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Molecular and Cellular Biology, May 1999, p. 3704-3713, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transcription Factor E2F-1 Is Upregulated in
Response to DNA Damage in a Manner Analogous to That of p53
Christine
Blattner,
Alison
Sparks, and
David
Lane*
Cancer Research Campaign Cell Transformation
Group, Department of Biochemistry, Medical Sciences Institute,
University of Dundee, Dundee DD1 4HN, United Kingdom
Received 12 October 1998/Returned for modification 30 November
1998/Accepted 27 January 1999
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ABSTRACT |
The transcription factor E2F-1 directs the expression of genes that
induce or regulate cell division, and a role for E2F-1 in driving cells
into apoptosis is the subject of intense discussion. Recently it has
been shown that E2F-1 binds and coprecipitates with the mouse
double-minute chromosome 2 protein (Mdm2). A domain of E2F-1 (amino
acids 390 to 406) shows striking similarity to the Mdm2 binding domain
of the tumor suppressor protein p53. It is known that interaction of
Mdm2 with p53 through this domain is required for Mdm2-dependent
degradation of p53. We show here that E2F-1 protein is upregulated in
response to DNA damage. The kinetics of induction are dependent upon
the source of DNA damage, i.e., fast and transient after irradiation
with X rays and delayed and stable after irradiation with UVC, and thus
match the kinetics of p53 induction in response to DNA damage. We show
further that E2F-1 is also upregulated by treatment with the
transcription inhibitor actinomycin D and with the kinase inhibitor
DRB, as well as by high concentrations of the kinase inhibitor H7, all conditions which also upregulate p53. In our experiments we were not
able to see an increase in E2F-1 RNA production but did find an
increase in protein stability in UVC-irradiated cells. Upregulation of
E2F-1 in response to DNA damage seems to require the presence of
wild-type p53, since we did not observe an increase in the level of
E2F-1 protein in several cell lines which possess mutated p53. Previous
experiments showed that p53 is upregulated after microinjection of an
antibody which binds to a domain of Mdm2 that is required for the
interaction of Mdm2 with p53. Microinjection of the same antibody also
increases the expression of E2F-1 protein, while microinjection of a
control antibody does not. Furthermore, microinjection of Mdm2
antisense oligonucleotides upregulates E2F-1 protein, while
microinjection of an unrelated oligonucleotide does not. These data
suggest that E2F-1 is upregulated in a similar way to p53 in response
to DNA damage and that Mdm2 appears to play a major role in this pathway.
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INTRODUCTION |
E2F-1 is a member of a family of
transcription factors whose target genes control entry into and
progression through the S phase of the cell cycle (11). For
high-affinity binding to the E2F-1 consensus sites at promoters of
target genes, E2F-1 needs to heterodimerize with a member of the DP
family of transcription factors. Binding to pRb, on the other hand,
regulates the activity of E2F-1 (reviewed in reference
24): hypophosphorylated pRb binds to the activation
domain of E2F-1, rendering the protein inactive (14, 20,
25). Before entry into S phase, pRb becomes phosphorylated by
cyclin-dependent kinases, disrupting the interaction between pRb and
E2F-1 and allowing the transcription of E2F-1 target genes
(54). Deregulation of the activity of E2F family members
appears to be a hallmark of all human cancers (2, 52). Transcription of E2F-1 is induced in late G1, and Johnson
et al. have shown that overexpression of E2F-1 is sufficient for entry into S phase and DNA synthesis (32). However, unscheduled
E2F-1 activity during S phase leads to cell cycle arrest and apoptosis (28, 31, 35, 51), and studies of mice nullizygous for E2F-1
suggest that E2F-1 can exert tumor-suppressing activity (13, 57,
58).
Recent studies have shown that E2F-1 is regulated by the ubiquitin
proteasome pathway (8, 22, 27), and the carboxyl terminus is
thought to control protein stability. Interestingly, E2F-1 physically
interacts with Mdm2 (41), a protein which is known to target
the tumor suppressor protein p53 for rapid degradation by the ubiquitin
proteasome pathway (23, 36).
When normal cells are exposed to DNA-damaging agents, p53 accumulates
and transcription of p53-responsive target genes is activated. This
leads to upregulation of WAF/p21, GADD45, cyclin G, Bax, and Mdm2
(1, 12, 21, 33, 44, 47, 56); induction of cell cycle arrest
(37); or apoptosis. The mechanism of p53 accumulation in
response to DNA damage is still not understood, but it requires, at
least in part, protection against proteolysis, since the half-life of
the protein is prolonged (40). p53 degradation is regulated
by the ubiquitin-proteolysis system (39), which requires a
ubiquitin-target protein adduct formation. This complex is built up by
the activity of three enzymes: a ubiquitin-activating enzyme, a
ubiquitin-conjugating enzyme, and a ubiquitin ligase. In cells infected
with human papillomavirus type 16 (HPV-16) or HPV-18, papillomavirus E6
protein and cellular E6-AP protein form a complex and function as a
ubiquitin ligase for p53 (50), abolishing the normal
p53-dependent stress response in HPV-infected cells.
Since tight regulation of p53 is critical not only for various stress
responses but also for normal cell growth and genetic stability, the
ubiquitin ligase for p53 was of intense interest until it was recently
discovered that the oncoprotein Mdm2 was the missing piece of the
puzzle (29). Thus, p53 abundance seems to be regulated by an
autoregulatory feedback loop, involving Mdm2: the mdm2 gene
is transcriptionally activated by binding of p53 to an internal
promoter within the gene (1, 56), and Mdm2 protein binds to
p53 and targets it for degradation. Support for the importance of this
autoregulatory loop in regulating p53 expression levels comes from
experiments in which the p53-Mdm2 interaction has been interrupted,
with the result that p53 protein accumulated and p53-responsive
reporter genes were activated (6, 43). Recently it has been
shown that p53 has to be exported to the cytoplasm in order to be
degraded and that Mdm2 acts as the scavenger for this process by
providing the nuclear export signal (17, 49). The presence
of Mdm2 alone, however, seems to be insufficient for degradation of
p53. In cells, most of the endogenous Mdm2 protein is complexed with
the histone acetylase p300, and Grossman et al. have shown that the
specific interaction between p300 and Mdm2 is required for the
degradation of p53 (18). Interestingly, p300 also binds to
and acetylates p53 (19). It is, however, still unclear if
the formation of a ternary complex is sufficient for p53 degradation or
if Mdm2 needs to be acetylated by p300.
