Báo cáo khoa học: BRCA1 accumulates in the nucleus in response to hypoxia and TRAIL and enhances TRAIL-induced apoptosis in breast cancer cells pdf

BRCA1 accumulates in the nucleus in response to hypoxia
and TRAIL and enhances TRAIL-induced apoptosis in
breast cancer cells
Latricia D. Fitzgerald
1,
*, Charvann K. Bailey
1
, Stephen J. Brandt
2,3
and Marilyn E. Thompson
1,3
1 Division of Cancer Biology, Meharry Medical College, Nashville, TN, USA
2 Departments of Medicine, Cell and Developmental Biology, and Cancer Biology, Vanderbilt University Medical Center and VA Tennessee
Valley Healthcare System, Nashville, TN, USA
3 Vanderbilt-Ingram Cancer Center, Nashville, TN, USA
The breast cancer susceptibility gene BRCA1 encodes a
tumor suppressor protein that is located predominantly
within the nucleus. However, BRCA1 also contains
two nuclear export sequences [1,2] and shuttles
between the nucleus and cytoplasm. BRCA1 has been
implicated in several cellular processes, including cell
cycle regulation [3], transcription [4], DNA repair [5]
and apoptosis [6]. Full understanding of BRCA1’s role
in these processes is lacking; however, it is likely that
some functions overlap. For example, the ability of
BRCA1 to regulate the cell cycle and enhance apopto-
sis could be due to its role as a transcriptional coacti-
vator. BRCA1 induces the transcription of GADD45a
[7], which is involved in the cell cycle G
2
–M check-

point and in the control of apoptosis.
BRCA1 can also enhance apoptosis via a pathway
involving H-Ras, mitogen-activated protein kinase
kinase kinase 4, Jun N-terminal kinase, Fas ligand ⁄ Fas
and caspase-9 [8]. The ability of BRCA1 to enhance
apoptosis mediated through Jun N-terminal kinase
Keywords
apoptosis; BRCA1; hypoxia; localization;
TRAIL
Correspondence
M. E. Thompson, Division of Cancer
Biology, WBSC 2137, Meharry Medical
College, 1005 D.B. Todd Blvd, Nashville,
TN 37208-3599, USA
Fax: +1 615 327 6442
Tel: +1 615 327 6787
E-mail:
*Present address
University of California Davis Medical
Center, Sacramento, CA, USA
(Received 25 April 2007, revised 1 August
2007, accepted 7 August 2007)
doi:10.1111/j.1742-4658.2007.06033.x
A major contributing factor to the development of breast cancer is
decreased functional expression of breast cancer susceptibility gene 1,
BRCA1. Another key contributor to tumorigenesis is hypoxia. Here we
show that hypoxia increased the nuclear localization of BRCA1 in MCF-7
and MDA-MB-468 human breast cancer cell lines without changing its
steady-state expression level. Nuclear accumulation of BRCA1 was not
evident in MCF-12A or HMEC (human mammary epithelial cell) nonma-

lignant mammary epithelial cells under the same conditions. Hypoxia also
increased the cell surface expression of TRAIL on MDA-MB-468 cells.
Neutralization of TRAIL precluded the hypoxia-induced accumulation of
BRCA1 in the nucleus, whereas exogenously administered TRAIL mim-
icked the effect. Treatment of MDA-MB-468 cells with TRAIL resulted in
a dose- and time-dependent increase in apoptosis. Furthermore, TRAIL-
induced apoptosis in HCC1937 cells, which harbor a BRCA1 mutation,
increased synergistically when wild-type BRCA1 was reconstituted in the
cells, and downregulation of BRCA1 expression in MDA-MB-468 cells
reduced the apoptotic response to TRAIL. These data provide a novel link
between hypoxia, TRAIL and BRCA1, and suggest that this relationship
may be especially relevant to the potential use of TRAIL as a chemothera-
peutic agent.
Abbreviations
BRCA1, breast cancer susceptibility gene 1; FACS, fluorescence-activated cell sorting; GADD45, growth arrest and DNA damage-inducible
gene; HIF, hypoxia-inducible factor; HMEC, human mammary epithelial cell; shRNA, short hairpin RNA; TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand.
FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS 5137
[8,9], a stress-activated kinase, as well as data from
studies with genotoxic agents [10], suggest a role for
BRCA1 in the cellular response to stress. Stress
induced by genotoxic drugs or UV radiation decreases
the steady-state mRNA levels and enhances the phos-
phorylation of BRCA1 [11]. Thus, these data provide
a precedent for the involvement of BRCA1 in the cel-
lular response to stress.
One stress common to multiple solid tumors is
hypoxia. During tumor growth, increased oxygen con-
sumption in the presence of inadequate oxygen deliv-
ery results in decreased intratumoral pO

