Molecular Biology of the Cell
Vol. 21, 2987–2995, September 1, 2010
Selective Association of Peroxiredoxin 1 with Genomic
DNA and COX-2 Upstream Promoter Elements in Estrogen
Receptor Negative Breast Cancer Cells
Xuemei Wang, Shihua He, Jian-Min Sun, Geneviève P. Delcuve,
and James R. Davie
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba R3E 0V9, Canada
Submitted February 24, 2010; Accepted June 28, 2010
Monitoring Editor: Kunxin Luo
In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and
breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen
receptor negative (ER?) MDA-MB-231 but not in estrogen receptor positive (ER?) MCF7 breast cancer cells. Indirect
immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and
breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that
PRDX1 association with the transcription factor nuclear factor-?B (NF-?B) in MDA-MB-231 but not in MCF7 cells
contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the
cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-?B in ER? but not in ER? breast cancer cells.
PRDX1 knockdown attenuated COX-2 expression by reducing NF-?B occupancy at its upstream promoter element in
MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER? breast cancer cells.
Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we
propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-?B in ER? breast cancer cells.
Breast cancer is the most commonly diagnosed cancer in
women in North America and Europe, second only to lung
cancer in mortality rate. It has been hypothesized that breast
tumorigenesis is a result of cumulative changes that lead to
the transformation of normal epithelium to abnormal cellu-
lar modifications resulting in hyperplasia, atypical hyper-
plasia, ductal carcinoma in situ, and invasive carcinoma.
Finally, all these changes culminate into metastasis (Allred et
al., 1993). Although breast cancer is characterized by heter-
ogeneity and various prognostic outcomes, it has been
shown that the wide diversity of genomic and transcrip-
tional abnormalities present in primary breast tumors is well
represented in human breast cancer cell lines (Neve et al.,
2006; Zhu et al., 2006; Vargo-Gogola and Rosen, 2007).
Transcription factors, cofactors, and architectural proteins
contribute to gene expression programming in normal and
disease states. Changes in the spectrum of these proteins
directly or indirectly bound to DNA impact gene program-
ming of normal and cancer cell types. A research tool to
identify the proteins associated with genomic DNA is in situ
cross-linking by cisplatin [cis-platinum(II)diamminedichlo-
ride; cis-DDP] (Ferraro et al., 1992; Spencer and Davie,
2002a). Cisplatin directly cross-links proteins to DNA, but
not to other proteins, and many DNA cross-linked proteins
are nuclear matrix proteins. We have shown that cisplatin
cross-links nuclear matrix-associated transcription factors
and cofactors (estrogen receptor, Hsp27 ERE-TATA binding
protein/scaffold attachment factor B, heterogeneous nuc-
lear ribonucleoprotein K [hnRNP K], histone deacetylases
[HDACs 1 and 2 but not 3]) to DNA in the MCF7 human
breast cancer cell line (Samuel et al., 1998; Davie et al., 1999;
Sun et al., 2002, 2007). These transcription factors and cofac-
tors are crucial to the organization and structure of chroma-
tin and to the regulation of genes involved in the prolifera-
tion of breast cancer cells. Histones are poorly cross-linked
to DNA by cisplatin. Nuclear proteins such as SRm160 and
carboxypeptidase are not cross-linked to DNA with cisplatin
(Samuel et al., 1998; Chichiarelli et al., 2002). Analyses of
two-dimensional gel patterns of proteins cross-linked to
DNA in situ with cisplatin led to the identification of pro-
teins that were differentially bound to DNA in hormone-
dependent and -independent human breast cell lines (Spen-
cer et al., 2000) or in hormone-dependent cell lines
representing different stages of malignant progression in
breast cancer (Spencer et al., 2001). Thus, cisplatin is a useful
cross-linking agent to identify transcription factors, cofac-
tors, and other DNA-associated proteins involved in DNA
organization and transcription of cancer cells.
In this study, we analyzed the proteins cross-linked to
nuclear DNA by cisplatin in situ in a panel of human breast
normal and cancer cell lines, including the nontumorigenic
MCF10A1 cell line; the estrogen receptor positive (ER?),
estrogen-dependent MCF7 and T-47D cancer cell lines;
and the estrogen receptor negative (ER?), estrogen-inde-
This article was published online ahead of print in MBoC in Press
on July 14, 2010.
Address correspondence to: James R. Davie (email@example.com).
© 2010 X. Wang et al. This article is distributed by The American Society
for Cell Biology under license from the author(s). Two months after
cial–Share Alike 3.0 Unported Creative Commons License (http://cre-
pendent MDA-MB-231, MDA-MB-468, and BT-20 cancer
The research objective was to identify proteins differen-
tially cross-linked to the DNA of these cell lines, to pinpoint
aberrant molecular behaviors involved in the differential
gene programming of estrogen-responsive or estrogen-non-
responsive breast tumors. We identified peroxiredoxin 1
(PRDX1) as a protein differentially cross-linked in ER? ver-
sus ER? cell lines. In an attempt to identify the molecular
mechanism that drives the selective recruitment of PRDX1 to
the genomic DNA of ER? cell lines, we found that PRDX1
associates with nuclear factor-?B (NF-?B), and both proteins
are bound together to the cyclooxygenase (COX)-2 upstream
promoter region in ER? but not ER? breast cancer cells.
Knocking down PRDX1 resulted in the attenuation of COX-2
expression in ER? but not ER? breast cancer cells. In the
PRDX1 knockdown ER? cells, NF-?B occupancy of the
COX-2 upstream promoter element was reduced. We further
present evidence suggesting that this interaction with NF-?B
is independent of PRDX1 peroxidase activity.
MATERIALS AND METHODS
The human breast cancer cell lines, ER? (MCF7 and T-47D) and ER? (MDA-
MB-231, MDA-MB-468, and BT-20) were grown as described previously
(Samuel et al., 1997). Immortalized breast epithelial MCF10A1 cells were
grown as described in Coutts et al. (1999). Normal human mammary epithelial
cells (HMECs) were purchased from Lonza Walkersville (Walkersville, MD)
and grown according to the manufacturer’s instructions. For some studies,
MCF7 and MDA-MB-231 cells were treated with 1 mM H2O2for 30 min.