Since E2F-1 is, like p53, targeted by the ubiquitin-dependent
degradation system and binds to Mdm2 (22, 41), we asked if
it was also regulated like p53, in response to DNA damage. We found
induction of the E2F-1 protein following DNA damage. There were
striking differences in the kinetics and magnitude in response to X
rays and UVC that were equivalent to the different p53 responses to
these signals (38). Induction of E2F-1 was not the result of
increased transcription of the E2F-1 gene; instead, E2F-1 protein was
stabilized in UVC-irradiated cells. In our experiments, not only was
E2F-1 expression induced by DNA damage, but also inhibition of protein
kinase activity could upregulate E2F-1. This upregulation of E2F-1 was
accompanied by downregulation of Mdm2 and supported the idea that
interruption of Mdm2-E2F-1 complexes could upregulate E2F-1
expression, just as interruption of Mdm2-p53 complex formation
upregulates p53 expression (6, 43). The induction of E2F-1
protein after microinjection of mdm2 antisense oligonucleotides and anti-Mdm2 antibody 3G5 (which binds to an epitope
at the amino terminus of Mdm2 and inhibits binding of Mdm2 to p53)
supported this concept. These observations suggest that E2F-1 may be
regulated similarly or even identically to p53. Both proteins are
stabilized in response to DNA damage, both proteins are degraded by the
ubiquitin-dependent degradation system, and both proteins bind to Mdm2,
suggesting that Mdm2 is a key regulator of p53 and E2F-1 expression in
response to DNA damage.
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MATERIALS AND METHODS |
Cell lines and their treatment.
U2-OS cells (6),
A431 cells (43), T47D cells (45), HAKAT cells
(7), MCF-7 cells (43), and OSA cells
(6) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml),
and streptomycin (100 µg/ml). GM02184D cells (NIGMS, Camden, N.J.),
were grown in RPMI medium supplemented with 15% heat-inactivated FCS
and antibiotics (100 U of penicillin per ml, 100 µg of streptomycin per ml). All cells were maintained at 37°C and 6% CO2 in
a humidified atmosphere. They were treated 3 days after plating, at
80% confluence.
X-ray irradiation was performed in culture medium with 5 Gy in a TORREX
150D X-ray source (Astrophysics Research Corp., Long Beach, Calif.) at
a dose rate of 5 Gy/min with settings at 5 mA and 145 kV. Prior to UVC
irradiation, the culture medium was removed, and the cell layer was
then irradiated with 30 J/m2 with a Stratalinker
(Stratagene) and further cultured in the original conditioned medium.
Actinomycin D, solubilized in ethanol, was added to the culture medium
at a final concentration of 2 µg/ml or 60 ng/ml.
1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7), solubilized in
H2O, was added at a final concentration of 10 or 100 µM.
5,6-Dichloro-1-
-D-ribofuranosylbenzimidazol (DRB), solubilized in dimethyl sulfoxide, was added at a final concentration of 250 µM, and cycloheximide, solubilized in water, was added at a
final concentration of 20 µg/ml.
Western blotting.
Cells were washed twice in ice-cold
phosphate-buffered saline (PBS), scraped into PBS, and centrifuged at
1,000 × g for 5 min. The cells were lysed in 250 mM
Tris (pH 7.8)-60 mM KCl-1 mM EDTA-1 mM dithiothreitol-1 mM
phenylmethylsulfonyl fluoride by three cycles of freezing and thawing.
The cell extracts were centrifuged at 13,000 × g for
10 min at 4°C, and the protein concentration of the supernatant
(protein extract) was determined by the Bradford method (Bio-Rad). A
45-µg portion of total protein (unless otherwise indicated) was
boiled for 5 min in sample buffer (2% sodium dodecyl sulfate [SDS],
80 mM Tris [pH 6.8], 10% glycerol, 5% 2-mercapthoethanol, 0.001%
bromophenol blue), separated on an SDS-10% polyacrylamide gel, and
transferred onto a polyvinylidene difluoride membrane (Millipore). The
membrane was blocked for 30 min in 5% dry milk-0.2% Tween 20 in PBS.
Primary antibodies C-20 (anti-E2F; Santa Cruz), diluted 1:500, CM-1
(anti-p53) (42) diluted 1:1,000, PC-10
(anti-proliferating-cell nuclear antigen [anti-PCNA] ascites)
(53) diluted 1:3,000, and 4B2 (anti-Mdm2) (9) at
2.9 µg/ml were used. Horseradish peroxidase-conjugated anti-mouse and
anti-rabbit immunoglobulin G (IgG) (DAKO) diluted 1:1,000 were used as
secondary antibodies. All antibodies were diluted in 5% dry
milk-0.2% Tween 20 in PBS at the indicated concentrations, incubated
for 90 min, and given three 5-min washes with PBS-0.2% Tween 20. The
Western blots were developed by the enhanced chemiluminescence method.
Northern blotting.
Cells were washed twice in ice-cold PBS,
scraped in PBS, and centrifuged at 1,000 × g for 5 min. Poly(A)+ mRNA was prepared with the QuickPrepR Micro
mRNA purification kit (Promega) as specified by the manufacturer. A
4.5-µg portion of poly(A)+ mRNA was resolved on a 1.4%
agarose-formaldehyde gel, transferred onto a Hybond N+
nylon membrane, and hybridized with a SalI-XhoI
fragment of the human E2F-1 gene (provided by Ed Harlow, Boston, Mass.)
or with a HindIII fragment of human mdm2. The
filters were reprobed with a PstI fragment of the open
reading frame of mouse glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)
cDNA (16).
Microinjection into cells.