2
[12]. In
addition, malignant cells express elevated levels of
hypoxia-inducible factor (HIF)-1a [13]. Under norm-
oxic conditions, the expression of HIF-1a is low, due
to its rapid degradation by the ubiquitin–proteasome
pathway. Upon exposure to hypoxia, HIF-1a is stabi-
lized, translocates to the nucleus and heterodimerizes
with HIF-1b. The heterodimer binds to the hypoxia-
responsive element of target genes that encode proteins
involved in various cellular processes, including inhibi-
tion of apoptosis.
Hypoxia has been reported to inhibit apoptosis
induced by tumor necrosis factor-related apoptosis-
inducing ligand (TRAIL) [14,15], which selectively
induces apoptosis in malignant, but not nonmalignant,
cells [16]. TRAIL binds to death receptors, DR4 and
DR5, and recruits Fas-associated death domain pro-
tein to the receptors. Caspase-8 is activated and subse-
quently activates downstream caspases. Hypoxia
inhibits TRAIL-mediated apoptosis in HCT116 human
colon carcinoma cells by inhibiting the translocation of
Bax from the cytosol to the mitochondria, thereby pre-
venting the release of cytochrome c and progression of
apoptosis [14].
Studies have also demonstrated that hypoxia
decreases the BRCA1 mRNA level in prostate cells
[17]. Hypoxia-induced downregulation of BRCA1
expression is due to changes in the relative distribution
of activating and repressive E2Fs on the BRCA1 pro-

moter [18]. Furthermore, in response to hypoxia,
BRCA1 is phosphorylated in a checkpoint kinase
1-dependent manner [19] and DNA repair is impaired
[17]. Here, we report that under conditions that do not
decrease BRCA1 expression, hypoxia increases the
nuclear content of BRCA1 in malignant mammary epi-
thelial cell lines while having no effect on nonmalig-
nant cell lines. This localization change was dependent
on TRAIL, and exogenous TRAIL mimicked the
effect. Furthermore, TRAIL, but not hypoxia, induced
apoptosis, and TRAIL-mediated apoptosis was
enhanced by BRCA1.
Results
Hypoxia induces nuclear accumulation of BRCA1
in breast cancer cells
To investigate the effects of hypoxia on the localiza-
tion of BRCA1 in mammary epithelial cells, we sub-
jected malignant (MDA-MB-468 and MCF-7) and
nonmalignant (MCF-12A and HMEC) cells to hypoxia
and examined the protein and relative nuclear and
non-nuclear levels of BRCA1. As it had been previ-
ously reported that a 48 h exposure to 0.01% oxygen
resulted in decreased BRCA1 expression [18], we used
a milieu of 1% oxygen for 24 h to assess changes in
BRCA1 localization in response to hypoxia without
concurrent changes in its expression. In MDA-MB-468
and MCF-7 cells exposed to normoxia, BRCA1 local-
ized within the nucleus, cytoplasm and in a concen-
trated area around the nucleus (Fig. 1A). Under
low-oxygen conditions, the nuclear content of BRCA1

increased, whereas the non-nuclear (cytoplasmic and
perinuclear) level decreased. In MCF-12A and HMEC
cells, there was no difference in BRCA1 localization in
cells exposed to hypoxia as compared to cells exposed
to normoxia.
We next performed subcellular fractionation of cells,
followed by western blot analysis, to substantiate and
quantify the changes between the nuclear and non-
nuclear compartments in malignant cells. Exposure of
MDA-MB-468 cells to hypoxia for 24 h resulted in an
approximately 70% increase in nuclear levels of
BRCA1 relative to those in cells exposed to normoxia
(Fig. 1B). Non-nuclear levels decreased in hypoxic cells
to approximately 65% of the level in normoxic cells.
There was no change in the steady-state level of
Fig. 1. Hypoxia induces nuclear accumulation of BRCA1 in breast cancer cells but not in nonmalignant cells. MDA-MB-468 cells, MCF-7
cells, MCF-12A cells or HMECs were exposed to normoxia or hypoxia (1% O
2
) for 24 h prior to (A) immunostaining for BRCA1 (magnifica-
tion, 400·) or (B) nuclear ⁄ non-nuclear fractionation. One hundred micrograms of whole cell lysate (wcl) or nuclear protein or 200 lg of non-
nuclear protein was immunoblotted for BRCA1. Densitometric values were normalized to actin. *P < 0.05; mean ± SE; n ¼ 3. N, normoxia;
H, hypoxia. The value for each normoxic sample was arbitrarily set at 1. (C) One hundred micrograms of protein from each cell line was im-
munoblotted for HIF-1a. Membranes were stripped and probed for MSH-6, a DNA mismatch repair protein, as a loading control as previously
described [24].
Novel association between hypoxia, TRAIL and BRCA1 L. D. Fitzgerald et al.
5138 FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS
A
B
C
L. D. Fitzgerald et al. Novel association between hypoxia, TRAIL and BRCA1

FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS 5139
BRCA1. Similar results were obtained with MCF-7
cells. Hypoxia enhanced the nuclear levels of BRCA1
by 40% and decreased the level of non-nuclear
BRCA1 by 20% when compared to levels in cells
exposed to normoxia. There was no change in the
steady-state protein level in these cells as well. Interest-
ingly, neither MCF-12A nor human mammary epithe-
lial cell (HMEC) cell lines exhibited a change in the
expression level or nuclear ⁄ non-nuclear distribution of
BRCA1 when exposed to hypoxia. Western blot analy-
sis of HIF-1a (Fig. 1C) demonstrated that each cell
line responded to hypoxia with increased expression of
HIF-1a. As similar changes were observed in both
malignant cell lines and no change occurred in either
nonmalignant cell line, subsequent experiments were
performed with MDA-MB-468 and MCF-12A cells as
the malignant and nonmalignant models, respectively.
Hypoxia-induced BRCA1 localization changes are
TRAIL-dependent
The selective cytotoxicity of TRAIL to tumor cells in
the absence of an effect in many nontransformed
cells has been widely reported. Therefore, as hypoxia
induced changes in BRCA1 localization in malignant,
but not in nonmalignant, mammary epithelial cells, we
sought to determine whether TRAIL was involved in
the localization change. First, we assessed whether
there was a change in TRAIL expression in MDA-
MB-468 cells in response to hypoxia by analyzing its
cell surface expression. Immunofluorescent analysis of

TRAIL in nonpermeabilized cells demonstrated an
 40% increase in TRAIL fluorescence in cells exposed
to hypoxia relative to cells exposed to normoxia
(Fig. 2A). These data indicate that hypoxia upregulates
cell surface expression of TRAIL.
Second, we reasoned that if the hypoxia-induced
change in BRCA1 localization was TRAIL-dependent,
blocking the TRAIL signaling pathway should abro-
gate this change. To test this, we preincubated MDA-
MB-468 cells with a neutralizing antibody against
human TRAIL, 2E5, before exposure to hypoxia, and
evaluated the nuclear⁄ non-nuclear distribution of
BRCA1. Neutralization of TRAIL blocked the nuclear
accumulation of BRCA1 induced by hypoxia in
MDA-MB-468 cells (Fig. 2B), resulting in a 35%
decrease in nuclear BRCA1 relative to the level in
normoxic cells in the presence of 2E5. The decrease in
AB C
Fig. 2. Hypoxia-induced changes in BRCA1 localization are mediated through TRAIL. (A) MDA-MB-468 cells were exposed to normoxia or
hypoxia for 24 h prior to fixation and immunostaining for the extracellular C-terminus of TRAIL. Cells were not permeabilized. Fluorescence
intensities of cells in captured images were quantified using
ALPHAIMAGER 2000 software. The values graphed are the average integrated den-
sity values of the cells. *P < 0.05; mean ± SE; n ¼ 90 cells. Magnification, 400·. (B) One microgram of neutralizing antibody against TRAIL
was added to MDA-MB-468 cells for 1 h before initiation of hypoxia for 24 h. Cells were fractionated and analyzed by western blot. Densito-
metric values were normalized to actin. *P < 0.05; mean ± SE; n ¼ 3. Values for each normoxic sample in the presence of normal IgG or
anti-TRAIL IgG were arbitrarily set at 1. (C) 50 ngÆmL
)1
KillerTRAIL was administered to MDA-MB-468 cells for 1.5 h before harvesting, frac-
tionation, and western blot analysis. Densitometric values were normalized to actin. * P < 0.05; mean ± SE; n ¼ 3. wcl, whole cell lysate;
V, vehicle; T, TRAIL. The value for the normoxic sample was arbitrarily set at 1.

