From the Department of Molecular and Cellular
Oncology and the § Department of Experimental Radiation
Oncology, University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030
Received for publication, August 2, 2000, and in revised form, September 26, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to extracellular signals, the
mechanisms utilized to transduce nuclear apoptotic signals are not well
understood. Characterizing these mechanisms is important for predicting
how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death
domain-containing protein p84N5 can induce apoptosis that is inhibited
by association with Rb. The pattern of caspase and NF- Programmed cell death is essential for normal development, tissue
homeostasis, and host defense mechanisms. Programmed cell death is
recognized by a collection of distinctive morphological and biochemical
characteristics termed apoptosis (1). The pathways leading to apoptosis
in response to extracellular stimuli, such as tumor necrosis factor, or
in response to mitochondrial apoptotic signals, such as cytochrome
c release, are well characterized (2, 3). Initiator caspases
are typically recruited to protein complexes composed of death
receptors and/or adapter molecules. These proteins contain signature
protein interaction motifs like the death domain, the death effector
domain, or the CARD domain. The locally high concentration of recruited
caspase proenzyme triggers its activation by proteolytic processing
thereby initiating a caspase proteolytic cascade. Apoptotic signals can
also originate from within the nucleus. For example, DNA damage caused
by radiation triggers a stress response that can result in apoptotic
cell death (4). The mechanisms utilized by nuclear signals to initiate apoptosis are not well understood.
A number of nuclear transcription factors can induce apoptosis,
including p53 (5, 6). Although p53 has a well documented role in the
response of the cell to DNA damage, the mechanism used by p53 to
initiate apoptosis is controversial. Although it may trigger apoptosis
from within the nucleus by altering the expression of genes directly
involved in the execution of apoptosis (7), non-nuclear mechanisms
unrelated to transcriptional regulation have also been proposed (8).
Few proteins other than transcription factors are known to require
nuclear localization to initiate apoptosis. Nuclear localization of
expanded polyglutamine repeat proteins is required for their ability to
induce apoptosis that causes progressive neurodegenerative diseases
like Huntington's disease or spinocerebellar ataxia (9, 10).
Activation of caspase-8 is a required step in this process (11).
Activation of caspase-8 apparently occurs by a novel mechanism
involving recruitment of the proenzyme to characteristic protein
aggregates that are associated with these diseases.
Recently, we demonstrated that the nuclear protein encoded by
the N5 gene (p84N5)1 was
capable of initiating p53-independent apoptosis (12). Since p84N5
contains a death domain that is required for its ability to induce
apoptosis, it may participate in a nuclear apoptotic pathway.
Consistent with this hypothesis, p84N5-induced apoptosis has a pattern
of caspase and NF- Cell Culture--
SAOS-2, 293, and C-33A cell lines were
obtained from American Type Culture Collection and maintained in
Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal
bovine serum and antibiotics (100 units/ml penicillin, 100 µg/ml
streptomycin) in a 5% CO2 incubator at 37 °C. The
AT22IJE-T cell line and the ATM-expressing derivative were cultured
under the same conditions except for the addition of hygromycin as
described (17). Viable cells were counted after trypan blue staining
using a hemocytometer. Caffeine was added to the culture media to a
final concentration of 2 mM.
Plasmids and Adenovirus--
The full-length p84N5 cDNA was
subcloned into the pCEP4 (Invitrogen, Carlsbad, CA) expression vector
as described previously (12) to create the expression vector pCMVN5.
This plasmid was used to express p84N5 upon calcium phosphate-mediated
transfection. The recombinant p84N5-expressing adenovirus (AdN5) was
made as described previously (13, 18) by cloning the p84N5 cDNA
into the pAdCMV (AS)-BGHpa vector. The green fluorescent
protein-expressing adenovirus (AdGFP) was made similarly and was
provided by Dr. T. J. Liu (M. D. Anderson Cancer Center). The
Ad/E1 Transfections and Western Blotting--
For transfections, 293 cells were seeded on 100-mm dishes and transfected the following day by
calcium phosphate precipitation (20) using 10 µg of total DNA.
Transfections typically included 1 µg of the pEGFP-C1 plasmid to
measure the transfection efficiency the following day under
fluorescence microscopy. Transfected cells were extracted in a buffer
containing 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin on
ice for 10 min. The total protein concentration of the soluble extract
was determined by Bradford assay according to manufacturer's
instructions (Bio-Rad). 25 µg of total protein for each sample,
normalized for transfection efficiency, was resolved by 10%
SDS-polyacrylamide gel electrophoresis, blotted, and stained as
described previously (13). Antibody directed against p84N5 (14) was
described previously. All other antibodies were used as directed by the
manufacturer (Santa Cruz Biotechnology, Santa Cruz, CA). Primary
antibodies were detected using a peroxidase-conjugated secondary
antibody and Enhanced Chemiluminescence according to manufacturer's
recommendations (Amersham Pharmacia Biotech).
