(Received for publication, August 17, 1996, and in revised form, January 17, 1997)
From the Division of Hemopoiesis, E2F is a heterodimeric transcription factor that
controls transcription of several growth-regulatory genes including
cdc2. To investigate the mechanism of interferon- Interferons (IFNs)1 are a family of
biological response modifiers with a broad spectrum of action on
cellular proliferation as well as immunoregulation (1, 2). It is well
known that IFNs can effectively inhibit the growth of some types of
hematopoietic cells, and now they have been used as a therapeutic
reagent for a variety of hematological malignancies including chronic
myeloid leukemia, hairy cell leukemia, and low grade B-cell lymphomas (3). Although IFNs have an established status as a first-line drug for
chronic myeloid leukemia and hairy cell leukemia, some problems remain
unresolved, including drug resistance and numerous adverse effects.
Insights into the molecular mechanisms of IFN action are helpful in
resolving these problems. However, the mechanisms regulating the
antiproliferative effect of IFNs have not been elucidated fully despite
extensive investigations.
Recent investigations revealed that IFNs can induce activation of a
group of IFN-stimulated gene products including a double-stranded RNA-activable protein kinase (4), the IFN regulatory factors IRF-1 and
IRF-2 (5), gene 200 cluster proteins (6), and RNase L (7). Direct and
indirect evidence indicates that several of these proteins may have
tumor-suppressive activities. Among the growth-regulatory genes and
gene products, IFNs are known to induce down-regulation of
c-myc expression (8), dephosphorylation of the
retinoblastoma (RB) susceptibility gene product (9), and inhibition of
both the expression and activity of cyclin-dependent kinases (10-12). Although the antiproliferative effect of IFN is thought to be mediated through these events, further investigation should be required to elucidate fully the direct mechanisms of its
action. For example, it is difficult to determine whether these events
are the cause of the growth arrest or simply a consequence of
IFN-induced failure of cell cycle progression. Furthermore, through the
study of transcriptional activation in response to IFNs, the direct
involvement of Jak-Stat (Janus kinases-signal transducers and
activators of transcription) pathways in signal transduction of IFNs
has recently been proved (13). The role of the Jak-Stat system in
IFN-mediated growth suppression of hematopoietic cells is not yet
clarified.
E2F is a heterodimeric transcription factor that was originally
identified as an element needed for the E1A-dependent
activation of a specific adenoviral E2 promoter (14). E2F is composed
of each member of the E2F and DP families. Currently, five distinct E2F
family members (E2F-1 to E2F-5) and three DP proteins (DP-1 to DP-3)
are known (15). E2F-1 is the most characterized component of E2F and
binds to DNA as a heterodimer with DP-1. Although DP-1 itself has
little or no affinity for DNA, the association of E2F-1 and DP-1 leads
to enhanced DNA binding and is required for E2F site-dependent transcriptional activation (16). E2F binding sites were detected in the promoter regions of many growth-responsive or growth-promoting genes such as c-myc, c-myb,
cdc2, and genes for dihydrofolate reductase, thymidine
kinase, DNA polymerase- In this study, with this background in mind, investigations were
carried out to clarify the involvement of E2F in IFN-induced growth
suppression of hematopoietic cells. We have also studied the mechanisms
of the inhibition of E2F activity by IFN- Highly purified natural IFN- Burkitt lymphoma cell line Daudi was
maintained in RPMI 1640 medium supplemented with 10% fetal calf serum.
Cellular DNA and RNA synthesis was monitored by pulse labeling the
cells for a final 1 h of the culture with 5 µCi/ml
[3H]thymidine and [3H]uridine (Amersham
Corp.), respectively. Cell cycle distribution was determined by
analyzing the samples of 106 cells stained with propidium
iodide in a flow cytometry with FACScan/CellFIT system
(Becton-Dickinson, San Jose, CA).
The following cDNA clones were used in this
study: a 1.4-kb EcoRI-BamHI fragment of human
E2F-1 cDNA (provided by Drs. William G. Kaelin, Jr.,
James A. DeCaprio, and David M. Livingston, Dana-Farber Cancer
Institute, Boston, MA) (23); a 0.9-kb KpnI-PvuII
fragment of human cdc2 cDNA (provided by Dr. Paul Nurse,
Oxford University, Oxford, U. K.); a 1.8-kb
ClaI-EcoRI fragment of c-myc exon 3 cDNA (Oncor Inc., Gaithersburg, MD); and a 2.2-kb
EcoRI-BamHI fragment of p53 cDNA
(provided by Japanese Cancer Research Resources Bank). A 1.3-kb
full-length fragment of murine DP-1 cDNA and a 1.2-kb full-length fragment of human E2F-4 cDNA were generated
by reverse transcription-polymerase chain reaction based on the
published sequences, respectively (24, 25).