Novel association between hypoxia, TRAIL and BRCA1 L. D. Fitzgerald et al.
5140 FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS
non-nuclear expression of BRCA1 in response to
hypoxia was also attenuated by 2E5.
Finally, we hypothesized that if hypoxia was
activating a TRAIL-dependent mechanism leading to
enhanced nuclear BRCA1 levels, exogenously adminis-
tered TRAIL under normoxic conditions should mimic
the effects of hypoxia. To determine whether human
soluble recombinant TRAIL could regulate BRCA1
subcellular localization, we treated MDA-MB-468 or
MCF-12A cells with KillerTRAIL for 1.5 or 4 h,
respectively. TRAIL resulted in an  1.7-fold increase
in nuclear BRCA1 (Fig. 2C) with a significant decrease
in non-nuclear BRCA1 in MDA-MB-468 cells. Expo-
sure to TRAIL did not alter the steady-state level of
BRCA1 protein. Likewise, in MCF-12A cells, TRAIL
had no effect on the steady-state levels of BRCA1;
however, there also was no effect on the subcellular
distribution of the protein (data not shown). Collec-
tively, the data in Fig. 2 support the involvement of a
TRAIL-dependent process in mediating hypoxia-
induced changes in BRCA1 localization.
TRAIL, but not hypoxia, induces apoptosis in
breast cancer cells
As our data suggested that hypoxia was stimulating a
TRAIL signaling pathway and TRAIL activates apop-
totic processes, we assessed the ability of hypoxia to
induce apoptosis in our model systems. Approximately
4% of MDA-MB-468 cells were present within the

subdiploid population of cells as determined by flow
cytometry of normoxic cells. This did not change fol-
lowing exposure to hypoxia for 8 or 24 h (Table 1).
As hypoxia failed to induce apoptosis in MDA-MB-
468 cells, we assessed the ability of TRAIL to do so.
Exposure of MDA-MB-468 cells to increasing concen-
trations of TRAIL from 5 to 100 ngÆmL
)1
resulted in
a dose-dependent increase in apoptosis, with the maxi-
mally effective dose being 50–100 ngÆmL
)1
(Fig. 3A).
Treatment of cells with 50 ngÆmL
)1
TRAIL resulted in
a time-dependent increase in apoptosis that was detect-
able within 2 h of treatment and stabilized at 6 h
(Fig. 3B). To confirm that the subdiploid population
of cells detected via flow cytometry was an accurate
index of apoptosis, we measured caspase-3 activity
(Fig. 3C). Cells treated with TRAIL exhibited a six-
fold increase in caspase-3 activity relative to that in
cells treated with vehicle. As it has been previously
reported that MDA-MB-468 cells do not respond to
TRAIL [20], we assessed the selectivity of Killer-
TRAIL. MCF-12A cells were treated with 50 ngÆmL
)1
KillerTRAIL and assessed for apoptosis by flow
cytometry and caspase-3 activity. Neither assay

Table 1. Effect of hypoxia on the induction of apoptosis in MDA-
MB-468 cells.
Percentage of apoptotic cells
8 h 24 h
Normoxia 4.49 ± 1.41 3.48 ± 1.05
Hypoxia 4.52 ± 0.89 3.53 ± 0.36
Fig. 3. TRAIL induces apoptosis in breast cancer cells. MDA-MB-468 cells were treated with (A) increasing concentrations of KillerTRAIL for
4 h or (B) with 50 ngÆmL
)1
KillerTRAIL for varying lengths of time. Cells were analyzed by FACS. Mean ± SE; n ¼ 3. (C) Caspase-3 activity
confirmed apoptosis in MDA-MB-468 cells. **P < 0.01, mean ± SE; n ¼ 3. (D, E) MCF-12A cells were treated with 50 ngÆmL
)1
KillerTRAIL
for 4 h, harvested, and analyzed by FACS (D) or assayed for caspase-3 activity (E). Mean ± SE; n ¼ 3.
L. D. Fitzgerald et al. Novel association between hypoxia, TRAIL and BRCA1
FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS 5141
revealed an induction of apoptosis in TRAIL-treated
cells, indicating that the TRAIL effect on MDA-MB-
468 cells was selective for malignant cells.
TRAIL does not alter the localization of truncated
BRCA1 or induce apoptosis in a cell line
expressing mutated BRCA1
The data in Figs 2 and 3 suggest that TRAIL can alter
BRCA1 localization and induce apoptosis in breast
cancer cells. Therefore, we assessed the requirement
for functional BRCA1 in TRAIL-stimulated apoptosis.
HCC1937 cells have a 5382insC mutation in BRCA1,
resulting in the expression of a truncated, nonfunc-
tional protein [21]. HCC1937 cells were exposed to
hypoxia for 24 h (Fig. 4A) or treated with 50 ngÆmL