Cells grown on 22-mm glass
coverslips were injected with protein A-purified monoclonal antibodies
3G5 and SMP 14 (1 mg/ml) or mdm2 antisense and control
oligonucleotides (1 mg/ml) (10) together with an unrelated
monoclonal antibody (1 mg/ml) by using the Eppendorf 5242 microinjector
and 5170 micromanipulator mounted to an Axiovert 35 M with heated
stage. After a 20-h incubation, the cells were fixed for 10 min in 1%
paraformaldehyde, permeabilized in 1% Nonidet P-40 (NP-40) in PBS, and
blocked in 10% FCS in PBS for 30 min. They were incubated for 1 h
with C-20 diluted 1:300 in 10% FCS in PBS, washed with PBS, and
incubated for 1 h with fluorescein isothiocyanate
(FITC)-conjugated donkey anti-rabbit IgG and Texas red-conjugated goat
anti-mouse IgG (Jackson Immunochemicals) diluted 1:500 in 10% FCS in
PBS. They were then washed four times in PBS, stained with
4',6-diamidino-2-phenylindole (DAPI; 0.5 µg/ml; Sigma) for 2 min, and
washed again with PBS; the coverslips were mounted on microscope slides
with Hydromount (National Diagnostics)-2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma).
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RESULTS AND DISCUSSION |
Principle of the study.
Since the 1984 study by Maltzman and
Czyzyk (40), it has been known that the tumor suppressor
protein p53 is upregulated in response to irradiation by prolongation
of the half-life of the protein. Today the mechanism leading to this
stabilization is still not completely understood. Recently, it has been
shown that overexpression of Mdm2 reduces the amount of endogenous p53 (36) and that cotransfection of mdm2 and p53 into
human cell lines reduces p53 expression compared to transfection of p53
alone (23). These findings suggest that Mdm2 can
downregulate p53 expression. Honda et al. showed that Mdm2 is a
ubiquitin ligase for p53, thus marking p53 for rapid degradation by
proteasomes (29), and we found that expression of a
synthetic miniprotein that competes with p53 for Mdm2 binding induces
p53 expression and activates the p53 response (6).
Furthermore, increased stability of exogenous mutated p53 stably
expressed in tumor cells turned out not to be dependent on individual
mutations but to depend strictly on the binding of p53 to Mdm2
(43). The mdm2 gene is transcriptionally
activated by binding of p53 to an internal promoter within the gene
(5, 56). However, p53 is frequently mutated in tumor cells,
and many mutations target the function of p53 as a transcription
factor. As a consequence, Mdm2 expression is downregulated in many
tumors and it can no longer target p53 for rapid degradation. This
evidence led to the idea that Mdm2 might be a key regulator of p53
stability and that inhibition of the interaction of p53 and Mdm2, e.g.,
by posttranslational modifications, could result in the stabilization
of p53, such as that observed after DNA damage. It should be noted that
all the above observations were obtained with genetically engineered cells and that one should be careful interpreting the data in relation
to the processes occurring in normal cells.
The recent analysis of p53 constructs in which all known
phosphorylation sites have been point mutated or deleted and which
have
been expressed in eucaryotic cells showed that phosphorylation
of p53
is not likely to be required for the stabilization of p53
in response
to DNA damage (
4,
26). However, if p53 itself
is not
modified by DNA damage in a way that abolishes its degradation
by the
ubiquitin-dependent degradation process, another protein
of this system
may potentially be modified in such a way as to
impair the degradation
of p53. Mdm2 is a very attractive candidate
as a target for such
modifications. If, however, modifications
of Mdm2 interfere with the
degradation of p53, other proteins
which bind to Mdm2 could be
regulated similarly. We were therefore
particularly interested in
identifying a protein which binds to
Mdm2 and which is regulated in a
way similar to p53, because this
would further support our idea that
binding to Mdm2 is crucial
for p53 instability and that inhibition of
this interaction stabilizes
p53, e.g., after DNA damage. A recent
report showed that Mdm2
functionally cooperates with the transcription
factor E2F-1; moreover,
it was reported that the p53 binding domain of
Mdm2 also binds
directly and specifically to E2F-1 (
41). The
E2F-1 protein is
therefore a good candidate for Mdm2-dependent
regulation of
stability.
E2F-1 is differentially upregulated in response to X-ray and UVC
irradiation.
We wished to investigate whether E2F-1 might be
upregulated following DNA damage in a similar way to the upregulation
of p53. We therefore irradiated U2-OS cells either with 30 J of
UVC light per m2 or with 5 Gy of X rays, harvested the
cells at different time points after irradiation, and analyzed the cell
extracts for E2F-1 expression by Western blotting. We found that both
ionizing radiation and UVC irradiation increased the expression level
of E2F-1 (Fig. 1), but we noted a
striking difference in the kinetics of E2F-1 expression following the
two forms of radiation. The increase in the level of E2F-1 protein was
detectable 4 to 6 h after exposure to UVC, increased with time,
and remained high for at least 24 h (Fig. 1A.I). In contrast,
induction after X-ray application was more rapid, showing a response as
early as 1.5 to 2 h and reaching a plateau after 2 h. The
most striking difference, however, was the decrease in E2F-1 levels 6 to 8 h after irradiation with X rays (Fig. 1B.I). The overall
increase in E2F-1 protein expression after UVC irradiation was much
higher than the response to X rays. E2F-1 expression in response to
irradiation thus showed a striking similarity to p53 expression in
response to the same stimuli, which is also differentially induced
after ionizing and UVC irradiation (38). To confirm that p53
and E2F-1 show the same kinetics in response to irradiation, we
rehybridized the Western blot membranes with an antibody recognizing
p53. As expected, the kinetics of p53 induction were very similar to
the kinetics of induction of E2F-1, showing a rapid and transient
increase after irradiation with X rays and a delayed and persistent
induction after irradiation with UVC, which exceeded the induction of
p53 at the peak of the X-ray response. Overall, the p53 induction
seemed to be slightly faster than the E2F-1 induction, since it was
already clearly detectable 2 to 4 h after irradiation with UVC.