Total cellular RNA was isolated by cesium
chloride centrifugation using CS120FX ultracentrifuge and S100AT5
fixed-angle rotor (Hitachi Koki, Co. Ltd.). 10-µg samples were
electrophoresed in a 1% agarose gel containing 6% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA, and blotted onto Hybond N+ synthetic
nylon membranes (Amersham Corp.). The membranes were hybridized with
each cDNA probe, which was labeled with [32P]dCTP
(DuPont-NEN) with the oligonucleotide random priming method.
Cells were washed with ice-cold TBS buffer
(25 mM Tris-HCl, pH 8.0, 150 mM NaCl), and
lysed in EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 100 mM sodium
fluoride, 200 mM sodium orthovanadate) containing protease
inhibitors. An equal amount (150 µg) of the samples was separated on
a 7.5% SDS-polyacrylamide gel and transferred onto nitrocellulose
filters (Bio-Rad). After blocking in TBS buffer with 4% bovine serum
albumin (fraction V, Sigma), the membranes were incubated for 16 h
with 1 µg/ml anti-pRB monoclonal antibody PMG3-245 (Pharmingen, San
Diego). The membranes were developed as described previously (26). For c-Myc protein detection, anti-c-Myc monoclonal antibody (Pharmingen) was used as a primary antibody, and the bands were visualized by the
enhanced chemiluminescence system (Amersham Corp.).
IFN-treated
Daudi cells were incubated in methionine-free RPMI 1640 medium
supplemented with 150 µCi/ml [35S]methionine for 3 h. Then the cells were washed three times and lysed in EBC buffer.
Incorporation of [35S]methionine was monitored by a
scintillation counting of the trichloroacetic acid-insoluble fraction.
An equal amount (7.5 × 107 cpm) of the lysates was
immunoprecipitated overnight with anti-E2F-1-specific monoclonal
antibodies KH95 (Santa Cruz Biotechnology, Santa Cruz, CA) and SQ41
(Upstate Biotechnology, Lake Placid, NY). Immune complexes were
collected on protein A-Sepharose beads (Pharmacia Biotech Inc.) and
separated by 7.5% SDS-polyacrylamide gel electrophoresis. The signal
was detected by fluorography. To monitor the efficiency of
immunoprecipitation and the integrity of protein, the samples were
subsequently immunoprecipitated with anti- Nuclear extract was prepared
according to the method of Dignam et al. (27). 5-µg
samples were incubated with approximately 0.5 ng (10,000 cpm) of
32P-labeled DNA fragment containing the E2F binding site
(5 Double-stranded oligonucleotides containing the AP-1 binding site
(5 For antibody perturbation experiments, 1 µg of each antibody was
added to the above noted reaction mixture. Specific antibodies used in
this study were as follows: anti-E2F-1, C-20; anti-E2F-4, C-108;
anti-pRB, C36; anti-pRB, XZ55; anti-p107, SD9; and anti-p130, C-20 (all
purchased from Santa Cruz Biotechnology except C36 and XZ55 from
Pharmingen). Densitometric analysis of the results was performed by a
Shimazu flying spot scanner CS-9000 (Shimazu Seisakusyo, Tokyo,
Japan).
Whole cell
lysates of Daudi cells (5 mg) were incubated for 60 min at 4 °C with
anti-pRB antibody C36 in IP-DOC buffer (20 mM Hepes, pH
7.9, 40 mM KCl, 6 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, and 0.1% Nonidet
P-40) containing protease inhibitors. Immune complexes were recovered
on protein A-Sepharose beads and washed three times in IP-DOC buffer.
The associated proteins were released by the addition of 16 µl of
0.8% deoxycholate and then neutralized with 4 µl of 6% Nonidet P-40
(28). The supernatants (4 µl/assay) were used for E2F gel shift
assays as described above.
Plasmids were introduced into Daudi cells by
electroporation as described previously (29). Briefly, cells (2 × 107 cells/transfection) were resuspended in 500 µl of
RPMI 1640 medium containing 20% fetal calf serum with 40 µg of
linearized plasmid DNA. Electropulse was delivered at 250 V, 960 microfarads by a Gene Pulser (Bio-Rad). The cells were placed on ice
for 15 min, resuspended at 5 × 105 cells/ml in RPMI
1640 containing 10% fetal calf serum, and split equally into two
flasks. IFN- Among hematopoietic cells, Daudi is the most
sensitive to IFN and easily arrests in G0/G1
phase of the cell cycle in response to a relatively low amount of
IFN-
Total cellular RNA
was isolated from Daudi cells at various time points after IFN-
Next,
we examined the effect of IFN-
To
investigate whether this reduction of the amount of E2F-1 protein
affects the transcriptional activity of E2F, we first examined the DNA
binding ability of E2F in IFN-treated Daudi cells. Nuclear extracts
were prepared at various time points after IFN treatment and tested for
E2F activity by gel retardation assays using an E2F consensus
oligonucleotide from the adenovirus E2 promoter as a probe (32). As
shown in Fig. 4A, when the nuclear extract
from untreated Daudi cells was used, multiple bands with retarded
mobilities were resolved on a 4% native polyacrylamide gel. By
oligonucleotide competition assays, seven distinct bands (designated as
A to G) were found to be specific E2F complexes. These bands were
considered to represent the baseline E2F activity of proliferating
Daudi cells. Two other fast migrating bands were considered to be
nonspecific, since they were not eliminated by the addition of 100 molar excess of unlabeled E2F oligonucleotide (indicated by
stars in Fig. 4). Then we performed antibody perturbation experiments to identify the nature of each complex. Specific antibodies against E2F-1, E2F-4, pRB, p107, and p130 were included in the reaction
mixture, and the E2F gel shift assay was carried out as above (Fig.