Cell Cycle and Apoptosis Analysis--
Routine analysis of cell
cycle distribution was determined by propidium iodide (PI) staining and
flow cytometry. Infected cells were harvested by trypsinization,
resuspended in growth media, washed once in phosphate-buffered saline
(PBS), resuspended in 0.5 ml PBS, and fixed by addition of
ice-cold 95% ethanol to 60% while vortexing. Cells were stored in
ethanol overnight at
For kinetic analysis of the cell cycle, bromodeoxyuridine (BrdUrd, 1 µM) was added to the culture media at the indicated times after infection. After 20 min BrdUrd media were removed, and the cells
were washed three times with pre-warmed and gassed media, and then
either fixed with ethanol as above (time 0) or incubation was continued
for 6 h before fixation. After fixation, the cells were prepared
for kinetic flow cytometric analysis. Fixed cells were digested in
0.04% pepsin (EM Science, Cherry Hill, NJ) in 0.1 N HCl
for 20 min while rocking at room temperature. After incubation with 2 N HCl for 20 min at 37 °C, 0.1 M sodium
borate (twice the HCl volume) was added. The nuclei were then
centrifuged and washed with PBS containing 0.5% Tween 20 and 0.5%
bovine serum albumin (PBTB). After centrifugation the nuclei were
resuspended in anti-BrdUrd monoclonal antibody IU-4 (1:100 v/v, Caltag,
South San Francisco, CA) in PBS plus 0.5% Tween 20 (PBT) and then
incubated at room temperature for 1 h in the dark. Another washing
with PBTB followed, and then the nuclei were incubated for 1 h in
the dark at room temperature with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG; 1:100 v/v; Sigma) in PBT and 1%
normal goat serum. After a final wash in PBTB, the nuclei were
resuspended in 10 µg/ml PI (Sigma) at a concentration of 106 nuclei/ml in PBTB.
Bivariate distributions of BrdUrd content (fluorescein isothiocyanate)
versus DNA content (PI) were measured using an Epics 752 flow cytometer (Coulter Corp., Hialeah, FL) equipped with narrow beam
(5-µm) excitation optics, a low velocity quartz flow cell and Cicero
data acquisition and display electronics (Cytomation, Fort Collins,
CO). Excitation was at 488 nm using a 5-watt argon ion laser operating
at 200 milliwatts. After blocking incident laser light, BrdUrd was
measured using a logarithmic amplifier with a 530-nm short pass filter
and DNA content collected after a 610-nm long pass filter. There was no
spectral overlap of the emitted fluorescence using this optical
configuration. Doublets and clumps were excluded from the analysis by
gating on a bivariate distribution of the red peak versus
integral signal. Data from 30,000 events were collected in the final
gated histograms. Bivariate DNA versus BrdUrd histograms
were analyzed using the "Summit" software (Cytomation), and
one-dimensional DNA histograms were fitted using Modfit LT (Verity
Software House, Topsham, ME).
The analytical methodology for calculation of kinetic parameters has
been described in detail elsewhere (21-24). The analysis is based on
our established approach that we introduced to compute cell kinetic
parameters using the extra information inherent in a bivariate DNA
versus incorporated BrdUrd flow cytometry histogram together
with our more recent method for the simultaneous estimation of the
durations of G2 and Mitosis
(TG2+M), and S phase (TS), and the potential doubling time
(Tpot) using single sample dynamic data from
bivariate DNA-thymidine analogue cytometry (25). In this study labeling
index (LI) was measured directly using data taken immediately (20 min)
after pulse labeling and computed for the 6-h time points from LI = ec(TG2 + M)(e
Apoptotic cells were identified by staining with
Annexin-V-AlexaTM 568 according to the manufacturer's
recommendations (Roche Molecular Biochemicals). At least 150 cells for
each infection were counted under fluorescence microscopy using a Texas
Red filter, and the percentage of cells staining with
Annexin-V-AlexaTM 568 was determined.
