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Oncogene (2000) 19, 6386 ± 6391
2000 Nature Publishing Group All rights reserved 0950 ± 9232/00 $15.00
www.nature.com/onc
SHORT REPORT
A novel role for IRF-1 as a suppressor of apoptosis
Rachel S Chapman1, Eleanor K Du€1, Paula C Lourenco1, Elizabeth Tonner2, David J Flint2,
Alan R Clarke3 and Christine J Watson*,4
1
Cancer Research Campaign (CRC) Laboratories, Department of Pathology, University of Edinburgh, Medical School, Edinburgh,
EH8 9AG, UK; 2Hannah Research Institute, Ayr, Scotland, KA6 5HL, UK; 3School of Biosciences, Cardi€ University, Cardi€,
CF10 3US, UK; 4Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK
The tumour suppressor IRF-1 is a transcription factor
involved in the induction of apoptosis in several in vitro
systems. Post-lactational involution of the mammary
gland is characterized by extensive apoptosis of the
epithelial cells. We have previously shown that signal
transducer and activator of transcription (Stat) 3 drives
apoptosis and involution in the mouse mammary gland.
Since one of the downstream targets of the Stat
signalling pathway is IRF-1, we have used IRF-1
knockout mice to address the potential role of this
transcription factor in involution. Surprisingly, in the
absence of IRF-1 signi®cantly higher numbers of
apoptotic cells were found in involuting glands at 48 h
compared to control glands. In addition, the alveolar
structure in IRF-1 null mammary glands had collapsed
whereas in control glands the alveoli remained intact and
distended. However, by 72 h control and null glands were
morphologically similar suggesting that IRF-1 suppresses
apoptosis only during the early, reversible, stage of
involution. This suggests a survival role for IRF-1 in
mammary epithelia and demonstrates a novel role for
IRF-1 in vivo ± suppression of premature epithelial
apoptosis during mammary gland involution. Oncogene
(2000) 19, 6386 ± 6391.
Keywords: IRF-1; apoptosis; mammary gland; involution
The transcription factor interferon regulatory factor
(IRF)-1 was originally identi®ed by its ability to bind
to the virus-inducible `enhancer-like' elements of the
human interferon-b gene and to activate interferoninducible genes (reviewed by Taniguchi et al., 1997). It
is now known to regulate many cellular responses
including the suppression of growth (Nguyen et al.,
1997; Kirchho€ and Hauser, 1999), susceptibility to
oncogenic transformation (Tanaka et al., 1994), and
induction of apoptosis by a p53 independent mechanism (Horiuchi et al., 1997; Tamura et al., 1995). Loss
of IRF-1 has recently been shown to contribute to cHa-ras-induced tumour development and to accelerate
and increase tumour development and to alter the
tumour spectrum in p53 null mice, thus formally
demonstrating its ability to function as a tumour
suppressor (Nozawa et al., 1999).
*Correspondence: CJ Watson
Received 15 June 2000; revised 12 October 2000; accepted 12
October 2000
Developmental studies using IRF-1 knockout mice
have focused on the role of this factor in regulating
the immune system where it has been shown to be
required for T cell and NK cell development
(Matsuyama et al., 1993; Ogasawara et al., 1998). A
function for IRF-1 in epithelial cells has not yet been
described. Given the importance of IRF-1 in inducing
apoptosis in vitro we have used the IRF-1 knockout
mice to examine whether this transcription factor is
involved in the regulation of epithelial cell apoptosis
in vivo.
Following weaning of the young, the mammary
gland undergoes a dramatic remodelling where the
epithelial cells are removed by apoptosis and the
extracellular matrix is degraded. At the onset of
involution there is a speci®c activation of the signal
transducer and activator of transcription, Stat3. We
have previously shown that Stat3 is required to drive
epithelial apoptosis and involution in the murine
mammary gland (Chapman et al., 1999). p53 has also
been shown to be increased at the start of involution
in some mice (Strange et al., 1992; Jerry et al., 1998).