Isolation and Analysis of Proteins Cross-Linked to DNA
In situ cross-linking of proteins to DNA by cisplatin or formaldehyde, their
subsequent isolation and resolution by two-dimensional (2D) electrophoresis
were described previously (Spencer et al., 2000; Sun et al., 2002; Spencer and
Davie, 2002a,b). Proteins samples on 2D gels were either silver stained,
imaged using Molecular Imager Fx (Bio-Rad Laboratories, Hercules, CA), and
analyzed with the PDQuest 2-D analysis software, version 7.3.1 (Bio-Rad
Laboratories) (50 ?g) or stained with CBB G250 (Sigma-Aldrich, St. Louis,
MO) and in-gel digested for identification (500 ?g), or transferred onto
nitrocellulose membranes and immunochemically stained with anti-PRDX1
antibodies (20 ?g) (Abcam, Cambridge, MA). In-gel digestion, nano-liquid
chromatography, and tandem mass spectrometry were performed essentially
as described previously (Meng and Wilkins, 2005), except that the gel pieces
dehydrated in acetonitrile were reduced in 10 mM dithiothreitol and subse-
quently alkylated with 55 mM iodoacetamide. To identify peptides, the
MSDB, version 20060831 database was searched using the Global Proteome
Machine (http://www.thegpm.org) search engine.
Cellular Fractionation, Immunoblotting, and
Total cell lysates and nuclear and cytosolic extracts were obtained as
described previously (Sun et al., 2001). Immunoblot analysis and immunopre-
cipitation were performed as described previously (Sun et al., 2002). Poly-
clonal antibodies against human PRDX1 (Abcam), glyceraldehyde-3-phos-
phate dehydrogenase (GAPDH; Abcam), HDAC1 (Affinity BioReagents,
Golden, CO), PRDX1-SO3(Abfrontier, Seoul, Korea), and NF-?B p65 (Milli-
pore, Billerica, MA), and monoclonal antibodies against human ?-actin (Sig-
ma-Aldrich) or TATA binding protein (TBP; Abcam) were used.
Indirect immunofluorescence was performed as described previously (He et
al., 2005). Antibodies against PRDX1 (Abcam) or SC35 (Abcam) and promy-
elocytic leukemia (PML) protein (Santa Cruz Biotechnology, Santa Cruz, CA)
were used. The 100 z-stack images of each color channel were taken at 200-nm
increments with corresponding filters (Chroma Technology, Bellows Falls,
VT) and an Axio Imager Z1 microscope and AxioCam HR charge-coupled
device camera (Carl Zeiss, Oberkochen, Germany). Constrained iterative de-
convolution of all color channels was performed for each color channel with
AxioVision 4.4 software (Carl Zeiss).
Chromatin Immunoprecipitation (ChIP)
ChIP and re-ChIP assays were done on MCF7 and MDA-MB-231 cells as
described previously (Li and Davie, 2008). The ChIP and input DNA concen-
trations were determined with the Quant-iT Picogreen dsDNA kit (Invitro-
gen, Carlsbad, CA). Equal amounts (2 ng) of ChIP and input DNAs were
quantitated by real-time polymerase chain reaction (PCR), by using the two
following pairs of primers: pair 1, forward, 5?-GTCAGCCTTTCTTAACCT-
TAC-3? and reverse, 5?-CAGTCTTTGCCCGAGCGCTTC-3? to amplify a 238-
base pair fragment including the -439 NF-?B binding site; and pair 2, forward,
5?-GCCCTCCCCCGGTATCCCATC-3? and reverse, 5?-AAAAAATTGCGTA-
AGCCCGGT-3? to amplify a 262-base pair fragment including the -214 NF-?B
binding site, in the promoter region of the COX-2 gene. The enrichment
values (ChIP DNA vs. input DNA) were calculated as follows: fold enrich-
ment ? R(Ct input–Ct ChIP), where R is the rate of amplification.
Protein Phosphatase Digestion
Total cell lysates or DNA cross-linked protein fractions were incubated with
or without calf intestinal alkaline phosphatase (CIP; GE Healthcare, Little
Chalfont, Buckinghamshire, United Kingdom) at 37°C for 1 h, resolved on
two-dimensional gels, and immunoblotted with anti-PRDX1 antibodies.
Generation and Maintenance of PRDX1 Stable
Knockdown MDA-MB-231 and MCF7 Cells
Empty GIPZ lentiviral vector, GIPZ scramble vector, and the GIPZ Lentiviral
microRNA-adapted short hairpin RNA (shRNA) clones for human PRDX1
(clone V2LHS_152610 (G1) and clone V2LHS_152606 (G2) (Thermo Scientific
Open Biosystems, Huntsville, AL) were obtained from the Biomedical Func-
tionality Resource at University of Manitoba. PRDX1 stable knockdown
MDA-MB-231 and MCF7 cell lines were obtained as described previously
(Drobic et al., 2010).
RNA Isolation and Real-Time Reverse Transcription
Total RNA was isolated using RNeasy Mini kit (QIAGEN, Valencia, CA)
following the manufacturer’s instructions. The isolated RNA was used to
synthesize the first-strand cDNA with the Moloney murine leukemia virus
reverse transcriptase kit and oligo(dT)12-18primer (Invitrogen). Real-time
PCR analysis was performed on iCycler IQ5 (Bio-Rad Laboratories) by using
SYBR Green for labeling. Primer sequences are as follows: 5?-AAGAAACT-
CAACTGCCAAGTG-3? (forward) and 5?-CAGCCTTTAAGACCCCATAAT-
-3? (reverse) for PRDX1; 5?-CTGATTCAAATGAGATTGTGG-3? (forward)
and 5?-CCCTCGCTTATGATCTGTCT-3? (reverse) for COX-2; and 5?-CAAGGCT-
GTGGGCAAGGTCATCC-3? (forward) and 5?-GAGGAGTGGGTGTCGCTGTT-
GAAGT-3? (reverse) for human GAPDH. The relative levels of PRDX1 and COX-2
gene expression were normalized to GAPDH levels.