4B). The results were quantitated by densitometric analysis
and are summarized in Table I. Given that any specific antibody against RB family proteins (pRB, p107, and p130) did not
affect the intensity of the bands E, F, and G, these complexes represent so-called free E2F. Furthermore, anti-E2F-4- but not anti-E2F-1-specific antibody could eliminate band E completely, indicating that this band is composed mainly of E2F-4 (Fig.
4B). On the other hand, two faster migrating free E2F bands
(F and G) were not supershifted by anti-E2F-4 antibody, but the signal intensities were slightly but significantly decreased in the presence of anti-E2F-1 antibody (Fig. 4B and Table I). This suggests
that complexes F and G contain E2F-1 and other components of the E2F family such as E2F-2 and E2F-3. Four other bands (A to D) were found to
be complexes of E2F and RB family proteins. Anti-pRB antibody
effectively supershifted both bands C and D, and the former was
eliminated by anti-E2F-4. These results therefore indicate that band C
represents a complex containing E2F-4 and pRB. In contrast, band D was
not abolished by either anti-E2F-1 or anti-E2F-4, suggesting that other
E2F family members such as E2F-2 and E2F-3 form a complex with pRB in
D. Similarly, the complex A was found to contain E2F-4, p107, and p130.
Complex B disappeared with the addition of anti-p130, but it was not
affected by either anti-E2F-1 or anti-E2F-4. Thus, complex B may be
composed of p130 and a member of the E2F family other than E2F-1 and
E2F-4. E2F-5 is a strong candidate as the E2F molecule present in this
complex, as suggested by recent investigations (33, 34).
Table I.
Quantitation of the effect of specific antibodies on signal intensities
of the E2F complexes
Department of Internal Medicine (Aoto) and
the Department of Molecular Genetics,
(IFN-
)-mediated growth suppression of hematopoietic cells, we
examined the effect of IFN-
on the expression and function of E2F
using IFN-sensitive Daudi cells. Down-regulation of E2F-1, a subunit of
E2F, was observed after 8 h of culture with IFN-
; expression of
E2F-4, another subunit of E2F, and DP-1, a heterodimeric partner of
E2F, was unaffected. Gel shift assays revealed that the DNA binding
activity of free E2F, which is composed of E2F-1 and E2F-4, was
inhibited by IFN-
. In contrast, IFN-
did not affect the DNA
binding ability of E2F-1 and E2F-4 in a complex with retinoblastoma
(RB) susceptibility gene family proteins including pRB, p107, and p130.
IFN-
could induce dephosphorylation of pRB, thereby turning
active E2F-pRB complexes into transcriptional repressors.
Transient chloramphenicol acetyltransferase assays revealed
that the activity of the E2F-dependent cdc2
promoter was suppressed by IFN-
. These results suggest that the
antiproliferative action of IFN-
is mediated through the modulation
of E2F activity in two different ways: down-regulation of
transcriptionally active free E2F and conversion of E2F-pRB complexes
into transcriptional repressors.
, and cyclin A (17). The role of E2F in the
transcriptional regulation of the c-myc, dihydrofolate
reductase, and cdc2 genes has been confirmed (18-20).
Moreover, E2F is now considered to be a relevant target of RB family
proteins including p107, p130, and RB protein (pRB) itself in their
activity as growth suppressors (21). These observations strongly
suggest that E2F can be a target of the action of growth-inhibitory
factors such as IFNs and transforming growth factor-
.
with a special reference
to its interaction with RB protein.
Reagents
derived from
Sendai virus-infected Nawalwa cells (22) was provided by Sumitomo
Pharmaceutical Co. Ltd. (Osaka, Japan). 20 IFN-
components were
included in this preparation with specific activities of 1.3 × 108-2.6 × 108 IU/mg of protein (22).
-actin antibody (Oncogene
Science Inc., Uniondale, NY).
-ATTTAAGTTTCGCGCCCTTTCTCAA-3
) in the presence of 1 µg
of sonicated salmon sperm DNA in a final volume of 25 µl. Incubations
were carried out at room temperature for 30 min in 20 mM
Hepes, pH 7.9, 0.1% Nonidet P-40, 40 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 10% glycerol. The protein-DNA
complexes were resolved on a 4% polyacrylamide gel (0.15 × 16 × 20 cm; acrylamide:bisacrylamide ratio, 86:1 w/w) in
0.25 × Tris borate-EDTA buffer at 4 °C.