However, its function has remained equivocal as p53
null BALB-C mice displayed delayed involution (Jerry
et al., 1998) whereas on an outbred background no
di€erence was seen between p53 wild type and
knockout animals (Li et al., 1996). In the absence of
Stat3, levels of p53 were increased suggesting that
Stat3 may regulate alternative transcription factors
and signalling molecules for the induction of apoptosis
(Chapman et al., 1999). One putative target of Stat3 is
IRF-1 (Yuan et al., 1994) so to establish whether
IRF-1 plays a role in regulating epithelial apoptosis
we have examined mammary gland involution in
IRF-1 null mice.
IRF-1 null mice were kindly provided by Professor
Tak Mak (Toronto, Canada). Mice were allowed to
lactate for 10 days and then pups were withdrawn to
initiate involution. The level of IRF-1 protein present
in mammary glands at day 10 of lactation and
during involution (days 1 ± 4 and 6) was measured by
Western blot (Figure 4). IRF-1 was detected at all
time points in wild type mammary glands but
densitometric analysis of glands from three independent mice for each time point revealed no signi®cant
di€erence in the average level of IRF-1 over the time
course. DNA binding activity was assessed using a
consensus IRF-1 binding site. IRF-1 DNA binding
was detected in KIM-2 mammary epithelial cells
treated for 30 min with IFN-g (as a positive control)
but not following LIF treatment (which activates
Stat3) or in extracts from lactating or involuting
IRF1 suppresses apoptosis
RS Chapman et al
mammary glands (data not shown) demonstrating
that in the mammary gland IRF-1 is not regulated
by Stat3. The lack of detectable IRF-1 DNA binding
activity during involution could re¯ect the fact that
in the mammary gland IRF-1 does not bind to a
consensus DNA binding element but may require a
speci®c DNA sequence (such as a composite
element). Alternatively IRF-1 could be part of a
multiprotein complex ± an IRF association domain
has been identi®ed in IRF-1 that may be responsible
for such an interaction with other transcription
factors (Meraro et al., 1999). Of note, the transcrip-
tion factor Stat1 has also been shown to regulate
apoptosis independently of direct transcriptional
activation (Kumar et al., 1997).
Lactating and involuting mammary glands were
sectioned and stained with haematoxylin and eosin,
Figure 1 shows representative glands from IRF-1 wt
(A,C,E,G) and IRF-1 null (B,D,F,H) mice. At day 10
of lactation the glands were composed of alveoli lined
by secretory epithelial cells, no phenotypic di€erence
was seen between wild type (Figure 1A) and null
(Figure 1B) animals at this stage. No di€erence was
seen earlier during lactation (days 0 and 5) and the
6387
Figure 1 Accelerated involution in IRF-1 null mammary glands. Pups were removed at 10 days of lactation to initiate involution.
Mammary glands were ®xed in formalin and embedded in paran for sectioning. Representative photos of haematoxylin and eosin
stained sections of wild type mammary glands (A,C,E,G) and IRF-1 null glands (B,D,F,H) are shown at day 10 of lactation (A,B),
24 h of involution (C,D), 48 h of involution (E,F) and 72 h of involution (G,H). Abdominal glands from three mice were examined
for each time point. Scale bar 100 mm
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IRF1 suppresses apoptosis
RS Chapman et al
6388
Oncogene
mothers were able to raise and feed their pups (data
not shown). Twenty-four hours following removal of
pups the alveoli of both the wild type (Figure 1C) and
null (Figure 1D) glands were expanded due to the
accumulation of unsuckled milk. At 48 h many of the
alveoli of the control animals remained expanded
although a few small areas were apparent where the
alveoli had started to collapse (Figure 1E). A low
number of apoptotic epithelial cells were seen shed
into the alveolar lumina. In contrast, by 48 h the IRF1 null glands were in a more advanced stage of
involution with breakdown of the structure of the
glands and collapse of the majority of the alveoli
(Figure 1F). By 72 h the control glands had undergone signi®cant involution with the collapse of the
alveoli and the reappearance of fat which makes up
the majority of tissue in a resting gland (Figure 1G).
