From the Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
Received for publication, September 19, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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MCF-7 and ZR-75 breast cancer cells infected with
an adenovirus constitutively expressing high levels of cyclin D1
demonstrated widespread mitochondrial translocation of Bax and
cytochrome c release that was approximately doubled after
the addition of all-trans retinoic acid (RA) or Bcl-2
antisense oligonucleotide. By comparison, the percentage of cells in
Lac Z virus-infected cultures containing translocated Bax and
cytoplasmic cytochrome c was markedly less even after RA
treatment. Despite this, RA-treated Lac Z and untreated cyclin D1
virus-infected cultures contained similarly low proportions of cells
with active caspase or cells that were permeable to propidium iodide.
Bax activation was p53-dependent and accompanied by arrest in G2 phase. Although constitutive Bcl-2 expression
prevented Bax activation, it did not alter cyclin D1-induced cell cycle arrest, illustrating the independence of these events. Both RA and
antisense Bcl-2 oligonucleotide decreased Bcl-2 protein levels and
markedly increased caspase activity and apoptosis in cyclin D1-infected
cells. Thus amplified cyclin D1 expression initiates an apoptotic
signal inhibited by different levels of cellular Bcl-2 at two points.
Whereas high cellular levels of Bcl-2 prevent mitochondrial Bax
translocation, lower levels can prevent apoptosis by inhibition of
caspase activation.
The D-type cyclins govern the activity of
cyclin-dependent kinases
(CDKs),1 CDK-4 and CDK-6,
which phosphorylate targets including the retinoblastoma protein that
are responsible for progression through G1 (1, 2).
The importance of cyclin D1 in oncogenesis is reflected in numerous
tumor types that display amplification and/or overexpression of the
cyclin D1 gene (1). On the other hand, high levels of cyclin D1 can
also result in growth suppression and apoptosis (reviewed in Refs. 3
and 4). Similar to other protooncogene products such as Myc,
high level expression of cyclin D1 elicits an apoptotic response in the
absence of trophic factors, although apoptosis can occur even in the
presence of serum (5). Thus increased cyclin D1 expression can also
present a survival challenge to the cell.
The Bcl-2 family of proteins plays an integral role in control of
programmed cell death. There are now numerous members of this family
defined by the presence of four conserved The caspase family of cysteine proteases plays an integral role in
apoptosis. The apoptosome constituent, caspase-9, is the apical enzyme
in the mitochondrial death pathway responsible for activation of
effector caspases including caspase-2, -3, -6, -7, and -10 (16).
Regulation of caspase activity is afforded by a growing family of
inhibitor of apoptosis (IAP) gene products, which contain from one to
three domains homologous to the initially described baculovirus IAP
repeat domain (17, 18). IAP activity may be inhibited by mitochondrial
release of the Smac/DIABLO protein, which then allows for full
activation of caspase-9 within the Apaf-1 cytochrome c
complex (19, 20). Although apoptosis is usually defined as a
caspase-dependent process, an inefficient apoptosis or
necrotic cell death can also occur as a result of mitochondrial
dysfunction (21, 22).
In the present work, we have acutely expressed cyclin D1 by
adenoviral-mediated gene transfer into breast cancer cell lines, resulting in G2/M phase arrest, p53-dependent
Bax mitochondrial translocation, and cytochrome c release
without accompanying caspase activation. The addition of RA or
antisense Bcl-2 to these cells markedly increased caspase activation.
The results support the notion that Bcl-2 can not only prevent Bax
recruitment to the mitochondrial membrane but can also interfere with
caspase activation during the course of an apoptotic signal.
Cell Culture and Transfection--
MCF-7 cells and ZR-75 human
breast cancer cell lines were grown in Dulbecco's modified Eagle's
medium (high glucose) at 37 °C/5% CO2 with 5% fetal
bovine serum, 1% non-essential amino acids, 110 µg/ml sodium
pyruvate, and 10 µg/ml gentamicin. For treatment with RA,
100-mm plates were seeded with 5 × 105 cells and made
1 µM with RA or the equivalent amount of vehicle after
attachment. Cells were harvested at the indicated times. To generate
stable HPV-16 E6 clones, CMV-HPV-16 E6 was
cotransfected with CMV-puromycin into MCF-7 cells using the
calcium phosphate precipitate method as described previously (23).
Cells were selected in 2 µg/ml puromycin, and individual clones were
isolated and expanded and then evaluated for the expression of the
E6 gene by Northern analysis of RNA. p53 protein was
assessed in E6-positive clones by immunoblot analysis. Two clones were
chosen for experimentation based on very low levels of p53 protein as
compared with control MCF-7 cells.
Plasmids and Antibodies--
The HPV-16 E6 oncoprotein cDNA
was obtained from Peter Howley (Harvard University). Anti-cyclin D1
monoclonal (HD11) and polyclonal (M20), anti-p53 (Pab 240), anti-Bcl-2,
and anti-Bax were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA); anti-Apaf-1 was purchased from Chemicon (Temecula, CA); monoclonal
(mAb) anti-Bax 6A7 was purchased from Trevigen (Gaithersburg, MD);
anti-cytochrome c monoclonal antibody was purchased
from Pharmingen; and polyclonal antibody (H-104) was obtained from
Santa Cruz Biotechnology. Anti-cytochrome c oxidase was
purchased from Molecular Probes (Eugene, OR); anti-mitochondrial hsp 70 was purchased from ABR (Golden, CO); anti-Smac/DIABLO was purchased
from Prosci (Poway, CA); and secondary antibodies conjugated to
horseradish peroxidase, CY3, and fluorescein were obtained from Jackson
ImmunoResearch Laboratories (West Grove, PA).
Viral Infection--
Lac Z-expressing adenovirus was obtained
from Dr. Ruth Slack (Ottawa, ON), and adenovirus expressing human
cyclin D1 was obtained from Dr. J Albrecht (Minnesota) (24). Viruses
were generated by ligation of either Lac Z or cyclin D1 into
pACCMV.pLpA followed by cotransfection with pJM17 into 293 cells.
Briefly, MCF-7 and ZR-75 cells were split and plated on coverslips.
