Oncogenic Potential of a Dominant Negative Mutant of Interferon
Regulatory Factor 3*
Tae Young
Kim
,
Kyoung-Hu
Lee
,
Seungwoo
Chang
,
Cheolho
Chung§,
Han-Woong
Lee¶,
Jeongbin
Yim
, and
Tae Kook
Kim
**
From the
Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, Taejon 305-701, Korea,
§ Department of Microbiology, Seoul National University,
Seoul 151-742, Korea, ¶ Samsung Biomedical Research Institute,
Sungkyunkwan University School of Medicine, Suwon 440-746, Korea, the
National Creative Research Initiative Center for Genetic
Reprogramming, Institute for Molecular Biology and Genetics, Seoul
National University, Seoul 151-742, Korea, and the
** Institute of Chemistry and Cell Biology, Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115
Received for publication, June 11, 2002, and in revised form, January 16, 2003
 |
ABSTRACT |
Interferon regulatory factor 3 (IRF3) is
activated in response to various environmental stresses including viral
infection and DNA-damaging agents. However, the biological function of
IRF3 in cell growth is not well understood. We demonstrated that IRF3 markedly inhibited growth and colony formation of cells. IRF3 blocked
DNA synthesis and induced apoptosis. Based on this negative control of
cell growth by IRF3, we examined whether functional loss of IRF3 may
contribute to oncogenic transformation. IRF3 activity was specifically
inhibited by expression of its dominant negative mutant. This mutant
lacks a portion of the DNA binding domain like IRF3a, an alternative
splice form of IRF3 in the cells. This dominant negative inhibition
blocked expression of specific IRF3 target genes. Mutant IRF3
efficiently transformed NIH3T3 cells, as demonstrated by
anchorage-independent growth in soft agar and tumorigenicity in nude
mice. These results imply that IRF3 may function as a tumor
suppressor and suggest a possible role for the relative levels of IRF3
and its dominant negative mutant in tumorigenesis.
 |
INTRODUCTION |
Inhibition of cell growth is critical for the maintenance of
normal tissue homeostasis. Disruption of this negative regulation can
clearly contribute to the multistep process of tumorigenesis, as
evidenced by the frequent mutation of tumor suppressor genes in human
cancer (reviewed in Ref. 1). The p53 tumor suppressor gene is
frequently mutated or functionally inactivated in human cancers of many
different tissue types. The mechanisms by which p53 loss contributes to
tumorigenesis have been substantiated by extensive studies of p53
function (2-4). For example, p53 plays a role in cell cycle checkpoint
control by inducing cell growth arrest in response to DNA damage.
Consistent with this function, cells lacking p53 fail to arrest in the
cell cycle following DNA damage, which may lead to genetic instability.
Thus, as a checkpoint regulator, p53 can directly and indirectly
suppress tumor induction by preventing the propagation of oncogenic
mutations in other genes.
Interferon regulatory factor 3 (IRF3)1 is known to be
induced by viral infection (reviewed in Ref. 5). We found that IRF3, like p53, can also be induced by DNA-damaging agents including doxorubicin (6-8). Recently, Weaver et al. (9) have show
that the DNA-damaging agent, etoposide, activates IRF3 and induces specific target genes including ISG54 in the absence of the action of
interferon. Interestingly, Karpova et al. (10) demonstrate that IRF3 is an in vivo target of DNA-dependent protein
kinase, which is involved in cell cycle checkpoint control in
response to DNA damage. A similar regulatory mechanism was described
for p53; DNA damage induced phosphorylation of Ser-15 in p53,
and this serine site is the known target for the DNA-dependent
protein kinase family of kinases (2-4). Furthermore, virus-inducible double-stranded RNA-dependent protein kinase, PKR, may be
involved in the DNA damage-induced phosphorylation of p53 through
regulating the activity of the DNA-PK family of kinases (11). These
results, together with possible cross-talk between DNA damage and other stress response pathways, suggest that IRF3, like p53, may play a role
in a variety of host defenses that have evolved to counter environmental stresses.
An interesting possibility emerging from these studies is that IRF3 can
directly control cell growth and the functional loss of IRF3 may
contribute to oncogenic transformation. In an effort to test this
possibility and to determine the physiological function of IRF3, we
induced IRF3 activity with its ectopic overexpression, and we inhibited
IRF3 activity using a dominant negative mutant that contains a
defective DNA binding domain like IRF3a, an alternative splice form of
IRF3 in the cells (12, 13). These studies revealed the anti-oncogenic
and oncogenic potentials of IRF3 and a dominant negative IRF3 mutant.
 |
EXPERIMENTAL PROCEDURES |
Plasmids and Antibodies--
Human IRF3 was expressed under the
control of the constitutive cytomegalovirus promoter in pcDNA3
(Amersham Biosciences). A deletion mutant for IRF3 lacking the
DNA binding domain (IRF3
) was made by PCR amplification of the
region encoding amino acid residues 58-427. This fragment of IRF3
(IRF3
) and an oncogenic version of Ha-ras
(Ha-ras G12V) (14) were also cloned into pcDNA3 (Amersham Biosciences). The plasmid used as a negative control was an
empty plasmid, pcDNA3, containing no corresponding gene in each
experiment. To generate anti-human IRF3 antibody, recombinant glutathione S-transferase (GST)-IRF3 was expressed in
Escherichia coli, purified by glutathione-Sepharose
4B (Amersham Biosciences), and then used to immunize rabbits.