PRDX1 Is Cross-Linked to DNA by Cisplatin In Situ in
ER? but Not ER? Human Breast Cancer Cell Lines
To identify proteins differentially bound to genomic DNA in
ER?, ER?, and pseudonormal breast cancer cell lines, we
compared the two-dimensional-gel patterns of proteins
cross-linked with cisplatin to genomic DNA. Proteins cross-
linked to DNA in cells with cisplatin were captured on
hydroxyapatite. Protein–DNA cross-links were reversed
with thiourea, and the proteins were isolated and resolved
by two-dimensional polyacrylamide gel electrophoresis
(PAGE). Figure 1 shows a protein, BC13, that was present
among the proteins associated with the nuclear DNA of
MDA-MB-231, an ER?, hormone-independent breast cancer
cell line with high metastatic potential, but absent from the
proteins associated with the nuclear DNA of MCF7, an ER?,
hormone-dependent breast cancer cell line and absent from
the proteins associated with the nuclear DNA of the nontu-
morigenic MCF10A1 cell line. Using mass spectrometry (in-
gel digestion, nano-liquid chromatography, and tandem
mass spectrometry), the BC13 protein, with a molecular
mass of 22 kDa and a pI of 8.3, was identified as PRDX1. To
verify the identification of BC13 as PRDX1, an immunoblot
analysis using anti-PRDX1 antibodies was performed on
samples of proteins isolated by cross-linking to DNA with
cisplatin in a panel of human breast cells (Figure 2A).
HDAC1, which we have shown previously to be cross-
linked to DNA by cisplatin or formaldehyde (Sun et al.,
2007), also was detected by immunoblot, whereas the cyto-
X. Wang et al.
Molecular Biology of the Cell2988
plasmic protein GAPDH was not (Figure 2B). Figure 2A
shows that PRDX1 was bound to DNA in ER? breast cancer
MDA-MB-231 cells but not in normal HMEC cells obtained
from reductive mammoplasty, pseudonormal MCF10A1
cells, or ER? breast cancer MCF7 cells. PRDX1 also was
differentially cross-linked in situ with formaldehyde to
the DNA of MCF10A1, MCF7, and MDA-MB-231, with
PRDX1 bound to the DNA of MDA-MB-231 but not MCF7
or MCF10A1 cells (Figure 2C). The lack of cisplatin cross-
linking of PRDX1 to DNA also was observed with MCF7
cells cultured under estrogen-deplete conditions (data not
To establish the generality of our observations, we de-
termined whether PRDX1 was differentially cross-linked
with cisplatin in ER? and ER? cells. Figure 2D shows
that PRDX1 was cross-linked in situ by cisplatin to the
DNA of MDA-MB-468 and BT20 cells, two other ER? cell
lines, whereas PRDX1 was not cross-linked to the DNA of
the ER? T47D cell line.
PRDX1 Is Found in the Cytoplasm and Nucleus
It has been reported that PRDX1 was overexpressed in hu-
man breast cancer in comparison with normal tissues (Noh
et al., 2001). To compare the PRDX1 levels in our panel of cell
lines, we did an immunoblot analysis on total cell extracts,
using anti-PRDX1 antibodies. Figure 3A shows that the ER?
MCF7 and ER? MDA-MB-231 cancer cell lines had higher
PRDX1 levels than the MCF10A1 pseudonormal cell line,
which itself displayed higher PRDX1 levels than the HMEC
normal cell line. Next, an immunoblot analysis with anti-
PRDX1 antibodies was performed on nuclear extracts, using
the TBP as a reference protein. Nuclear PRDX1 levels were
cross-linked proteins (50 ?g) from cells treated with 1 mM cisplatin were electrophoretically resolved on two-dimensional gels. The gels were
stained with silver. LA, HNRNPK, CK8, CK18, and CK19 point to the positions of lamin A; heterogeneous nuclear ribonucleoprotein K; and
cytokeratins 8, 18, and 19, respectively. Data are representative of seven independent experiments.
Proteins cross-linked to DNA by cisplatin in situ in MCF10A1, MCF7, and MDA-MB-231 human breast cell lines. DNA
MB-231 and other ER? human breast cell lines. DNA cross-linked
proteins (20 ?g) from cells treated with 1 mM cisplatin (A and D) or
1% formaldehyde (C) were resolved by SDS-10% PAGE and
immunoblotted with anti-PRDX1 or anti-HDAC1 antibodies. (B)
DNA cross-linked proteins (20 ?g) from MDA-MB-231 cells
treated with 1 mM cisplatin in the left lane, and proteins (20 ?g)
from MDA-MB-231 cell lysates. The blots were immunostained
with anti-PRDX1, anti-GAPDH, or anti-HDAC1 antibodies. Data
are representative of two and three independent experiments for
formaldehyde and cisplatin cross-linking, respectively.
PRDX1 cross-linked to DNA by cisplatin in situ in MDA-
normal and cancer cell lines. Proteins (20 ?g) from total cell lysates
(A), nuclear extracts (B), and total cell lysates and cellular fractions
(C) were resolved by SDS-10 or 15% PAGE (with equal volumes of
cytosol and nuclear samples loaded) and immunoblotted with anti-
PRDX1, anti-?-actin, anti-TBP, anti-GAPDH, or anti-HDAC1 anti-
bodies. Data are representative of three independent experiments.
PRDX1 found in cytosol and nucleus of human breast
PRDX1 Association with COX-2 Promoter
Vol. 21, September 1, 20102989
greater in the ER? MCF7 and ER? MDA-MB-231 cancer cell
lines than in the MCF10A1 pseudonormal cell line (Figure 3B).