Adenoviral-mediated Expression of p84N5 Induces a G2/M
Phase Cell Cycle Arrest--
The full-length, wild-type N5 cDNA
was used to generate a recombinant, E1-deleted, replication-defective
adenovirus (AdN5) that expressed wild-type p84N5 under control of the
cytomegalovirus early promoter and the bovine growth hormone
polyadenylation signal (13, 18). This adenovirus was used to drive
expression of p84N5 in infected cells. The cell cycle distribution of
cells at different times after infection was determined by propidium iodide staining and flow cytometry. Two days after infection, the percentage of cells containing 2 N DNA content
indicative of the G2/M phase increased substantially in
AdN5-infected SAOS-2 and C33-A cells relative to cells infected with an
adenovirus designed to express the green fluorescent protein (AdGFP)
(Fig. 1, A and B).
AdGFP-infected SAOS-2 cells had a mean of 15.8% of cells in the
G2/M phase of the cell cycle 2 days after infection, whereas AdN5-infected cells had a mean of 29.0% of cells in the G2/M phase of the cell cycle at the same time point. The
mean percentage of AdGFP-infected C33-A cells with G2/M DNA
content 2 days after infection was 13.6% compared with 32.8% for
AdN5-infected cells. To ensure that the changes in cell cycle
distribution observed were caused by expression of p84N5, similarly
treated SAOS-2 cells were extracted, and the protein extracts were
analyzed for p84N5 by Western blotting. AdN5-infected cells show a
3-5-fold increase in the accumulation of p84N5 compared with
AdGFP-infected cells (Fig. 1C).
These results are consistent with induction of a G2/M cell
cycle delay caused by p84N5. However, cell death or an alteration in
the duration of other cell cycle phases may cause similar changes in
cell cycle distribution. To confirm that p84N5 expression increases the
duration of G2/M phase, we compared the kinetic parameters of the cell cycle in AdN5- and AdGFP-infected cells. S phase cells were
pulse-labeled with BrdUrd at various times after infection. Cell
populations at 0 and 6 h after labeling were analyzed by bivariate
flow cytometry. The histograms were used to calculate the potential
doubling time, the duration of S phase, and the duration of
G2/M phase (21-25). Consistent with the hypothesis that
AdN5 infection triggers a G2/M cell cycle delay, the
duration of G2/M phase for both SAOS-2 and C33-A
AdN5-infected cells increased compared with AdGFP-infected cells (Table
I). For both cell lines, the duration of
G2/M phase increased approximately 2-fold. There was no
consistent difference observed in the duration of S phase or the
potential doubling time between AdN5- and AdGFP-infected cells.
To assess the extent of this G2/M delay, we infected cells
and subsequently treated them with aphidicolin to block new DNA synthesis. If cells were delayed through G2/M, then the
G2/M peak should disappear over time in the presence of
aphidicolin leading to an increase in the fraction of G1
phase cells. If, however, the cells were more permanently arrested
prior to mitosis, the fraction of G1 and G2/M
cells would remain constant unless there is loss due to cell death.
Cells infected with Ad/E1
We have also compared the expression levels of various regulatory
proteins that serve as markers of the cell cycle in pCMVN5 versus pCMV-transfected cells. Consistent with
G2/M cell cycle arrest, the level of both cyclin A and
cyclin B protein increase in pCMVN5-transfected cells relative to
pCMV-transfected cells (Fig. 3). Cyclin A
and B are normally expressed beginning in S phase, and protein levels
peak during G2 phase. The levels of cyclin D1, cyclin E,
cdk4, p16, and p27 are not significantly different in pCMVN5- or
pCMV-transfected cells. As expected, the amount of p84N5 increases
3-5-fold upon pCMVN5 transfection relative to pCMV transfection.
The G2/M Checkpoint Activated by p84N5 Expression Is
Sensitive to Caffeine but Does Not Require Functional ATM
Protein--
Caffeine has been demonstrated to abrogate cell cycle
checkpoint controls that are normally activated in response to DNA
damage (26, 27). However, the type of DNA damage and the cell cycle phase in which it occurs influence whether caffeine will affect the
subsequent cell cycle checkpoint (28). Abrogation of these checkpoints
facilitates subsequent cell death by apoptosis. In particular, caffeine
blocks G2/M cell cycle arrest and increases sensitivity to
ionizing irradiation or other genotoxic treatments. The mechanisms
utilized by caffeine to block activation of the G2/M cell
cycle checkpoint are not completely defined. However, caffeine has been
demonstrated to inhibit the ataxia telangiectasia-mutated (ATM) kinase
(29, 30). ATM kinase can phosphorylate and activate the cell cycle
regulator Chk2/Cds1. Activation of Chk2/Cds1, in turn, enforces a
G2/M checkpoint by phosphorylating and inactivating Cdc25C.
Cdc25C is normally required to remove an inhibitory phosphate on the
mitotic cyclin-dependent kinase Cdk1. Loss of ATM function, therefore, compromises cell cycle checkpoints that are triggered in
response to genotoxic stress (31).