)1
TRAIL for 4 h (Fig. 4B), and BRCA1 localization was
assessed. There was no change in protein level or sub-
cellular distribution of BRCA1 under these conditions.
Cells were also exposed to TRAIL for up to 10 h, and
apoptosis was not increased above that in cells exposed
to vehicle during this time period (Fig. 4C).
BRCA1 enhances TRAIL-mediated apoptosis
Having demonstrated that HCC1937 cells were resis-
tant to TRAIL-mediated apoptosis, we investigated
whether introduction of wild-type BRCA1 could sensi-
tize the cells to TRAIL. Routinely, we were able to
obtain  20% transfection efficiency of BRCA1 into
HCC1937 cells. This resulted in an increase in BRCA1
expression as evidenced by a more intense band recog-
nized by BRCA1 antibody Ab-1 in cells transfected
with pEGFPC1BRCA1 when compared to the inten-
sity in cells transfected with empty vector (Fig. 5A).
The slightly slower migration of the predominant band
in BRCA1-transfected cells was consistent with the
expression of a tagged, full-length protein. Transfec-
tion of BRCA1 into these cells followed by treatment
with vehicle did not alter the percentage of apoptotic
cells relative to that in cells transfected with vector
alone and treated with vehicle (Fig. 5A). Interestingly,
in contrast to untransfected cells, the administration of
TRAIL to cells transfected with vector alone resulted
in a consistent, although not significant, increase in
apoptosis relative to cells treated with vehicle. This
effect was routinely observed, and we attribute it to

the transfection procedure. Nevertheless, the expres-
sion of BRCA1 in HCC1937 cells resulted in a 22-fold
enhancement in apoptosis relative to cells expressing
BRCA1 in the absence of TRAIL and a three-fold
enhancement of apoptosis over that induced by
TRAIL in the presence of the empty vector. Thus, the
data indicate that expression of full-length BRCA1
augments TRAIL-induced apoptosis.
We also investigated the effects of reduced BRCA1
expression on TRAIL-mediated apoptosis in MDA-
MB-468 cells. Figure 5B demonstrates the decrease in
BRCA1 in cells transfected with BRCA1-targeted
short hairpin RNA (shRNA) relative to the level in
cells transfected with control shRNA. The levels of
active caspase-3 between cells treated with vehicle and
those treated with TRAIL were assessed via immuno-
fluorescence of cells transfected with either negative
control shRNA or BRCA1-targeted shRNA. Transfec-
tion efficiency was  10%. Integrated density values of
ABC
Fig. 4. Neither hypoxia nor TRAIL alters BRCA1 localization or induces apoptosis in HCC1937 cells. Cells were (A) exposed to normoxia or
hypoxia for 24 h or (B) treated with 50 ngÆmL
)1
KillerTRAIL for 4 h prior to harvesting, fractionation, and western blot analysis. Densitometric
values were normalized to actin. Mean ± SE; n ¼ 3. wcl, whole cell lysate; N, normoxia; H, hypoxia; V, vehicle; T, TRAIL. Values for normox-
ic and vehicle samples were arbitrarily set at 1. (C) Cells were treated with vehicle or 50 ngÆmL
)1
KillerTRAIL for various lengths of time prior
to FACS analysis. Mean ± SE; n ¼ 2.
Novel association between hypoxia, TRAIL and BRCA1 L. D. Fitzgerald et al.

