--
p53(+) HMEC-P parental cells (passage 8), p53(+)
HMEC-LXSN controls (passage 10), early passage p53( p53( Tamoxifen but Not 4-Hydroxytamoxifen Induces Apoptosis in Early
Passage p53(
Annexin V exhibits anti-phospholipase activity and binds to
phosphatidylserine (41). Cells undergoing apoptosis acquire annexin
V-binding sites during apoptosis and provide a convenient method for
detection of cells undergoing apoptosis. We utilized FITC-conjugated
annexin V followed by FACS analysis to detect the presence or absence
of apoptosis in cells treated with tamoxifen. Ninety nine percent of
early passage p53(
Electron microscopy of tamoxifen-treated early passage p53(
The diphenylamine assay measures endonuclease-fragmented DNA present in
the cytoplasm of apoptotic cells (42). Time points were obtained from 0 to 24 h after treatment of the target cells with either 1.0 µM tamoxifen or 4-hydroxytamoxifen. Early passage p53(
These observations indicate that tamoxifen but not
4-hydroxytamoxifen induces apoptosis in early passage p53( Mitochondrial Ultrastructure in Tamoxifen-sensitive and -resistant
HMECs--
Changes in mitochondrial ultrastructure correlated with the
development of resistance to tamoxifen-induced apoptosis in p53(
Sequential changes in mitochondrial ultrastructure were identified in
tamoxifen-treated early passage p53( Caspase Activation in Tamoxifen-sensitive and -resistant
HMECs--
Caspases are thought to be responsible, in part, for
cellular changes that occur during apoptosis such as DNA fragmentation, chromatin condensation, and formation of apoptotic bodies. Effector caspases are constitutively expressed in their inactive form and are
activated through intracellular caspase cascades. We investigated the
relationship between caspase-9 and caspase-3 activation in tamoxifen-treated, apoptosis-sensitive and -resistant HMECs.
Recent evidence suggests that apoptosis can be triggered by inducing
mitochondrial release of cytochrome c. The initiator caspase, caspase-9, is activated by mitochondrial release cytochrome c and that in turn activates caspase-3 (9). Activation of
caspase-9 was observed in apoptosis-sensitive, early passage p53(
Caspase-3 is an active cell death protease involved in the execution
phase of apoptosis. A 5-fold activation of caspase-3 was observed in
apoptosis-sensitive, early passage p53( Relationship between Mitochondrial Transmembrane (
The relative mitochondrial mass was similar in p53(+) HMEC-LXSN cells
(passage 12) and early passage p53(
As measured by rhodamine 123 and JC-1 red fluorescence,
To Compare the Base-line Values of Apoptosis is a dynamic process that involves initiation by a
pharmacologic or DNA-damaging agent, activation of proteolytic enzymes,
and execution of characteristic morphologic changes. Mitochondria serve
as sensors and amplifiers of the apoptotic process (43). Diverse
apoptotic stimuli converge at the mitochondria resulting in activation
of the caspase proteolytic cascade that ultimately leads to cellular
disassembly (9). In this study, we present evidence that the acute
suppression of p53( We observe that early passage p53( To date, most studies have investigated tamoxifen action using human
breast cancer cell lines that contain complex chromosomal rearrangements and are a poor model for chemoprevention. Although the
importance of p53 as a tumor suppressor is well documented, little is
known about the fate of normal human cells that acutely lose p53
function in the context of tamoxifen chemoprevention. A majority of
cellular studies investigating the role of p53 in tamoxifen sensitivity
have been made in experimentally transformed cells lines or in cancer
cell lines. Loss of p53 function confers genetic instability, and
studies of p53 function in these model systems may be complicated by
mutations acquired subsequent to p53 inactivation. We observe that
p53( It has been observed recently that the acute loss of p53 function
results in enhanced sensitivity to apoptosis in normal human fibroblasts expressing HPV-16 E6, human placental cells expressing SV40
T antigen, and mouse embryonic fibroblasts isolated from p53 Tamoxifen has been extensively studied as a chemotherapeutic agent;
however, the mechanism of tamoxifen chemoprevention in normal mammary
tissue is poorly understood. Tamoxifen chemotherapy is felt to involve
the following mechanism: ligand-bound steroid receptors bind to
specific promoter elements and thereby activate or inhibit the
expression of target genes (47). This "genomic" mechanism of
tamoxifen action requires the presence of the estrogen receptor and
both transcription and translation. However, normal proliferating
luminal mammary epithelial cells exhibit both low levels of estrogen
receptor expression and estrogen binding (48). We observe that
tamoxifen, but not an equimolar concentration of 4-hydroxytamoxifen,
induces apoptosis in early passage p53( It is hypothesized that patients treated with tamoxifen may exhibit
de novo or acquired resistance through changes in drug metabolism. Recently it has been observed that there is an accumulation of 4-hydroxytamoxifen in primary breast cancers that have acquired resistance to tamoxifen chemotherapy (49). In addition, significantly higher levels of 4-hydroxytamoxifen, relative to tamoxifen, were observed in the plasma samples taken from the acquired resistance group
(49). These observations lead the investigators to hypothesize that
perhaps the appearance of increased levels of 4-hydroxytamoxifen could
account for tamoxifen resistance and are consistent with observations
in our in vitro system.