Interestingly, p53 displayed a second wave of induction at 24 to
32 h after irradiation with X rays in this cell line, which is
clearly distinct from E2F-1 expression (Fig. 1B.I). While p53 and E2F-1
accumulated during the time course after UVC irradiation, Mdm2
expression decreased continually in U2-OS cells, becoming undetectable
6 h after UVC irradiation. Thus, the decrease in Mdm2 expression exactly preceded the increase in E2F-1 and p53 expression (Fig. 1A.II).
Although the mdm2 gene is a p53 target gene, we were not able to detect induction of Mdm2 by p53 in response to UVC irradiation during the time course in these cells. This result is consistent with
the observation of Wu and Levine (55) that the induction of
Mdm2 in response to high-dose UV irradiation is quite late and delayed
compared, e.g., to the induction of p21, another p53 target gene, which
is already accumulating 2 to 5 h after UVC irradiation in some
cell lines. In response to irradiation with X rays, there was also a
slight initial decrease in Mdm2 expression in U2-OS cells. This
decrease in Mdm2 expression preceded the induction of E2F-1 and p53. At
4 to 6 h after X-ray irradiation, Mdm2 expression increased,
probably due to activated p53, whose expression reached maximal levels
just before the increase in Mdm2 protein became visible. This increased
Mdm2 expression then correlated with a decrease in the expression of
E2F-1 and p53 8 h after X-ray irradiation. These data suggest that
high expression of p53 and E2F-1 and high expression of Mdm2 are
mutually exclusive, pointing to a potential function of Mdm2 not only
in the regulation of p53 but also in the regulation of E2F-1. However,
the question whether the initial decrease in Mdm2 expression in
response to X rays is sufficient for the upregulation of E2F-1 and p53
or whether further modifications of the proteins involved are required remains to be elucidated in further experiments.
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FIG. 1.
Time course of E2F-1 induction in response to DNA
damage. U2-OS cells were irradiated either with UVC (A) or X rays (B).
At the indicated time points, the cells were harvested and 45 µg of
protein was separated on a 10% polyacrylamide minigel. After Western
transfer, the membranes were consecutively hybridized with antibodies
recognizing E2F-1 (C-20), p53 (CM-1), and PCNA (PC10) (A.I and B.I) or
with antibodies recognizing Mdm2 (4B2) and PCNA (A.II and B.II). The
Western blots were developed by the enhanced chemiluminescence method.
The time course of E2F-1 induction and the time course of p53 induction
in response to irradiation are almost identical.
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Rehybridization of the membranes with an antibody recognizing PCNA
showed no major differences in PCNA signals during the
time courses,
confirming that approximately equal amounts of proteins
had been loaded
onto the gel (Fig.
1). In our experiment, we were
not able to confirm
the results of Huang et al. (
30), who detected
upregulation
of E2F-1 from 6 to 24 h after X-ray treatment. The
reason for this
might be the different cell types
used.
We extended our study to another cell line to confirm that the
upregulation of E2F-1 in response to irradiation is not restricted
to
U2-OS cells. We used a human lymphoblastoid cell line (GM02184D)
originating from a healthy donor, irradiated the cells, and analyzed
them at different times after irradiation for E2F-1, p53, and
Mdm2
expression. From previous experiments, we knew that p53 was
strongly
induced in this cell line both at 1.5 and 4 h after irradiation
with X rays and at 4 and 9 h after irradiation with UVC. As
expected,
E2F-1 was upregulated in this cell line at both time points
after
UVC and X-ray irradiation (Fig.
2).
We probed also for p53 expression
and found that p53 was induced
simultaneously (Fig.
2). The analysis
of Mdm2 expression in these cells
showed that Mdm2 expression
was also increased at both 1.5 and 4 h
after X-ray irradiation
and at 9 h after UVC irradiation (Fig.
2)
and thus that induction
of Mdm2 is much faster in these cells than in
U2-OS cells.
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FIG. 2.
E2F-1 upregulation in response to DNA damage is not
restricted to a particular cell line. GM02184D cells (human lymphoblast
cell line from a healthy donor) were irradiated with X rays (5 Gy) or
UVC (30 J/m2) or treated with actinomycin D (Act. D; 60 ng/ml or 2 µg/ml, as indicated). The cells were harvested 0, 1.5, and
4 h after irradiation with X rays or 0, 5, and 9 h after
irradiation with UVC or after treatment with actinomycin D. Proteins
(75 µg) were separated on a 10% polyacrylamide gel and analyzed by
the Western blot method for expression of Mdm2, E2F-1, and p53 protein
by using the 4B2 anti-Mdm2, C-20 anti-E2F-1, and CM-1 anti-p53
antibodies.
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p53 is very strongly induced not only by DNA damage caused by
irradiation but also by actinomycin D, an agent which intercalates
into
DNA, resulting in DNA strand breaks. The kinetics of p53
induction by
actinomycin D is very similar, if not identical,
to the kinetics of p53
induction in response to UVC (
4). If
p53 and E2F-1 are
induced after DNA damage by the same mechanism,
E2F-1 should also be
upregulated by actinomycin D. To test this
prediction, we treated the
cells with two different concentrations
of actinomycin D, harvested the
cells at two different time points
after treatment, and analyzed the
cell extracts for both E2F-1
and p53 expression. Figure
2 shows that
both p53 and E2F-1 are
strongly induced by both concentrations of
actinomycin D at both
time points and that Mdm2 protein levels are
decreased after treatment
with actinomycin D and are undetectable
9 h after the cells were
treated with 60 ng of actinomycin D per
ml and 5 and 9 h after
the cells were treated with 2 µg of
actinomycin D per
ml.
Immunofluorescence staining of cells showed that p53 upregulation in
response to irradiation is very heterogeneous among individual
cells
(
38). While most cells show very little enhanced nuclear
p53
staining, in a small percentage of the cells there is an intense
accumulation of p53. We analyzed E2F-1 expression in individual
cells
in response to UVC irradiation by immunofluorescence staining
and found
that E2F-1 expression in response to DNA damage is as
heterogeneous as
is p53 expression. Moreover, intense nuclear
E2F-1 and p53 staining
colocalized in the very same cell (data
not shown). This observation
strongly indicates that the two proteins
are coregulated: whatever
factor causes variation between cells
in the p53 response causes
exactly the same variation in the E2F-1
response.