5142 FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS
active caspase-3 fluorescence in cells treated with vehi-
cle or TRAIL are shown in Fig. 5B. In cells transfect-
ed with control shRNA, administration of TRAIL
resulted in a three-fold increase in active caspase-3 flu-
orescence relative to fluorescence in vehicle-treated
cells. This increase was not evident in cells transfected
with BRCA1 shRNA. Thus, these data support the
hypothesis that BRCA1 enhances TRAIL’s ability to
mediate apoptosis in breast cancer cell lines.
Discussion
There is increasing interest in the association between
hypoxia and BRCA1. Recent studies showed that pro-
longed exposure (48 h) of cells to severe hypoxia
(0.01% oxygen) resulted in decreased levels of BRCA1
[18] and impaired ability of cells to repair damaged
DNA [17]. However, no data are available on the
effects of short-term exposure (£ 24 h) to hypoxia,
which does not alter protein levels, on the localization
or function of BRCA1 in mammary cells.
As solid tumors grow, their oxygen consumption
can exceed oxygen delivery, resulting in decreased
intratumoral oxygen. The reported pO
2
values within
breast tumors range from 0 to 100 mmHg, with the
highest frequency occurring between 0 and 16 mmHg.
In noncancerous tissue, the highest frequency of values
occurred between 60 and 80 mmHg [12]. We exposed
cells to 1% oxygen, which would exert a pressure of

7.6 mmHg at sea level. Thus, our studies are consistent
with oxygen levels in the range of values occurring
with high frequency in breast tumors. We monitored
the sustained oxygen concentration in all experiments
using an oxygen sensor within the Billups–Rothenburg
hypoxia chamber. Furthermore, if cells were responsive
to a low-oxygen environment, oxygen-sensitive mark-
ers would be expected to increase. We observed an
increase in HIF-1a protein, and thus were confident
that the cells were being maintained in a low-oxygen
environment and were responsive to low oxygen.
There is also increasing interest in the effects of
hypoxia on TRAIL-mediated apoptosis. Available data
suggest that hypoxia blocks TRAIL-mediated apopto-
sis by blocking the translocation of Bax to the
mitochondria [14] and by activation of lysosomal cath-
epsins [20]. In contrast, Lee et al. [22] reported that
hypoxia synergistically enhanced TRAIL-mediated
apoptosis in the DU-145 prostate cancer cell line.
Furthermore, Kwon & Choi [23] reported that
hydrogen peroxide increases the expression of TRAIL
AB
Fig. 5. BRCA1 enhances TRAIL-mediated
apoptosis. (A) Approximately equimolar
amounts of pEGFPC1 or pEGFPC1BRCA1
were transfected into HCC1937 cells.
Expression levels were assessed by immu-
noblotting lysates with BRCA1 antibody
Ab-1. Membrane was stripped and reprobed
for MSH-6 to ensure equal loading. Forty-

eight hours post-transfection, cells were
treated with KillerTRAIL for 4 h before being
harvested and analyzed by FACS. *P < 0.05
compared to pEGFPC1 vehicle, pEGFPC1
TRAIL or pEGFPC1BRCA1 vehicle;
mean ± SE; n ¼ 3. (B) Negative control or
BRCA1-targeted shRNA was transfected
into MDA-MB-468 cells. Twenty-four hours
later, cells were treated with vehicle or
50 ngÆmL
)1
TRAIL before fixation and immu-
nofluorescence of active caspase-3. The val-
ues graphed are densitometric values for
fluorescence intensity of active caspase-3 in
cells from at least 12 fields. BRCA1 immuno-
blot shows BRCA1 levels in cells transfected
with control or BRCA1-targeted shRNA.
Loading variability was assessed by probing
for MSH-6 as previously reported [24].
L. D. Fitzgerald et al. Novel association between hypoxia, TRAIL and BRCA1
FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS 5143
in astroglial cells, providing a precedent for oxidative
stress-mediated regulation of TRAIL expression.
The results presented here provide the first evidence
linking hypoxia, TRAIL signaling and BRCA1 in
mammary epithelial cells. Our data demonstrate that
hypoxia induces nuclear accumulation of BRCA1 with-
out altering its protein level, and that this effect is
mediated, at least in part, by TRAIL. Our results also

indicate that hypoxia can upregulate TRAIL cell sur-
face expression and that the apoptotic effects of
TRAIL are enhanced by BRCA1. Thus, our studies
are consistent with others demonstrating a role for
BRCA1 in stress-induced apoptotic signaling.
Our results further indicate that whereas both
hypoxia and TRAIL effect a change in BRCA1 locali-
zation, only TRAIL induces apoptosis. The inability of
hypoxia to cause apoptosis was not surprising, consid-
ering the number of reports demonstrating that
hypoxia blocks TRAIL-induced apoptosis. There are
two possible explanations for our results. First,
hypoxia could stimulate TRAIL signaling, leading to
the activation of two distinct pathways. One pathway
might lead to increased nuclear localization of BRCA1
and the other to apoptosis. Hypoxia could block the
apoptotic arm (Fig. 6A). Alternatively, hypoxia could
stimulate TRAIL signaling, resulting in nuclear accu-
mulation of BRCA1 and potentially leading to the
induction of TRAIL-mediated apoptosis. However,
hypoxia might also block this pathway downstream of
the change in BRCA1 localization (Fig. 6B).
Our data are also consistent with studies demon-
strating TRAIL’s specificity for malignant cells [16].
However, it has been reported that MDA-MB-468 cells
are resistant to TRAIL [20]. Our data contradict those
findings, and there are several possible reasons for the
discrepancy. Previous studies used 50 nm TRAIL,
whereas our results were obtained with 50 ngÆmL
)1