Although the tamoxifen metabolite, 4-hydroxytamoxifen, is detected in
the plasma and tissue of women treated with tamoxifen, we do not detect
4-hydroxytamoxifen in tamoxifen-treated HMECs by HPLC. This observed
lack of tamoxifen metabolism allows us to assess independently the
ability of tamoxifen and 4-hydroxytamoxifen to induce apoptosis. Since
1) normal mammary epithelial tissue exhibits low levels of estrogen
receptor expression and 2) treatment of p53( Recent evidence suggests that estrogens and perhaps anti-estrogens may
also act through nongenomic, calcium-mediated signaling pathways (47).
Estrogen has been shown to induce extremely rapid increases in
intracellular calcium and cAMP (50-52) and thereby activate
mitogen-activated protein kinase (52). We observe that induction of
apoptosis in early passage p53( Cumulatively, the data presented in this study support the hypothesis
that tamoxifen-mediated apoptosis in early passage p53()
HMEC-E6 cells as evidenced by characteristic morphologic changes,
annexin V binding, and DNA fragmentation. We observed that a
decrease in mitochondrial membrane potential, mitochondrial
condensation, and caspase activation preceded the morphologic
appearance of apoptosis in tamoxifen-treated early passage p53(
)
HMEC-E6 cells. p53(
) HMEC-E6 cells rapidly developed resistance to
tamoxifen-mediated apoptosis within 10 passages in vitro.
Resistance to tamoxifen in late passage p53(
) HMEC-E6 cells
correlated with an increase in mitochondrial mass and a lack of
mitochondrial depolarization and caspase activation following tamoxifen
treatment. We hypothesize that an early event in the induction of
apoptosis by tamoxifen involves mitochondrial depolarization and
caspase activation, and this may be important for effective chemoprevention.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m). Associated with this
decrease in
m, cytochrome c is translocated from the intermembrane compartment of the mitochondria to the cytosol.
Cytosolic cytochrome c forms an essential part of the "apoptosome," composed of cytochrome c, Apaf-1, and
procaspase-9 (11). This results in activation of procaspase-9 and that,
in turn, activates other caspases, such as caspase-3, to orchestrate the execution phase of apoptosis (10).
) HMECs. Tamoxifen-induced apoptosis was
associated with a fall in mitochondrial potential, mitochondrial
condensation, and caspase activation suggesting a critical role for
mitochondrial targeting in mediating sensitivity to tamoxifen-induced apoptosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Control cultures received equivalent
volumes of the ethanol solvent. Stocks were used under reduced light.
Cell culture plasticware was from Corning Glass (Corning, NY).
5 M isoproterenol (Sigma), 10 mM HEPES buffer (Sigma) (Standard Medium). G418 (Life
Technologies, Inc.) containing medium was prepared by the addition of
300 µg/ml of G418 to Standard Medium. Cells were cultured at 37 °C
in a humidified incubator with 5% CO2, 95% air.
Mycoplasma testing was performed as reported previously (33).
) HMEC-E6 cells were plated
in a T-75 flask on Day
5 in Standard Medium and grown for 4 days (Day
1). We previously observed that on Day
1 greater than 85% of the
cells that had become growth factor-depleted are in G1/0
phase, trypsinize without difficulty, and rapidly resume proliferation
in the presence of fresh Standard
Medium.2 Cells were
synchronized by this method prior to each experiment.
) HMEC-E6, and vector control clones are designated p53(+)
HMEC-LXSN.
) HMEC-E6 (passage
10), and p53(
) HMEC-E6 (passage 20) cells were treated for 2, 12, and 24 h with 0.1 µM [3H]tamoxifen
combined with 0.9 µM unlabeled tamoxifen. The cells were
then washed twice with ice-cold PBS, removed from the flask by scraping
into 5 ml of ice-cold PBS, and pelleted. The pellet was extracted twice
with 1 ml of methanol/ethyl acetate (1:3 v/v). The extracts were
combined, dried under a stream of argon, and redissolved using the same
solvent. Analysis of the extract was by HPLC using a C18 narrow bore
column (Vydac). The gradient used was as follows: 1) 75% solvent A
(aqueous 1% triethylamine (Aldrich)), 25% solvent B (acetonitrile
(Burdick & Jackson) containing 1% triethylamine) that was held
for 5 min after sample injection; 2) a linear gradient to 80% solvent
B over 15 min; and 3) a continuation of 80% solvent B for 10 min. The
flow rate was 0.3 ml/min, and 20 µl of extract, containing ~50,000
dpm, was injected. Samples were held in amber vials at 4 °C, handled
under low light conditions, and monitored with an on-line scintillation
detector (Packard Instrument Co.). Unlabeled tamoxifen and
4-hydroxytamoxifen were used as standards and monitored by UV absorption.
)
HMEC-E6 cells were plated in duplicate at 1 × 104
cells per 12-well tissue culture plates on Day
1 and allowed to
adhere. On Day 0 the medium was replaced with Standard Medium with or
without 1.0 µM tamoxifen or 4-hydroxytamoxifen. Untreated controls received an equivalent volume of ethanol solvent (0.1% final
concentration). Cells were trypsinized at 24-h time intervals and
counted in triplicate.