-CTAGTGATGAGTCAGCCGGATC-3
) and E2F-1 site (shown above) were used
for competition assays.
was added into one of them at the final concentration
of 100 IU/ml. Cell extracts were prepared after 24 h of the
culture, and the protein concentration was determined by the Bradford
method (60). CAT activity was assayed with 50 µg of each sample. CAT
assays were carried out according to the standard procedure, and the
activities were measured quantitatively by a liquid scintillation
counting (30). The 5
-untranslated sequence of the cdc2
promoter up to nucleotide
383 relative to the transcription start
site was linked to the CAT gene in pCAT-basic vector (Promega, Madison,
WI) and was used as a reporter plasmid (30). pCAT-control vector
(Promega), which contains SV40 promoter and enhancer sequences, was
transfected simultaneously and used as a positive control. As a
negative control, pCAT-basic vector containing the cdc2
promoter (up to
383) with the mutated E2F binding site was used (30).
All plasmids were purified by cesium chloride gradient
ultracentrifugation, linearized by appropriate restriction enzymes, and
purified again by ethanol precipitation before transfection. Each
result was adjusted according to the value obtained with the
transfection of pCAT-control vector into corresponding cells.
Effect of IFN- on Cell Cycle Distribution and DNA Synthesis of
Daudi Cells
(31). We first examined the effect of IFN-
on cell cycle
distribution and DNA synthesis of Daudi cells. The cells were seeded at
an initial concentration of 5 × 105 cells/ml and
grown in the absence or presence of 100 IU/ml IFN-
. The change of
cell cycle distribution was monitored by serial analysis of the DNA
histogram. The proportion of S phase was approximately 45% in
untreated Daudi cells (Fig. 1A, T-0). When
they were cultured with IFN-
, the S phase fraction was decreased
gradually and reached to 21% after 24 h of culture (Fig.
1A, T-24). Conversely, approximately 60% of the cells were
found to be arrested in G0/G1 phase after 24 h. Cellular DNA synthesis, as determined by
[3H]thymidine uptake, correlated well with the portion of
the cells in S phase, i.e. it was suppressed to be about
40% of the untreated control at 24 h (Fig. 1B). RNA
synthesis was also suppressed by IFN-
in a similar manner (Fig.
1C). Using this culture system, we investigated the effect
of IFN-
on E2F.
Fig. 1.
Effect of IFN- on proliferation of Daudi
cells. Daudi cells were cultured at an initial concentration of
5 × 105 cells/ml in the absence (
) or presence (+)
of 100 IU/ml IFN-
for 24 h. Panel A, DNA histogram
was obtained by flow cytometric analysis of propidium iodide-stained
cells (106 cells/sample). The percentage of S phase was
calculated with the CellFIT program. Panel B, cellular DNA
synthesis was monitored by pulse labeling the cells for a final 1 h of the culture with 5 µCi/ml [3H]thymidine. The data
were corrected for the number of viable cells at each time point and
expressed as cpm/105 cells. Panel C, cellular
RNA synthesis was monitored simultaneously with the same procedure
using [3H]uridine. Data shown are representative of three
independent experiments.
[View Larger Version of this Image (26K GIF file)]
on mRNA Expression of Major Subunits of E2F
Transcription Factor, E2F-1, E2F-4, and DP-1
treatment, and the expression of E2F-1, E2F-4,
and DP-1 mRNA was examined by Northern blot
hybridization. In agreement with our previous observation (30), both
E2F-1 and DP-1 mRNA transcripts were detected
readily in untreated Daudi cells, reflecting the proliferative state of
the cells. As shown in Fig. 2A, the amount of
E2F-1 mRNA decreased significantly after 8 h of
culture with IFN-
. It is of note that [3H]thymidine
incorporation, a sensitive marker of cell proliferation, was not
suppressed at this time point (Fig. 2B). This clearly indicates that down-regulation of E2F-1 mRNA is not a
simple consequence of the growth arrest and may have a causative role
in IFN-induced growth arrest. In striking contrast, the E2F-4
mRNA level was unchanged during this culture period.
DP-1 mRNA expression was also unaffected by the IFN
treatment, although a minor decrease was observed after 8 h (Fig.
2A).
Fig. 2.
Effect of IFN- on mRNA expression of
major subunits of E2F transcription factor, E2F-1, E2F-4, and
DP-1. Daudi cells were cultured in the absence (
) or presence
(+) of 100 IU/ml IFN-
for 24 h. Panel A, total
cellular RNA was isolated at the indicated time points and subjected to
sequential analysis for E2F-1, E2F-4, and
DP-1 mRNA expression. Ethidium bromide-stained 28 S
rRNA is shown as a loading control. Panel B, cell
proliferation was monitored simultaneously by an
[3H]thymidine incorporation assay as described in the
legend of Fig. 1. The results are shown as the percent of the untreated control (([3H]thymidine uptake of IFN-
-treated
cells)/([3H]thymidine uptake of the untreated cells at
the same time point) × 100). Data shown are representative of three
independent experiments.