TRAIL, which is approximately five times higher. A
dose–response curve demonstrated this concentration
to be a near maximally effective dose. We also used
commercially available KillerTRAIL, which does not
require a crosslinker or enhancer for its biological
activity. Finally, our experiments were performed in
the presence of 1% serum, whereas other studies were
performed in 10% serum. With the low level of in vivo
toxicity reported for TRAIL and its selective cytotoxic-
ity, it has been targeted as a desirable therapeutic tool,
and its development as a therapeutic modality is
underway. Our data suggest that BRCA1 expression
may be critical to the success of TRAIL as a chemo-
therapeutic agent. BRCA1 is mutated in approximately
half of hereditary breast cancer cases, and its expres-
sion is reduced in sporadic cancers. Therefore, the level
of functional protein is decreased, and this may hinder
the response of cells to TRAIL.
In conclusion, this study provides novel evidence
that BRCA1 is critical in the transduction of a
TRAIL-mediated apoptotic signal. It also suggests that
hypoxia may not only block apoptosis resulting from
exogenously administered TRAIL, but also blocks
apoptosis induced by endogenously expressed TRAIL.
Investigation of the mechanism for this inhibition is
ongoing. However, regardless of the mechanism, this
article establishes a connection between hypoxia,
TRAIL and BRCA1 in regulating apoptosis in breast
cancer cells.
Experimental procedures

Cell culture
MCF-7, MDA-MB-468, HCC1937 and MCF-12A cells
were from the American Type Culture Collection (Manas-
sas, VA, USA). Unless otherwise specified, cell culture
reagents were from Invitrogen (Carlsbad, CA, USA) or
Sigma (St Louis, MO, USA). MCF-7 cells were cultured as
previously described [24]. MDA-MB-468 cells were cultured
in DMEM and 10% Fetal Clone III (fetal bovine serum)
(Hyclone, Logan, UT, USA). HCC1937 cells were cultured
in RPMI 1640 (Hyclone), 1% insulin ⁄ transferrin ⁄ selenium
A, and 10% fetal bovine serum. MCF-12A cells were
cultured in DMEM ⁄ F-12, 20 ngÆmL
)1
human epidermal
growth factor, 100 ngÆmL
)1
cholera toxin, 0.01 mgÆmL
)1
bovine insulin, 500 ngÆmL
)1
hydrocortisone, and 5% horse
serum. HMECs (provided by S. Eltom, Meharry Medical
College) were cultured in DMEM ⁄ F-12, 10 mm Hepes,
1 lgÆmL
)1
bovine insulin, 1 lgÆmL
)1
hydrocortisone,
10 lgÆmL
)1

ascorbic acid, 12.5 ngÆmL
)1
EGF, 10 lgÆmL
)1
transferrin, 0.1 mm phosphoethanolamine, 2 nm b-estradiol,
10 nm triiodo-l-thyronine, 15 nm sodium selenite, 0.1 mm
ethanolamine, 1 ngÆ mL
)1
cholera toxin, 1% fetal bovine
serum, and 35 lgÆmL
)1
bovine pituitary extract.
Fig. 6. Schematic representation of two possible mechanisms by
which hypoxia could preclude TRAIL-mediated apoptosis.
Novel association between hypoxia, TRAIL and BRCA1 L. D. Fitzgerald et al.
5144 FEBS Journal 274 (2007) 5137–5146 ª 2007 The Authors Journal compilation ª 2007 FEBS
For hypoxia and TRAIL studies, 3 · 10
6
cells were pla-
ted in 150 mm dishes for 24 h. Cells were incubated in
media containing 1% serum for 1 h before continuation of
normoxia or initiation of hypoxia (1% oxygen, 5% carbon
dioxide, 94% nitrogen in a Billups–Rothenberg chamber;
Billups Rothenberg, Inc., Del Mar, CA, USA) for 24 h or
treatment with KillerTRAIL (Alexis Biochemicals, San
Diego, CA, USA) or vehicle (20 mm Hepes, pH 7.4,
300 mm NaCl, 0.006% Tween-20, 1% sucrose, 0.05 mm
dithiothreitol). For TRAIL neutralization experiments,
monoclonal antibody to human TRAIL (2E5; Alexis Bio-
chemicals) was added 1 h before hypoxia initiation.