We analyzed the effects of caffeine treatment on the p84N5-induced
G2/M cell cycle delay and apoptosis. C33-A cells were
infected with AdN5, AdGFP, or Ad/E1
Since caffeine abrogated the p84N5-induced G2/M cell cycle
arrest and caffeine can inhibit the ATM kinase, we tested the
hypothesis that ATM may be required for activation of this checkpoint.
Immortalized ataxia telangiectasia (AT) fibroblasts that lack wild-type
ATM and the same cells reconstituted for ATM function by expression of
recombinant ATM (AT/ATM) were infected with AdN5 or AdGFP, and the cell
cycle distribution of infected cells was determined. AdGFP infection
had little effect on the cell cycle distribution of these cells
although a consistent small increase in the fraction of
G2/M cells 2 days following infection was observed (Fig.
5). As in C33-A and SAOS-2 tumor cell
lines, AdN5 caused a large increase in the percentage of cells in the
G2/M phase of the cell cycle 2 days after infection. On
average, the percentage of G2/M phase AdN5-infected cells
was about 2-fold greater than AdGFP-infected cells for both the AT and
AT/ATM cell lines. The presence of reconstituted ATM function did not
appear to influence the extent or the rate of the accumulation of
G2/M phase cells since the cell cycle distributions for AT
and AT/ATM cells were very similar.
The results presented here confirm the hypothesis that p84N5
expression activates an authentic G2/M cell cycle
checkpoint prior to the onset of apoptosis. This conclusion is
supported first by the fact that AdN5 causes an accumulation of cells
with G2/M phase DNA content relative to infection with the
control virus. Since similar results have been obtained in two
different tumor cell lines and an immortalized human AT fibroblast cell line, the effects observed are unlikely to be cell line- or tumor cell-specific. Neither p53 nor Rb is required for this G2/M
arrest since SAOS-2 cells are null for both. Furthermore, functional ATM is not required for activation of the G2/M checkpoint
by p84N5 since AT cells lacking ATM still accumulate in the
G2/M phase of the cell cycle after infection with AdN5.
Second, the accumulation of cells in G2/M phase is due to
an increase in the duration of this phase of the cell cycle. Based on
analysis kinetic data obtained from bivariate flow cytometry of BrdUrd
pulse-labeled cells, the calculated duration of G2/M
doubles upon AdN5 infection relative to AdGFP infection. In contrast,
the duration of S phase and the potential doubling time are not
consistently dissimilar in AdN5- and AdGFP-infected cells. Third, the
relative increase in cyclin A and cyclin B expression observed in
pCMVN5- versus pCMV-transfected cells is consistent with an
accumulation of cells in the G2/M phase of the cell cycle
since the expression of these proteins peak during this phase. The
accumulation of G2/M phase cells upon pCMVN5
transfection also indicates that the effects observed are not dependent
on adenovirally mediated gene transfer. Finally, treatment of
AdN5-infected cells with caffeine, a known inhibitor of the
G2/M cell cycle checkpoint, prevents accumulation of
G2/M phase cells.
DNA damage induced by ionizing radiation also activates a
G2/M checkpoint that is sensitive to caffeine. Although
caffeine abrogates radiation-induced G2/M cell cycle
arrest, it sensitizes cells to radiation-induced cell death (32). This
indicates that G2/M cell cycle arrest is not a prerequisite
for subsequent apoptosis. Similarly, caffeine abrogates p84N5-induced
G2/M checkpoint activation but not apoptosis. The
percentage of apoptotic AdN5-infected cells is similar in the presence
or absence of caffeine. Although the percentage of apoptotic cells at
the 48-h time point is not significantly altered by caffeine, caffeine
does reduce the total number of viable AdN5-infected cells. These
observations suggest that, like radiation, caffeine sensitizes cells to
the effects of AdN5. The G2/M arrest induced by DNA damage
is persistent. In the absence of normal G1 and S phase
checkpoints, such as in Rb or p53 mutant cells, G2/M cells
arrested by DNA damage will initiate another round of DNA synthesis
without completing the intervening mitosis. Polyploid cells are also
observed after infection with AdN5 but not control virus Ad/E1 Caffeine can inhibit ATM protein kinase activity and subsequent
activation of Chk2/Cds1 (29, 30). However, AdN5- and radiation-induced (data not shown) G2/M cell cycle arrest still occurs in the
absence of ATM, indicating that ATM-independent mechanisms must exist to enforce a G2/M checkpoint. This finding is consistent
with the observations that Chk2 is dispensable for initiation of the G2/M phase checkpoint (35) and that ATM-independent
mechanisms may exist to regulate Chk2 (28). How p84N5 expression leads to maintenance of Cdk1 in the inactive state required to prevent mitotic entry is unclear. One possibility is that forced p84N5 expression stresses the cell in a manner that induces an
ATM-independent response analogous to that observed in response to DNA damage.