After attachment, cultures were infected with the Lac Z adenovirus or
cyclin D1 adenovirus at a multiplicity of infection of 75 for 3 h
in a minimum of medium without serum. Following infection, medium with
5% fetal bovine serum and 1 µM RA or vehicle was added
to cultures for 96 h unless otherwise stated. For antisense Bcl-2
experiments, cells were transfected in serum-free medium at
37 °C/5% CO2 with the oligonucleotide
5'-TCTCCCAGCGTGCGCCAT-3' from Trilink Bio Technologies Inc. at a final
concentration of 150 nM using Lipofection reagent
(Invitrogen). A control oligonucleotide contained the same base
composition in a random order. Four h later, the transfection medium
was replaced with complete medium for the final 48 h of culture.
Mitochondrial Fractionation--
Subconfluent cultures were
collected after trypsinization, centrifuged in medium containing 5%
serum, and washed in phosphate-buffered saline (PBS) (137 mM NaCl, 27 mM KCl, 10 mM
Na2HPO4, KH2PO4). The insoluble
microsomal fraction containing mitochondria was isolated exactly as
described (23) except that final supernatants were transferred to
ultrafuge tubes and further clarified following a 1-h centrifugation at
100,000 × g.
Immunoblot--
Cultures were harvested into prechilled
microfuge tubes in 500 µl of radioimmune precipitation assay buffer
containing a protease inhibitor mixture. Lysates were processed,
subjected to SDS-PAGE, and transferred to polyvinylidene difluoride
membranes for immunodetection and visualization by chemiluminescence as
described previously (23). Densitometric analysis was performed using a
Kodak Image Station 440 CF.
Immunocytochemistry--
For Bax 6A7 staining, cells were
cultured in 35-mm dishes on coverslips, and at the appropriate times,
they were fixed with 3% formaldehyde in PBS at room temperature
and then permeabilized for 2 min with 0.2% CHAPS in PBS. Mouse
anti-Bax monoclonal 6A7 was used at a 1:300 dilution in 3% bovine
serum albumin in PBS and incubated with coverslips for 1 h at room
temperature. For double-staining, the anti-cyclin D1 polyclonal
antibody was used at a dilution of 1:50. Immunostained cells were
detected following a 1-h incubation at room temperature with
CY3-labeled goat anti-mouse or fluorescein-labeled goat anti-rabbit
secondary antibody. Cytochrome c staining (1:50) was
performed on cold methanol-fixed cells, and antibody was detected with
CY-3-conjugated donkey anti-rabbit secondary antibody. Coverslips were
mounted with anti-fade (glycerol containing 1 mg/ml
p-phenylene-diamine). For Hoechst staining, cells were fixed
on coverslips with cold methanol at Analysis of Caspase Activity--
Cells were trypsinized for
96 h following viral infection and culture in the presence or
absence of RA, and 3 × 105 cells were resuspended in
300 µl of complete Dulbecco's modified Eagle's medium. 2 µl of
propidium iodide solution (250 µg/ml) was added to the suspension
just prior to flow analysis. Detection of caspase activity was
performed using the CaspaTag fluorescein caspase VAD activity kit
(Intergen, Purchase, NY) containing the inhibitor FAM-VAD-FMK according
to the manufacturer's instructions. Caspase activity in
camptothecin-treated cells was used as a positive control. Flow
analysis of ungated fluorescence-labeled cells was performed on a
Coulter Epics Altra cytometer (Hialeah, FL) equipped with an Argon
laser and EXPO II software (Applied Cytometry Systems). Background
levels of FAM-VAD-FMK and PI staining in uninfected cells were
subtracted from experimental values.
Cell Cycle Analysis--
Cells were collected by centrifugation
at 4 °C in PBS with 1 mM EDTA and then resuspended in 1 ml of ice-cold PBS/EDTA and fixed by the addition of 3 ml of 80%
ice-cold ethanol. For flow cytometric analysis, an aliquot of cells was
pelleted at 500 × g for 5 min, washed with PBS/EDTA,
and then resuspended in PBS/EDTA containing 100 µg/ml RNase A for 20 min at room temperature and made 32 µM with propidium
iodide. Following flow cytometry, data analysis was performed using the
Multicycle AV program for Windows (Phoenix Flow Systems, San Diego, CA).
Assessment of Mitochondrial Membrane Potential--
For the last
2 h of culture, infected cells on coverslips that had been treated
with 1 µM RA or vehicle for 96 h were incubated in
medium containing 250 nM Mitotracker Red
CM-H2XRos (Molecular Probes), which fluoresces only
in cells with an intact mitochondrial membrane potential. Coverslips
were then rinsed twice in PBS, fixed, and permeabilized as described
above for immunostaining with the Bax 6A7 antibody and then stained
with Hoechst.
Amplified Cyclin D1 Expression Results in a Conformational Change
in Bax without Inducing Apoptosis--
To investigate the effects of
amplified expression of cyclin D1, we infected MCF-7 and ZR-75 cells
with an adenovirus expressing human cyclin D1 (adeno-cyclin D1) or the
same virus expressing Lac Z (adeno-Lac Z). The immunoblot in Fig.
1A shows that cells infected
with adeno-cyclin D1 express cyclin D1 at more than 20-fold (determined
densitometrically) above the endogenous level assayed in adeno-Lac
Z-infected cells at 96 h following infection. Staining for Lac Z
in the adeno-Lac Z-infected cultures revealed Lac Z activity in at
least 75% of the cells at 96 h after infection (not shown).
Initially, we determined the sensitivity to RA-induced cell death and
the availability of the N-terminal epitope of Bax in adeno-cyclin
D1-expressing cells. Monoclonal antibody 6A7 (25) recognizes the N
terminus of Bax associated with its mitochondrial membrane insertion
and oligomerization early in apoptosis but does not bind to soluble
Bax. Fig. 1B contains micrographs of Bax 6A7
immunoreactivity and Hoechst staining of MCF-7 cells infected with
either adeno-Lac Z or cyclin D1. Numerous mAb 6A7-positive cells can be
seen in the adeno-cyclin D1 cultures but not in the adeno-Lac
Z-infected cultures. Strikingly, almost no apoptotic nuclei as
determined morphologically following Hoechst stain were present in
adeno-cyclin D1-infected cells despite the widespread Bax activation in
these cultures. Fig. 1C shows that there were few
6A7-positive cells in adeno-Lac Z-infected MCF-7 and ZR-75 populations.