[View Larger Version of this Image (66K GIF file)]
on De Novo Synthesis of E2F-1 Protein
on de novo synthesis of
the E2F-1 gene product by metabolic labeling and subsequent immunoprecipitation. IFN-treated Daudi cells were pulse labeled for
3 h with [35S]methionine, and whole cell lysates
were subjected to immunoprecipitation with anti-E2F-1-specific
monoclonal antibodies to monitor the amount of a newly synthesized
E2F-1 protein. As a control, the samples were subsequently
immunoprecipitated with anti-
-actin antibody. In accordance with the
marked reduction of the amount of mRNA transcript, a newly
synthesized E2F-1 protein was barely detected after 8 h of the
treatment with IFN-
, whereas the amount of
-actin was unaffected
(Fig. 3). A minor increase in E2F-1 protein was observed
in untreated cells after 8 h of culture. This may be the result of
a small increase in the amount of E2F-1 mRNA (see Fig.
2A) during spontaneous cell growth (see Fig.
1B).
Fig. 3.
Effect of IFN- on de novo
synthesis of E2F-1 protein. Daudi cells were cultured in the
absence (
) or presence (+) of 100 IU/ml IFN-
for 8 h. The
cells were pulse labeled with [35S]methionine for 3 h. An equal amount (7.5 × 107 cpm) of the lysates was
immunoprecipitated with anti-E2F-1-specific monoclonal antibodies and
subsequently with anti-
-actin antibody. Immune complexes were
separated by 7.5% SDS-polyacrylamide gel electrophoresis, and the
signal was detected by fluorography. 14C-Labeled standard
proteins (Amersham Corp.) were used as molecular weight markers
(M.W.). The position of each band is shown on the left. Data shown are representative of three independent
experiments.
[View Larger Version of this Image (48K GIF file)]
on DNA Binding Activity of E2F
Fig. 4.
Characterization of E2F complexes present in
Daudi cells by gel retardation assays with antibody perturbation.
Panel A, nuclear extracts were prepared from untreated Daudi
cells and incubated with 32P-labeled DNA fragment
containing E2F binding sites. The protein-DNA complexes were resolved
on a 4% polyacrylamide gel in 0.25 × Tris borate-EDTA buffer.
Double-stranded oligonucleotides containing the AP-1 binding site and
E2F binding site were used for competition assays. The bands indicated
by stars were found to be nonspecific. Panel B,
E2F gel shift assays were performed in the absence or presence of
antibodies against E2F-1, E2F-4, pRB, p107, and p130. For quantitation
of the results, see Table I. Data shown are representative of three
independent experiments.
[View Larger Version of this Image (70K GIF file)]
Additions
None
E2F-1
E2F-4
pRB
p107
p130
A
0.58
0.72
0.03a
1.22
NDa,b
0.03a
B
0.13
0.20
0.62
0.48
0.03a
C
0.48
0.48
0.18a
NDa,b
0.36
0.31
D
0.31
0.44
0.39
ND a,b
0.28
0.30
E
1.46
1.20
0.19a
1.28
1.18
1.39
F
2.35
1.78a
2.42
2.05
2.20
2.13
G
0.59
0.12a
0.45
0.46
0.38
0.61
*
1.0
1.0
1.0
1.0
1.0
1.0
a
More than 20% decrease in intensity compared with the
control (None).
b
ND, not detected (under the detection limit).
The effect of IFN- on E2F activity was analyzed next by comparing
the intensities of these bands before and after treatment. Fig.
5A displays the representative result of gel
retardation assays with nuclear extracts from Daudi cells isolated
after 0, 8, and 24 h of culture with 100 IU/ml IFN-
. The
intensity of bands F and G (corresponding to free E2F-1) was decreased
significantly by IFN-
after 8 h of IFN treatment, which is
consistent with the reduction of E2F-1 mRNA and protein. After
24 h, the amounts of free E2F-1 were reduced to approximately 25%
of the untreated control by densitometric comparison. The abundance of
the band E, which corresponds to free E2F-4, was unaltered at 8 h
but was reduced markedly after 24 h of treatment (Fig.
5A). IFN also reduced the amounts of E2F-pRB complexes (C
and D) and the E2F-4-p107 complex (A). The decrease in the E2F-p107
complex may correspond to the reduction of S phase cells by IFN, since
this complex was reported to be preferentially formed in S phase
(35).
Next, we performed an immunoprecipitation-deoxycholate release assay to
confirm that E2F-4 really made a complex with pRB and also to see more
precisely the effect of IFN on the E2F-4-pRB complex. pRB-associated
proteins were immunoprecipitated from whole cell lysates of Daudi cells
before and after the IFN treatment, and E2F gel shift assays with
antibody perturbation were carried out after deoxycholate release of
the supernatants. As shown in Fig. 5B, two bands (designated
as X and Y) were observed, and both of them were eliminated completely
by the cold competitor (lanes 2 and 6).