DNA damage is one apoptotic signal that unambiguously originates from
within the nucleus. The nuclear localized Rb protein negatively
regulates DNA damage-induced apoptosis (36, 37). How Rb influences DNA
damage-induced apoptosis is not completely understood. The observation
that an amino-terminal domain of Rb that is dispensable for cell cycle
regulation may be required to inhibit some forms of
apoptosis2 (38) suggests that
the ability of Rb to regulate apoptosis may be a novel function. The
amino-terminal domain of Rb is also required to bind p84N5, and this
binding inhibits p84N5-induced apoptosis (12) and G2/M
arrest.2 We hypothesize that p84N5 plays a role in a
cellular stress response that is similar to that triggered by
radiation-induced DNA damage and, therefore, that it is a good
candidate for mediating the effects of Rb on this response.
B activation
during p84N5-induced apoptosis is similar to p53-independent cellular
responses to DNA damage. One hallmark of this response is the
activation of a G2/M cell cycle checkpoint. In this
report, we characterize the effects of p84N5 on the cell cycle.
Expression of p84N5 induces changes in cell cycle distribution and
kinetics that are consistent with the activation of a G2/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine
blocks p84N5-induced G2/M arrest but not subsequent
apoptotic cell death. The p84N5-induced checkpoint is functional in
ataxia telangiectasia-mutated kinase-deficient cells. We
conclude that p84N5 induces an ataxia telangiectasia-mutated kinase
(ATM)-independent, caffeine-sensitive G2/M cell cycle
arrest prior to the onset of apoptosis. This conclusion is consistent
with the hypotheses that p84N5 functions in an Rb-regulated cellular
response that is similar to that triggered by DNA damage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation that is similar to radiation-induced
apoptosis (13). The N5 gene was originally isolated based on the
ability of p84N5 to bind an amino-terminal domain of the retinoblastoma
tumor suppressor protein (Rb) (14). Rb can regulate both
p53-dependent and p53-independent apoptotic responses to
DNA damage (15). Association with pRb inhibits p84N5-induced apoptosis,
identifying p84N5 as a potential mediator of the inhibitory effects of
pRb on p53-independent apoptosis. One characteristic feature of DNA
damage-induced apoptosis, especially in the absence of wild-type p53,
is the activation of a G2/M cell cycle checkpoint prior to
cell death (16). In the current study, we test whether p84N5 activates
a G2/M cell cycle checkpoint.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
adenovirus is made from the pXCJL and pJM17 plasmids and lacks
adenoviral E1 but contains no foreign gene. Recombinant adenovirus was
purified by CsCl equilibrium density gradient centrifugation and viral particle numbers estimated by A260 in the
presence of SDS as described (19). Infectious titer was determined by
an end point dilution of the viral stock on 293 cells. Viral infections
were typically performed by adding an appropriate number of infectious
units to cells at a multiplicity of infection of 10 and incubating
under normal growth conditions overnight.
20 °C prior to staining. Cells were then
washed in PBS and resuspended in PBS containing 10 µg/ml propidium
iodide and 250 µg/ml RNase A. Cells were incubated at 37 °C for 15 min prior to flow cytometric analysis using a Coulter EPICS Profile
instrument (Beckman Coulter Inc., Fullerton, CA). Cell cycle
distributions were determined from histograms using Multicycle (Phoenix
Flow Systems, San Diego, CA).
v
1) where c is the growth rate of the population, and
is a dimensionless quantity based on the division status of labeled cells, which may be measured from these labeled populations (22, 23,
25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Adenovirally mediated p84N5 expression causes
cells to accumulate in the G2/M phases of the cell
cycle. SAOS-2 (A) or C33-A (B) cells were
infected with the indicated adenovirus and then fixed and processed for
PI staining and flow cytometry at the indicated times. Histograms of
cell number versus PI staining intensity were generated by
flow cytometric analysis of at least 10,000 cells, and the cell cycle
profiles were calculated as described under "Experimental
Procedures." The shaded areas show the cell cycle profiles
calculated from a representative experiment repeated at least three
times. C, protein from SAOS-2 cells infected with the
indicated virus was extracted 2 days after infection and analyzed for
p84N5 or -actin by Western blotting. The positions of molecular
weight standards are shown at left. The position of the
p84N5 and
-actin bands are shown at right.