Transfection--
Cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing fetal bovine serum or calf serum. All
of the cell growth media contained 100 units/ml penicillin and
100 µg/ml streptomycin. Cells (typically 0.5-1 × 106; otherwise as indicated in the figure legends) were
transfected with specific amounts of plasmids as indicated in the
figure legends, using LipofectAMINE reagents (Invitrogen) in accordance
with the manufacturer's instructions. Transfection efficiency was
analyzed by cotransfection with pEGFP-N1 (Clontech)
followed by monitoring under a fluorescence microscope. To select
transiently transfected cells, we utilized the Capture-Tec kit
(Invitrogen) according to the manufacturer's instructions. Cells were
cotransfected with either control or IRF3 expression plasmids and phOx
SFV expression plasmid (pHook-1, Invitrogen). Transfected cells were
then selected through incubation with magnetic beads coated with hapten
for 1 h as suggested (Invitrogen).
Cell Division Assay--
Cells (0.5-1 × 106)
were cotransfected with either control or IRF3 (and IRF3
) expression
plasmids and GFP expression plasmid (pEGFP-N1,
Clontech). After transfection, the number of
GFP-expressing cells was monitored for 5 days under fluorescent
microscopy. Cell division was observed as an increase in the number of
aggregates of GFP-expressing cells.
Stable Colony Formation Assay--
For stable colony formation
assays, cells (1 × 106) were transfected with
plasmids containing the neomycin resistance gene. At 24 h
post-transfection, G418 (1 mg/ml) was added to the transfected cells
and cells were cultured for 2 weeks. The plates were then stained with crystal violet in 20% ethanol, and colonies containing more than 30 cells were counted from representative fields.
BrdUrd DNA Synthesis Assay--
DNA synthesis assay was
performed with the 5-bromo-2'-deoxyuridine (BrdUrd) labeling and
detection kit (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Cells were incubated with BrdUrd for
24 h. After the labeling medium was removed, the cells were fixed
and the DNA was denatured by adding FixDenat (Roche Molecular
Biochemicals). Then, anti-BrdUrd-POD antibody is added and BrdUrd
incorporated into the newly synthesized DNA was detected by a substrate
reaction (Roche Molecular Biochemicals). The DNA synthesis rate was
quantified with a microplate reader (EG&G Wallac).
Flow Cytometry--
For fluorescence-activated cell sorter
(FACS) analysis, cells were washed with phosphate-buffered saline. The
harvested cells were fixed with 70% ethanol for 1 h on ice. Cells
were then washed three times with phosphate-buffered saline. After
incubation with RNase (1 mg/ml), the DNA in fixed cells was stained
with propidium iodide (10 mg/ml) for 30 min. Cells were analyzed by
FACSCaliber (BD Biosciences).
Western Blot--
Whole cell extracts were prepared by lysis in
buffer containing 50 mM Tris (pH 7.4), 150 mM
NaCl, 0.5% Nonidet P-40, 5 mM EDTA, 50 mM NaF,
and protease inhibitors. Following the centrifugation, proteins in the
extracts were separated by 10% SDS-PAGE and transferred to
nitrocellulose membranes (Millipore). The membranes were blocked with
5% nonfat milk and probed with anti-human IRF3 antibody. Membranes
were then incubated with horseradish peroxidase-conjugated anti-rabbit
IgG (Santa Cruz Biotechnology) and visualized using the enhanced
chemiluminescence system (ECL; Amersham Biosciences).
Reverse Transcriptase PCR--
Cells were incubated with IFN-
(1000 units/ml) or Sendai virus (200 hemagglutinin unit/ml) as
described previously (6-8). Total RNA was prepared from cells treated
with Trizol reagent (Invitrogen). For each sample, 5 µg of isolated
RNA was used for the reverse transcriptase reaction with the
SuperScript System (Invitrogen) in accordance with the manufacturer's
instructions. The resultant cDNA was used for PCR with
primers specific for IFN-
, 2',5'-oligoadenylate synthetase
(2',5'-AS), and GBP-1 as described previously (15). Amplification was
carried out for 28 cycles, and 5 µl of the resulting reaction
mixtures were separated and analyzed on a 2% agarose gel.
Soft Agar Assay--
Soft agar assays in 6-cm dishes were
performed as previously described (16). Cells (1, 3, and 5 × 105) were seeded in 0.3% agarose solution in DMEM
containing 10% calf serum over a cushion of 0.5% agarose solution
also in DMEM growth medium. Cells were fed with fresh DMEM growth
medium containing 0.3% agarose every week. After 3 weeks, plates were
checked and photographed.