Previous studies reported that PRDX1 was primarily lo-
cated in the cytosol (Wood et al., 2003). To assess the parti-
tioning of PRDX1 between cytosol and nucleus, total cellular
extracts from MCF7 and MDA-MB-231 were prepared and
then fractionated into cytosol and nuclear extracts. All frac-
tions were subjected to immunoblot analysis with anti-
PRDX1 and anti-HDAC1 antibodies. HDAC1 was primarily
found in the nuclear fractions as expected, whereas PRDX1
was present in both the cytosol and nucleus of MCF7 and
MDA-MB-231 cells, with the cytosol levels being slightly
lower than the nuclear levels (Figure 3C). However, the
cytoplasmic GAPDH also was present in the nuclear frac-
tion, demonstrating that nuclear fraction still had cytoplas-
To further explore the cellular distribution of PRDX1,
indirect immunofluorescence imaging was performed on
cells grown and fixed on coverslips. In Figure 4A, PRDX1
detected with the anti-PRDX1 (ab41906) antibody appeared
mostly as nuclear foci in MCF10A1, MCF7, and MDA-MB-
231 cells, with the intensity of the foci being the greatest
in MDA-MB-231 cells. On longer exposure, cytoplasmic
PRDX1 also was observed. The specificity of the ab41906
antibody was demonstrated by the absence of immunoflu-
orescent signal when the labeling was done in the presence
of a peptide block (data not shown). When PRDX1 was
visualized with another anti-PRDX1 antibody (ab15571),
PRDX1 staining in the cytosol and nucleus was visible in
MCF10A1, MCF7, and MDA-MB-231 cells, with the stained
bodies seeming bigger and more diffuse in MCF7 cells (Fig-
ure 4A). Figure 4A also shows that, regardless of the anti-
PRDX1 (ab41906 or ab15571) antibodies, PRDX1 nuclear foci
were more intense in MDA-MB-231 than in MCF7 cells.
Anti-PRDX1 (ab41906 or ab15571) antibodies, PRDX1 nu-
clear foci were more intense in MDA-MB-231 than in MCF7
cells. Figure 4B shows a large number of MCF7 and MDA-
MB-231 cells in which PRDX1 was detected with the anti-
PRDX1 (ab41906) antibody. Again, nuclear foci in MDA-MB-
231 cells were more intense than in MCF7 cells. Moreover,
every MDA-MB-231 cell displayed PRDX1 nuclear foci,
whereas only a subset of MCF7 cells had them (Figure 4B).
Indirect immunofluorescence imaging also was performed,
using the anti-PRDX1 (ab41906) antibody on MDA-MB-468
and BT-20 cells (Figure 4C). For each of these ER? breast
cancer cell lines, PRDX1 intense nuclear foci and cytosolic
staining were apparent.
As shown in Figure 4, we observed variability in immu-
nofluorescence imaging, particularly with MCF7 cells and
less so for MDA-MB-231 cells. With different lots of the
anti-PRDX1 antibody (ab15571, ab41906, and 07-609 [Milli-
pore]; data not shown), we observed nucleolar staining in
MCF7 cells (Figure 4A, ab15571), similar to that reported
previously (Immenschuh et al., 2003). However, regardless
of the anti-PRDX1 antibody used, we always observed
bright staining foci in the nucleus of MDA-MB-231 cells and
less intense staining nuclear foci in MCF10A1 cells.
immunofluorescence labeling with anti-PRDX1 ab41906 or ab15571 antibodies, costained with 4,6-diamidino-2-phenylindole (DAPI), and
digitally imaged. (A) PRDX1 distribution was visualized in MCF10A1, MCF7, and MDA-MB-231 cells, with indicated antibodies. (B) PRDX1
distribution in MCF7 and MDA-MB-231 cells was detected with anti-PRDX1 ab41906 antibody, and is shown at a lower amplification. (C)
PRDX1 distribution in MDA-MB-468 and BT-20 cells was detected with anti-PRDX1 ab41906 antibody. Bar, 10 ?m. Data are representative
of three independent experiments.
PRDX1 nuclear foci are more intense in MDA-MB-231 than in MCF7 cells. Human breast cells were subjected to indirect
X. Wang et al.
Molecular Biology of the Cell2990
To identify the nuclear body that PRDX1 was associated
with, we first determined whether PRDX1 was localized
with nuclear speckles, also called interchromatin granule
clusters (IGCs). IGCs are dynamic structures enriched in
pre-mRNA splicing factors and number 25–50 per cell (Spec-
tor, 2006). MDA-MB-231 cells grown and fixed on coverslips
were double-labeled with anti-PRDX1 and anti-SC35 anti-
bodies and visualized by fluorescence microscopy and im-
age deconvolution (Figure 5). There was no colocalization of
the PRDX1 and SC35 signals. However, the PRDX1-asso-
ciated bodies were localized on the boundaries of the
IGCs, which is a common feature of the PML bodies
(Ishov et al., 1997; Batty et al., 2009). To determine whether
nuclear PRDX1 was associated with PML bodies, we com-
pared their spatial distributions in MDA-MB-231 cells by
using indirect immunofluorescence microscopic analyses.
Figure 5 shows that all PRDX1-associated nuclear bodies
colocalized with PML bodies.
PRDX1 and NF-?B Are Bound Together to the COX-2
Upstream Promoter Region in MDA-MB-231 Cells
Because PRDX1 is not a DNA-binding protein, we consid-
ered a likely mechanism of PRDX1 recruitment to genomic
DNA was by association with a transcription factor. It has
been demonstrated that PRDX1 increased the transactiva-
tion potential of NF-?B in HeLa cells (Hansen et al., 2007).
Thus, we investigated whether PRDX1 was associated with
NF-?B and whether the interaction between these two pro-
teins was observed in ER? but not in ER? breast cancer
cells. MCF7 and MDA-MB-231 cell lysates were incubated
with antibodies raised against PRDX1 or the p65 subunit of
NF-?B, and the total cell lysates and immunoprecipitated
fractions were analyzed by immunoblot. With MCF7 cell
extracts, p65 was not coimmunoprecipitated with PRDX1
and vice versa, PRDX1 did not coimmunoprecipitate with
p65 (Figure 6A). However, in MDA-MB-231 cell extracts,
PRDX1 and p65 were coimmunoprecipitated with either
anti-PRDX1 or anti-p65 antibodies (Figure 6B). Thus, PRDX1
and NF-?B are associated with each other in ER? MDA-MB-
231 but not in ER? MCF7.