The duration of G2 + M phases of the cell cycle lengthens
upon expression of p84N5
, a nonrecombinant replication-defective
adenovirus, did show a decrease in the fraction of G2/M
phase cells and an increase in G1 phase cells in the
presence of aphidicolin (Fig. 2). The
G1 fraction increased from 37 ± 1 to 54 ± 2%
with aphidicolin, and the G2/M fraction decreased from 32 ± 1 to 11 ± 1%. As expected, AdN5-infected cells
exhibited an increase in G2/M phase cells and cells with
sub-G1 DNA content relative to Ad/E1
cells.
Interestingly, 26 ± 5% of AdN5-infected cells were aneuploid,
suggesting that these cells had reinitiated DNA synthesis without
completing the intervening mitosis. In the presence of aphidicolin, the
fraction of G2/M phase cells decreased from 41 ± 3 to
22 ± 3%. However, aphidicolin did not cause a significant increase in the fraction of G1 phase cells (25 ± 3 versus 32 ± 4%). Furthermore, the apparent fraction
of S phase cells increased from 38 ± 1 to 60 ± 2% in cells
prevented from synthesizing DNA by aphidicolin treatment. This
suggested that the decrease in the fraction of G2/M cells
upon aphidicolin treatment was not due to completion of mitosis and
entry into G1 phase but rather was due to loss of DNA
content in G2/M-arrested cells during apoptotic cell
death.
View larger version (25K):
[in a new window]
Fig. 2.
Cells G2/M arrested by AdN5
re-replicate their DNA and/or undergo apoptosis without completion of
mitosis. C33-A cells were infected with the indicated virus at a
multiplicity of infection of 10. Thirty two hours after infection,
cells were incubated in the presence or absence of 5 µg/ml
aphidicolin for an additional 16 h. Cells were then stained with
propidium iodide and analyzed by flow cytometry. The data shown are the
raw propidium iodide distribution with the DNA content scale compressed
to show both polyploid and sub-G1 cells. The data shown are
representative of three different infections for each sample.
View larger version (69K):
[in a new window]
Fig. 3.
Changes in the expression level of cell cycle
regulatory proteins upon p84N5 expression. Protein from 293 cells
transfected with the indicated expression plasmids was extracted 2 days
after transfection. Equal mass of total cell protein was analyzed by
Western blotting for the indicated proteins. Blots were re-probed for
-actin to serve as a protein loading control.
in the presence or absence of
caffeine. The cell cycle distribution of cells was analyzed by
propidium iodide staining and flow cytometry at varying times after
infection. The percentage of apoptotic cells was determined by staining
with annexin-V. As expected, AdN5 infection induced a significant
accumulation of cells in the G2/M phase of the cell cycle
and a 2-3-fold increase in the percentage of apoptotic cell by 2 days
after infection (Fig. 4). Treatment with
caffeine prevented the accumulation of G2/M phase cells
normally observed upon AdN5 infection (Fig. 4A). In the
experiment shown, the fraction of G2/M phase cells 2 days after infection with AdN5 was 36.3% in the absence of caffeine and
18.5% in the presence of caffeine. Similar results were obtained with
SAOS-2 cells (data not shown). However, caffeine treatment did not
alter the percentage of apoptotic cells observed (Fig. 4B).
Given the experimental conditions, it is not possible to determine
whether caffeine increases the rate of apoptosis. However, the number
of remaining viable cells 48 h after AdN5 infection is typically
reduced by 40% in the presence of caffeine (1 × 106
viable AdN5-infected cells in the absence of caffeine and 6.1 × 105 viable AdN5-infected cells in the presence of
caffeine), suggesting that caffeine may sensitize cells to AdN5-induced
apoptotic cell death.
View larger version (17K):
[in a new window]
Fig. 4.
Caffeine abrogates the p84N5-induced
G2/M cell cycle arrest but not apoptosis.
A, cell cycle profiles were calculated for AdN5- or
AdGFP-infected cells at the indicated time either in the absence or
presence of caffeine. The cell cycle profiles were calculated based on
plots of cell number versus PI staining intensity (DNA
content) obtained from flow cytometric analysis of at least 10,000 cells. The data shown are representative of at least three independent
experiments. B, C33-A cells infected with the indicated
adenovirus either in the presence or absence of 2 mM
caffeine were harvested 48 h post-infection and stained with
annexin-V. The percentage of annexin-V staining cells is the mean and
S.D. of three different infections.
View larger version (29K):
[in a new window]
Fig. 5.