E2F-1 upregulation is due to prolongation of the protein
half-life.
We investigated whether the increase in E2F-1
expression is preceded by an upregulation of E2F-1 RNA. Accumulation of
p53 protein after DNA damage is due to protein stabilization and not to
enhanced synthesis of the protein or to increased transcription of p53
RNA (4, 40). If the two proteins are indeed coregulated, we
would not expect an upregulation of E2F-1 RNA after DNA damage. To test
this prediction, we irradiated U2-OS cells with X rays or UVC or
treated the cells with actinomycin D and analyzed poly(A)+
mRNA for E2F-1 expression. Simultaneously, we analyzed the proteins for
upregulation of E2F-1 in Western blots (data not shown). In our
experiments, the E2F-1 RNA level remained constant after irradiation with X rays (Fig. 3). The slight
induction of E2F-1 RNA in Fig. 3B was not consistently seen in all
experiments. Also, after irradiation with UVC or treatment with
actinomycin D, we did not observe any detectable accumulation of E2F-1
RNA. Instead, we repeatedly found a slight reduction in E2F-1 RNA
expression after these treatments. The signal produced by the GAPDH RNA
displays the amount of poly(A)+ mRNA that was transferred
onto the membrane, confirming that there is no enhanced expression of
E2F-1 RNA as a result of the various treatments.
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FIG. 3.
No induction of E2F-1 RNA in response to DNA damage. (A)
U2-OS cells were irradiated with X rays (5 Gy) and harvested after
2.5 h, irradiated with UVC (30 J/m2) and harvested
after 8 h, or treated with actinomycin D (Act. D; 60 ng/ml) and
harvested after 8 h or mock treated for the same times for control
experiments. Poly(A)+ mRNAs were prepared, and 4.5 µg was
resolved on a 1.4% agarose-formaldehyde gel. After transfer to a
Hybond N+ blotting membrane, the membrane was probed consecutively with
32P-labelled E2F-1 and GAPDH cDNAs and exposed to X-ray
film to approximately the same band intensity. (B) The relative levels
of E2F-1 RNA were normalized by using the GAPDH RNA signal and plotted
in arbitrary units.
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Since p53 is upregulated after DNA damage by prolongation of the
half-life of the protein, we attempted to test if E2F-1 is
also
upregulated by stabilization of the protein in response to
DNA damage.
Due to the very low abundance of E2F-1 protein in
the cell lines we
have analyzed, we were not able to label E2F-1
metabolically and
monitor the decay of the protein in the normal,
untreated cellular
environment. Instead, we blocked the overall
protein synthesis of the
cells by addition of cycloheximide and
monitored the degradation of
E2F-1 protein by Western blot analysis
in UVC-irradiated and
nonirradiated cells (Fig.
4). While E2F-1
protein disappeared with a half-life of 100 to 120 min in nonirradiated
cells, it was stable throughout the experiment when the cells
had been
irradiated with UVC 6 to 16 h before the addition of
cycloheximide. p53 behaved accordingly under these experimental
conditions, with the difference that in nonirradiated cells it
disappeared with a half-life of 20 min (data not shown). Although
the
difference in the half-life of E2F-1 in nonirradiated cells
compared to
UVC-irradiated cells is striking, one should be aware
that
determination of the half-life of E2F-1 under these conditions
can be
only a rough estimation. By treating the cells with cycloheximide,
not
only is the de novo synthesis of E2F-1 blocked but also the
synthesis
of proteins required for proteolysis, which influences
the half-life of
all cellular proteins, is blocked.
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FIG. 4.
Induction of E2F-1 in response to UVC irradiation is
mediated by prolongation of the half-life of E2F. (A) U2-OS cells
irradiated with UVC (30 J/m2) or an unirradiated control
was further incubated for 16 h before addition of 20 µg of
cycloheximide (CHX) per ml. After a further incubation for the
indicated periods, the cells were harvested and 45 µg (control) or 20 µg (UVC irradiated) of cellular protein was resolved on a 10%
polyacrylamide minigel. The blots were probed for E2F-1 expression by
using the rabbit polyclonal anti-E2F-1 antibody C-20. The blots were
reprobed with an anti-PCNA antibody (PC10) to show loading of the
samples. (B) E2F-1 expression was quantified, and the mean value of
E2F-1 expression of two independent experiments was plotted.
|
|
Downregulation of Mdm2 correlates negatively with p53 and E2F-1
expression.
Recent reports have shown that p53 is not induced only
by DNA damage but also by the protein kinase inhibitor H7
(48). Interestingly, concentrations which inhibit protein
kinase C specifically are not able to upregulate p53, whereas
concentrations at least 1 log unit above the concentration required to
inhibit protein kinase C can upregulate p53. These observations point
to the involvement of a protein kinase distinct from protein kinase C
which maintains low expression of p53. In previous experiments, we
found that the protein kinase inhibitor DRB also upregulates p53
expression, supporting the idea that inhibition of a particular protein
kinase activity can increase p53 stability without damaging the DNA. We
wanted to know if inhibition of kinase activity by H7 or DRB is also
able to upregulate E2F-1. We treated GM02184D cells with 10 µM H7, a
concentration that specifically inhibits protein kinase C and should
not induce p53, with 100 µM H7, a concentration that has been shown
to induce p53 expression (48), and with 250 µM DRB.
Actinomycin D was used as an internal control. The differentially treated cells were harvested 18 h after stimulation. While
treatment with 10 µM H7 had no effect on E2F-1 expression, we found a
strong induction of E2F-1 after using 100 µM H7 or 250 µM DRB.
Rehybridization of the membranes with an anti-p53 antibody showed the
same dose dependency of H7 for p53 as well as upregulation of p53 by
DRB and actinomycin D (Fig. 5A). We
analyzed Mdm2 abundance under these conditions, which upregulated p53
and E2F-1, by rehybridizing the membranes from our experiments with the
kinase inhibitors with an anti-Mdm2 antibody. We found that the basal
Mdm2 expression was only marginally reduced after treating the cells
with concentrations of H7 sufficient to block protein kinase C
activity. However, when we treated the cells with concentrations of H7
which upregulated p53 and E2F-1, Mdm2 disappeared completely (Fig. 5A).