IRF-1 null glands at this stage had a very similar
appearance to control glands with areas of collapsed
alveoli surrounded by areas of fat (Figure 1H). Thus
IRF-1 appears to regulate the early stages of
involution and prevents a premature collapse of the
alveoli.
To quantify the degree of alveolar collapse that
had occurred in the absence of IRF-1 the cross
sectional area of alveoli in the gland was measured
(Lund et al., 1996), Figure 2A shows the distribution
of the alveolar area. At 24 h a range of alveolar
areas were recorded, a small number of alveoli were
collapsed but the variation in size also re¯ects the
position in space of the alveoli when the section was
cut. There was no signi®cant di€erence between
control and knockout mice at this point. By 48 h
the percentage of alveoli in the IRF-1 null glands
with an area of less than 2 mm26103 had signi®cantly
increased from 28% (+13%) at 24 h to 81% (+5%,
n=3 mean+s.e.m., Mann ± Whitney U test P50.05)
whereas alveoli in the control glands had a similar
size distribution to that observed at 24 h. As
expected, IRF-1 null glands also had signi®cantly
fewer alveoli larger than 2 mm26103 compared with
control glands at 48 h. By 72 h, alveoli in the control
glands had also started to collapse with a marked
increase seen in the percentage of alveoli with an
area of less than 2 mm26103, by this stage there was
no signi®cant di€erence between control and null
animals.
During the later stages of involution there is a
reappearance of fat tissue (see Figure 1G,H), the
area of the gland occupied by adipocytes was
measured as previously described (Chapman et al.,
1999) and is shown in Figure 2B. Less than 5% of
the area of the gland was occupied by adipocytes
after 24 h of involution, a small increase was
observed at 48 h and 72 h and by 96 h more than
50% of the gland area was ®lled with adipocytes.
No signi®cant di€erence was seen between control
and IRF-1 null animals at any of the time points
measured (Mann ± Whitney U test, P40.05 n=3).
This supports our observations that early stages of
involution are accelerated in the absence of IRF-1
but later stages occur with the same kinetics as in
control mice.
During the early stages of involution, prior to
generalized degradation of the gland, the secretory
epithelial cells undergo apoptosis and are shed from
Figure 2 Measurement of alveolar collapse and gland remodelling in the absence of IRF-1. (A) Distribution of alveolar cross
sectional area at 24, 48 and 72 h of involution. The area of
alveolar lumina was measured using haematoxylin and eosin
stained slides and the general morphometry (object) programme
at 406 magni®cation on the AxioHOME microscope. The
internal circumference of 200 intact alveoli was drawn around
and their area calculated. (B) Proportion of gland occupied by
adipocytes at 24, 48, 72 and 96 h of involution was measured as
previously described (Chapman et al., 1999). Wild type glands ±
open bars, IRF-1 null glands ± solid bars. Each bar represents
the mean of data collected from three mice, error bars represent
the standard error of the mean. (*) P50.05 Mann ± Whitney U
test
the epithelial wall (Walker et al., 1989; Strange et
al., 1992). IRF-1 has been shown to be required for
apoptosis in some systems (Tamura et al., 1995;
IRF1 suppresses apoptosis
RS Chapman et al
Horiuchi et al., 1997; Kirchho€ and Hauser, 1999)
but the accelerated involution seen in the absence of
this factor could be due to premature engagement of
the apoptotic programme. Apoptosis was scored by
both morphological assessment of condensed chromatin (Figure 3A) and TUNEL analysis of DNA
strand breaks (Figure 3B). At 24 h of involution
there were very few apoptotic cells in the control
and IRF-1 null glands scored by either method. By
48 h a small increase in the number of cells
undergoing apoptosis was seen in control glands
(morphology 1.6+0.2%, TUNEL 1.9+0.5%). However, a signi®cantly greater number of cells were
apoptotic in the absence of IRF-1 scored by both
morphology (3.9+0.5%) and TUNEL (6.5+0.8%,
mean+s.e.m., n=3, P50.05 Mann ± Whitney U
test). After 72 h the number of apoptotic cells
increased in the control glands to similar levels
found in the IRF-1 null glands. Thus the accelerated
involution seen in the absence of IRF-1 at 48 h is
characterized by premature apoptosis of the epithelial cells resulting in the breakdown of the lobularalveolar structure.