Tumor Growth in Nude Mice--
Tumorigenicity assays were
performed as described previously (16). Nude mice (BALB/c
nu/nu) were injected subcutaneously with 1 × 106 cells resuspended in 100 µl of phosphate-buffered
saline. Injected mice were observed every 3 days. Subcutaneous tumors
were grossly visible at the site of injection after 2-3 weeks. Each
cell line was tested for tumorigenicity in 3-12 different mice.
 |
RESULTS |
To examine the function of IRF3 in cell growth, we analyzed the
effect of its overexpression on cell growth (Fig.
1). To this end, HeLa cells were
transfected with the IRF3 expression plasmid, together with the GFP
expression plasmid as a marker of transfection. Using GFP, we were able
to examine the division of live cells over time as evidenced by the
increasing number of GFP-expressing cell aggregates. Examination by
fluorescence microscopy for 5 days following transfection showed that
IRF3-transfected cells failed to divide, whereas control-transfected
cells continued to divide (Fig. 1A). In control-transfected
cells, we found increased numbers of GFP-expressing cell aggregates
over time, whereas only a few cells expressing GFP were observed in
IRF3-transfected cells (Fig. 1B).

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Fig. 1.
IRF3 inhibits growth of HeLa cells.
HeLa cells were cotransfected with either control or IRF3
expression plasmids (2 µg) and a GFP expression plasmid (2 µg).
A, the number of GFP-expressing cells was monitored for 5 days after transfection under fluorescent microscopy. B,
cells in this representative field were observed under fluorescent
microscopy on the fifth day.
|
|
We also used a colony formation assay to evaluate the growth-inhibitory
effect of IRF3 (Fig. 2). HeLa cells were
transfected with the IRF3 expression plasmid with a neomycin resistance
gene. After transfection, G418 was added and transfected cells were selected for 2 weeks. Ectopic expression of IRF3 markedly inhibited stable colony formation in HeLa cells (Fig. 2A). IRF3
inhibited by nearly 90% of the stable clones as compared with
control-transfected cells (Fig. 2B). Furthermore, the
average size of the colonies was much smaller when transfected with the
expression plasmid for IRF3 (data not shown). Taken together with data
from Fig. 1, these results indicate that IRF3 inhibits cell division
and stable colony formation in HeLa cells.

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Fig. 2.
IRF3 inhibits colony formation of HeLa
cells. HeLa cells were transfected with 2 µg of either control
or IRF3 expression plasmids containing a neomycin resistance gene. At
24 h post-transfection, G418 (1 mg/ml) was added to transfected
cells. G418 selection was continued for 2 weeks. A, plated
clones were stained with crystal violet. B, the
relative numbers of clones with more than 30 cells, from five
representative fields, are shown.
|
|
Next, we tried to gain insight into the mechanisms for the observed
cell growth inhibition by IRF3. Ectopic expression of IRF3 induced
chromatin condensation and fragmentation of the nucleus in a small
percentage of transfected cells stained with
4',6-diamidino-2-phenylindole (data not shown). To test the
significance of this observation, transfected cells were verified by
cotransfection with GFP expression plasmids, and then cell survival was
assayed by a trypan blue exclusion assay. Consistently, we detected a
small decrease in the number of live cells upon expression of IRF3
(Fig. 3A). Thus, apoptosis is
probably not the major cause for the observed growth-inhibitory effect
of IRF3 under our conditions. Based on these results, we examined
whether the IRF3-mediated growth inhibition was also due to a block in
DNA synthesis (Fig. 3B). DNA synthesis was monitored by
measuring BrdUrd incorporated into newly synthesized DNA. Ectopic expression of IRF3 dramatically reduced BrdUrd incorporation into DNA
(Fig. 3B). These results indicate that IRF3 can inhibit cell growth by blocking DNA synthesis and, to some extent, inducing apoptosis.

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Fig. 3.
IRF3 induces apoptosis and inhibits DNA
synthesis. A, HeLa cells were transfected with 2 µg
of either control or IRF3 expression plasmids. Transfected cells were
verified by cotransfection with GFP expression plasmids (2 µg). At
30 h post-transfection, cell survival was determined by trypan
blue exclusion assay. B, HeLa cells were cotransfected with
either control (2 µg) or IRF3 (1, 2, and 4 µg) expression plasmid
and phOx SFV expression plasmid (2 µg). Cells were incubated with
BrdUrd for 24 h. Transfected cells were selected with magnetic
beads coated with hapten, and BrdUrd incorporated into the newly
synthesized DNA was detected by an assay system (Roche Molecular
Biochemicals) and quantified with a plate reader (EG&G Wallac).
Experiments were repeated twice with similar results.