Hypoxia was initiated by flushing a Billups–Rothenburg
chamber, which housed the cells, with a gas mixture of 1%
oxygen, 5% carbon dioxide and 94% nitrogen until an oxy-
gen sensor (Micropac O
2
; Drager, Leubeck, Germany),
which was also sealed within the chamber, stabilized at 1.0–
1.2% oxygen. The oxygen meter remained in the chamber
throughout the experiment, and experiments were completed
only if the oxygen level remained between 1.0% and 1.2%.
Subcellular fractionation and western blotting
Cell fractionation and BRCA1 immunoblotting were per-
formed as previously described [24]. HIF-1a and MSH-6
antibodies (BD Biosciences, San Jose, CA, USA) were
diluted 1 : 500.
Caspase activity assays
Cells were plated at 2–3 · 10
6
cells per 100 mm dish for
24 h before treatment. Two hundred micrograms of protein
was assayed using a Caspase-3 Colorimetric Assay Kit
(Sigma).
Fluorescence-activated cell sorting (FACS)
After treatment, cells and media were collected and centri-
fuged at 500 g at 4 °C with an Eppendorf 5810 centrifuge
and A-4-62 rotor. Cells were permeabilized in 0.1% sodium
citrate, 0.1% Triton X-100, and 50 lgÆmL
)1
propidium
iodide, and analyzed by FACS using a Becton-Dickinson

(Franklin Lakes, NJ, USA) FACScan Benchtop Analyzer
and winlist software.
Transfections
Modified pEGFPC1 vector and pEGFPC1BRCA1 have
been described previously [2]. HCC1937 cells were plated at
1 · 10
6
cells per 100 mm dish and transfected with approxi-
mately equimolar amounts of pEGFPC1 (5 lg) or pEG-
FPC1BRCA1 (10 lg) using GeneJammer (Stratagene, La
Jolla, CA, USA). Forty-eight hours later, cells were treated
and harvested for flow cytometry.
Immunofluorescence
Immunofluorescence and quantification were performed as
previously described [24]. Immunofluorescence of cells in
captured images was quantified using AlphaImager2000
(Alpha Innotech, San Leandro, CA, USA) or the image j
processing and analysis program (NIH, Bethesda, MD,
USA). For detection of cell surface TRAIL, Triton X-100
was omitted. TRAIL antibody (ProSci Incorporated,
Poway, CA, USA) was diluted 1 : 100.
shRNA studies
Cells were plated at 120 000 cells per well in a six-well cul-
ture dish and transfected with 1 lg of pEGFPC1 green
fluorescent protein expression plasmid (Clontech, Mountain
View, CA, USA) and 3 lg of negative control shRNA or
BRCA1 shRNA (SuperArray Bioscience, Frederick, MD,
USA) using GeneJammer. Twenty-four hours post-transfec-
tion, cells were treated. Three hours after treatment, cells
were fixed in 2% paraformaldehyde and immunostained for

active caspase-3 (Promega, Madison, WI, USA; 1 : 500)
using Cy-3-conjugated donkey anti-(rabbit IgG) (Jackson
Immunoresearch, West Grove, PA, USA). Transfected cells
were assessed by the presence of green fluorescence,
and Cy-3 immunofluorescence was quantified by imagej
software.
Statistical analysis
Student’s t-test and anova were performed using graphpad
prism version 4 for Windows (GraphPad Software, San
Diego, CA, USA).
Acknowledgements
We thank Drs Hal Moses, Lynn Matrisian and Lee
Limbird for helpful comments. This work was sup-
ported by Public Health Service Grants 2G12RR03032
from NCRR-supported Research Centers in Minority
Institutions, KO1 CA89494 and U54 CA91408 from
the National Cancer Institute, Research Initiative for
Scientific Enhancement Grant 2R25 G59994, and
T32 HL007735-11 from the National Heart, Lung and
Blood Institute.
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