) HMEC-E6 cells
were plated in T-75 flasks on Day
1 and allowed to adhere. On Day 0 the medium was replaced with fresh Standard Medium, and tamoxifen or
4-hydroxytamoxifen was added for a final concentration of 1.0 µM. Untreated controls received an equivalent volume of
ethanol solvent (0.1%). Cells were harvested after 24 h (Day 1)
and did not exceed 25% confluency. For the tamoxifen-apoptosis time
course cells were treated on Day 0 with 1.0 µM tamoxifen
in Standard Medium and harvested 0, 1, 3, 6, 12, or 24 h after
treatment. Cells were trypsinized, washed in PBS, resuspended in
binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; filtered
through a 0.2-µm pore filter). Cell density was adjusted to 2-5 × 105 cells/ml. Five µl of recombinant human annexin
V-FITC/a (BMS306F/a) was added to 195 µl of cell suspension, and the
mixture was briefly mixed and incubated for 10 min at room temperature
in the dark. Cells were washed once and resuspended in 190 µl binding
buffer. Cells were analyzed by FACScan as described below.
) HMEC-E6 cells and
p53(+) HMEC-LXSN vector control cells were plated on Day
1 in 6-well
tissue culture plates. On Day 0 cells were treated with 1.0 µM tamoxifen or 4-hydroxytamoxifen for 0, 1, 3, 6, 12, 18, and 24 h. Cells were then fixed in half-strength Karnovsky's
fixative (37) for 6 h, rinsed in 0.1 M sodium
cacodylate buffer, and post-fixed in 1% collidine-buffered osmium
tetroxide. Dehydration in graded ethanol and propylene oxide was
followed by infiltration and embedding in Epon 812. Approximately
70-90-nm sections were stained using saturated aqueous uranyl acetate
and lead tartrate. Photographs were taken using a JEOL 100 SX
transmission electron microscope operating at 80 kV. Approximately 200 cells were surveyed per data point following treatment for the presence or absence of apoptosis.
) HMEC-E6 cells and p53(+)
HMEC-LXSN vector control cells were plated on Day
1 and treated with
1.0 µM tamoxifen or 4-hydroxytamoxifen in duplicate. Cells were trypsinized and counted, and 1 × 106 cells
were pelleted and frozen at
80 °C. On the day of assay, the pellet
was thawed, resuspended in 50 ml of chilled cell Lysis Buffer
(CLONTECH), and incubated on ice for 10 min. Cell
lysates were centrifuged for 3 min at 4 °C to remove debris, and
lysates were then transferred to a new microcentrifuge tube. Fifty µl of 2× Reaction Buffer (CLONTECH), with 10 µl of
1 M dithiothreitol, and 5.0 µl of either 1.0 mM caspase-3 substrate (DEVD-AFC; 50 µM final
concentration) or 5 mM caspase-9 substrate (LEHD-AMC; 250 µM final concentration) was added to each tube and
incubated for 1 h at 37 °C. To confirm the correlation between
protease activity and product formation, either 1.0 µl of
caspase-3 inhibitor (DEVD-CHO) or 2.0 µl of caspase-9 inhibitor
(LEHD-CHO) was added to the reaction mixture of an induced sample and
incubated for 1 h at 37 °C before adding the caspase-3 or
caspase-9 substrate, respectively. Samples were read in a Shimzdzu
RF-1501 spectrofluorophotometer with at a 400 nm excitation filter and
a 505 nm emission filter (caspase-3) or 380 nm excitation filter and a
460 nm emission filter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
HMEC-E6 transduced cells (passages 10 and 18) to determine the relative
levels of p53 protein expression. Expression of p53 protein was
observed in p53(+) HMEC-LXSN vector controls but was not detectable by
Western analysis in early or late passage p53(
) HMEC-E6 transduced
cells (Fig. 1).
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Fig. 1.
p53 protein expression is suppressed in HMECs
transduced with HPV-16 E6. p53(+) HMEC-LXSN vector controls (LXSN)
(passages 10 and 18) and p53( ) HMEC-E6-transduced cells (E6)
(passages 10 and 18) are analyzed for p53 protein expression as
described under "Experimental Procedures." Equal amounts of protein
lysate were loaded per lane. An unknown 45-kDa protein band was used as
a loading control.
) HMEC-E6 cells
(passage 10), and late passage p53(
) HMEC-E6 cells (passage 20) were
treated with 1.0 µM radiolabeled tamoxifen and analyzed
by HPLC at 24 h. There was no difference in tamoxifen metabolism
in p53(+) or p53(
) HMECs (data not shown). No tamoxifen was
metabolized to 4-hydroxytamoxifen, and all radioactivity was recovered
in the tamoxifen peak.