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To further examine the basis for IRF3-mediated cell growth inhibition,
we analyzed the cell cycle progression upon the ectopic expression of
IRF3 (Fig. 4). To this end, HeLa cells
were transfected with the IRF3 expression plasmid, together with the
phOx SFV expression plasmid as a marker of transfection. Transfected
cells were stained with propidium iodide for FACS analysis. Western
blot analysis showed the induction of IRF3 protein levels in the
transfected cells (Fig. 4A). Under these conditions, IRF3
significantly increased the number of G1 cells, reducing
that of cells in the S-phase (Fig. 4B). Consistent with the
results shown in Fig. 3A, we detected no such marked
increase in the number of apoptotic cells upon expression of IRF3.
These results further support the conclusion from our results shown in
Figs. 1-3 that IRF3 plays a negative role in cell division and
growth.

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Fig. 4.
IRF3 inhibits cell cycle progression.
HeLa cells were cotransfected with either control or IRF3 expression
plasmid (2 µg) and sFV expression plasmid (2 µg). Transfected cells
were selected with magnetic beads coated with hapten. A,
IRF3 protein levels were measured by Western blotting with anti-human
IRF3 antibody. Actin was used to normalize protein levels in whole cell
extracts. B, the cell cycle was analyzed by FACSCaliber (BD
Biosciences) after transfected cells were stained with propidium
iodide. The cell population was indicated in each cell cycle stage
(M1, M2, and M3).
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HeLa cells were utilized in various cell growth assays with IRF3 (Figs.
1-4). Next, we determined the inhibitory role of IRF3 in cell growth
using other cell types (HepG2, NIH3T3, and REF52) (Fig.
5). These different types of cells were
transfected with the IRF3 expression plasmid, together with the GFP
expression plasmid as a marker of transfection. The cell growth was
monitored by the increasing number of GFP-expressing cells under the
fluorescence microscopy for 5 days after transfection. As in HeLa
cells, IRF3-transfected cells failed to divide in all of the tested
cells, whereas control-transfected cells continued to divide (Fig. 5,
A (HepG2), B (NIH3T3), and C (REF52)).
The slight decrease in the number of IRF3-transfected HeLa (Fig.
1A), HepG2, and NIH3T3 (Fig. 5, A and
B) cells may support the significance of
IRF3-dependent apoptotic response demonstrated in Fig.
3A (9, 17). Thus, IRF3 can inhibit cell growth in various
kinds of cells.

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Fig. 5.
IRF3 inhibits cell growth. HepG2
(A), NIH3T3 (B), and REF52 (C) cells
were cotransfected with either control or IRF3 expression plasmids (2 µg) and GFP expression plasmid (2 µg). The number of GFP-expressing
cells was monitored for 5 days after transfection under fluorescent
microscopy.
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Based on the function of IRF3 in negative control of cell growth, we
next examined how the loss of IRF3 activity affects on cell growth. To
this end, we attempted to utilize mutant IRF3
lacking a portion of
the DNA binding domain at its amino terminus like IRF3a, which is an
alternative splice isoform of IRF3 in the cells (12, 13). As a first
step, we examined the possible dominant negative effects of IRF3
on
the observed IRF3-induced attenuation of cell growth (Fig.
6). The cell growth was monitored by the
increasing number of GFP-expressing cells under fluorescence microscopy
for 5 days after cotransfection with IRF3 and IRF3
expression
plasmids. Consistent with our results (Figs. 1 and 5), IRF3 markedly
attenuated the cell growth of NIH3T3 and HeLa. This attenuation was
significantly inhibited by coexpression of IRF3
(Fig. 6). These
results suggest that IRF3
can be functional as a dominant negative
mutant of IRF3 in the cell growth.

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Fig. 6.
A dominant negative IRF3 mutant inhibits
IRF3-mediated attenuation of cell growth. NIH3T3 (A)
and HeLa (B) cells were cotransfected with either control or
IRF3 (and IRF3 ) expression plasmids (2 µg) and GFP expression
plasmid (2 µg). IRF3 lacks the DNA binding domain of human IRF3.
The number of GFP-expressing cells was monitored for 5 days after
transfection under fluorescent microscopy.
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|
To further examine the function of dominant negative mutant of IRF3 in
cell growth, we generated NIH3T3 cell clones overexpressing IRF3
.
The plasmid pcDNA3, which expresses the human IRF3 protein (58-427
amino acid residues) without its DNA binding domain under the control
of the constitutive cytomegalovirus promoter, was transfected into
NIH3T3 cells. As a positive control, some cells were transfected with
an expression plasmid for an oncogenic version of Ha-ras
(Ha-ras G12V) in which glycine at amino acid position 12 was
changed to valine (14). A control expression plasmid, pcDNA3, was
transfected into NIH3T3 cells as a negative control. After selection
for G418 resistance, we obtained several clones that expressed some
levels of truncated human IRF3 protein. One such clone (2) was
chosen for initial experiments because of its high level of expression
(Fig. 7A).

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Fig. 7.