Next, we investigated whether PRDX1 was associated
with NF-?B in an upstream promoter region containing a
NF-?B binding site. We selected the COX-2 gene for study
because NF-?B has a pivotal role in the expression regula-
tion of COX-2, a gene that has a role in breast cancer (Singh-
Ranger et al., 2008). First, we determined the expression
levels of COX-2 in MCF7 and MDA-MB-231cells. Figure 7A
shows that, relative to GAPDH levels, COX-2 levels were
greater in MDA-MB-231 than in MCF7 cells. To determine
whether PRDX1 was associated with the COX-2 upstream
promoter element, ChIP assays were performed in which
MCF-7 and MDA-MB-231 cells were treated with formalde-
hyde, chromatin isolated, fragmented, and immunoprecipi-
tated by either anti-p65 or anti-PRDX1 antibodies. Equal
amounts of input DNA and immunoprecipitated DNA were
quantified by real-time PCR. Each of the amplified DNA
regions, named -439 and -214, includes a NF-?B binding site
and are depicted in Figure 7B. Figure 7C shows a similar
enrichment of the -439 and -214 sequences in the DNA
immunoprecipitated by anti-p65 antibodies from MCF7 and
MDA-MB-231 cell lines, demonstrating that NF-?B was
bound to its two cognate sites in the COX-2 promoter region
in both cell lines. In contrast, only the chromatin immuno-
precipitated from MDA-MB-231 cells by anti-PRDX1 anti-
bodies was enriched in the -439 and -214 sequences, with the
chromatin immunoprecipitated from MCF7 cells showing
bodies in MDA-MB-231 cell line. Top, MDA-
MB-231 cells, grown on coverslips, were fixed
and double labeled with antibodies raised
against SC35 and PRDX1. Bottom, MDA-MB-
231 cells, grown on coverslips, were fixed and
double labeled with antibodies raised against
PML and PRDX1. DNA was stained with 4,6-
diamidino-2-phenylindole (DAPI). SC35, PML,
and PRDX1 distributions were visualized by
fluorescence microscopy and image deconvolu-
tion as described in Materials and Methods. Sin-
gle optical sections are shown. Yellow in the
merged images signifies colocalization. Bar, 5
?m. Data are representative of three indepen-
PRDX1 is colocalized with PML
MCF7 cells. Aliquots of 500 ?g of MCF7 (A) or MDA-MB-231 (B) cell
lysates were incubated with anti-PRDX1, anti-NF-?B p65, or rabbit
preimmune immunoglobulin G (IgG) antibodies. An input aliquot
corresponding to 20 ?g of total cell extracts, and the whole immu-
noprecipitated (IP) fractions were resolved by SDS-12% PAGE and
immunoblotted with anti-NF-?B p65 or anti-PRDX1 antibodies.
Data are representative of three independent experiments.
PRDX1 associated with NF-?B in MDA-MB-231 but not
PRDX1 Association with COX-2 Promoter
Vol. 21, September 1, 2010 2991
no enrichment in these sequences (Figure 7C). These results
demonstrate that PRDX1 was associated with the COX-2
promoter region in ER? MDA-MB-231 cells but not in ER?
MCF7 cells. To determine whether NF-?B and PRDX1 co-
occupied the same COX-2 promoter region, we performed a
re-ChIP assay in which formaldehyde cross-linked and frag-
mented chromatin preparations from MCF7 and MDA-MB-
231 cells were incubated with anti-p65 antibodies. The im-
munoprecipitated chromatin fraction was then subjected to
a second immunoprecipitation by using anti-PRDX1 anti-
bodies. Again, equal amounts of input DNA and immuno-
precipitated DNA were quantified by real-time PCR. Figure
7D shows that the -439 and -214 sequences were not en-
riched in the DNA immunoprecipitated from MCF7. Con-
versely, DNA immunoprecipitated from MDA-MB-231 cells
was enriched in both sequences, demonstrating that in ER?
MDA-MB-231 cells, NF-?B and PRDX1 are associated simul-
taneously to the each of the NF-?B recognition sites on the
Knockdown of PRDX1 Attenuates the Expression of
COX-2 in MDA-MB-231 but Not in MCF7 Cells
To evaluate the importance of the interaction between
PRDX1 and NF-?B in the expression of the COX-2 gene, we
knocked down the levels of PRDX1 in MDA-MB-231 and
MCF7 cells. We used a lentiviral vector system stably ex-
pressing shRNA to generate PRDX1 stable knockdown
MDA-MB-231 and MCF7 cell lines. The transfection of an
empty lentiviral vector or a vector containing a scrambled
DNA sequence provided negative control cell lines. Immu-
noblot analysis demonstrated that the level of PRDX1 in the
PRDX1 knockdown cells was reduced by 65 and 78% in
clone G1-MCF7 and clone G2-MCF7 cells, respectively (Fig-
ure 8A). In the G1 and G2-MDA-MB-231 clones, PRDX1 was
reduced by 77 and 73%, respectively (Figure 8B). In the G1
and G2 MDA-MB-231 clones, expression of COX-2 was re-
duced to ?30% of that observed in the control-transfected
cells (Figure 8D). In marked contrast, knockdown of PRDX1
expression in MCF7 cells resulted in increased expression of
COX-2 (Figure 8C).
To determine the impact of reduced expression of PRDX1
on the occupancy of NF-?B in the upstream promoter region
of the COX-2 gene in MDA-MB-231 cells, we performed
ChIP assays with the control and PRDX1 knockdown MDA-
MB-231 cell lines. Figure 8E shows that occupancy of p65 at
the NF-?B sites (-439 and -214 regions) was reduced in the
PRDX1 knockdown but not control MDA-MB-231 cell lines.
Together, these observations suggest that the PRDX1 asso-
ciation with NF-?B has a role in the retention of NF-?B at the
COX-2 upstream promoter region in MDA-MB-231 cells.
PRDX1 Is Phosphorylated in MDA-MB-231 Cells
Because PRDX1 was expressed in both ER? and ER? breast
cancer cells, we investigated what properties of PRDX1 may
promoter region. (A) Total RNA was isolated from MCF7 and MDA-MB-231 cells and amplified by RT-PCR. The PCR products were resolved
on a 1.5% (wt/vol) agarose gel and stained with ethidium bromide to visualize the expression levels of COX-2 relative to GAPDH. Data are
representative of two independent experiments. (B) Schematic representation of COX-2 promoter showing regions amplified in the ChIP
assays. (C) ChIP experiments were performed using antibodies against NF-?B p65 or PRDX1 on formaldehyde cross-linked and fragmented
chromatin prepared from MCF7 and MDA-MB-231 cells. Equal amounts of input and immunoprecipitated DNA were quantified by real-time
quantitative PCR for the two NF-?B binding sites. The relative enrichment in the ChIP DNA compared with the input DNA is shown.