) HMEC-E6 Cells Exhibit Increased Tamoxifen
Cytotoxicity--
p53(
) HMEC-E6 cells and p53(+) HMEC-LXSN vector
controls were cultured in Standard Medium containing 1.0 µM tamoxifen or 4-hydroxytamoxifen. HPV-16 E6 inhibition
of p53 expression was associated with a marked increase in sensitivity
of early passage p53(
) HMEC-E6 cells (passage 10) to tamoxifen
cytotoxicity relative to early passage p53(+) HMEC-LXSN vector controls
(passage 10) (Fig. 2A). In
contrast, early passage p53(
) HMEC-E6 cells and p53(+) HMEC-LXSN
controls treated with 1.0 µM 4-hydroxytamoxifen exhibited
similar cytotoxicity (Fig. 2A). Late passage p53(
) HMEC-E6
cells (passage 18) were resistant to both tamoxifen and 4-hydroxytamoxifen (Fig. 2B). Although p53(+) HMEC-LXSN
vector controls (passage 16) demonstrated a decreased rate of
proliferation as they neared in vitro senescence, they
remained sensitive to tamoxifen and 4-hydroxytamoxifen-mediated growth
arrest (Fig. 2B). These observations suggest that the acute
loss p53 function by the targeted expression of HPV-16 E6 in HMECs is
associated with enhanced sensitivity to tamoxifen-mediated cytotoxicity
but not that of 4-hydroxytamoxifen. p53(
) HMEC-E6 rapidly acquire resistance to both tamoxifen and 4-hydroxytamoxifen cytotoxicity after
serial passaging in vitro.
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[in a new window]
Fig. 2.
Acute suppression of p53 protein
expression in HMECs is associated with increased tamoxifen but not
4-hydroxytamoxifen cytotoxicity. Growth curves of p53(+) HMEC-LXSN
vector controls passage 10 (A) and passage 16 (B)
and p53( ) HMEC-E6 cells at passage 11 (A), and passage 18 (B) treated with and without 1.0 µM tamoxifen
(TAM) and 4-hydroxytamoxifen (4-OH) are shown.
Cells were plated on Day
1 in Standard Medium in duplicate at 1 × 104 cells per well and treated on Day 0 with 0 or 1.0 µM tamoxifen or 4-hydroxytamoxifen. Untreated controls
received an equivalent volume of ethanol (0.1% final concentration).
Cells were trypsinized and counted in triplicate. These data are
representative of three separate experiments.
) HMEC-E6 Cells--
We investigated the mechanism by
which tamoxifen might potentiate increased cytotoxicity in early
passage p53(
) HMEC-E6 cells. We observed that early passage p53(
)
HMEC-E6 cells treated with tamoxifen underwent apoptosis as evidenced
by annexin V binding, characteristic morphologic changes, and by the
presence of fragmented cytoplasmic DNA. In contrast, early passage
p53(
) HMEC-E6 cells did not demonstrate evidence of apoptosis when
treated with 4-hydroxytamoxifen.
) HMEC-E6 cells (passage 10) treated with 1.0 µM tamoxifen for 24 h demonstrated evidence of
apoptosis as demonstrated by annexin V staining (Fig.
3D). In contrast, early
passage p53(
) HMEC-E6 cells did not undergo apoptosis by this measure
when treated with 1.0 µM 4-hydroxytamoxifen (Fig.
3E). In addition p53(+) HMEC-LXSN vector controls (passage 10) and late passage p53(
) HMEC-E6 cells (passage 18) were resistant to tamoxifen-mediated apoptosis in vitro as evidenced by
lack of annexin V binding (Fig. 3, B and G).
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Fig. 3.
Tamoxifen but not 4-hydroxytamoxifen induces
apoptosis in early passage HMECs lacking p53 expression.
A and B, p53(+) HMEC-LXSN vector controls
(LXSN); C E, early passage p53(
) HMEC-E6 cells
(passage 10) (E6(E)); and F and G,
late passage p53(
) HMEC-E6 cells (passage 18) (E6(L)) and
are treated with 1.0 µM tamoxifen (TAM)
(B, D, and G) or 1.0 µM
4-hydroxytamoxifen (OHT) (E) for 24 h.
Untreated control cells (A, C, and F) received an
equivalent volume of ethanol. Detection of apoptotic cells was with
FITC-conjugated annexin V as described under "Experimental
Procedures." These data are representative of three
experiments.
) HMEC-E6
cells (passage 10) revealed morphologic changes characteristic of the
effector phase of apoptosis including margination of chromatin, cell
shrinkage, and formation of apoptotic bodies (5). Margination of
chromatin was the first morphologic change detected and was observed
12 h after treatment with 1.0 µM tamoxifen (Fig.
4E). After 24 h, 99% of
cells exhibited cell shrinkage, condensed chromatin, and formation of
apoptotic bodies (Fig. 3D). In contrast, early passage
p53(
) HMEC-E6 cells treated with 1.0 µM
4-hydroxytamoxifen did not exhibit evidence of apoptosis by morphologic
criteria (data not shown). In addition, neither p53(+) HMEC-LXSN vector controls (passage 10) nor late passage p53(
) HMEC-E6 cells (passage 21) demonstrated morphologic evidence of apoptosis after treatment with
1.0 µM tamoxifen for 24 h (Fig. 4, B and
H, and data not shown).
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Fig. 4.