Mdm2 expression was also not detectable in cells which had been treated
with DRB. In cells which had been treated with 60 ng of actinomycin D
per ml, Mdm2 was detectable in some experiments only as a faint band (Fig. 5A) while it was undetectable in others, and this band was lost
in every experiment when we used higher concentrations of actinomycin D
(Fig. 2). As in irradiated U2-OS cells (Fig. 1), accumulation of E2F-1
and p53 correlated with the disappearance of Mdm2.
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FIG. 5.
Inhibitors of transcription and protein kinase
inhibitors upregulate E2F-1 expression. GM02184D cells were treated
with H7 (10 and 100 µM, as indicated), DRB (250 µM), and
actinomycin D (60 ng/ml). (A) At 18 h after treatment, the cells
were harvested and 45 µg of protein extract was separated on an
SDS-10% polyacrylamide gel. The proteins were transferred to a
polyvinylidene difluoride membrane and analyzed for Mdm2, E2F-1, and
p53 expression and for expression of PCNA for the loading control. (B)
Cells were harvested after the indicated time and analyzed for the
expression of Mdm2, E2F-1, p53, and PCNA for the loading control. (C)
Cells were harvested at the indicated time points, and
poly(A)+ mRNAs were prepared. mRNAs (4.5-µg portions)
were resolved on a 1.4% agarose-formaldehyde gel and transferred to a
Hybond N+ blotting membrane. The membrane was probed
consecutively with 32P-labelled mdm2 and GAPDH
cDNAs and exposed to X-ray film. The relative levels of mdm2
RNAs were normalized by using the GAPDH RNA signal and plotted in
arbitrary units. Upregulation of E2F-1 and p53 correlates with the
disappearance of Mdm2.
|
|
We further investigated whether the virtual mutual exclusion of Mdm2
expression and E2F-1/p53 accumulation was also reflected
in a time
course experiment. We treated GM02184D cells with 100
µM H7, 60 ng of
actinomycin D per ml, or 250 µM DRB and harvested
them after
increasing time intervals. We found that all three
agents decreased
Mdm2 expression to barely detectable levels in
less than 5 h. As
soon as Mdm2 protein was decreased to an almost
undetectable level, we
saw an increase in E2F-1 expression (Fig.
5B). The increase in p53
expression occurred slightly earlier,
exactly as it did after UVC
irradiation (Fig.
1A.I). We measured
the half-life of Mdm2 protein in
cells which had been treated
with these inhibitory agents; however, we
found no increased degradation
of Mdm2 protein under these conditions
(data not shown), which
could be responsible for the rapid
disappearance of the protein.
Northern analysis, however, revealed that
the decrease in Mdm2
protein expression was strictly correlated with a
rapid decrease
in
mdm2 RNA levels (Fig.
5C). DRB inhibits
mRNA elongation by
inhibiting the transcription factor IIH
(TFIIH)-associated protein
kinase (
59), and the decrease in
mdm2 RNA levels is probably
due to this activity. H7 at 100 µM inhibits various protein kinases
quite nonspecifically, and it is
conceivable that it can inhibit
the TFIIH-associated kinase as well. H7
could therefore easily
cause the same phenotype as DRB, as we see it in
our experiments.
It should be noted that the signal for GAPDH RNA
increases during
the time course when cells have been treated with DRB
or high
concentrations of H7 but also with 60 ng of actinomycin D per
ml. This is probably due to inhibition of transcription or inhibition
of RNA elongation. De novo synthesis is blocked under these conditions,
while the degradation, at least at early time points after treatment,
is not affected. Thus, transcripts with a short half-life are
depleted
from the RNA pool while transcripts with a long half-life,
like the
GAPDH RNA, are
enriched.
In all our experiments, we found an absolute conformity between p53 and
E2F-1 induction and Mdm2 disappearance when using
different
concentrations and different inhibitory agents. Mdm2
is supposed to be
the ubiquitin ligase for p53 (
29), and thus
the
disappearance of the p53 ubiquitin ligase could well be the
reason for
the increased stability of
p53.
Our results which show that not only p53 but also E2F-1 is stabilized
when Mdm2 disappears, together with the evidence from
Martin et al.
(
41) for a physical interaction between Mdm2 and
E2F-1,
speak persuasively in favor of both p53 and E2F-1 being
targeted by
Mdm2 for
degradation.
E2F-1 upregulation in response to DNA is impaired in cells with
mutated p53.
To further support our theory that Mdm2 plays a
regulatory role in the accumulation not only of p53 but also of E2F-1,
we sought to analyze more cell lines with different levels of Mdm2 expression. We used GM02184D and U2-OS cells as cell lines with wild-type p53 and normal expression levels of Mdm2. We also used MCF-7
cells, which are derived from a breast tumor and which overexpress Mdm2
at the protein level (5), and OSA cells, a human
osteosarcoma cell line with highly elevated Mdm2 levels due to gene
amplification (15), as cell lines which possess wild-type
p53 but which express Mdm2 at unusually high levels. A431 cells, HAKAT
cells, and T47D cells were used as cell lines which possess mutated p53
and virtually no detectable Mdm2. We confirmed the relative Mdm2 levels
in these different cell lines by immunoprecipitation and Western
blotting (data not shown).
The cells were irradiated with 30 J of UVC per m
2 or
treated with actinomycin D at a final concentration of 60 ng/ml and
harvested
18 h posttreatment, and the protein extracts were
analyzed in
Western blots for E2F-1 and p53 expression (Fig.
6). As expected,
U2-OS cells and GM02184D
cells accumulated both p53 and E2F-1
in response to UVC irradiation and
in response to treatment with
actinomycin D. Although MCF-7 cells and
OSA cells overexpress
Mdm2, they still accumulated p53 in response to
DNA damage, confirming
earlier experiments (
6a,
43). Both
cell lines also induced
E2F-1 protein in response to UVC irradiation or
treatment with
actinomycin D. HAKAT cells, A431 cells, or T47D cells,
in which
the p53 gene is mutated, failed repeatedly to upregulate E2F-1
or p53 in response to these agents. We used three different tumor
cell
lines because cell lines with mutated p53 are genomically
unstable and
quite frequently acquire secondary mutations. By
analyzing three
different cell lines with mutant p53, we wanted
to rule out the
possibility that other mutations, apart from the
mutation of p53, are
responsible for the failure to induce E2F-1.