Enrichment values are the mean of three independent experiments, and the error bars represent the SE. (D) Formaldehyde cross-linked and
fragmented chromatin prepared from MCF7 and MDA-MB-231 cells was subjected to immunoprecipitation first by anti-NF-?B p65 antibodies
and then by anti-PRDX1 antibodies.
Increased level of COX-2 gene expression coupled with PRDX1 and NF-?B co-occupancy of the two NF-?B recognition sites in the
X. Wang et al.
Molecular Biology of the Cell2992
differ in these two types of breast cancer cells that may
confer the ability to associate with DNA in one cell type but
not the other. To determine whether PRDX1 was differen-
tially modified in the breast cancer cell lines, total cell ex-
tracts from MCF10A1, MCF7, and MDA-MB-231 cells were
electrophoretically resolved on two-dimensional gels and
analyzed by immunoblot with anti-PRDX1 antibodies. Fig-
ure 9A shows that a single PRDX1 species was detected in
the MCF10A1 and MCF7 cell extracts, whereas a second
more acidic species also was detected in the MDA-MB-231
cell extract. Two PRDX1 species also were detected in a
preparation of proteins cross-linked to the DNA of MDA-
MB-231 cells in situ by cisplatin (Figure 9B). The extract from
MDA-MB-231 cells was treated with CIP before the electro-
phoresis to determine whether the presence of the more
acidic PRDX1 species was the result of a phosphorylation
event. Indeed, Figure 9C shows that the CIP treatment re-
sulted in the disappearance of the second species, indicating
that it represented a phosphorylated form of PRDX1. More-
over, Figure 9B provides evidence that phosphorylated
PRDX1 was bound to DNA in MDA-MB-231 cells.
Previous reports demonstrated that oxidation of PRDX1
results in an acidic shift in two-dimensional gels. Treatment
of MDA-MB-231 and MCF7 cells with H2O2results in the
oxidation of PRDX1, which is readily detected on immu-
noblots stained with an anti-PRDX1-SO3antibody (Sup-
plemental Figure S1A). To determine whether oxidation is
in part responsible for the acidic shift of PRDX1 isolated
from MDA-MB-231 cells, two-dimensional immunoblots
were stained with an anti-PRDX1-SO3antibody. No im-
munostaining with this antibody was observed (Supple-
mental Figure S1B).
Proteins cross-linked to DNA by cisplatin are involved in
regulating the function and structure of DNA. Thus, pro-
teins differentially cross-linked in breast cancer and normal
mammary epithelial cells are likely to have roles in the cell
type-specific regulation of chromatin structure and function
and the biology of that cell type. In this study, we found that
PRDX1 was bound to DNA in ER? cell lines, but not in ER?
human breast cancer cells and nontumorigenic mammary
epithelial cell lines.
PRDX1, also known as natural killer cell enhancing factor
A and proliferation-associated protein (Wood et al., 2003), is
a multifunctional protein. It operates with thioredoxin to
detoxify hydrogen peroxide, thus preventing the buildup of
expression of the COX-2 gene in MDA-MB-
231 but not in MCF7 cells. Cellular proteins
isolated from the control and PRDX1 knock-
down (G1 and G2) MCF7 (A) and MDA-MB-
231 (B) cells were resolved on SDS-12% PAGE
and immunoblotted with anti-PRDX1 anti-
bodies. Total RNA was isolated from the con-
trol and PRDX1 knockdown (G1 and G2)
MCF7 (C) and MDA-MB-231 (D) cells and
quantified by real-time RT-PCR. Fold change
values, normalized to GAPDH levels are rep-
resentative of experiments done twice. (E)
ChIP assays were performed using antibodies
against NF-?B p65 on formaldehyde cross-
linked and fragmented chromatin prepared
from control (empty vector control; E) and
PRDX1 knockdown (G1 and G2) MDA-MB-231
cells as described in the legend to Figure 7.
Knockdown of PRDX1 reduces the
PRDX1 Association with COX-2 Promoter
Vol. 21, September 1, 20102993
reactive oxygen species, an accumulation of which results in
oxidative stress. Alternatively, phosphorylation of PRDX1
Thr90promotes the formation of a high-molecular-weight
complex, and this quaternary structure change comes with a
functional switch from peroxidase to chaperone activity
(Jang et al., 2006). Moreover, although there is evidence that
PRDX1 is a tumor suppressor, it also has proliferative and
antiapoptotic properties (Noh et al., 2001; Neumann and
Although PRDX1 was more abundant in breast cancer cell
lines (ER? and ER?) than in normal or pseudonormal
breast cell lines, we did not find a significant difference in
PRDX1 levels between ER? and ER? breast cancer cell
lines. This result is consistent with previous data showing
overexpression of PRDX1 in breast cancer tissues from most
patients (87.5%) compared with normal tissues but showing
no correlation between PRDX1 levels and pathological fac-
tors, including estrogen receptor status (Noh et al., 2001).
Similarly, breast cancer cell lines had higher nuclear PRDX1
levels than a pseudonormal cell line, but there was no sig-
nificant difference in nuclear PRDX1 levels between ER?
and ER? breast cancer cell lines. Nuclear PRDX1 levels were
higher than cytoplasmic levels in both ER? and ER? breast
cancer cell lines, which was somewhat unexpected because
PRDX1 was initially believed to be primarily located in the
cytosol (Wood et al., 2003). However, more recent evidence
revealed a PRDX1 nuclear presence (Immenschuh et al.,
2003; Park et al., 2007). During the course of this study, when
we performed cellular fractionations, the cellular lysis buffer
was free of nonionic detergent to prevent the leaking from
nuclei of PRDX1, because we had observed that PRDX1 was
readily extracted from nuclei with Triton X-100.
Using indirect immunofluorescence microscopic analyses,
PRDX1 was colocalized with PML nuclear bodies in ER?
breast cancer cell lines. This colocalization is interesting,
because PML bodies have been implicated in a variety of
cellular functions, including transcriptional regulation (Ber-
nardi and Pandolfi, 2007).