Morphologic evidence of apoptosis in
tamoxifen-treated but not in 4-hydroxytamoxifen-treated early passage
p53( ) HMEC-E6 cells or in tamoxifen-treated p53(+) HMEC-LXSN vector
controls or late passage p53(
) HMEC-E6 cells. A and
B, electron micrographs of early passage p53(+) HMEC-LXSN
vector control cells (passage 10) (LXSN) treated with
(B) and without (A) 1.0 µM
tamoxifen (TAM) for 24 h do not demonstrate apoptosis
as evidenced by absence of cell shrinkage, presence of normal
chromatin, and absence of apoptotic bodies. C-F, early
passage p53(
) HMEC-E6 cells (passage 10) (E6(E)) treated
with and without 1.0 µM tamoxifen. D, at
1 h after treatment there is no evidence of apoptosis in
tamoxifen-treated early passage p53(
) HMEC-E6 cells. E, by
12 h treatment with tamoxifen, early passage p53(
) HMEC-E6 cells
exhibit margination of chromatin (m). F,
tamoxifen-treated early passage p53(
) HMEC-E6 cells exhibit
morphologic evidence of apoptosis based on the presence of cell
shrinkage (s), formation of apoptotic bodies containing
cellular organelles (ap), and margination of chromatin
(m). G and H, in contrast, late
passage p53(
) HMEC-E6 cells (passage 21) do not undergo apoptosis
when treated with tamoxifen. Late passage p53(
) HMEC-E6 cells
(E6(L)) are treated with (H) and without
(G) tamoxifen. Late passage p53(
) HMEC-E6 cells exhibit a
qualitative increase in the number of mitochondria relative to p53(+)
HMEC-LXSN controls and early passage p53(
) HMEC-E6 cells
(G) and do not undergo apoptosis when treated with 1.0 µM tamoxifen as evidenced morphologic criteria
(H). Magnification is × 1500.
)
HMEC-E6 cells (passage 12) treated with tamoxifen demonstrated increased DNA fragmentation starting at 18 h (Fig.
5B). The percentage of
fragmented DNA for tamoxifen-treated early passage p53(
) HMEC-E6 cells was 17% at 18 h and 56% at 24 h. In contrast, early
passage p53(
) HMEC-E6 cells treated with 4-hydroxytamoxifen did not
demonstrate fragmented DNA (Fig. 5B). In addition, no
significant increase in DNA fragmentation was observed in p53(+)
HMEC-LXSN vector controls (passage 12) and late passage p53(
) HMEC-E6
cells (passage 18) treated with 1.0 µM tamoxifen for
24 h (Fig. 5, A and B).
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Fig. 5.
Increased cytoplasmic DNA fraction provides
evidence of apoptosis in tamoxifen-treated but not in
4-hydroxytamoxifen-treated early passage p53( ) HMEC-E6 cells.
p53(+) HMEC-LXSN vector controls (LXSN) (passage 12 and
passage 16) (A) and p53(
) HMEC-E6 cells (E6) (passage 10 and passage 18) (B) were treated for 0, 6, 12, 18, and
24 h with 1.0 µM tamoxifen (TAM) or
4-hydroxytamoxifen (4OHT). Cells were treated in triplicate.
Cytoplasmic DNA was determined by the diphenylamine assay as described
under "Experimental Procedures." This experiment is representative
of three separate experiments.
) HMEC-E6
cells. Expression of HPV-16 E6 appears to sensitize HMECs to apoptosis as p53(+) HMEC-LXSN cells do not undergo apoptosis. Resistance to
tamoxifen-mediated apoptosis develops rapidly (within 10 passages) as
apoptosis is not observed in tamoxifen-treated late passage p53(
)
HMEC-E6 cells.
) HMEC-E6 cells. Mitochondria in untreated p53(+) HMEC-LXSN cells (passage 10) appear similar to those in untreated early passage p53(
)
HMEC-E6 cells (passage 10) (Fig. 6,
A and C). Electron micrographs depict oval-shaped
mitochondria with a finely granular matrix. Cristae are few in number.
In contrast, mitochondria in apoptosis-resistant, late passage
untreated p53(
) HMEC-E6 cells (passage 21) are increased in number,
are longer, and exhibit a branched morphology (Fig. 6G).
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Fig. 6.
Mitochondrial changes in apoptosis-sensitive
and -resistant tamoxifen-treated cells. High magnification
electron microscopy images of p53(+) HMEC-LXSN (passage 10)
(LXSN) (A and B), apoptosis-sensitive,
early passage p53( ) HMEC-E6 cells (passage 10) (E6(E))
(C-F), and apoptosis-resistant, late passage p53 HMEC-E6
cells (passage 21) (E6(L)) (G and H) treated with
(B, D-F and H) and without (A,
C, and G) 1.0 µM tamoxifen
(TAM). p53(
) HMEC-LXSN cells do not exhibit mitochondrial
changes after 24 h of tamoxifen treatment (B).
Mitochondria in early passage p53(
) HMEC-E6 cells treated with
tamoxifen exhibit mitochondrial matrix condensation starting at 1 h (D). At 6 h after tamoxifen treatment, mitochondria
exhibit increased mitochondrial matrix condensation (E) that
is evident also at 24 h (F). Mitochondria in
apoptosis-resistant, late passage p53(
) HMEC-E6 are increased in
number and exhibit a branching morphology (G). There
is no evidence of mitochondrial condensation in late passage p53(
)
HMEC-E6 cells after 24 h treatment with 1.0 µM
tamoxifen. Magnification is × 20,000.