RA increased the percentage 4-5-fold in both cell lines. In remarkable
contrast, percentages of 6A7-positive cells in adeno-cyclin D1-infected
MCF-7 and ZR-75 cells were increased 10- and 36-fold, respectively,
over those observed in Lac Z virus-infected cells. Although RA further
increased the percentage of 6A7-reactive cells, this increase was
fractional as compared with the severalfold increase in apoptotic
nuclei following RA treatment of adeno-cyclin D1-infected cells.
Surprisingly, less than 2% of both untreated Lac Z and adeno-cyclin
D1-infected MCF-7 and ZR-75 cells contained morphologically apoptotic
nuclei. Only small increases in apoptotic nuclei were obtained after RA
treatment of adeno-Lac Z-infected cultures. This contrasts with the
much larger increase in percentages of apoptotic cells induced by RA in
the cyclin D1 virus-infected cells. Thus cyclin D1 induces Bax
translocation but not apoptosis in these breast cancer cell lines.
Moreover, RA acts synergistically with cyclin D1 to increase apoptosis
by a factor that far exceeds that by which it increases Bax
translocation. When vehicle-treated adeno-cyclin D1-infected cells were
left in culture past 96 h, what appeared morphologically to be
necrotic cell death occurred involving cell swelling without blebbing
beginning at about day 6 after infection.
To ensure that cyclin D1 overexpression was coincident with activated
Bax, we co-immunostained cells with both mAb 6A7 and anti-cyclin D1.
Fig. 2A shows intense nuclear
staining for cyclin D1 in cells indicated by the arrowheads.
The accompanying panel shows that the same cells stain
positively with mAb 6A7. A time course of cyclin D1 expression is shown
in Fig. 2B, indicating that 24 h after viral infection,
cyclin D1 protein levels began to increase and were markedly increased
96 h after adeno-cyclin D1 infection. A time course of the
progressive increase in immunoreactivity with mAb 6A7 in Fig.
2C indicated that Bax activation began at levels of cyclin
D1 expressed at ~72 h in both control and RA-treated cultures and
increased further by 96 h. Together, these data indicate that high
cyclin D1 expression results in a conformational change in Bax that
does not itself cause apoptosis.
Presence of Cytoplasmic Cytochrome c and Variable Loss of
Mitochondrial Membrane Potential in Cyclin D1-overexpressing
Cells--
The insertion of Bax into the mitochondrial membrane
results in release of cytochrome c to the cytosol,
potentially through Bax-associated pore formation (7, 26). To see
whether the large increase in 6A7 immunostaining was associated with
cytochrome c translocation, we treated MCF-7 cells with
vehicle or RA for 96 h following infection with either the Lac Z
or cyclin D1 virus and performed immunocytochemistry for cytochrome
c. Fig. 3A shows punctate staining that was predominantly perinuclear in control Lac Z
virus-infected cells. After RA treatment, about 5% of these cells
showed evidence of more uniform staining throughout the cytoplasm,
which often obscured the nucleus. By contrast, in control cyclin D1
virus-infected cultures, between 20 and 25% of the cells appeared to
lose the perinuclear staining as immunoreactivity became more
widespread throughout the cell, and this percentage appeared increased
after the addition of RA. To more quantitatively compare the relative
levels of cytochrome c release in these treatment groups, we
next isolated membrane and cytosolic fractions from both RA-treated and
untreated adeno-cyclin D1- and adeno-Lac Z-infected cultures for
immunoblot analysis of cytochrome c. The results in Fig.
3B indicate that 96 h after infection, cytochrome
c is clearly present in the cytoplasmic fractions from
untreated adeno-cyclin D1-infected ZR-75 and MCF-7 cultures, and this
level is slightly increased in RA-treated cultures. Immunoblots were
reacted with anti-cytochrome c oxidase as a control for the
integrity of the fractionation. Ratios of cytoplasmic:mitochondrial
cytochrome c from two separate experiments each using MCF-7
and ZR-75 cells are shown below.
Depending on the cell type and the mode of induction of apoptosis, a
number of studies have shown that the release of cytochrome c can precede or follow the loss of mitochondrial membrane
potential. For example, although tumor necrosis factor- Cyclin D1 Overexpression Induces Bax Translocation but Does Not
Alter Bax Protein Levels--
Overexpression of Bax can result in
formation of Bax homodimers and mitochondrial membrane insertion;
therefore, cyclin D1 overexpression could increase activated Bax by
inducing an increase in Bax expression. The immunoblots in Fig.
4A indicate that after a 96-h
infection, Bax or Bcl-2 levels were equivalent in Lac Z- and cyclin
D1-expressing cell extracts. Concomitant treatment with RA resulted in
either no change (MCF-7) or a slight increase (ZR-75) in Bax levels
while producing a profound decrease in Bcl-2 levels, which we have seen
previously in MCF-7 cells (23). Thus Bax activation in cyclin
D1-infected cells is not the result of altered levels of Bax or Bcl-2.
To assess Bax and Bcl-2 protein in mitochondria, the same membrane
fractions used in the immunoblot in Fig. 3A were subjected
to analysis for Bax and Bcl-2. Fig. 4B shows that
mitochondrial Bax levels are more than doubled in cyclin D1-expressing
cells as compared with those expressing Lac Z. The blot was reincubated
with Bcl-2 antibody and, as expected, cyclin D1 had no effect on
mitochondrial Bcl-2 levels. Importantly, although RA decreased
mitochondrial Bcl-2 in the cyclin D1-expressing cultures, it had no
effect on Bax levels. Mitochondrial heat shock protein (mt hsp) 70 is
exclusively localized to mitochondria in most cells (30) and was used
as a marker for intact mitochondria and a gel-loading control. These
data support the notion that RA-induced apoptosis in cells ectopically
expressing cyclin D1 is not the result of increased mitochondrial Bax
but rather is due to a reduction in Bcl-2 levels.
Ectopic Cyclin D1 Expression Induces p53 and
G2/M Phase Arrest--
p53 can mediate both
G1 and G2 arrest as well as apoptosis.