The p84N5-induced G2/M cell cycle
arrest is independent of ATM. AT or AT/ATM fibroblasts were
infected with AdN5 or AdGFP and harvested for PI staining and flow
cytometry at the indicated times. The cell cycle profiles were
calculated based on plots of cell number versus PI staining
intensity (DNA content) obtained from flow cytometric analysis of at
least 10,000 cells. The data shown are representative of two
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Like
DNA damage-induced checkpoint activation, this suggests that
AdN5-infected cells arrested at G2/M can reinitiate DNA
synthesis without completing the intervening mitosis. Blocking DNA
synthesis by treatment with aphidicolin does not cause an increase in
the percentage of AdN5 cells in G1 phase as is observed in
cells infected with Ad/E1
, but rather causes an increase in the
fraction of cells with S phase DNA content. Since new DNA synthesis is
blocked by aphidicolin, the increased fraction of apparent S phase
cells is likely due to loss of DNA content during apoptotic cell death
of G2/M or polyploid cells. Hence, it appears as if cells
arrested at G2/M by AdN5 either re-initiate DNA synthesis and/or die by apoptotic cell death before completing mitosis and reentering G1 phase.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Ta-Jen Liu for providing AdGFP. Dr. Wen-Hwa Lee kindly provided the 5E10 anti-N5 antibody. We thank other members of the Goodrich laboratory for helpful discussions. The M. D. Anderson FACS Core Facility is the recipient of National Institutes of Health Grant CA-16672.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA-70292 (to D. W. G.) and CA-06294 (to N. H. A. T.) and the M. D. Anderson Physicians Referral Service.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 Molecular and Cellular Oncology, Box 108, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-745-1391; Fax: 713-794-0209; E-mail: goodrich@odin.mdacc.tmc.edu.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M006944200
2 B. S. Poe and D.W. Goodrich, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: p84N5, the protein encoded by the N5 cDNA; Rb, the retinoblastoma tumor suppressor protein; AdN5, recombinant adenovirus designed to express p84N5; AdGFP, recombinant adenovirus designed to express the green fluorescent protein; PI, propidium iodide; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; ATM, ataxia telangiectasia-mutated kinase; AT, ataxia telangiectasia.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wyllie, A. H. (1980) Nature 284, 555-556[Medline] [Order article via Infotrieve] |
2. | Yuan, J. (1997) Curr. Opin. Cell Biol. 9, 247-251[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Wolf, B. B.,
and Green, D. R.
(1999)
J. Biol. Chem.
274,
20049-20052 |
4. | Haimovitz-Friedman, A. (1998) Radiat. Res. 150, 102-108 |
5. | Sheikh, M. S., and Fornace, A. J., Jr. (2000) J. Cell. Physiol. 182, 171-181[CrossRef][Medline] [Order article via Infotrieve] |
6. | Sionov, R. V., and Haupt, Y. (1999) Oncogene 18, 6145-6157[CrossRef][Medline] [Order article via Infotrieve] |
7. | Yin, C., Knudson, C. M., Korsmeyer, S. J., and Van Dyke, T. (1997) Nature 385, 637-640[Medline] [Order article via Infotrieve] |
8. |
Bennett, M.,
Macdonald, K.,
Chan, S. W.,
Luzio, J. P.,
Simari, R.,
and Weissberg, P.
(1998)
Science
282,
290-293 |
9. | Klement, I. A., Skinner, P. J., Kaytor, M. D., Yi, H., Hersch, S. M., Clark, H. B., Zoghbi, H. Y., and Orr, H. T. (1998) Cell 95, 41-53[Medline] [Order article via Infotrieve] |
10. | Saudou, F., Finkbeiner, S., Devys, D., and Greenberg, M. E. (1998) Cell 95, 55-66[Medline] [Order article via Infotrieve] |
11. | Sanchez, I., Xu, C. J., Juo, P., Kakizaka, A., Blenis, J., and Yuan, J. (1999) Neuron 22, 623-633[Medline] [Order article via Infotrieve] |
12. |
Doostzadeh-Cizeron, J.,
Evans, R.,
Yin, S.,
and Goodrich, D. W.
(1999)
Mol. Biol. Cell
10,
3251-3261 |
13. |
Doostzadeh-Cizeron, J.,
Yin, S.,
and Goodrich, D. W.