) HMEC-E6 cells undergoing
apoptosis but not in tamoxifen-treated p53(+) HMEC-LXSN cells nor in
late passage p53(
) HMEC-E6 cells. One hour after treatment with 1.0 µM tamoxifen the mitochondrial matrix in early passage
p53(
) HMEC-E6 cells (passage 10) is condensed, and the cristae are
well formed, transversing the mitochondrion (Fig. 6D). Six
hours following treatment with tamoxifen, mitochondria in early passage
p53(
) HMEC-E6 cells exhibit further morphologic changes. Mitochondria
are small, and the outer mitochondrial membrane appears indistinct, and
internal structures are obscured by highly electron-dense material
(Fig. 6E). Overall, these changes are consistent with
mitochondrial matrix condensation and volume loss. After 6 h of
treatment, nuclear chromatin is normal, and there is no morphologic
evidence of apoptosis (data not shown). At 12 h, a majority of
cells exhibited margination of chromatin and mitochondrial condensation
(Fig. 4E and data not shown). In contrast, tamoxifen-treated
p53(+) HMEC-LXSN vector controls (passage 10) and late passage p53(
)
HMEC-E6 cells (passage 21) do not exhibit changes in mitochondrial
morphology after 24 h of treatment (Fig. 6, B and
H). These observations indicate that mitochondrial matrix condensation, starting at 1 h, precedes execution phase
morphologic changes in early passage p53(
) HMEC-E6 cells treated with
tamoxifen but not 4-hydroxytamoxifen.
)
HMEC-E6 cells (passage 11) treated with 1.0 µM tamoxifen
but not 1.0 µM 4-hydroxytamoxifen for 6 h (Fig.
7A). In contrast, p53(+)
HMEC-LXSN vector controls (passage 12) and apoptosis-resistant, late
passage p53(
) HMEC-E6 cells (passage 19) did not exhibit caspase-9
activation when treated with tamoxifen (Fig. 7A). Caspase-9
was maximally activated 1 h after tamoxifen treatment in early
passage p53(
) HMEC-E6 cells and correlated with appearance of
mitochondrial condensation first detected by electron microscopy at
1 h. Caspase-9 activation significantly preceded the detection of
apoptosis by annexin V binding at 12 h (data not shown) and the
detection of chromatin condensation, cell shrinkage, and formation of
apoptotic bodies at 24 h (Fig. 7B). These observations
indicate that casapse-9 activation may be an early event in
tamoxifen-induced apoptosis.
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Fig. 7.
Caspase-9 activity increases during tamoxifen
treatment in early passage p53( ) HMEC-E6 undergoing apoptosis.
A, caspase-9 activity in tamoxifen-treated (1.0 µM) apoptosis-sensitive, early passage p53(
) HMEC-E6
cells (passage 11) (E6(E)), p53(+) HMEC-LXSN vector controls (passage
12) (LXSN), and apoptosis-resistant, late passage p53(
) HMEC-E6 cells
(passage 19) (E6(L)) at 6 h. B, caspase activity in
early passage p53(
) HMEC-E6 cells (passage 10) treated with either
1.0 µM tamoxifen (Tam) or 1.0 µM
4-hydroxytamoxifen (4OHT) for 0-24 h. The negative control
was obtained by pretreatment with the caspase-9 inhibitor LEHD-CHO.
Experiments were performed in duplicate. Data are an average of three
separate experiments.
) HMEC-E6 cells (passage 11)
treated with 1.0 µM tamoxifen but not 1.0 µM 4-hydroxytamoxifen for 24 h (Fig.
8A). In contrast,
apoptosis-resistant, late passage p53(
) HMEC-E6 cells (passage 19)
did not exhibit activation of caspase-3 when treated with tamoxifen
(Fig. 8A). We next tested whether the temporal activation of
caspase-3 correlated with morphologic changes observed in early passage
p53(
) HMEC-E6 cells undergoing tamoxifen-mediated apoptosis. Caspase
activation was first detected at 12 h after tamoxifen treatment
and preceded the detection of apoptosis by electron microscopy at
24 h (Fig. 8B).
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Fig. 8.
Caspase-3 is activated during
tamoxifen-mediated apoptosis in p53( ) HMEC-E6 cells.
A, caspase-3 activity in apoptosis-sensitive, early passage
p53(
) HMEC-E6 cells (passage 10) (E6(E)) and late passage
p53(
) HMEC-E6 cells (passage 19) (E6(L)) treated with
either 1.0 µM tamoxifen (Tam) or 1.0 µM 4-hydroxytamoxifen (4OHT) for 24 h.
B, caspase-3 activity in early passage p53(
) HMEC-E6 cells
treated with 1.0 µM tamoxifen for 0-24 h. Negative
controls were obtained by omitting the DEVD-AFC from the reaction
mixture or by pretreatment with the caspase-3 inhibitor DEVD-CHO. Data
are an average of three separate experiments.
m)
Potential and Sensitivity to Tamoxifen-induced
Apoptosis--
Disruption of mitochondrial electron transport is an
early feature of apoptosis. The mitochondrial abnormalities observed by
electron microscopy during tamoxifen-induced apoptosis were further
evaluated using fluorescent measures of mitochondrial mass and membrane
potential. Mitochondrial mass was measured by staining with NAO, a
fluorescent dye that specifically binds to the mitochondrial inner
membrane independent of the transmembrane potential (40). To measure
mitochondria potential (
m), cells were stained with
rhodamine 123 (38) or the J-aggregate forming cationic dye JC-1 (39).