A dominant negative IRF3 mutant inhibits
specific IRF3 target gene expression. NIH3T3 cells were stably
transfected with expression plasmids for a dominant negative mutant
form of human IRF3 (IRF3 ). One of the clones (2) was chosen for
these experiments. A control expression plasmid was transfected as a
negative control. A, IRF3 protein levels were measured by
Western blotting with anti-human IRF3 antibody in whole cell extracts
derived from control and an IRF3 cell line. B, control
and an IRF cell line were control-treated, infected with virus, or
stimulated with IFN- (1000 units/ml). Total RNA was subjected to
reverse transcriptase PCR analysis of IFN- , 2',5'-AS, GBP-1, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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To address the specific effect of the IRF3 mutant, we assayed the
induction of three endogenous genes having promoters that contain IRF
binding sites: IFN-
, 2',5'-AS, and GBP-1 (Fig. 7B). These
promoters are activated by viral infection through specific activation
of IRF3 (18-25). IFN treatment also induces certain promoters but
fails to stimulate transcriptional activity of IRF3; thus, IFN may
induce the promoters through activation of the other IRF family factors
(e.g. IRF1 and ISGF3) (26). Overexpression of a dominant
negative IRF3 mutant (IRF3
) sharply reduced viral induction of three
tested genes including IFN-
(Fig. 7B). In contrast,
overexpression of IRF3
did not affect induction of the 2',5'-AS and
GBP-1 genes in response to IFN treatment. Furthermore, under these
conditions, expression of a control gene, GAPDH
(glyceraldehyde-3-phosphate dehydrogenase), was not affected by
expression of IRF3
. These results strongly suggest that expression
of IRF3
inhibits endogenous genes that are specifically regulated by
IRF3 but not those regulated by other IRF family factors.
The demonstrated role of IRF3 in cell growth inhibition raised the
possibility that its inactivation might play a role in oncogenic
transformation in a manner analogous to mutations of the p53 tumor
suppressor gene. NIH3T3 cells are highly susceptible to oncogenic
transformation by inactivation of p53 or activation of
Ha-ras. Indeed, microscopic examination of the cells
expressing Ha-ras G12V revealed multilayer growth typical of
transformed cells (Fig. 8A).
Similar morphological changes were observed with expression of IRF3
.
To assess their oncogenic potential, clones overexpressing IRF3
and
Ha-ras G12V were tested for colony formation in soft agar
(Fig. 8B). Anchorage-independent growth in soft agar often
correlates with malignant transformation. At 3 weeks, we detected the
formation of tight colonies in soft agar with expression of both
IRF3
and Ha-ras G12V. In contrast, control-transfected control cells showed almost no colony formation under these conditions. Thus, cells overexpressing a dominant negative IRF3 mutant (IRF3
) display anchorage-independent growth in soft agar. Furthermore, whole
populations of cells transfected with IRF3
were plated directly into
soft agar, and the results confirmed the ability of IRF3
to form colonies (Fig. 8C).

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Fig. 8.
Oncogenic transformation by inhibition of
IRF3. NIH3T3 cells were stably (A, B, and
D) or transiently (C) transfected with 2 µg of
expression plasmids for a dominant negative human IRF3
(IRF3 ) and an oncogenic version of Ha-ras
(Ha-ras G12V). A control expression plasmid was transfected
into NIH3T3 cells as a negative control. A, cells were
observed by light microscopy for morphological changes. B,
cells were assayed for colony formation on soft agar. C,
cells (1 × 105 and 3 × 105) were
seeded in 0.3% agarose over a cushion of 0.5% agarose for soft agar
assays. Colonies were scored 3 weeks after plating. D, tumor
formation in nude mice was assayed as described under "Experimental
Procedures."
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To further confirm the tumorigenic potential of cells expressing
IRF3
, we injected these cells and cells expressing Ha-ras G12V subcutaneously into nude mice (Fig. 8D). In all mice
injected with cells overexpressing IRF3
or Ha-ras G12V,
tumors developed within 2 to 3 weeks and continued to grow
unrestrictedly (12 mice for IRF3
and 3 mice for Ha-ras
G12V). No tumors developed in nude mice injected with cells from
control-transfected control cells during the same period of time. These
results suggest that the expression of a dominant negative IRF3 mutant
(IRF3
) imposes altered growth properties and tumorigenicity in
NIH3T3 cells.
Finally, we analyzed the tumorigenic potentials of several other clones
expressing some levels of truncated IRF3 protein (Fig. 9). Selected clones (4, 5, and 7) showed
less amounts of truncated IRF3 mutant protein (IRF3
) than the clone
(2) used for the experiments in Figs. 7 and 8 (Fig. 9A).
Under these conditions, all of these clones facilitated tight colony
formations in soft agar (Fig. 9B). Furthermore, tumors were
developed within 2-3 weeks in all the mice injected with cells
expressing IRF3
mutant protein (Fig. 9C). Alterations in
growth properties were observed consistently in all of the tested
clones with expression of a dominant negative IRF3 mutant (IRF
), and
therefore transformed phenotypes were not the result of clonal
variations.