The observation that PRDX1 contributed to NF-?B tran-
scriptional activity (Hansen et al., 2007) prompted us to
investigate whether PRDX1 was in complex with NF-?B in
ER? breast cancer cells. In addition to finding that PRDX1
was in complex with NF-?B in ER?, but not ER? cells, we
demonstrated that PRDX1 and NF-?B co-occupied NF-?B
recognition sites in the COX-2 upstream promoter region in
ER? but not ER? cells. Thus, the selective binding of
PRDX1 to NF-?B in MDA-MB-231 cells provides a mecha-
nism by which PRDX1 would be associated with nuclear
DNA in MDA-MB-231 cells but not in MCF7 cells. The
results of the PRDX1 knockdown studies further highlights
a differential function of PRDX1 in ER? MDA-MB-231 and
ER? MCF7 cells. Previous reports have shown that in-
creased oxidative stress results in increased COX-2 expres-
sion (Wu et al., 2009). A similar response may be occurring in
MCF7 cells as a consequence of knocking down PRDX1.
However, in MDA-MB-231 cells knocking down PRDX1
results in decreased expression of the COX-2 gene, consis-
tent with a role of PRDX1 in increasing the activity, reten-
tion, or both of NF-?B on the upstream promoter region of
the COX-2 gene.
To elucidate the molecular basis of the exclusive cross-
linking by cisplatin and formaldehyde of PRDX1 to the DNA
of ER? cells, we analyzed PRDX1 from ER? and ER? breast
cancer cells for posttranslational modifications. A phosphor-
ylated form of PRDX1 was present in ER?, but not ER?,
breast cancer cells. This phosphorylated form was cross-
linked to DNA by cisplatin. Phosphorylation of PRDX1 on
Thr90was shown to reduce PRDX1 peroxidase activity by
80% (Chang et al., 2002) and to promote oligomerization and
enhance PRDX1 chaperone activity (Jang et al., 2006). Thus,
it is possible that the chaperone activity of PRDX1 rather
than its antioxidant activity plays a role in the co-occupancy
of PRDX1 and NF-?B in upstream promoter elements of the
COX-2 and perhaps other NF-?B–responsive genes in ER?
breast cancer cells. It was reported that, in aggressive pros-
tate cancer cell lines, PRDX1 interacted with the androgen
receptor (AR), and was bound to the androgen-responsive
element (ARE) of the prostate-specific antigen (PSA) gene.
The stimulation of AR transactivation by PRDX1 was inde-
pendent of the PRDX1 peroxidase activity (Park et al., 2007).
However, this binding of PRDX1 to the PSA gene ARE only
occurred under hypoxia/reoxygenation, a cell culture con-
dition mimicking the variable oxygenation typical of tumor
tissues (Park et al., 2007). In contrast, PRDX1 and NF-?B
were loaded simultaneously onto the COX-2 upstream pro-
moter element in normally cycling MDA MB 231 ER? breast
NF-?B, as a critical transcription factor regulating the
expression of a plethora of genes, interacts with a number
of regulators controlling its activity, including several
chaperones (Mankan et al., 2009). Deregulation of NF-?B
activity has been implicated in the development of vari-
ous inflammatory and autoimmune diseases, as well as in
the development of tumors. This study has identified
PRDX1 as a protein that interacts with NF-?B at the DNA
level and presumably modulates its transcriptional activ-
ity in ER? breast cancer cells. As such, PRDX1 is a po-
tential target in the treatment of endocrine-nonresponsive
MCF7 or MCF10A1 cells. Proteins (20 ?g) from MCF10A1, MCF7, or
MDA-MB-231 total cell lysates (A) or proteins cross-linked to DNA
by cisplatin in MDA-MB-231 (B) were resolved on two-dimensional
gels and immunoblotted with anti-PRDX1 antibodies. MDA-MB-
231 cellular extract was treated with CIP before electrophoresis (C).
Data are representative of two independent experiments.
PRDX1 is phosphorylated in MDA-MB-231 but not
X. Wang et al.
Molecular Biology of the Cell2994
ACKNOWLEDGMENTS Download full-text
We thank Dr. Sam Kam-Pun Kung for help with the production of the
lentiviral vectors. This research was supported by grants from CancerCare
Manitoba Foundation, Canadian Breast Cancer Foundation, and a Canada
Research Chair (to J.R.D.).
Allred, D. C., O’Connell, P., and Fuqua, S.A.W. (1993). Biomarkers in early
breast neoplasia. J. Cell. Biochem. 17G, 125–131.
Batty, E., Jensen, K., and Freemont, P. (2009). PML nuclear bodies and their
spatial relationships in the mammalian cell nucleus. Front. Biosci. 14, 1182–
Bernardi, R., and Pandolfi, P. P. (2007). Structure, dynamics and functions of
promyelocytic leukaemia nuclear bodies. Nat. Rev. Mol. Cell Biol. 8, 1006–
Chang, T. S., Jeong, W., Choi, S. Y., Yu, S., Kang, S. W., and Rhee, S. G. (2002).
Regulation of peroxiredoxin I activity by Cdc2-mediated phosphorylation.
J. Biol. Chem. 277, 25370–25376.
Chichiarelli, S., Coppari, S., Turano, C., Eufemi, M., Altieri, F., and Ferraro, A.
(2002). Immunoprecipitation of DNA-protein complexes cross-linked by cis-
diamminedichloroplatinum. Anal. Biochem. 302, 224–229.
Coutts, A. S., Leygue, E., and Murphy, L. C. (1999). Variant estrogen receptor-
alpha messenger RNA expression in hormone-independent human breast
cancer cells. J. Mol. Endocrinol. 23, 325–336.
Davie, J. R., Samuel, S. K., Spencer, V. A., Holth, L. T., Chadee, D. N., Peltier,
C. P., Sun, J.-M., Chen, H. Y., and Wright, J. A. (1999). Organization of
chromatin in cancer cells: role of signalling pathways. Biochem. Cell Biol. 77,
Drobic, B., Perez-Cadahia, B., and Davie, J. R. (2010). Promoter chromatin
remodeling of immediate-early genes is mediated through H3 phosphoryal-
tion at either serine 28 or serine 10 by the MSK1 multi-protein complex.
Nucleic Acids Res. 38, 3196–3208.
