(Received for publication, March 10, 1997, and in revised form, April 23, 1997)
From the The c-erbB-2 gene overexpression
plays a major role in the pathogenesis of breast cancer. Binding
studies detected a nuclear matrix protein (NMP) in human breast tumor
tissues that recognizes a matrix attachment region (MAR) in the
immediate vicinity of the c-erbB-2 gene promoter. This NMP
is expressed in breast tumor tissues and cell lines along with
c-erbB-2, but is not found in corresponding normal tissues.
Furthermore, when NMP purified from the breast tumors by its affinity
to the MAR sequence is added to nuclear extracts of breast cancer
cells, it selectively stimulates the binding of the NF- Invasive breast cancer is the most common serious malignancy and a
leading cause of death among women. It is generally believed that the
overexpression of the c-erbB-2 gene (also known as
HER-2 and Neu; Refs. 1-3) leads to abnormal
growth, cellular transformation, and neoplasia (4-6); but the function
of c-erbB-2 and the role of overexpression in tumor
progression are still obscure.
Some functional hints have come from the detection of a
c-erbB-2 promoter-specific DNA-binding nuclear protein that
is present only in malignant human breast tissues (9) and induces
mitogenesis and cell surface expression of the c-erbB-2
protein in resting NIH/3T3 cells. Many cellular factors are involved in
such specific gene regulation (e.g. Refs. 7-9) and can, in
some cases, elevate the expression of c-erbB-2 at the
transcriptional level by a transactivating effect at a promoter-vicinal
DNA sequence in the gene. To better understand the progression of
breast tumorigenicity in c-erbB-2-expressing tumors, an
attempt was made to study its mechanism within the innermost nuclear
structure, the nuclear matrix, through DNA-protein interaction. Nuclear
matrix is believed to play critical roles in regulating many key
biological reactions in the nucleus such as: gene transcription, DNA
replication, DNA organization, and RNA splicing and processing. In this
study, we show that a nuclear DNA-binding protein is a breast
tumor-specific nuclear matrix protein
(NMP)1 that recognizes a nonconventional
matrix attachment region (MAR) in the c-erbB-2 gene
promoter. The NMP protein stimulates the binding of NF- Human normal (benign) and
malignant (tumor) breast tissues were obtained from Pathology
Department of Wayne State University Medical School, Detroit, MI. Only
confirmed sets of c-erbB-2-expressing as well as negative
breast tissue samples were used.
Human breast carcinoma cell lines MCF-10A and
BT-20 were obtained from ATCC (Rockville, MD). MCF-10A was grown in
Dulbecco's modified Eagle's medium, while BT-20 was grown in minimum
Eagle's medium, with 10% fetal bovine serum, 1%
penicillin/streptomycin, 1% glutamine in a humidified 5%
CO2 atmosphere.
Nuclear
matrices from breast tissues and cell lines were prepared by a standard
procedure (33) with certain modifications. Tissues were powdered under
liquid N2, and nuclei were purified according to the
protocol. Nuclei were extensively treated with 200 µg/ml DNase I
enzyme for 2 h with constant agitation, at room temperature. DNase
I-treated residual nuclear pellets were extracted several times with 2 M NaCl containing high-salt buffer and finally washed with
RSB (10 mM NaCl, 10 mM Tris-HCl, pH 7.5, 3 mM MgCl2, 0.5 mM phenylmethylsulfonyl
fluoride)-0.25 M sucrose buffer.
For the NMP isolation, the method of Fey and Penman (29) was used with
certain modifications. The nuclear matrix were solubilized in 8 M urea containing disassembly buffer for 30 min at room
temperature and then dialyzed in a renaturation buffer (100 mM KCl, 25 mM HEPES, pH 7.6, 5 mM
MgCl2, 2 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, 0.125 mM EGTA, and 2%
glycerol) overnight at room temperature. The dialyzed material was
centrifuged at high speed to remove the intermediate filaments (the
pelleted material). The clear supernatant thus obtained is the
solubilized NMP pool, which was further dialyzed in low salt buffer (25 mM HEPES, pH 7.6, 100 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.2 mM phenylmethylsulfonyl fluoride) and used for DNA-protein
gel binding and SouthWestern assays as well as for affinity
purification.
Corresponding to different
regulatory regions of the c-erbB-2 gene as well as
BRCA1, NF- The oligonucleotide probes used are as follows: 1) Nuclear extracts from
breast tissues and cell lines were prepared according to a method
described earlier (9). Nuclear extracts were finally dialyzed against
buffer (25 mM HEPES, pH 7.6, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.2 mM phenylmethylsulfonyl fluoride) and stored at
MAR binding assays were performed
according to a standard procedure (33) with some modifications. A DNase
I-treated, high-salt-extracted residual nuclear pellet was washed three
times in RSB-0.25 M sucrose buffer and once in MAR binding
buffer. The resulting pellet (nuclear matrix) was mixed with
5 DNA isolated from
5A260 units of nuclear matrix were
electrophoresed on agarose gel, transferred to the nitrocellulose
paper, and hybridized with GGA-specific ( EMSA reactions were carried out with NMPs and nuclear
extracts and with 32P-labeled double-standard
oligonucleotide probes in the presence of 3 µg of poly[d(I·C)]
and KCl containing binding buffer, exactly as described earlier (8, 9).