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Fig. 9.
Oncogenic transformation by inhibition of
IRF3 in various clones. NIH3T3 cells were stably transfected with
expression plasmids for a dominant negative human IRF3 (IRF3 ).
Several clones (4, 5, and 7) were
chosen for these experiments. Clone 2 was used for the initial
experiments shown in Figs. 7 and 8. A control expression plasmid was
transfected into NIH3T3 cells as a negative control. A, IRF3
protein levels were measured by Western blotting with anti-human IRF3
antibody in whole cell extracts derived from control and several
IRF3 cell lines (clones 2, 4, 5,
and 7). B, cells were assayed for colony
formation on soft agar. C, tumor formation in nude mice was
assayed as described under "Experimental Procedures."
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 |
DISCUSSION |
Our present studies have demonstrated the tumor suppressor
function of IRF3 and the oncogenic potential of a dominant negative IRF3 mutant that specifically inhibits the expression of IRF3 target
genes. Ectopic expression of IRF3 markedly attenuated cell growth in
various kinds of cells including HeLa, HepG2, REF52, and NIH3T3 cells.
Expression of the IRF3 mutant (IRF3
) facilitated oncogenic
transformation in NIH3T3 cells, and its tumorigenic potential was
clearly demonstrated by anchorage-independent growth in soft agar and
tumor formation in nude mice. Impaired IRF3 may be responsible for the
observed transformation, because IRF3
inhibited expression of
specific IRF3 target genes (Fig. 7) and ectopic expression of IRF3
suppressed the colony formation by IRF3
in NIH3T3 cells (data not shown).
The physiological relevance of a dominant negative IRF3 mutant
(IRF3
) can be supported by the identification of IRF3a in the cells
(10, 12, 13). IRF3a is generated by alternative splicing of IRF3
pre-mRNA. As demonstrated with IRF3
, IRF3a can selectively and
potently inhibit virus-induced activation of specific target genes
including IFN-
and it contains defective DNA binding domain. The
expression of IRF3a is ubiquitous, but the levels of IRF3a, compared
with those of IRF3, can vary in different cellular contexts. Such
regulated production of the IRF3a protein would result in the
controlled inhibition of IRF3 activity at the specific target
promoters. Because the present studies indicate the role of IRF3 in the
cell growth control, it is important to elucidate the mechanisms
showing how the ratio of IRF3a to IRF3 is maintained and
controlled in response to various signals and that the ratio can be
deregulated to alter cell growth control during tumorigenesis.
The ability of an IRF3 mutant to transform cells was nearly as great as
that of the oncogenic Ha-ras G12V. Furthermore, mutational inactivation of the p53 tumor suppressor gene can also facilitate oncogenic transformation under similar conditions (27-30). One interesting possibility emerging from the present studies is that expression of mutant IRF3 protein may confer tumorigenic phenotype upon
the cells, similar to the gain-of-function associated with p53 mutants
(31). The p53 mutant loses its tumor suppressor function as a
consequence of the functional inactivation of p53. However, certain
types of p53 mutations exert additional gain-of-function phenotypes
with dominant negative effects on wild type p53. The mechanisms for
these gain-of-function of p53 mutants have not been well characterized.
The relevance of these mechanisms remains to be determined in
IRF3, with further elucidation of how tumorigenesis may be enhanced by
IRF3
.
It is well known that most spontaneously immortalized rodent cells such
as NIH3T3 cells can be transformed efficiently by activation of one
oncogene (or inactivation of one tumor suppressor gene) alone, in
contrast to primary rodent and human cells. Interestingly, NIH3T3 cells
were shown to contain the deleted p16 tumor suppressor gene,
which plays a critical role in Rb regulation pathways (32). Thus,
inactivation of IRF3 might cooperate with other oncogenic events for
tumorigenesis in normal cells. To understand these tumorigenesis
mechanisms (including gain-of-function effects), it is critical to
determine which genes play a role in the inhibition and promotion of
cell growth with up- and down-regulation of IRF3, respectively. Related
to this concern, the profiling of genome activities is in progress
with conditional expression of IRF3 and IRF3
using DNA
microarray methods.
Another member of the IRF family, IRF2, blocks the activation of
promoters containing IRF binding sites. When IRF2 was overexpressed in
NIH3T3 cells, the cells became transformed and displayed enhanced tumorigenicity in nude mice (33). Consistently, overexpression of IRF2
impaired the activation of genes that are controlled by IRF family
factors (e.g. IRF1 and ISGF3). Based on our results, we
infer that the ability of IRF2, like an IRF3 mutant, to induce cell
transformation might also be due to suppression of the transcriptional activity of IRF3. Indeed, ectopic expression of IRF2 can inhibit IRF3-dependent transcriptional activation (data not shown).