G1 arrest is mostly attributed to p21 induction, whereas
arrest in G2 in response to damaged DNA or nucleotide
depletion in S phase involves induction of Gadd45, p21, and 14-3-3F,
all of which contribute to cdc2 inhibition (31). Induction of apoptosis
is facilitated by a number of p53 targets including Bax, Noxa, PUMA, as
well as components of the death receptor pathway (32). To determine
whether p53 was induced by cyclin D1 overexpression and might therefore
be involved in the response to cyclin D1 overexpression, we performed
immunoblot analysis of whole cell extracts from adeno-cyclin D1 and Lac
Z-infected MCF-7 and ZR-75 cells in the presence or absence of RA. The
results in Fig. 5A show that
p53 protein levels were induced in both cell lines by cyclin D1;
however, RA did not augment these levels. Although MCF-7 reportedly
contains wild-type p53, it remained possible that some highly passaged
isolates may have acquired p53 mutation. Fig. 5B shows that
adeno-cyclin D1 infection increased p53 expression by 24 h, and it
remained expressed throughout the duration of the experiment.
Transcriptionally competent p53 regulates the expression of the
oncoprotein, mdm2, which in turn facilitates ubiquitin-mediated
degradation of p53 (reviewed in Ref. 32). Incubation of the same blot
in mdm2 antibody shows that mdm2 expression after a short latency
period increased in parallel with that of p53. Thus p53 protein
produced in MCF-7 cells is transcriptionally active. We also noticed
that cells became enlarged to nearly twice their normal size,
suggestive of a potential premitotic arrest in G2/M. To
determine whether cyclin D1 levels mediated differential effects on the
cell cycle, we performed cell cycle analysis on adeno-cyclin
D1-infected MCF-7 cells at 24, 48, and 96 h after infection. Fig.
5C shows that at 24 h after infection, cultures
contained elevated percentages of S phase cells as compared with Lac Z
controls. However, by 48 h, more than half of cyclin D1 cells were
in G2/M as compared with only 20% of control cells. At
72 h, two-thirds of the cyclin D1-expressing cells were in
G2/M as compared with less than 30% of Lac Z control
cells. Although RA usually produces a G1 accumulation in
breast cancer cells (33), amplified cyclin D1 expression clearly
superceded its effect. Thus cyclin D1 induces p53 expression associated
with both Bax translocation and G2/M arrest.
Activation of Bax Following Cyclin D1 Overexpression Is
p53-dependent--
To assess whether or not p53 played a
key role in the activation of Bax adeno-cyclin D1 infection, we stably
expressed HPV-E6 in MCF-7 cells and assayed Bax 6A7 reactivity. Fig.
6A shows that the HPV-E6
protein significantly reduced the level of p53 protein in the stably
transfected MCF-7 cells (clones 3 and 9). Moreover, Fig. 6B
indicates that this expression was accompanied by a drastic reduction
in Bax 6A7-positive cells as compared with untransfected control cells
after cyclin D1 expression. Whereas RA induced 6A7 reactivity in
between 40 and 50% of cyclin D1-infected MCF-7 cells, less than 20%
of MCF-7(HPV-E6) cells were 6A7 immuno-positive. Thus p53 participates
in signaling the Bax conformational change following cyclin D1
overexpression. Although p53 was clearly required for Bax activation,
it was not possible to differentiate between p53-dependent
activation of Bax and a putative role for p53 in mitochondrial
cytochrome c release during cyclin D1 overexpression.
Bcl-2 Prevents Cyclin D1-induced Bax Activation but Not Cell Cycle
Arrest of Cyclin D1 Adenovirus-infected Cells--
Bcl-2
overexpression can prevent many types of cell death and has been shown
to prevent conformational changes in Bax associated with Fas-induced
cell death (34). Since cell death and activation of Bax was exacerbated
in adeno-cyclin D1-infected MCF-7 cells following RA treatment, and RA
decreases Bcl-2 levels in MCF-7 cells (23), we investigated whether
constitutive expression of Bcl-2 alone was sufficient to prevent Bax
activation and death in RA-treated cyclin D1-expressing MCF-7 cells.
Using previously characterized clones of MCF-7(Bcl-2) clones (35), we
infected pooled populations of clones with adeno-cyclin D1 or adeno-Lac Z and determined the effects of Bcl-2 overexpression on Bax
conformational change. Fig. 7A
shows that, in contrast to untransfected MCF-7 cells, activated Bax was
detected at very low levels in untreated adeno-cyclin D1-infected
cultures. Even when MCF-7(Bcl-2) cells were cultured in RA for 96 h, Bcl-2 strongly attenuated the increase in Bax N-terminal exposure to
the extent that the percentages were more than 10-fold less than those
observed in cyclin D1 virus-infected control MCF-7 cells. Cell death
was also reduced substantially by Bcl-2 as determined by Hoechst
staining, especially in RA-treated cultures of adeno-cyclin
D1-infected MCF-7(Bcl-2) cells. Therefore, in adeno-cyclin D1-infected
cells, overexpressed but clearly not endogenous levels of Bcl-2 can
prevent Bax N-terminal exposure in the absence or presence of
RA.
We next asked whether the inhibition of Bax activation by Bcl-2 also
prevented the G2/M block induced by cyclin D1. Results of
cell cycle analysis in Fig. 7B indicated that, similar to
untransfected MCF-7 cells, adeno-cyclin D1 infection of MCF-7(Bcl-2)
cells still induced a G2/M block, thus indicating that the cell cycle
effects were dissociable from the activation of Bax.