(2000)
J. Biol. Chem.
275,
25336-25341 |
14. | Durfee, T., Mancini, M. A., Jones, D., Elledge, S. J., and Lee, W.-H. (1994) J. Cell Biol. 127, 609-622[Abstract] |
15. | Wang, J. Y. J. (1997) Curr. Opin. Genet. & Dev. 7, 39-45[CrossRef][Medline] [Order article via Infotrieve] |
16. | Iliakis, G. (1997) Semin. Oncol. 24, 602-615[Medline] [Order article via Infotrieve] |
17. | Ziv, Y., Bar-Shira, A., Pecker, I., Russell, P., Jorgensen, T. J., Tsarfati, I., and Shiloh, Y. (1997) Oncogene 15, 159-167[CrossRef][Medline] [Order article via Infotrieve] |
18. | Yin, S., Hung, M.-C., and Goodrich, D. W. (2000) Cancer Gene Ther. 7, 985-990[CrossRef][Medline] [Order article via Infotrieve] |
19. | Huyghe, B. G., Liu, X., Sutjipto, S., Sugarman, B. J., Horn, M. T., Shepard, H. M., Scandella, C. J., and Shabram, P. (1995) Hum. Gene Ther. 6, 1403-1416[Medline] [Order article via Infotrieve] |
20. | Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979) Cell 16, 777-785[Medline] [Order article via Infotrieve] |
21. | Terry, N. H. A., White, R. A., Meistrich, M. L., and Calkins, D. P. (1991) Cytometry 12, 234-241[Medline] [Order article via Infotrieve] |
22. | White, R. A., Terry, N. H. A., and Meistrich, M. L. (1990) Cell Tissue Kinet. 23, 561-573[Medline] [Order article via Infotrieve] |
23. | White, R. A., Terry, N. H. A., Meistrich, M. L., and Calkins, D. P. (1990) Cytometry 11, 314-317[Medline] [Order article via Infotrieve] |
24. | Terry, N. H., and White, R. A. (1996) Clin. Immunol. News 16, 46-50[CrossRef] |
25. | White, R. A., Meistrich, M. L., Pollack, A., and Terry, N. H. A. (2000) Cytometry 41, 1-8[CrossRef][Medline] [Order article via Infotrieve] |
26. | Murnane, J. P. (1995) Cancer Metastasis Rev. 14, 17-29[Medline] [Order article via Infotrieve] |
27. | Harris, A. L. (1985) Cancer Surv. 4, 601-624[Medline] [Order article via Infotrieve] |
28. |
Darbon, J. M.,
Penary, M.,
Escalas, N.,
Casagrande, F.,
Goubin-Gramatica, F.,
Baudouin, C.,
and Ducommun, B.
(2000)
J. Biol. Chem.
275,
15363-15369 |
29. |
Zhou, B. B.,
Chaturvedi, P.,
Spring, K.,
Scott, S. P.,
Johanson, R. A.,
Mishra, R.,
Mattern, M. R.,
Winkler, J. D.,
and Khanna, K. K.
(2000)
J. Biol. Chem.
275,
10342-10348 |
30. | Blasina, A., Price, B. D., Turenne, G. A., and McGowan, C. H. (1999) Curr. Biol. 9, 1135-1138[CrossRef][Medline] [Order article via Infotrieve] |
31. | Rotman, G., and Shiloh, Y. (1997) Cancer Surv. 29, 285-304[Medline] [Order article via Infotrieve] |
32. | Bracey, T. S., Williams, A. C., and Paraskeva, C. (1997) Clin. Cancer Res. 3, 1371-1381[Abstract] |
33. |
Hong, F. D.,
Chen, J.,
Donovan, S.,
Schneider, N.,
and Nisen, P. D.
(1999)
Carcinogenesis
20,
1161-1168 |
34. | Bulavin, D. V., Tararova, N. D., Aksenov, N. D., Pospelov, V. A., and Pospelova, T. V. (1999) Oncogene 18, 5611-5619[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Hirao, A.,
Kong, Y. Y.,
Matsuoka, S.,
Wakeham, A.,
Ruland, J.,
Yoshida, H.,
Liu, D.,
Elledge, S. J.,
and Mak, T. W.
(2000)
Science
287,
1824-1827 |
36. | Haas-Kogan, D. A., Kogan, S. C., Levi, D., Dazin, P., T'Ang, A., Fung, Y.-K. T., and Israel, M. A. (1995) EMBO J. 14, 461-472[Abstract] |
37. |
Park, D. S.,
Morris, E. J.,
Bremner, R.,
Keramaris, E.,
Padmanabhan, J.,
Rosenbaum, M.,
Shelanski, M. L.,
Geller, H. M.,
and Greene, L. A.
(2000)
J. Neurosci.
20,
3104-3114 |
38. | Riley, D. J., Liu, C.-Y., and Lee, W.-H. (1997) Mol. Cell. Biol. 17, 7342-7352[Abstract] |