Thus, several IRF family members including IRF3a might interact
functionally to tightly modulate cell growth, and alterations in
their regulation could contribute to oncogenic transformation.
Thus far, the involvement of IRF3 in human cancer has not been studied
extensively, and no mutation in the IRF3 gene has been reported in a
human cancer. However, several viral oncoproteins, including human
papillomavirus 16 E6 and adenovirus E1A, can inhibit the activation of
IRF3 as well as p53 (18, 34). The v-IRF protein from Kaposi's
sarcoma-associated herpes virus is shown to inhibit IRF3
transcriptional activity by blocking the interaction of CBP
(CREB-binding protein)/p300 coactivator with IRF3 (35). On the basis of
the present studies, the interaction of these oncoproteins with IRF3
and the inhibition of its transcriptional activation function could
contribute directly to the oncogenic potential of these viruses
by altering cell growth control pathways. Quite interestingly,
and consistent with our idea of the tumor suppressing function of IRF3,
ectopic expression of IRF3 has recently been found to suppress the
growth of B16 melanoma tumors in the context of gene therapy
(36).
 |
FOOTNOTES |
*
This work was supported by the Brain Korea 21 Project of the
Korean Ministry of Education, the Molecular Medicine Research Group
Program (M1-0106-00-01117), Creative Research Initiatives, and the
Center for Biological Modulators of the 21st Century Frontier R&D
Program (CBM-01-B-5) of the Korean Ministry of Science and Technology.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 Biological
Sciences, Korea Advanced Inst. of Science and Technology, Taejon
305-701, Korea. Tel.: 82-42-869-2634; Fax: 82-42-869-8160; E-mail:
tkkim@ mail.kaist.ac.kr.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M205792200
 |
ABBREVIATIONS |
The abbreviations used are:
IRF3, interferon
regulatory factor 3;
IFN, interferon;
GBP, guanylate-binding protein;
DMEM, Dulbecco's modified Eagle's medium;
BrdUrd, 5-bromo-2'-deoxyuridine;
GFP, green fluorescent protein;
FACS, fluorescence-activated cell sorter;
2', 5'-AS, 2',5'-oligoadenylate
synthetase.
 |
REFERENCES |
1.
|
Evan, G. I.,
and Vousden, K. H.
(2001)
Nature
411,
342-348[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Wahl, G. M.,
and Carr, A. M.
(2001)
Nat. Cell Biol.
3,
277-286
|
3.
|
Vogelstein, B.,
Lane, D.,
and Levine, A. J.
(2000)
Nature
408,
307-310[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Giaccia, A. J.,
and Kastan, M. B.
(1998)
Genes Dev.
12,
2973-2983[Free Full Text]
|
5.
|
Maniatis, T.,
Falvo, J. V.,
Kim, T. H.,
Kim, T. K.,
Lin, C. H.,
Parekh, B. S.,
and Wathelet, M. G.
(1998)
Cold Spring Harbor Symp. Quant. Biol.
63,
609-620[Medline]
[Order article via Infotrieve]
|
6.
|
Kim, T.,
Kim, T. Y.,
Song, Y.-H.,
Min, I. M.,
Yim, J.,
and Kim, T. K.
(1999)
J. Biol. Chem.
274,
30686-30689[Abstract/Free Full Text]
|
7.
|
Kim, T. K.,
Kim, T.,
Kim, T. Y.,
Lee, W. G.,
and Yim, J.
(2000)
Cancer Res.
60,
1153-1156[Abstract/Free Full Text]
|
8.
|
Kim, T.,
Kim, T. Y.,
Lee, W. G.,
Yim, J.,
and Kim, T. K.
(2000)
J. Biol. Chem.
275,
16910-16917[Abstract/Free Full Text]
|
9.
|
Weaver, B. K.,
Ando, O.,
Kumar, K. P.,
and Reich, N. C.
(2001)
FASEB J.
15,
501-514[Abstract/Free Full Text]
|
10.
|
Karpova, A. Y.,
Trost, M.,
Murray, J. M.,
Cantley, L. C.,
and Howley, P. M.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
2818-2823[Abstract/Free Full Text]
|
11.
|
Cuddihy, A. R.,
Li, S.,
Tam, N. W.,
Wong, A. H.,
Taya, Y.,
Abraham, N.,
Bell, J. C.,
and Koromilas, A. E.
(1999)
Mol. Cell. Biol.
19,
2475-2484[Abstract/Free Full Text]
|
12.
|
Karpova, A. Y.,
Howley, P. M.,
and Ronco, L. V.
(2000)
Genes Dev.
14,
2813-2818[Abstract/Free Full Text]
|
13.
|
Karpova, A. Y.,
Ronco, L. V.,
and Howley, P. M.
(2001)
Mol. Cell. Biol.
21,
4169-4176[Abstract/Free Full Text]
|
14.
|
Barbacid, M.
(1987)
Annu. Rev. Biochem.