Cyclin D1 Fails to Induce Caspase Activity in the Absence of RA or
Antisense Bcl-2--
Release of cytochrome c from
mitochondria constitutes one of the terminal steps in the activation of
caspase-9 within the apoptosome. However, release of cytochrome
c does not necessarily result in caspase activation
since IAP molecules are capable of inhibiting the activity of caspase-9
even in the presence of cytochrome c and ATP. The absence of
high levels of cell death in the adeno-cyclin D1-infected cells
suggested that release of cytochrome c in these cultures was
insufficient to activate apoptosis. To assess terminal apoptotic
events, we measured caspase activity using a fluorescence-tagged FAM-VAD-FMK inhibitor that binds irreversibly to all active
caspases except caspase-4 and -10 in MCF-7 and ZR-75 cells infected
with either adeno-Lac Z or cyclin D1 virus. Table
I displays the results of flow cytometric
analysis of fluorescent cells 96 h after infection in the presence
or absence of RA. Concomitant propidium iodide staining allowed
evaluation of cells at various stages of death including those that
were only VAD-positive (early apoptosis), both VAD and PI-positive
(mid-apoptosis), and only PI-positive (late apoptosis). Caspase
activity was clearly present in both cell lines infected with Lac Z,
although these levels were ~15% higher in MCF-7 cells as compared
with ZR-75 cells, demonstrating a higher level of adenoviral toxicity
in MCF-7 cells. The addition of RA to Lac Z-infected cells only weakly
reduced the percentage of healthy cells. Importantly, in neither cell
line did the expression of cyclin D1 alone result in an increase in
VAD-positive cells over Lac Z controls. Only in the presence of RA was
there a considerable increase in the percentage of VAD-positive cells
from either cell line. Most of these cells were present in the
VAD/PI-positive population, suggesting that apoptosis was not acute
at 96 h. Since a clear proapoptotic effect of RA is the reduction
of Bcl-2 levels, we asked whether antisense Bcl-2-mediated
down-regulation of Bcl-2 would produce a similar effect on caspase
activation. Fig. 8A shows that
transfection of the oligonucleotide resulted in a greater than
50% decrease in Bcl-2 levels. This effect persisted for at least
48 h after transfection (not shown); therefore, transfection of
the oligonucleotides into virus-infected cells was for the last 48 h of culture. Transfection of control oligonucleotide into cyclin
D1-infected cells resulted in nearly 40% 6A7 mAb positivity (Fig.
8B), whereas antisense Bcl-2 increased this percentage to 55%. Thus the effect of reduction of Bcl-2 protein levels using antisense Bcl-2 closely paralleled the effects of the addition of RA on
Bax activation. Importantly, 6A7-positive cells increased following
antisense Bcl-2 transfection into Lac Z-infected cells considerably
more than after RA treatment, indicating a direct effect of the Bcl-2
antisense on Bax activation. To assess the associated induction of
caspase activity, we performed flow cytometry on CaspaTag-labeled Bcl-2
antisense-treated cells. The result of this analysis in the
lower panel of Table I indicates that, similar to RA, antisense Bcl-2
increased the percentage of cells containing active caspase. Moreover,
these values are likely an underestimate of VAD- and/or PI-positive
cells, based on the fact that many cells were floating or not intact by
the end of the study. Thus, on one level, Bcl-2 controls the activation
of Bax following the cyclin D1-induced death signal, and on another
level, it acts as a determinate of caspase activation.
Ectopic Cyclin D1 Induces Smac Release from
Mitochondria--
One reason for the lack of caspase
induction following cytochrome c release in cells
ectopically expressing cyclin D1 might be either a lack of or a weak
release of the Smac/DIABLO protein from the mitochondrial compartment.
This is reasonable since Smac/DIABLO is thought to be contained in
submitochondrial locations separate from cytochrome c (36).
To assay Smac release, we subjected mitochondrial and cytoplasmic
extracts to immunoblotting with an anti-Smac/DIABLO antibody. Using the
same extracts as in Fig. 3, Fig.
9A shows that Smac protein is
found predominately in the membrane fraction of Lac Z virus-infected
cells in the presence or absence of RA, although some is also present
in the cytoplasm, likely as a result of low level viral toxicity. The
ratio of membrane to soluble Smac decreased by half after ectopic
cyclin D1 expression and even further when cells were treated with RA.
Given the effect of RA on Bcl-2 levels, we predicted that antisense
Bcl-2 oligonucleotide transfection into cyclin D1 virus-infected cells
would achieve a similar release of Smac as did RA. The results in Fig.
9B indicate that although control oligonucleotide
transfection of cyclin D1-expressing cells caused Smac release to a
similar extent as in untransfected cells, transfection of Bcl-2
antisense caused a profound induction of Smac release comparable with
that after RA treatment of control oligonucleotide-transfected cells,
which was further augmented by RA. Notably, although mt hsp 70 was
retained almost entirely in the membrane fraction after RA treatment of
control oligonucleotide-transfected cells ectopically expressing cyclin
D1, release or leakage of this protein from mitochondria occurred after
Bcl-2 antisense treatment, perhaps reflecting a complete loss of
mitochondrial membrane integrity in some cells as a result of radical
Bcl-2 protein reduction. Thus, although adeno-cyclin D1 infection
causes Smac release, it is insufficient to induce caspase activation in
the absence of a decrease in Bcl-2 protein levels.
Most proapoptotic agents or stimuli do not simply result in
formation of Bax dimers but also intersect with other facets of the
death pathway, resulting in the activation of a complete apoptotic program. This has made it difficult to evaluate the relative
importance of individual factors in the mitochondrial death pathway
without ectopic expression or overexpression of either engineered or
wild-type Bcl-2 family members. Rossé et al. (37)
showed that overexpression of transiently transfected Bax resulted in
its mitochondrial translocation, cytochrome c release,
caspase activation, and cell death. Although Bax-induced cytochrome
c release was not prevented by simultaneous overexpression
of Bcl-2 in that study, the activation of caspase-3 was inhibited.
Taken together with our data, this suggests that the ratio of Bcl-2 to
Bax required to prevent cytochrome c release must be higher
than that needed to prevent caspase activation regardless of the mode
of activation of cytochrome c release. Thus the
post-mitochondrial steps in the death pathway are more difficult to
achieve than the initiating events for a given level of Bcl-2.