In order to investigate the mechanism of this
accelerated involution we analysed a series of
apoptosis-regulatory proteins which are known to be
altered during involution and which therefore might
have been predicted to mediate the IRF-1 dependent
survival (see Philp et al., 1996; Liu et al., 1996;
Strange et al., 1992; Lund et al., 1996; Heermeier et
al., 1996; Jerry et al., 1998; Tonner et al., 1997).
Figure 4 shows Western blot analysis. No di€erences
were seen between control and IRF-1 null glands in
the regulation of Stats1 and 3, SGP-2, Bcl-xL, Bax
(not shown), p53, p21 or c-myc. Levels of IGFBP-5,
which is proposed to induce apoptosis by sequestering
IGF-1, started to increase in the IRF-1 null glands at
24 h. However, the variation between mice (a common
feature of IGFBP-5) meant that the di€erence between
wild type (11 385, 20 372 and 27 556 arbitrary units)
and IRF-1 null glands (19 791, 41 495 and 140 226
arbitrary units) was not signi®cant. By 48 h higher
levels of IGFBP-5 were actually found in the wild
type glands than the IRF-1 null glands ± this result
probably re¯ects the earlier engagement of the
apoptotic pathway and collapse of the glands seen in
the absence of IRF-1. Transcriptional inhibition of
IGFBP-5 may be the mechanism by which IRF-1
suppresses apoptosis ± delayed involution in the
absence of Stat3 was accompanied by signi®cantly
lower levels of IGFBP-5 (Chapman et al., 1999) ±
however more experiments will need to be performed
to clarify whether there are early signi®cant changes in
IGFBP-5.
In the absence of any signi®cant changes in
involution-associated proteins IRF-1 may be acting
6389
Figure 3 Accelerated apoptosis in the absence of IRF-1. (A) Morphological assessment of cells exhibiting condensed chromatin at
24, 48 and 72 h of involution. Apoptotic cells were identi®ed on haematoxylin and eosin stained slides using light microscopy and
classical morphological criteria (condensation and fragmentation of chromatin, cell shrinkage/separation from neighbours). A
running mean was established and a minimum of 1800 cells were scored per section, split between at least 15 randomly chosen ®elds
and calculated as a percentage of the total cell count. A representative photograph at 48 h of involution is shown, arrows indicate
examples of apoptotic cells. (B) TUNEL analysis at 24, 48 and 72 h of involution. TUNEL staining was carried out on formalin
®xed paran embedded sections using the ApopTag1 kit (Intergen, NY, USA) according to the manufacturer's instructions. A
running mean was established and a minimum of 1700 cells were scored per section, split between at least 15 randomly chosen ®elds
and calculated as a percentage of the total cell count. A representative photograph at 48 h of involution is shown. Wild type glands,
open bars; IRF-1 null glands, solid bars. Each bar represents the mean of data collected from three mice, error bars represent the
standard error of the mean. (*) P50.05 Mann ± Whitney U test. Scale bar 10 mm
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IRF1 suppresses apoptosis
RS Chapman et al
6390
Figure 4 Western blot analysis of apoptosis-related proteins
during involution. Samples were taken at day 10 of lactation
(10L) and at 24, 48, 72 and 96 h of involution (24I, 48I, 72I, 96I).