56,
779-827[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Fujita, T.,
Reis, L. F.,
Watanabe, N.,
Kimura, Y.,
Taniguchi, T.,
and Vilcek, J.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
9936-9940[Abstract]
|
16.
|
Galaktionov, K.,
Lee, A. K.,
Eckstein, J.,
Draetta, G.,
Meckler, J.,
Loda, M.,
and Beach, D.
(1995)
Science
269,
1575-1577[Medline]
[Order article via Infotrieve]
|
17.
|
Heylbroeck, C.,
Balachandranm, S.,
Servant, M. J.,
DeLuca, C.,
Barber, G. N.,
Lin, R.,
and Hiscott, J.
(2000)
J. Virol.
74,
3781-3792[Abstract/Free Full Text]
|
18.
|
Juang, Y.,
Lowther, W.,
Kellum, M.,
Au, W. C.,
Lin, R.,
Hiscott, J.,
and Pitha, P. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9837-9842[Abstract/Free Full Text]
|
19.
|
Lin, R.,
Heylbroeck, C.,
Pitha, P. M.,
and Hiscott, J.
(1998)
Mol. Cell. Biol.
18,
2986-2996[Abstract/Free Full Text]
|
20.
|
Navarro, L.,
Mowen, K.,
Rodems, S.,
Weaver, B.,
Reich, N.,
Spector, D.,
and David, D.
(1998)
Mol. Cell. Biol.
18,
3796-3802[Abstract/Free Full Text]
|
21.
|
Sato, M.,
Tanaka, N.,
Hata, N.,
Oda, E.,
and Taniguchi, T.
(1998)
FEBS Lett.
425,
112-116[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Schafer, S. L.,
Lin, R.,
Moore, P. A.,
Hiscott, J.,
and Pitha, P. M.
(1998)
J. Biol. Chem.
273,
2714-2720[Abstract/Free Full Text]
|
23.
|
Wathelet, M. G.,
Lin, C. H.,
Parekh, B. S.,
Ronco, L. V.,
Howley, P. M.,
and Maniatis, T.
(1998)
Mol. Cell
1,
507-518[Medline]
[Order article via Infotrieve]
|
24.
|
Weaver, B. K.,
Kumar, K. P.,
and Reich, N. C.
(1998)
Mol. Cell. Biol.
18,
1359-1368[Abstract/Free Full Text]
|
25.
|
Yoneyama, M.,
Suhara, W.,
Fukuhara, Y.,
Fukuda, M.,
Nishida, E.,
and Fujita, T.
(1998)
EMBO J.
17,
1087-1095[Abstract/Free Full Text]
|
26.
|
Darnell, J. E., Jr.
(1997)
Science
277,
1630-1635[Abstract/Free Full Text]
|
27.
|
Jenkins, J. R.,
Rudge, K.,
and Currie, G. A.
(1984)
Nature
312,
651-654[Medline]
[Order article via Infotrieve]
|
28.
|
Zambetti, G. P.,
Olson, D.,
Labow, M.,
and Levine, A. J.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3952-3956[Abstract]
|
29.
|
Eliyahu, D.,
Raz, A.,
Gruss, P.,
Givol, D.,
and Oren, M.
(1984)
Nature
312,
646-649[Medline]
[Order article via Infotrieve]
|
30.
|
Parada, L. F.,
Land, H.,
Weinberg, R. A.,
Wolf, D.,
and Rotter, V.
(1984)
Nature
312,
649-654[Medline]
[Order article via Infotrieve]
|
31.
|
Oijen, M.,
and Slootweg, P. J.
(2000)
Clin. Cancer Res.
6,
2138-2145[Abstract/Free Full Text]
|
32.
|
Linardopoulos, S.,
Street, A.,
Quelle, D. E.,
Parry, D.,
Peters, G.,
Sherr, C. J.,
and Balmain, A.
(1995)
Cancer Res.
55,
5168-5172[Abstract]
|
33.
|
Harada, H.,
Kitagawa, M.,
Tanaka, N.,
Yamamoto, H.,
Harada, K.,
Ishihara, M.,
and Taniguchi, T.
(1993)
Science
259,
971-974[Medline]
[Order article via Infotrieve]
|
34.
|
Ronco, L. V.,
Karpova, A. Y.,
Vidal, M.,
and Howley, P. M.
(1998)
Genes Dev.
12,
2061-2072[Abstract/Free Full Text]
|
35.
|
Lin, R.,
Genin, P.,
Mamane, Y.,
Sgarbanti, M.,
Battistini, A.,
Harrington, W. J., Jr.,
Barber, G. N.,
and Hiscott, J.
(2001)
Oncogene
20,
800-811[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Duguay, D.,
Mercier, F.,
Stagg, J.,
Martineau, D.,
Bramson, J.,
Servant, M.,
Lin, R.,
Galipeau, J.,
and Hoscott, J.
(2002)
Cancer Res.
62,
5148-5152[Abstract/Free Full Text]
|
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