The working hypothesis for regulation of the mitochondrial cell death
pathway is that the ratio between proapoptotic molecules such as Bax
and Bak and the antiapoptotic molecules, Bcl-2 and Bcl-xL, helps to
determine the susceptibility of cells to die in response to a death
signal (38). RA/antisense-mediated decreases in Bcl-2 expression could
contribute to the total number of cells in which cyclin D1-induced Bax
translocation occurs or could itself induce Bax activation in those
cells either infected with low numbers or not infected with the cyclin
D1 virus. However, neither agent was synergistic with cyclin D1 in
terms of the activation of Bax. Most importantly, the fold increases in
caspase activity and apoptosis after RA/Bcl-2 antisense treatment of
cyclin D1-infected cells were dramatically higher than in untreated
cells and in RA-treated Lac Z-infected cultures. This result
demonstrates pathway progression from the mitochondrial membrane to
caspase activation and death within the time frame of the study
primarily as a result of decreased Bcl-2.
Cyclin D1 is a key determinant in some forms of tumorigenesis, yet
similar to other proliferative protooncogenes such as c-Myc (39),
increased expression can also induce cell death. The series of
experiments presented herein demonstrates for the first time the
cellular response to high level expression of cyclin D1. Table II summarizes the effects of cyclin D1 on
Bax N-terminal exposure and caspase/death induction in breast cancer
cells, clearly illustrating that increased expression of cyclin D1
initiates apoptotic events up to but not including activation of
caspases and death. Thus Bax translocation and cytochrome c
release are insufficient for caspase activation. One reason may be that
endogenous levels of Bcl-2 are sufficiently high to prevent this
activation. To this end, it has been postulated that Bcl-2 may act at a
post-mitochondrial step to inhibit Apaf-1 activation of caspase-3,
although the most recent evidence suggests that they do not (40 and
references therein). Bcl-2 can also inhibit Bax-induced cytoplasmic
acidification, which would normally facilitate caspase activation (41)
and may do so by regulating mitochondrial proton flux (42). A failure to release the IAP inhibitor Smac/DIABLO from the mitochondria might
also provide a mechanism for lack of caspase activation. However, in
contrast to caspase activation, the release of mitochondrial Smac/DIABLO appeared to reflect the level of Bax activation and cytochrome c released in adeno-cyclin D1-infected cells.
Although caspase inhibitors can prevent the release of Smac/DIABLO
(36), there may be sufficient basal activity in cells to allow for its release. Consistent with this, both RA and Bcl-2 antisense caused increased Smac release in the present study.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical Bcl-2 homology
domains called BH-1-4. The central role of the Bcl-2 family in the
regulation of cell death is control over mitochondrial membrane
permeability and release of cytochrome c into the cytoplasm, where it is free to interact with Apaf-1 and caspase-9 to form an
active "apoptosome" complex, inducing a cascade of protease activity and ensuing cell death (6). Proapoptotic molecules including
Bax and Bak do not have the BH-4 domain, but their activity depends on
the presence of the BH-3 domain (7, 8). Bax is a cytoplasmic monomer in
thriving cells and undergoes mitochondrial translocation in response to
stress or damage signals following a conformational change in the
molecule. Cytochrome c release is thought to occur as a
result of the formation of large Bax conductance channels in the outer
mitochondrial membrane as multimers, alone or in protein/lipid
complexes, or in coordination with the voltage-dependent
anion channel (9). Although membrane integration of this
"activated" Bax requires the hydrophobic C terminus, the translocation/insertion process is also associated with exposure of the
normally occluded N terminus (10-12). Experimentally enforced dimerization of Bax results in mitochondrial translocation but no
cytochrome c release and death as a result of mitochondrial dysfunction (13). Other types of proapoptotic molecules are the BH3
domain-only proteins including Bid, Bim, Bad, and the p53-regulated
NOXA (14). Bid can activate Bax directly, whereas the latter three
proteins can be bound and sequestered by Bcl-2 and Bcl-xL, which is
thought to represent their major antiapoptotic activity (14).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for 5 min and then
incubated in a 1:1500 dilution of Hoechst 33258 at room temperature,
washed with PBS, and then mounted on coverslips with anti-fade.
Intensely stained nuclei of reduced size were scored as apoptotic.
Image capture and slide evaluations were performed using a Zeiss
Axiophot fluorescence microscope equipped with Northern Eclipse image
analysis software (EMPIX Imaging Inc., Mississauga, ON). All histogram
bars represent results from evaluation of at least 1000 cells
enumerated on coverslips from multiple microscopic fields.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amplified cyclin D1
expression results in N-terminal Bax epitope exposure in the absence of
cell death. A, immunoblot analysis with cyclin D1
antibody of 15 µg of whole cell extracts from ZR-75 and MCF-7 cells
infected with adenovirus expressing Lac Z or cyclin D1. As shown in
B, MCF-7 cells grown on coverslips in the presence or
absence of 1 µM RA for 96 h after infection with
either adeno-Lac Z or adeno-cyclin D1 were immunostained with
monoclonal antibody 6A7 or stained with Hoechst.
Arrows indicate apoptotic nuclei as described under
"Experimental Procedures." Bar, 100 µm. C,
percentages of MCF-7 and ZR-75 cells immunoreactive with Bax 6A7
antibody and Hoechst-stained apoptotic nuclei. Graphs depict
the mean of three separate experiments, and bars indicate
standard errors which were 5% or less.
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Fig. 2.
Cyclin D1 expression correlates with Bax 6A7
immunostaining. As shown in A, adeno-cyclin D1-infected
MCF-7 cells were fixed and co-immunostained with a polyclonal antibody
against cyclin D1 and Bax 6A7. Secondary antibodies were conjugated to
fluorescein isothiocyanate and CY3, respectively. Bar, 100 µm. B, Western blot analysis of cyclin D1 expression at
the indicated times after adeno-cyclin D1 infection. C, time
course of Bax 6A7 immunoreactivity in adeno-cyclin D1-infected cells.
MCF-7 and ZR-75 cells grown on coverslips were fixed and immunostained
with Bax 6A7 monoclonal antibody at the indicated times after infection
with adeno-cyclin D1. Open bars, adeno-Lac Z-infected
cultures; filled bars, adeno-cyclin D1-infected cultures.
Percentages were derived from enumeration of at least 1000 cells.
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Fig. 3.
Amplified cyclin D1 is associated with
cytochrome c release and variable loss of membrane
potential. As shown in A, Lac Z- or cyclin D1
virus-infected MCF-7 cells on coverslips were treated with vehicle or
RA for 96 h and then fixed and stained with anti-cytochrome
c as described under "Experimental Procedures."