JC-1 is a dye that normally exists as a monomer emitting green
fluorescence. JC-1 is taken up by mitochondria and in response to the
mitochondrial membrane potential forms multimers, which then emit red
fluorescence (39). Decreasing mitochondrial transmembrane potential
results in a decrease in JC-1 red fluorescence and an increase in JC-1
green fluorescence.
) HMEC-E6 cells (passage 11)
(1.0 ± 0.05 and 0.92 ± 0.07, respectively) (Table I). These data are consistent with the
observation made by electron microscopy that there is a qualitative
increase in the number of mitochondria in apoptosis-resistant p53(
)
HMEC-E6 cells relative to apoptosis-sensitive p53(
) HMEC-E6 cells and
p53(+) vector controls (Fig. 4). These observations suggest that an
increase in mitochondrial mass may be associated with the development
of resistance to tamoxifen-induced apoptosis.
Base-line mitochondrial membrane potential (m) is
decreased in early passage p53(
) HMEC-E6 cells relative to p53(+)
HMEC-LXSN vector controls
m) is measured by rhodamine 123 staining and by JC-1 (red) fluorescence and normalized to mitochondrial
mass, measured by NAO staining. Mitochondrial mass in
apoptosis-resistant, late passage p53(
) HMEC-E6 cells (passage 21) is
relative to p53(+) HMEC-LXSN vector controls (passage 11) and
apoptosis-sensitive, early passage p53(
) HMEC-E6 cells (passage 11).
Base-line mitochondrial potential normalized to mitochondrial mass
(JC-1 red (
m)/NAO or rhodamine (
m)/NAO
fluorescence) in p53(
) HMEC-E6 cells is relative to p53(+) HMEC-LXSN
vector controls. Fluorescent values are reported relative to
ethanol-treated controls. Reported values represent the average of
three separate experiments. Tam, tamoxifen.
m
did not decline in p53(+) HMEC-LXSN or late passage p53(
) HMEC-E6
cells (passage 19) after treatment with 1.0 µM tamoxifen for 6 h, and there was no change in JC-1 green fluorescence in late passage p53(
) HMEC-E6 cells treated with 1.0 µM
tamoxifen for 6 h (Fig. 9,
G and H). However,
m declined by
17-20% after early passage p53(
) HMEC-E6 cells (passage 11) were
treated with 1.0 µM tamoxifen (Table I). JC-1 red
fluorescence decreased 32-66% after 3 and 6 h of tamoxifen
treatment (data not shown). Increased JC-1 green fluorescence was
observed in these cells at 1, 3, and 6 h (Fig. 9, B-D)
in association with the decreased JC-1 red fluorescence. In contrast,
treatment of early passage p53(
) HMEC-E6 cells treated with 1.0 µM 4-hydroxytamoxifen showed no change in either
rhodamine 123 and JC-1 red fluorescence (data not shown) or JC-1 green
fluorescence (Fig. 9, E and F). p53(+) HMEC-LXSN
vector controls (passage 12) and apoptosis-resistant p53(
) HMEC-E6
cells (passage 19) did not undergo apoptosis when treated with
tamoxifen and did not exhibit a decline in
m. Changes in
m observed at 1 h in tamoxifen-treated early passage
p53(
) HMEC-E6 cells (passage 11) correlate with the detection at
1 h of mitochondrial condensation by electron microscopy (Fig. 6)
and caspase-9 activation (Fig. 7).
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Fig. 9.
Decreased JC-1 aggregates are observed in
tamoxifen-treated early passage p53( ) HMEC-E6 cells. Early
passage p53(
) HMEC-E6 cells (passage 11) (E6(E)) treated
with 1.0 µM tamoxifen (TAM) for 0, 1, 3, and
6 h (A-D, respectively) and 1.0 µM
4-hydroxytamoxifen (OHT) for 6 and 12 h (E
and F). Apoptosis-resistant, late passage p53(
) HMEC-E6
cells (passage 19) (E6(L)) treated with (H) and
without (G) 1.0 µM tamoxifen for 6 h.
m in
apoptosis-sensitive and -resistant cells,
m was
normalized to mitochondrial mass (
m/NAO fluorescence).
The normalized values of
m were decreased in both
untreated apoptosis-sensitive (passage 11) and untreated
apoptosis-resistant (passage 19) p53(
) HMEC-E6 cells relative to
p53(+) HMEC-LXSN vector control cells (passage 12) (Table I). The
decreased base-line
m and normal mitochondrial content in
apoptosis-sensitive p53(
) HMEC-E6 cells correlates with sensitivity
to tamoxifen-induced apoptosis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) function in HMECs, mediated by HPV-16 E6,
results in increased sensitivity to tamoxifen-induced apoptosis via a
signaling pathway that involves mitochondrial depolarization and
caspase activation. In contrast, the related metabolite,
4-hydroxytamoxifen fails to induce either mitochondrial depolarization
or caspase activation and subsequently does not induce apoptosis.