Protein was extracted from frozen mammary glands and run on
SDS-polyacrylamide gels as previously described (Chapman et al.,
1999). Phosphorylated Stat3 (PStat3), Stat1 and phosphorylated
Stat1 (PStat1) antibodies ± New England Biolabs; Stat3, SGP-2,
Bcl-x, p21, c-myc and IRF-1 antibodies ± Santa Cruz; p53 CM5
antibody ± a gift from David Lane (Dundee, UK). 20 mg of
protein was loaded per lane, SGP-2 5 mg per lane. Speci®cally
bound antibody was detected with horseradish peroxidaseconjugated secondary antibodies and ECL (Amersham) and
recorded using X-ray ®lm. (+) wild type glands, (7) IRF-1 null
glands. Samples from three di€erent mice were examined for each
time point using abdominal glands from the same mice used for
histological analysis. Graph shows Western ligand blot analysis of
IGFBP-5 using 125I-labelled IGF-1 as previously described
(Hossenlopp et al., 1986). Quantitative changes in IGFBP-5
concentrations were determined using Image Quant analysis of a
phospho image (Molecular Dynamics, Sunnyvale, CA, USA).
Wild type glands, open bars; IRF-1 null glands, solid bars. Each
bar represents the mean of data collected from three mice, error
bars represent the standard error of the mean. (*) P50.05
Mann ± Whitney U test
Oncogene
by a novel mechanism ± this hypothesis is more
consistent with the lack of any detectable IRF-1
DNA binding activity during involution. Mice
transgenic for Bcl-2 displayed delayed apoptosis
during involution but here also none of the
associated molecular events were altered suggesting
that if these signals are important for driving
apoptosis then Bcl-2 must act downstream of them
(Jager et al., 1997). Further work will focus on the
possible mechanism of action of IRF-1 in the
suppression of apoptosis.
In summary we have used an IRF-1 knockout
mouse to examine the role of IRF-1 in the mammary
gland. The absence of IRF-1 resulted in accelerated
apoptosis of epithelial cells leading to premature
involution following weaning. IRF-1 was found to
be expressed in KIM-2 mammary epithelial cells in
culture but it is possible that in the mammary gland
IRF-1 may not be having a direct e€ect in epithelial
cells. Stromal cells contribute to involution through
the production of proteases which degrade the
extracellular matrix resulting in collapse of the
lobuloalveolar structure (Lund et al., 1996). However,
epithelial apoptosis occurs prior to and independently
of the production of these proteases suggesting that
IRF-1 is not exerting its e€ect in these cells. It is also
possible that IRF-1 is mediating its e€ect through the
lymphocytes and macrophages that are found in the
gland (Walker et al., 1989). However, this possibility
again seems unlikely as the in®ltration of in¯ammatory cells seen during involution is delayed, occurring
in conjunction with the breakdown of the lobuloalveolar structure suggesting that these cells do not
contribute signi®cantly to the initial phase of involution.
The timing of involution is dependent on the
balance between apoptosis-inducing and apoptosissuppressing signals (Li et al., 1997). IRF-1 is
therefore an important component of the survival
signal provided to mammary epithelial cells at the
start of involution which allows the process to be
reversed during the ®rst 24 ± 48 h. This is a novel
function for this tumour suppressor gene and is in
stark contrast to its previously reported role in
initiating apoptosis either directly or following DNA
damage or serum withdrawal (Nguyen et al., 1997;
Tamura et al., 1995; Tanaka et al., 1994; Horiuchi et
al., 1997).
This study identi®es a new anti-apoptotic role for
IRF-1. We therefore show that in di€erent circumstances IRF-1 can act in apparently contradictory
ways (pro and anti apoptotic) and the challenge now
will be to understand the changes in context which
lead to such dramatically di€erent endpoints.
Acknowledgements
This work was supported by an AICR grant. AR Clarke is
a Royal Society University Research Fellow and CJ
Watson is funded by the Cancer Research Campaign.
IRF1 suppresses apoptosis
RS Chapman et al
6391
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