Arrows indicate cells with intact mitochondrial,
predominantly perinuclear, punctate staining.
Arrowheads indicate cells with cytoplasmic cytochrome
c. See "Results" for details. As shown in B,
10 µg of soluble (S) and membrane fraction (M)
from MCF-7 and ZR-75 cells infected with adeno-Lac Z or cyclin D1 virus
were immunoblotted with anti-cytochrome c. Cytochrome
c oxidase was used as a control for the integrity of the M
fraction, and a cross-reactive band at 20 kDa was present only in the S
fraction. Ratios derived from densitometric analysis of cytochrome
c in membrane and cytosolic fractions from two separate
experiments (expt 1 and expt 2) with each cell
line are shown below. Both gels depicted are from expt
2. As shown in C, mitochondrial membrane potential in
Bax 6A7-positive cells was detected using Mitotracker Red loading as
described under "Experimental Procedures." MCF-7 cells infected
with adeno-cyclin D1 were cultured for 96 h in the absence
(upper control panels, CON) or presence (lower
panel) of 1 µM RA. Cells were fixed and stained with
Hoechst and immunostained with Bax 6A7. Arrows indicate Bax
6A7-immunopositive cells across the panels. Bar,
50 µm. D, quantization of cells positive for Bax 6A7
reactivity and Mitotracker Red fluorescence. Each bar
represents the mean percentages derived from two separate
experiments.
treatment of
hepatocytes results in simultaneous release of cytochrome c
and mitochondrial membrane depolarization (27), UV- or
staurosporine-treated HeLa and HL-60 cells do not lose mitochondrial
membrane potential until well after the appearance of cytoplasmic
cytochrome c (28, 29). To determine the status of
mitochondrial membrane potential, we loaded cells for 1 h prior to
harvest with the reduced form of MitoTracker Red and then immunostained
with Bax 6A7 and counterstained with Hoechst. Fig. 3C shows
bright punctate MitoTracker labeling in untreated MCF-7 cells infected
with adeno-Lac Z. Adeno-cyclin D1-infected cultures also showed a
similar pattern of MitoTracker labeling in many of the cells that also
stained positive with the 6A7 antibody. Reduced staining was observed
in the balance of 6A7-positive cells. Both these populations were
devoid of apoptotic nuclei as determined by Hoechst staining. As
expected, apoptotic nuclei were abundant in the RA-treated 6A7-positive
adeno-cyclin D1-infected population, and a number of these cells showed
a more diffuse pattern of MitoTracker staining or highly reduced
staining. The percentages of mAb 6A7-positive cells displaying
MitoTracker Red labeling are shown in Fig. 3D and indicate
that over 50% of the untreated 6A7-positive cells retained
mitochondrial membrane potential, whereas it was drastically reduced
after RA treatment. Thus amplified expression of cyclin D1 causes
widespread Bax activation and cytochrome c translocation,
often without loss of mitochondrial membrane potential. Only after the
addition of RA is there a more complete loss of membrane potential.
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Fig. 4.
Bax translocates to mitochondria, but levels
are unchanged in cyclin D1 virus-infected cultures. A,
immunoblot analysis of Bax and Bcl-2 in cell extracts from vehicle and
RA-treated Lac Z or cyclin D1-overexpressing cells. Actin was used as a
control for gel loading. As shown in B, membrane fractions
from Lac Z- and cyclin D1 virus-infected cells were subjected to
immunoblot with a polyclonal anti-Bax and anti-Bcl-2. Subcellular
fraction integrity and loading was confirmed using anti-proliferating
cell nuclear antigen and anti-mt hsp 70 as markers for
nuclear/cytoplasmic contamination and mitochondria, respectively.
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Fig. 5.
Induction of p53 and G2 block following
cyclin D1 virus infection. As shown in A, MCF-7 and
ZR-75 cells infected with the Lac Z or cyclin D1 virus were cultured in
the presence or absence of 1 µM RA for 96 h as
described under "Experimental Procedures." Whole cell extracts were
immunoblotted for p53 and actin. As shown in B, MCF-7 cell
extracts were obtained at the indicated times after cyclin D1 virus
infection and immunoblotted with anti-p53, anti-mdm2, and anti-actin.
The Lac Z virus-infected cell extracts were obtained 96 h after
infection. C, cell cycle analysis of Lac Z- and cyclin
D1-infected MCF-7 cells. Cell cycle analysis was performed as described
under "Experimental Procedures" at the indicated times after
viral infection.
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Fig. 6.
Cyclin D1-induced Bax activation is
p53-dependent. As shown in A, MCF-7 cells
were transfected with an HPV-16 E6 expression plasmid. The resulting
levels of p53 assessed by immunoblot of whole cell extracts from
untransfected cells (con) and two clones (3 and
9) are shown. B, evaluation of mAb 6A7-positive
cells and apoptotic nuclei following Lac Z or cyclin D1 virus infection
and culture in the presence or absence of 1 µM RA.
Graphs represent the mean of percentages derived from three
separate experiments, and bars indicate standard errors
which were 6% or less.
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Fig. 7.
Bcl-2 prevents cyclin D1-induced Bax
activation and RA-induced cell death. As shown in A,
three pooled MCF-7 cell lines stably expressing Bcl-2 (Teixeira
et al. (35)) were infected with adeno-cyclin D1 or adeno-Lac
Z and then cultured in the presence or absence of 1 µM
RA. Cells were stained with Hoechst and immunostained with the Bax 6A7
monoclonal antibody as described under "Experimental Procedures."
The graphs show the results of enumeration of the
percentages of mAb 6A7-positive cells and apoptotic nuclei in adeno-Lac
Z and adeno-cyclin D1-infected cells. Graphs represent mean
percentages from two separate experiments. Con,
untransfected control cells. B, cell cycle analysis of
MCF-7(Bcl-2) clones. Cell cycle analysis was performed as described
under "Experimental Procedures" at the indicated times following
adeno-cyclin D1 infection of three pooled Bcl-2- expressing
clones.
Cyclin D1-induced caspase activity and cell death
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Fig. 8.