) HMEC-E6 cells treated with
tamoxifen for 12-24 h exhibit morphologic and biochemical changes
characteristic of the execution phase of apoptosis. These apoptotic
changes are preceded by mitochondrial matrix condensation, mitochondrial depolarization (
m), and caspase-9
activation, all first observed 1 h after tamoxifen treatment.
p53(
) HMEC-E6 cells rapidly developed resistance to tamoxifen-induced
apoptosis after about 10 passages in vitro. Resistance to
tamoxifen was associated with an increase in mitochondrial mass, the
appearance of a branching mitochondrial morphology, and lack of
membrane depolarization and caspase activation following tamoxifen
treatment. Taken together, these observations suggest a critical role
for mitochondrial signaling in mediating sensitivity to
tamoxifen-induced apoptosis.
) HMEC-E6 cells are genetically unstable and acquire major
chromosomal rearrangements and deletions within 10 passages of
transduction in
vitro.3
/
transgenic mice (44-46). We compared base-line mitochondrial membrane
potential (
m) standardized to mitochondrial mass
(Mm) in p53(+) and p53(
) HMECs to investigate whether a
decrease in base-line
m/Mm might
correlate with sensitivity to tamoxifen-induced apoptosis. We observed
that
m/Mm in apoptosis-insensitive
p53(+) HMEC-LXSN controls was significantly increased relative to
apoptosis-sensitive early passage p53(
) HMEC-E6 cells. This observed
base-line mitochondrial membrane depolarization may provide a mechanism
for the increased sensitivity of early passage p53(
) HMECs to
apoptotic stimuli. Interestingly, the development of tamoxifen
resistance in late passage p53(
) HMEC-E6 cells was not the result of
increased
m/Mm. This indicates that
although sensitivity to tamoxifen apoptosis correlates with a decrease
in baseline
m/Mm, resistance to
tamoxifen is not associated with a return to base-line mitochondrial
membrane potential.
) HMEC-E6 cells. Since
4-hydroxytamoxifen has a higher affinity for the estrogen receptor than
does tamoxifen, we expected that if apoptosis was initiated via an
estrogen receptor-derived signal, 4-hydroxytamoxifen would exhibit
equal or increased ability to induce apoptosis relative to tamoxifen
(18, 30). We observe, however, that whereas tamoxifen is able to induce
apoptosis in p53(
) HMEC-E6 cells, 4-hydroxytamoxifen induces growth
arrest alone.
) HMEC-E6 cells with an
agent that has increased in vitro affinity for the estrogen
receptor fails to induce apoptosis, this raises the possibility that
tamoxifen chemoprevention may be, in part, mediated by an estrogen
receptor-independent pathway.
) HMEC-E6 cells occurs within 1 h
of tamoxifen treatment. This rapid induction of apoptosis is consistent
with a nongenomic mechanism of anti-estrogen signaling. Tamoxifen and
4-hydroxytamoxifen are structurally and functionally very similar but
differ in their affinity for calmodulin (53). Calmodulin has been shown
recently to be a mediator of apoptosis in several cell systems (54,
55), and calcium/calmodulin-dependent protein kinase has
been shown to regulate apoptosis through the death-associated protein
kinase-2 (31, 57). We speculate that apoptosis mediated by
anti-estrogens in HMECs with low levels of estrogen receptor expression
may involve a calcium-mediated signaling pathway, perhaps modulated by
calmodulin binding.
) HMEC-E6
cells rapidly occurs via a mitochondrial signaling pathway, requiring
mitochondrial membrane depolarization and the activation of caspase-3
and caspase-9. Whereas tamoxifen readily induces apoptosis in early
passage p53(
), the tamoxifen metabolite 4-hydroxytamoxifen does not
induce mitochondrial changes and hence does not induce apoptosis. These
data suggest a need to investigate the relative contributions of
genomic and nongenomic mechanisms in the induction of apoptosis by
tamoxifen, an activity that is likely to support the development of
novel pharmacologic agents for breast cancer chemoprevention.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Judy Goombridge and Franque Remington for the preparation of electron microscopy specimens.
![]() |
FOOTNOTES |
---|
* This work was supported by NCI Grant R01CA88799 (to V. L. S.), NCI Grant 5-P30CA16058 (to V. L. S.) and NIDDK Grant 2P30DK 35816-11 (to V. L. S.) from the National Institutes of Health, a Susan G. Komen Breast Cancer award (to V. L. S. and E. C. D.), an American Cancer Society New Investigator award (to V. L. S.), a V-Foundation New Investigator award (to V. L. S.), and a Howard Hughes undergraduate award (to S. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Box 2628, Duke
University Medical Center, Durham, NC 27710. Tel.: 919-668-2455; Fax:
919-668-2458; E-mail: seewa001@mc.duke.edu.
Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M007915200
2 E. C. Dietze, L. E. Caldwell, S. L. Grupin, M. Mancini, and V. L. Seewaldt, unpublished observations.
3 K. Mrózek and V. L. Seewaldt, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HMEC, human mammary epithelial cells; HPV-16, human papillomavirus type 16; PBS, phosphate-buffered saline; ECL, enhanced chemiluminescent detection; FACS, fluorescent-activated cell sorting; RT, room temperature; FITC, fluorescein isothiocyanate; HPLC, high performance liquid chromatography; NAO, n-nonyl acridine orange.
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