Anti-E2F-4 antibody could reduce the intensity of band X, indicating
that E2F-4 really is present in E2F-pRB complexes (lane 5).
Similarly, E2F-1 was shown to be present in band Y (lane 4).
There was no difference in the amounts of pRB-bound E2F between pre-
and post-treatment samples (compare lanes 1 and 3) despite the decrease in the intensities of E2F-pRB
complexes (C and D in Fig. 5A) in gel retardation assays.
This suggests that E2F molecules associated with a single pRB molecule
are not decreased even after the IFN treatment. This is fully
consistent with the previous report by Melamed et al. (36),
wherein inhibition of E2F activity started after 8 h of treatment
with interleukin-6 or IFN, but it was not eliminated completely even
after 24 h. In keeping with the previous assumption that E2F binds
preferentially to unphosphorylated pRB (37, 38), this may be explained
by the change in phosphorylation status of pRB. Therefore, we then examined the effect of IFN- on the amount and phosphorylation status
of RB protein. This may be also helpful in characterizing further the
function of E2F-pRB complexes, since the activity of E2F was regulated
mainly by its association with pRB.
Whole cell lysates were prepared from Daudi cells at various time points after the IFN treatment and were subjected to immunoblot analysis for pRB expression. Previous reports have documented that RB protein is unphosphorylated in G0/G1 phase of the cell cycle and is specifically phosphorylated at the G1/S boundary; phosphorylated pRB is dominant throughout the S and G2/M phases (39, 40). Only underphosphorylated RB protein can function as a suppressor of cell growth. These two functionally distinct forms of pRB are clearly distinguishable on a 7.5% polyacrylamide gel.
As shown in Fig. 6A, RB protein was present
almost exclusively in heavily phosphorylated forms in untreated Daudi
cells, reflecting the active proliferative status of the cells. IFN-
induced the accumulation of an underphosphorylated form of pRB after
24 h of treatment (Fig. 6A). Recently, Weintraub
et al. (41) reported that unphosphorylated RB protein, upon
binding to E2F, switches E2F from a transcriptional activator to a
repressor. Thus, E2F-pRB complexes present after the IFN treatment may
function as a suppressor of cell growth. Taken together, these results
indicate that IFN-
suppresses transcriptional activity of E2F in two
ways: through reduction of transcriptionally active free E2Fs (E2F-1
and E2F-4) and through induction of unphosphorylated pRB to change
E2F-pRB complexes to transcriptional repressors.
Effect of IFN-
We next investigated the effect of IFN- on the
transactivating ability of E2F in vivo. For this purpose, we
chose to examine whether IFN-
could suppress the activity of the
cdc2 promoter by transient CAT assay. A 467-base pair
fragment of the 5
-flanking region of the cdc2 gene (
383
to +87) was linked to the CAT gene in pCAT-basic vector and used as a
reporter plasmid (hereinafter designated as pCAT-5
-cdc2). In our
previous study, it was shown that this segment had a strong promoter
activity that was dependent on the binding of E2F at nucleotide
positions
124 to
117 (30). A nonbinding mutation was introduced
into the E2F binding site of pCAT-5
-cdc2 (5
-TTTCGCGC-3
to 5
-TTTCGATC-3
) and used as a negative control. The
plasmid containing SV40 promoter-driven CAT (pCAT-control) was
transfected simultaneously into corresponding cells and used as a
positive control. These plasmids were transiently transfected into
Daudi cells by electroporation, and the cells were split equally into
two flasks. IFN-
was added into one of them, and the cells were
cultured for 24 h and harvested for measurement of intracellular
CAT activity. The promoter activity of pCAT-control was independent of
E2F and was shown to be unaffected by IFN-
(data not shown). In
contrast, IFN-
could suppress the CAT activity of pCAT-5
-cdc2 to
74 ± 4% of the untreated control in four independent experiments
(p < 0.01 by Student's t test, Fig.
7, left panel). As anticipated, IFN-
could
not inhibit the promoter activity of the mutant plasmid lacking a
functional E2F binding site (Fig. 7, right panel). This
indicates that E2F-dependent promoter activity was
regulated negatively by IFN-
in vivo. This result
facilitated further examination of the expression of the genes whose
promoter contains functional E2F binding sites in IFN-treated Daudi
cells.
IFN-
Both c-myc and cdc2 had functional
E2F binding sites in their promoter regions, and their transcription
was known to be regulated by E2F (18, 20, 30). The effect of IFN- on
expression of c-myc and cdc2 mRNAs was
investigated in IFN-treated Daudi cells. Expression of p53
mRNA, whose transcription is independent of E2F, was examined
simultaneously as a negative control. As shown in Fig.