Bcl-2 antisense reduces Bcl-2 and augments
the percentage of mAb 6A7-positive cells. A, immunoblot
analysis of Bcl-2 expression 48 h after transfection of MCF-7
cells with either control (con) oligonucleotide or antisense
Bcl-2. Actin reactivity was used as an internal gel loading control. As
shown in B, following infection with either Lac Z or cyclin
D1 virus and transfection of either control or Bcl-2 antisense
oligonucleotide for the final 48 h of culture, cells on coverslips
were immunostained with mAb 6A7. Graphs represent mean
percentages of positive cells from three separate experiments, and
bars indicate standard errors, which were 8% or less.
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Fig. 9.
Amplified cyclin D1 expression induces
mitochondrial Smac release. As shown in A, membrane
(M) and soluble (S) fractions used in Fig. 3 from
adeno-cyclin D1- and adeno-Lac Z-infected cells treated with vehicle or
1 µM RA were subjected to immunoblot with anti-Smac. The
ratios of membrane to soluble Smac were determined from densitometric
quantization of Smac levels in the two fractions. As shown in
B, adeno-cyclin D1-infected cells were transfected with
either control or Bcl-2 antisense oligonucleotide and membrane and
soluble fractions isolated as in panel A. Immunoblot
analysis was performed with antibodies to Smac and mt hsp 70 as a
control for mitochondrial integrity. The Smac M/S ratio was determined
as in panel A. Blots are representative of two independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparative analysis of cyclin D1-induced Bax and caspase
activation
Why overt apoptosis does not occur despite cytochrome c release remains in question, although Martinou et al. (9) have invoked scenarios wherein there remains sufficient cytochrome c associated with the electron transport chain complexes or reassociation of free cytochrome c to maintain respiration and polarization. Where caspase activation has been blocked (43, 44), cells can, in fact, recover from complete cytochrome c translocation. There is good evidence that even the loss of mitochondrial membrane potential is reversible and not a final commitment to cell death (45). In the case of Bid- or Bax-mediated cytochrome c release, only the outer mitochondrial membrane is permeable, whereas the inner membrane remains intact (46), an observation which corroborates our finding that MitoTracker staining remained intact in many of the Bax 6A7-positive cells. Cytochrome c release in individual cells is rapid and complete and independent of caspase activation (47). This is an important point, given that we did not detect a significant increase in caspase activity in adeno-cyclin D1-infected cells without the addition of RA or Bcl-2 antisense treatment.
Our data show that p53 is pivotal to Bax activation following cyclin D1 overexpression. There is growing evidence that p53 may act at the level of the mitochondria to promote cell death in a non-transcriptional mode (48, 49). From the transcriptional standpoint, a number of p53-responsive genes are thought to contribute to p53-mediated apoptosis including bax, bak, NOXA, and PUMA (32, 50). NOXA and PUMA may act in a Bid-like capacity to facilitate opening of the Bax molecule. Thus, although we did not observe any increase in Bax after cyclin D1 overexpression, it is conceivable that p53-mediated induction of the latter genes plays a role in the observed Bax activation. The lack of Bax activation in HPV-16 E6-expressing cells and the fact that cyclin D1 overexpression did not produce Bax activation in several breast cancer cell lines lacking functional p53 2 indicate that cyclin D1-induced Bax activation is p53-dependent. It is important to note that HPV-18 E6 can also induce degradation of Bak, thereby inhibiting apoptosis in a p53-independent manner (51). However, immunoblot analysis of Bak in MCF-7(HPV-18 E6) cells showed only a marginal decrease in Bak expression, and Bak was not present in the mitochondrial fraction before or after cyclin D1 overexpression (data not shown).
Evidently the level of cyclin D1 expression determines the cellular
response. Normally, cyclin D1 is required for G1
progression and is decreased in subsequent phases of the cell cycle (1, 2). Two groups have shown that constitutive cyclin D1 expression sensitizes cells to serum starvation-induced death (3, 52). We have
found that MCF-7 cells constitutively expressing cyclin D1 were
sensitized to RA (23). Pagano et al. (52) showed that acute
cyclin D1 overexpression prevented normal fibroblasts from entering S
phase, an effect that was prevented by coexpressed proliferating cell
nuclear antigen. Thus, although cyclin D1:proliferating cell nuclear
antigen associations form part of the G1 checkpoint in
normal cells, at least in breast cancer cells, progression into S phase
is clearly permitted since the block is now in the G2 phase. The
mechanism of cyclin D1-induced Bax translocation remains unclear.
Levels of cyclin D1 are normally reduced after G1 as
a result of glycogen synthase kinase-3 phosphorylation and
subsequent targeting for degradation or nuclear export (53). The high
cyclin D1 levels in the infected MCF-7 cells may have saturated the
glycogen synthase kinase-3
enzyme and/or the nuclear exporter,
resulting in constitutive nuclear cyclin D1. Alt et al. (53)
have speculated that nuclear cdk4-cyclin D1 complexes may phosphorylate
proteins in S phase that are normally substrates for cdk2, resulting in
perturbation of the timing of activation of the DNA synthesis
machinery. At moderate levels of cyclin D1, this may result in gene
amplification events. On the other hand, higher levels could induce
sufficient genetic instability to result in activation of the DNA
damage checkpoint and subsequent induction of p53. Thus cyclin
D1-initiated apoptosis provides the first example of a death signal,
which demonstrates that Bcl-2 can function at different levels to
regulate both mitochondrial and post-mitochondrial events.
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FOOTNOTES |
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* This work supported in part by Grant 98-B062 from The American Institute for Cancer Research (to M. A. C. P.).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: Dept. of Cellular and
Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, ON,
Canada K1H 8M5. Tel.: 613-562-5800 (ext. 8366); Fax: 613-562-5636;
E-mail: cpratt@uottawa.ca.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M209650200
2 M. A. C. Pratt and M.-Y. Niu, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; RA, retinoic acid; HPV, human papillomavirus; IAP, inhibitor of apoptosis; mAb, monoclonal antibody; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mt hsp, mitochondrial heat shock protein; PI, propidium iodide; FAM-VAD-FMK, carboxyfluorescein analog of benzyloxycarbonylvalylalanyl aspartic acid fluoromethyl ketone.
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