Ferraro, A., Grandi, P., Eufemi, M., Altieri, F., and Turano, C. (1992).
Crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum in
intact cells. Involvement of nuclear matrix proteins. FEBS Lett. 307, 383–385.
Hansen, J. M., Moriarty-Craige, S., and Jones, D. P. (2007). Nuclear and
cytoplasmic peroxiredoxin-1 differentially regulate NF-kappaB activities. Free
Radic. Biol. Med. 43, 282–288.
He, S., Sun, J. M., Li, L., and Davie, J. R. (2005). Differential intranuclear
organization of transcription factors Sp1 and Sp3. Mol. Biol. Cell 16, 4073–
Immenschuh, S., Baumgart-Vogt, E., Tan, M., Iwahara, S., Ramadori, G., and
Fahimi, H. D. (2003). Differential cellular and subcellular localization of
heme-binding protein 23/peroxiredoxin I and heme oxygenase-1 in rat liver.
J. Histochem. Cytochem. 51, 1621–1631.
Ishov, A. M., Stenberg, R. M., and Maul, G. G. (1997). Human cytomegalovi-
rus immediate early interaction with host nuclear structures: definition of an
immediate transcript environment. J. Cell Biol. 138, 5–16.
Jang, H. H., et al. (2006). Phosphorylation and concomitant structural changes
in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase
and molecular chaperone functions. FEBS Lett. 580, 351–355.
Li, L., and Davie, J. R. (2008). Association of Sp3 and estrogen receptor alpha
with the transcriptionally active trefoil factor 1 promoter in MCF-7 breast
cancer cells. J. Cell. Biochem. 105, 365–369.
Mankan, A. K., Lawless, M. W., Gray, S. G., Kelleher, D., and McManus, R.
(2009). NF-kappaB regulation: the nuclear response. J. Cell Mol. Med. 13,
Meng, X., and Wilkins, J. A. (2005). Compositional characterization of the
cytoskeleton of NK-like cells. J. Proteome. Res. 4, 2081–2087.
Neumann, C. A., and Fang, Q. (2007). Are peroxiredoxins tumor suppressors?
Curr. Opin. Pharmacol. 7, 375–380.
Neve, R. M., et al. (2006). A collection of breast cancer cell lines for the study
of functionally distinct cancer subtypes. Cancer Cell 10, 515–527.
Noh, D. Y., Ahn, S. J., Lee, R. A., Kim, S. W., Park, I. A., and Chae, H. Z. (2001).
Overexpression of peroxiredoxin in human breast cancer. Anticancer Res. 21,
Park, S. Y., Yu, X., Ip, C., Mohler, J. L., Bogner, P. N., and Park, Y. M. (2007).
Peroxiredoxin 1 interacts with androgen receptor and enhances its transacti-
vation. Cancer Res. 67, 9294–9303.
Samuel, S. K., Minish, M. M., and Davie, J. R. (1997). Nuclear matrix proteins
in well and poorly differentiated human breast cancer cell lines. J. Cell.
Biochem. 66, 9–15.
Samuel, S. K., Spencer, V. A., Bajno, L., Sun, J.-M., Holth, L. T., Oesterreich, S.,
and Davie, J. R. (1998). In situ cross-linking by cisplatin of nuclear matrix-
bound transcription factors to nuclear DNA of human breast cancer cells.
Cancer Res. 58, 3004–3008.
Singh-Ranger, G., Salhab, M., and Mokbel, K. (2008). The role of cyclooxy-
genase-2 in breast cancer: review. Breast Cancer Res. Treat. 109, 189–198.
Spector, D. L. (2006). SnapShot: cellular bodies. Cell 127, 1071
Spencer, V. A., and Davie, J. R. (2002a). Isolation of proteins cross-linked to
DNA by cisplatin. In: The Protein Protocols Handbook, ed. J. M. Walker,
Totowa, NJ: Humana Press, 747–752.
Spencer, V. A., and Davie, J .R. (2002b). Isolation of proteins cross-linked to
DNA by formaldehyde. In: The Protein Protocols Handbook, ed. J. M. Walker,
Totowa, NJ: Humana Press, 753–760.
Spencer, V. A., Samuel, S. K., and Davie, J. R. (2000). Nuclear matrix proteins
associated with DNA in situ in hormone-dependent and hormone-indepen-
dent human breast cancer cell lines. Cancer Res. 60, 288–292.
Spencer, V. A., Samuel, S. K., and Davie, J. R. (2001). Altered profiles in
nuclear matrix proteins associated with DNA in situ during progression of
breast cancer cells. Cancer Res. 61, 1362–1366.
Sun, J. M., Chen, H. Y., and Davie, J. R. (2007). Differential distribution of
unmodified and phosphorylated histone deacetylase 2 in chromatin. J. Biol.
Chem. 282, 33227–33236.
Sun, J. M., Chen, H. Y., Moniwa, M., Litchfield, D. W., Seto, E., and Davie, J. R.
(2002). The transcriptional repressor Sp3 is associated with CK2 phosphory-
lated histone deacetylase 2. J. Biol. Chem. 277, 35783–35786.
Sun, J.-M., Chen, H. Y., and Davie, J. R. (2001). Effect of estradiol on histone
acetylation dynamics in human breast cancer cells. J. Biol. Chem. 276, 49435–
Vargo-Gogola, T., and Rosen, J. M. (2007). Modelling breast cancer: one size
does not fit all. Nat. Rev. Cancer 7, 659–672.
Wood, Z. A., Schroder, E., Robin, H. J., and Poole, L. B. (2003). Structure,
mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28, 32–40.
Wu, N., Siow, Y. L., and O, K. (2009). Induction of hepatic cyclooxygenase-2
by hyperhomocysteinemia via nuclear factor-kappaB activation. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 297, R1086–R1094.
Zhu, Y., Wang, A., Liu, M. C., Zwart, A., Lee, R. Y., Gallagher, A., Wang, Y.,
Miller, W. R., Dixon, J. M., and Clarke, R. (2006). Estrogen receptor alpha
positive breast tumors and breast cancer cell lines share similarities in their
transcriptome data structures. Int. J. Oncol. 29, 1581–1589.
PRDX1 Association with COX-2 Promoter
Vol. 21, September 1, 20102995