8A, IFN-
could effectively inhibit the
expression of c-myc and cdc2, whereas
p53 mRNA expression was unaffected. IFN-induced suppression of cdc2 mRNA occurred after 12 h of
treatment, whereas down-regulation of c-myc mRNA became
evident after 72 h. The inhibition of cdc2 and
c-myc mRNA was apparently preceded by the suppression of
E2F activity. This is entirely compatible with the notion that IFN-induced suppression of these growth-related genes is mediated at
least in part through the inhibition of E2F activity. Down-regulation of these genes may contribute somewhat to the growth arrest of Daudi
cells, although many other factors should be involved in this process
(Fig. 8B).
Two decades ago the antiproliferative activity of IFNs was first
identified (42). However, the mechanism of IFN-mediated growth
suppression is not fully understood. Previous investigations have shown
that IFN causes G0/G1 arrest through the
reduction of c-myc proto-oncogene (8) and dephosphorylation
of pRB (9). Regarding the cell cycle-regulatory elements,
down-regulation of cyclin A and cdk2 mRNA transcripts by
IFN- was observed recently (11). Inhibition of cyclin E- and cyclin
D1- but not cyclin A-dependent cdk2 kinase
activity was also reported (12). Although the action of IFN is thought
to be mediated through these events, it is not clear whether or not
this is a direct action. It is possible that some of these events were
the result of IFN-induced growth arrest. Given the fact that IFN is now
used widely as a therapeutic reagent, further investigation should be
required to elucidate fully the direct mechanism of its action for
better clinical application.
In this work, we investigated the effect of IFN- on transcription
factor E2F, one of the most important regulators of G1/S transition. We found that expression of E2F-1 mRNA,
which encodes a major subunit of E2F, was reduced markedly after 8 h of the culture with IFN-
, whereas expression of DP-1,
another subunit of E2F, was unaffected. Down-regulation of E2F-1
mRNA was faster than that of any other genes whose expression
was regulated negatively by IFN (11). Because of the inhibition of
E2F-1 mRNA expression, a newly synthesized E2F-1 protein
became undetectable after 8 h, which was accompanied by the
suppression of its DNA binding activity. In addition, IFN-
also
diminished the DNA binding ability of E2F-4, another member of the E2F
family with an affinity to the same binding site as E2F-1. In contrast
to E2F-1, IFN-induced suppression of E2F-4 activity is suspected to be
post-transcriptional, since E2F-4 mRNA expression was
not inhibited by IFN. Gel shift assays revealed that IFN could
especially repress the DNA binding ability of E2F-1 and E2F-4 without a
complex with RB family proteins (free E2F). Since it has been
established that both free E2F-1 and free E2F-4 activate transcription
of many growth-promoting genes (43, 44), suppression of free E2F
activity may be implicated in IFN-induced cell growth arrest. By
contrast, IFN did not reduce the amounts of E2F in a complex with RB
protein in immunoprecipitation-deoxycholate release assays.
Accumulation of an underphosphorylated, growth-suppressive form of RB
protein was induced simultaneously by the IFN treatment. Recently, it
has been shown that unphosphorylated RB protein, upon binding to E2F,
switches E2F from a transcriptional activator to a repressor (41).
Therefore, E2F-pRB complexes present after the IFN treatment may
function as suppressors of cell growth. Taken together, these results
indicate that IFN-
suppresses E2F activity in two different ways:
through the reduction of transcriptionally active E2F-1 and E2F-4 and
through the induction of unphosphorylated pRB to generate
transcriptional repressor complexes containing E2F. As a result,
E2F-dependent cdc2 promoter activity was indeed suppressed by IFN-
in vivo. The suppression of E2F
activity was followed by down-regulation of the growth-regulatory genes
such as c-myc and cdc2 whose transcription is
controlled by E2F.
Recent studies using E2F-1 have demonstrated a central role for E2F in
cell cycle regulation. Expression of E2F-1 can prevent quiescence upon
serum deprivation and can induce quiescent cells into S phase (45).
Recombinant adenovirus-mediated overexpression of E2F-1 can activate
transcription of DNA synthesis-related and G1/S-regulatory
genes such as those for DNA polymerase-, thymidylate synthase,
proliferating cell nuclear antigen, cyclin A, and cdc2 in
REF52 cells without serum stimulation (46). Moreover, infection of
Mv1Lu mink lung epithelial cells with adenovirus vector containing E2F-1 cDNA overcomes transforming growth
factor-
-mediated growth suppression (47). Similarly, overexpression
of E2F-1 can bypass a G1 arrest caused by the inhibition of
G1-specific cyclin-dependent kinase activity
and by
-irradiation (48). Recently, Guy et al. (49)
reported that overexpression of E2F-1 in transgenic mouse under the
control of PF4 promoter resulted in the block of megakaryocytic
maturation and caused abnormal proliferation of megakaryocytes. Their
study clearly demonstrates the role of E2F-1 in cell proliferation and
differentiation in vivo. From these observations, E2F-1 is
thought to be a general target for both growth-stimulatory and
-inhibitory signals. Our present finding is in line with this view and
may provide a new concept on signaling pathways of IFN-mediated growth
suppression.