For competition studies, 50-fold molar excess of unlabeled
double-stranded oligonucleotides were added. In SouthWestern blot
assay, 10 µg of NMPs were usually resolved on a 10%
SDS-polyacrylamide gel, electrotransferred onto nitrocellulose
membrane, and renatured as described (8, 9). The membrane was
hybridized with 2.5 × 106 cpm of
32P-labeled oligonucleotide probes for 15 h at room
temperature, with constant agitation. The rest of the steps were as
described in the standard protocol.
An affinity resin with Large pools of NMPs from breast tumor tissues were used for the
purification purposes. All the steps of the purification protocols were
exactly as described earlier (9). Briefly, solubilized bulk NMP pools
from breast tumor tissues were first mixed with total 200 µg of
salmon sperm DNA for 15 min and then mixed with the nonspecific,
Twenty micrograms of
nuclear matrix from normal and tumor tissues were resolved on a 10%
SDS-polyacrylamide gel and electrotransferred on to nitrocellulose
membrane. Filters were incubated with NF- To better understand the progression of breast tumorigenicity in
c-erbB-2-expressing tumors, an attempt was made to study the
possible involvement of protein interactions with DNA within the
innermost nuclear structure, the nuclear matrix. To test for novel
factor(s) in the nuclear matrix that bind to a specific segment of the
c-erbB-2 gene regulatory sequences, MAR binding studies were
conducted with many distinct segments of 5
To reaffirm the results of Fig. 1, whether this DNA attachment onto the
nuclear matrix of breast tumors is mediated by any specific protein
factor(s), we performed DNA-protein binding gel shift assays with NMPs
from breast biopsy tissues. Solubilized NMPs devoid of intermediate
filaments (29) from several normal (benign), tumor breast biopsy
tissues and breast cancer cell line, BT-20, were mixed with the
radiolabeled GGA-rich DNA probe, in DNA binding assays. The DNA binding
EMSA clearly demonstrates a strong DNA-protein complex formation only
with the NMPs of breast tumors (Fig. 2A, panels
a-c, lanes 2, 4, 6) and not with NMPs of their normal breast
tissue counterparts (Fig. 2A, panels a-c, lanes 1, 3, 5).
The tumor cell line BT-20 displays the same complex as the tumor tissue
NMP (Fig. 2A, panel d, lane 8), while no such complex is
observed with a normal breast cell line MCF-10A (Fig. 2A, panel
d, lane 7). Different groups have reported the presence of
MAR-binding proteins, such as: SATB1, OCT-1, nucleolin, and recently
from human breast carcinomas (26, 28, 37-40).
To test whether this binding activity of NMPs from breast tumors is a
specific phenomenon, we examined the status of NMP binding with other
region of c-erbB-2 ( Together, the results of Figs. 1 and 2 clearly demonstrate the presence
of a MAR-like non-conventional (rich in GGA and not in AT) element in
the close vicinity of the c-erbB-2 gene transcription start
site, whose attachment to the breast nuclear matrix is mediated by a
sequence-specific DNA-binding NMP that is expressed only in the
malignant tissues and breast cancer cell line BT-20 and not in the
normal breast tissues.
To elucidate a functional role(s) of the identified NMP factor, we
affinity-purified the protein from the breast tumor NMP pools on the
GGA-rich DNA (
To ascertain a functional role that this specific NMP factor may be
contributing, a functional test was performed. The purified NMP was
added to the nuclear extracts from various tumor cell lines in an EMSA
binding reaction, using defined DNA probes from c-erbB-2,
BRCA1, exon 1 regulatory sequences, and NF-
To further evaluate whether NF-
The proteinaceous network of the NM is believed to be involved in
DNA organization, DNA replication, gene transcription, and RNA splicing
and processing (17-20). The protein components (NMPs) of the NM
provide the structural framework for loop domains of DNA, attached at
MARs (15, 21, 22).
Several groups (12-14, 25-28, 41) have demonstrated that
sequence-specific DNA-binding proteins can be components of nuclear matrix attachment sites. In recent years, intensive studies have also
suggested possibly related role(s) of the nuclear matrix in tumor
progression (10-16). In cancer cells, some transforming proteins
appear to be associated with the matrix (23, 24), and there are also
indications of specific alterations in the protein composition of the
matrix as cells undergo differentiation (25) and during the invasion
and proliferation of tumors (14, 15, 24, 25, 42, 43). Could a
"tumor-specific" NMP be involved in a transition associated with
tumor progression? The results reported here may provide such an
instance, based on a sequence-specific DNA-binding nuclear matrix
protein exclusively present in human breast tumor tissues.