It should be noted that
there was no obvious increase in basal
expression of E2F-1 in these
cell lines whereas p53 expression
was markedly increased in the cell
lines with mutated p53. We
do not completely understand the difference
in basal expression
of E2F-1 and p53 in cell lines in which p53 is
mutated, but since
several factors contribute to the basal expression
of a protein,
we assume that Mdm2 is only one of the regulators of
E2F-1 expression
which become important when the DNA of the cell is
damaged. Previous
experiments have shown that Mdm2 levels are
profoundly decreased
in MCF-7 and OSA cells after UVC irradiation (data
not shown)
and probably also after treatment with actinomycin D. This
reduction
in Mdm2 expression is presumably responsible for decreased
degradation
of p53 and E2F-1 in these cells after UVC irradiation or
addition
of actinomycin D. Therefore, the enhanced Mdm2 expression
under
normal conditions does not interfere with upregulation of p53
and
E2F-1 in response to irradiation with UVC or treatment with
actinomycin
D. A431, HAKAT, and T47D cells, on the other hand,
possess mutated p53
with a markedly decreased turnover of p53.
Interestingly, transfection
of wild-type p53 into one of these
tumor cell lines (A431) has been
reported to result in stable
endogenous and exogenous p53, although the
same product was obviously
degraded in cell lines harboring wild-type
p53. Other experiments
showed that mutant p53 accumulated in response
to DNA damage in
MCF-7 cells which possess endogenous wild-type p53
(
43). This
strongly indicates that point mutations of p53 do
not result in
intrinsically stable p53, which cannot accumulate
further. Moreover,
loss of induction of mutant p53 in response to DNA
damage in tumor
cell lines such as A431 is the result of the cell
environment.
Our data suggest that E2F-1 accumulation is dependent on
the same
cell environment as p53 and that it accumulates only in
response
to DNA damage in an environment possessing wild-type p53.
Experiments
reported by Midgley and Lane (
43) have shown
that expression
of Mdm2 after microinjection reduces p53 stability in
A431 cells.
We tried a similar approach by using A431 cells stably
transfected
with an inducible
mdm2 expression vector.
Unfortunately, the basal
E2F-1 expression turned out to be too low to
monitor a further
reduction by ectopic Mdm2 expression.
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FIG. 6.
E2F-1 is induced in response to UVC or actinomycin D in
cells with wild-type p53 but not in cells with mutated p53. (I) Cell
lines with wild-type p53 (GM02184D, U2-OS, MCF-7, and OSA cells). (II)
Cell lines with mutated p53 (HAKAT, T47D, and A431 cells). The cells
were irradiated with UVC (UV; 30 J/m2), treated with
actinomycin D (A; 60 ng/ml), or left untreated for control experiments
( ). At 18 h after treatment, the cells were harvested and
analyzed for the expression of E2F-1, p53, and PCNA as described in the
legend to Fig. 1.
|
|
Microinjection of 3G5 antibody or mdm2 antisense
oligonucleotides induce E2F-1 protein expression.
Although we
observed a striking correlation between downregulation of Mdm2 and
accumulation of both E2F-1 and p53 under various conditions, we were
not able to rule out the contribution of other cellular pathways to the
upregulation of E2F-1. If, however, Mdm2 is the molecule that targets
E2F-1 for degradation, as it does for p53, specific disruption of this
pathway would be predicted to cause accumulation of E2F-1. We used two
similar approaches to disrupt a predicted interaction of Mdm2 and
E2F-1: (i) microinjection of mdm2 antisense oligonucleotides
and (ii) microinjection of anti-Mdm2 antibody 3G5 as previously
described (6, 43).
Antisense oligonucleotides often act by inducing RNase H cleavage at
the heteroduplex region, resulting in increased degradation
of the
target mRNA and reduction in the expression of the target
protein.
Antisense phosphothiorate oligodeoxynucleotides which
specifically bind
to human
mdm2 RNA inhibit Mdm2 protein expression
and
Mdm2-p53 complex formation even in tumor cells containing
mdm2 gene amplifications (
10). We microinjected
antisense oligonucleotides
at 1 mg/ml in the presence of a mouse
monoclonal antibody, to
facilitate the detection of microinjected
cells, and analyzed
E2F-1 expression by immunofluorescence. Figure
7 shows that E2F-1
accumulated repeatedly
in cells which were injected with
mdm2 antisense
oligonucleotides (Fig.
7a and c) but not in cells which
were injected
with unrelated oligonucleotides (Fig.
7e and g).
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FIG. 7.
E2F-1 protein is upregulated after microinjection of
mdm2 antisense oligonucleotides. U2-OS cells were
microinjected with mdm2 antisense oligonucleotides (a to d)
or unrelated oligonucleotides for control experiments (e to h),
together with an unrelated mouse monoclonal antibody to identify
microinjected cells. At 20 h after microinjection, the cells were
fixed with 1% paraformaldehyde, permeabilized with 1% NP-40, and
stained with rabbit anti-E2F-1 antiserum (C-20) followed by
FITC-conjugated donkey anti-rabbit IgG and Texas red-conjugated goat
anti-mouse IgG. The right-hand panels (in red) show successful
injection of oligonucleotides by staining of coinjected mouse
monoclonal antibodies with Texas red-conjugated anti-mouse IgG (b, d,
f, and h). The left-hand panels (in green) show expression of E2F-1 (a,
c, e, and g). Arrowheads indicate microinjected cells. Cells
microinjected with mdm2 antisense oligonucleotides
upregulate E2F-1 protein, while microinjection with control
oligonucleotides has no effect on E2F-1 expression.