Convincing evidence has accumulated suggesting that E2F is a relevant
target of the action of RB protein as a growth suppressor. The
interaction of pRB, especially in its underphosphorylated form, with
E2F results in a switch of E2F from a transcriptional activator to a
repressor (41). The ability of pRB to interact with E2F correlates
directly with the ability of pRB to arrest cells in
G0/G1 (50). Mutant RB protein that lacks the
ability to cause a growth arrest also lacks the ability to bind E2F and to inhibit E2F-dependent transcription. In the
G0/G1 phase of the cell cycle, unphosphorylated
pRB binds directly to E2F and inhibits E2F-dependent
transcription (37, 38). As cells pass the G1/S boundary by
growth stimulation, pRB becomes phosphorylated, and E2F is released.
The unbound free E2F (mainly E2F-1 and E2F-4) is presumed to be
transcriptionally active. Conversely, some negative growth factors such
as transforming growth factor- can induce dephosphorylation of pRB,
thereby inhibiting E2F activity and leading cells to G1
arrest (26, 51). The accumulation of the underphosphorylated,
growth-suppressive form of RB protein and its negative effect on E2F
activity might be important for IFN-mediated growth suppression. In
this study, we demonstrated that E2F-4, in addition to E2F-1, formed a
specific complex with pRB in Daudi cells. E2F-4 is a recently
identified member of the E2F family which makes a complex with RB
family proteins (25). E2F-4-p107 and E2F-4-p130 complexes are present
in early to mid G1 phase of the cell cycle and are shown to
act as repressors of E2F site-directed transcription of
B-myb (52) and cdc2 (53). In previous reports, E2F-4 was shown to bind preferentially p107 and p130 and was believed not to bind pRB (43, 44). This is somewhat contradictory to our present
observation that E2F-4 is present in a complex with pRB. However, Ikeda
et al. (54) recently described that E2F-4 was the major
species of E2F in quiescent fibroblasts and in differentiated HL-60
cells, where E2F-4 made complexes with unphosphorylated pRB and p130 to
induce cell cycle arrest. They claimed that it is the ratio of free E2F
to pRB-bound E2F which is critical in the decision to pass through the
G1/S transition. Our finding is fully consistent with their
assertion and demonstrates that the relative increase in the ratio of
pRB-bound E2F to free E2F, mainly because of the decrease in free E2F,
is linked to IFN-induced suppression of E2F-mediated transcription and
cell cycle arrest.
How does IFN- repress E2F-1 mRNA expression? Transcriptional
activation in response to IFN-
is known to be mediated through the
activation of a multiprotein DNA-binding complex described as
IFN-stimulated gene factor 3 (ISGF3) (55). The components of ISGF3 were
purified and are recognized now as Stat1, Stat2, and p48 (56). Tyrosine
phosphorylation of Stat proteins by IFN-
/
receptor-associated Jak
kinases is necessary for activation of ISGF3. The phosphorylated Stat
proteins move to the nucleus and bind specific DNA elements called
IFN-stimulated response elements to direct transcription (57). However,
there is no binding site for ISGF3 in the promoter region of the
E2F-1 gene according to recent reports (58, 59). Therefore,
direct involvement of the Jak-Stat signaling pathway in IFN-induced
suppression of E2F-1 mRNA transcription is unlikely.
Recent investigations suggest that transcription of the E2F-1
gene is under autoregulatory control through the E2F binding sites
in its promoter (58, 59). Accordingly, transcription of E2F-1
is repressed when E2F binds to the promoter region of the
E2F-1 gene in a complex with underphosphorylated pRB, since
this complex acts as a transcriptional repressor. Although this is a
plausible explanation, down-regulation of E2F-1 mRNA started apparently prior to dephosphorylation of pRB in our
experiments. It is possible that other molecules in the E2F family play
a role in IFN-mediated inhibition of E2F-1 mRNA. E2F-5
may be a good candidate because it binds to the RB family protein p130
and is the predominant E2F complex in the early phases of the cell
cycle (G0 to mid G1) (34, 35). To understand
the precise mechanism of the repression of E2F-1 expression
requires a study of the effect of IFN-
on other members of the E2F
family. Detailed analysis of these subjects is currently under way in
our laboratory.
Finally, we have to mention the discrepancy between our study and the previous one by Melamed et al. (36) regarding the expression patterns of c-Myc after IFN treatment. As shown in Fig. 8, down-regulation of c-myc mRNA was observed after 72 h of IFN treatment in our study. However, they described that c-Myc protein levels were inhibited at 8 h in Daudi cells (36). Thus, we also performed Western blot analysis of c-Myc expression in IFN-treated Daudi cells. Consistent with their result, the c-Myc protein was down-regulated after 24 h of culture with IFN in our system (data not shown), whereas c-myc mRNA expression was unaffected at this time point. This suggests that there might be multiple distinct mechanisms for IFN-induced suppression of c-Myc including E2F-dependent inhibition of mRNA transcription and post-translational modification of the protein levels. The precise mechanism of the latter is at present unknown.