|
|
To further support our experimental evidence that E2F-1 is regulated
via its interaction with Mdm2, we microinjected two different
monoclonal antibodies recognizing two different domains of Mdm2
protein. The 3G5 anti-Mdm2 antibody binds to an epitope at the
amino
terminus of Mdm2, involving the p53 binding pocket, and
the SMP 14 anti-Mdm2 antibody is directed against an epitope in
the central part
of the Mdm2 protein. It has been shown that the
3G5 antibody is able to
block the interaction of p53 with Mdm2
(
5), and after
microinjection, 3G5 upregulates p53 expression
(
6,
43). p53
and E2F-1 were coregulated throughout our experiments,
and thus we
expected that Mdm2 would interact with E2F-1 via the
same domain as it
interacts with p53. To determine whether anti-Mdm2
antibodies are able
to disrupt the interaction of Mdm2 and E2F-1,
leading to the
accumulation of E2F-1, we microinjected U2-OS cells
with 1 mg of 3G5
per ml or with 1 mg of SMP 14 per ml and costained
them with anti-mouse
IgG, to facilitate detection of microinjected
cells, and with the C-20
anti-E2F-1 antibody. Only cells microinjected
with 3G5 showed increased
nuclear staining for E2F-1 (Fig.
8e
and
g), while cells microinjected with SMP 14 showed no difference
in E2F-1 expression in comparison with noninjected
cells (Fig.
8a and c). Microinjection of antibodies directed against
pRb were
used as a further control, but these antibodies were also
unable
to induce E2F-1 expression (data not shown). These experiments
demonstrate that it is specifically the interruption of Mdm2-E2F-1
complexes which induces E2F-1 expression, since it has been shown
that
interruption of p53-Mdm2 complexes, and not the microinjection
process
or the presence of antibodies in the nucleus, induces
p53 expression
(
43).
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FIG. 8.
Microinjection of anti-Mdm2 3G5 antibody induces the
expression of E2F-1. U2-OS cells were microinjected with mouse
monoclonal antibodies SMP 14 (a to d) or 3G5 (e to h), recognizing
different epitopes on Mdm2. At 20 h after microinjection, the
cells were fixed with 1% paraformaldehyde, permeabilized with 1%
NP-40, and incubated with a rabbit anti-E2F-1 antibody (C-20, diluted
1:300) followed by FITC-conjugated donkey anti-rabbit IgG (a, c, e, and
g) and Texas red-conjugated goat anti-mouse IgG (b, d, f, and h),
recognizing the microinjected anti-Mdm2 antibody. Cells microinjected
with 3G5 upregulate E2F-1 protein, while cells microinjected with SMP
14 do not affect E2F-1 expression.
|
|
Our data obtained by various microinjection experiments provide direct
evidence that E2F-1 and p53 display the same kinetics
after irradiation
and every inhibitory agent used and, moreover,
that the expression and
stability of both proteins are mediated
through their interaction with
cellular
Mdm2.
Conclusions.
The experiments presented here demonstrate that
p53 and E2F-1 are coregulated under various conditions. Previous
studies have shown that both proteins are degraded by the
ubiquitin-dependent degradation system (22, 39), and Mdm2
was identified as a ubiquitin ligase for p53 (29).
Additionally, Mdm2 is required for the export of p53 into the
cytoplasm, where it is degraded by proteasomes (17, 49).
Future studies must investigate whether Mdm2 is also a ubiquitin ligase
for E2F-1 and whether E2F-1, like p53, must be exported into the
cytoplasm in order to be degraded.
Despite repeated efforts, we were unable to detect any decrease in
E2F-1 expression in cells cotransfected with E2F-1 and
mdm2
or in cells cotransfected with DP-1, E2F-1, and
mdm2,
compared
to cells transfected with E2F-1 alone. This is in contrast to
the clear decrease seen in p53 expression when cotransfected with
mdm2 (
4a,
23). Microinjection of the 3G5
anti-Mdm2 antibody,
however, shows that the p53 interaction domain of
Mdm2 is required
for degradation of E2F-1. We know from previous
studies that only
p53 tetramers are targeted for degradation by Mdm2
(
4), and
so we assume that E2F-1 has to oligomerize with
other proteins
distinct from DP-1 to be targeted for degradation by
Mdm2. Alternatively,
Mdm2 could act more indirectly by regulating a
protein required
for E2F-1
degradation.
Since E2F-1 and p53 are both induced by DNA damage and since both
proteins interact physically with Mdm2, it becomes likely
that
upregulation of p53 and E2F-1 in response to DNA damage is
caused by
disruption of Mdm2-p53 and Mdm2-E2F-1 complexes. As
a consequence, p53
and E2F-1 are no longer subjected to rapid
degradation and accumulate
in the cell. Recently, Nip et al. have
shown that DNA damage caused by
topoisomerase II inhibition induces
apoptosis in a cell line
overexpressing E2F-1 (
46). Although
the authors did not
confirm the E2F-1 expression levels following
etoposide treatment,
which are presumably increased, their data
suggest that upregulation of
E2F-1 in response to DNA damage has
functional consequences.
Interestingly, E2F-1-specific induction
of apoptosis is blocked by
coexpression of Mdm2 (
34). Although
the authors did not
determine whether inhibition of E2F-1-specific
apoptosis by Mdm2 occurs
at the level of E2F-1 expression, E2F-1
activity, or both, they
confirmed the role of Mdm2 in the regulation
of E2F-1.
| |
ACKNOWLEDGMENTS |
This work was funded by the Cancer Research Campaign. D.P.L. is a
Gibb Fellow of the Cancer Research Campaign.
We thank Ed Harlow for providing the E2F-1 cDNA, Arnold Levine for
providing the 3G5 and 4B2 antibodies, and Sudhir Agrawal for providing
control and mdm2 antisense oligonucleotides. We are grateful
to our colleagues Carol Midgley for providing the A431 cell line with
an inducible mdm2 expression plasmid, Ralf Dahm for helping with the
confocal images, and Dimitris Xirodimas for being always ready to help.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Cancer Research
Campaign Cell Transformation Group, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, United Kingdom. Phone: 44-1382-344920. Fax: 44-1382-224117. E-mail:
dplane{at}bad.Dundee.ac.uk.
| |
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Molecular and Cellular Biology, May 1999, p. 3704-3713, Vol. 19, No. 5
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