Concerning the association with DNA, the protein is unlikely to be part
of one of the integrated structures that generates loop domains of
chromatin. Such binding sequences, found in DNase I-sensitive sites
near the bases of the loops, are generally rich in AT sequences. In
contrast, the sequence-specific DNA-binding protein studied here
mediates the attachment of a GGA-rich enhancer region of
c-erbB-2 to the nuclear matrix. This is the other recognized mode of attachment to the nuclear matrix: transient and based on
enhancer regions upstream (5 Based on these findings, the juxtaposition of DNA binding factors and
DNA regulatory elements at the matrix may increase the local
concentration of various transcription factors (25, 29, 30). We suggest
that the specific binding of the c-erbB-2 DNA element to the
breast tumor nuclear matrix thereby concentrates nuclear factor NF- We thank Drs. David Schlessinger, Nancy
Colburn, and Scott Durum for helpful comments and discussion on the
manuscript and Dr. Dan Longo for his consistent support of this
project.
Intramural Research Support Program,
B
transcription factor to DNA. A model is suggested in which the
association of the MAR-like sequence with the nuclear matrix raises the
local concentration of the specific NMP, which in turn interacts with
the nuclear factor NF-
B to increase its local level. Such a complex
could explain at a molecular level the "increase in NF-
B DNA
binding activity" often observed in c-erbB-2- and
BRCA1-positive human breast tumors. The increased NF-
B
activity could thereby contribute to breast cancer progression.
B to DNA in
nuclear extracts from breast cancers, suggesting a possible role in
breast cancer progression.
Human Breast Tissue Samples
B binding sequences were chemically synthesized, gel-purified, annealed to make double-stranded, and 5
-end-labeled with [
-32P]ATP and T4 polynucleotide
kinase, according to standard protocols.
79/
22,
c-erbB-2 sequences
(5
-TCCAATCACAGGAGAAGGAGGAGGTGGAGGAGGAGGGCTGCTTGAGGAAGTATAAG-3
); 2)
22/+9, c-erbB-2 sequences
(5
-AATGAAGTTGTGAAGCTGAGATTCCCCTCCA-3
); 3) NF-
B binding sequences
(5
-AGCTTCAGAGGGGACTTTCCGAGAGG-3
); 4)
78/
42, BRCA1
sequences (5
CAGAAGGCCTCCTGAGCGCAGGTTATCTGAGAAACCCCA-3
).
70 °C in small aliquots.
-end-labeled oligonucleotide probes corresponding to regions: A+T
(
450/
390), GGA-rich (
79/
22),
300/
280, and
22/+9 (34) of
the c-erbB-2 gene in 100 µl of MAR binding buffer for
4 h at room temperature, with constant agitation. After three washings with MAR binding buffer the bound DNAs were further processed and gel electrophoresed as described in Ref. 33. In most of MAR binding
assays, 5A260 of the NM and 50,000 cpm of 5
-DNA
probes in the presence of 150 µg/ml Escherichia coli DNA
were used, unless otherwise mentioned.
79/
22) and nonspecific
32P-nick-translated probes according to standard
protocol.
79/
22, GGA-rich, MAR-like
sequences of c-erbB-2 was generated as described earlier
(9). Complementary nucleotides corresponding to
79 to
22 sequences
of c-erbB-2 promoter were annealed, phosphorylated, ligated,
and coupled to CNBr-activated Sepharose 4B.
22/+9, DNA affinity resin for 6 h, all at 4 °C, with shaking.
The low-salt flow-through from nonspecific affinity column was then
mixed with the GGA-rich (
79/
22) DNA affinity column, and the rest
of the purification protocol was followed as described (9).
B antibodies (anti-p65 and
anti-p50 subunits of NF-
B) and anti-human nuclear matrix 45. The
rest of the procedure followed was as suggested by the manufacturers
(Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and Upstate
Biotechnology, Inc. (Lake Placid, NY).
-upstream regulatory regions
of c-erbB-2 enhancer-promoter (Fig.
1A) and nuclear matrix preparations from
human normal, c-erbB-2-expressing tumor breast biopsy
tissues and breast cancer cell lines (33). Results (Fig. 1B)
indicate that of the four regions tested, only one specific region,
rich in GGA repeats (
79/
22) and in the immediate vicinity of the
c-erbB-2 gene transcription start site, has the specific
binding affinity observed only with the nuclear matrices of tumor
tissues (Fig. 1B, lane 1) and not with the normal tissues
(Fig. 1B, lane 2). This observation was further confirmed in
the breast tumor cell line BT-20 (Fig. 1B, lane 3). The
upstream region that contains a stretch of AT-rich sequences
(conventionally a part of MAR) binds less effectively to the nuclear
matrix than the GGA-rich region (Fig. 1B, lane 4). Breast
tumor and BT-20 cell nuclear matrices binding with the GGA-rich DNA
element were tested with more stringent conditions by using increasing
amounts of a competitor E. coli DNA, where the GGA-rich DNA
element still appears to be the dominant bound material (Fig. 1C,
lane 3, breast tumor NM, lane 4, BT-20 NM with 200 µg/ml E. coli DNA versus lanes 1 and
2 of same NMs with 100 µg/ml E. coli DNA). A
Southern blot analysis with DNAs from tissue nuclear matrices further
demonstrates a strong binding with GGA-rich DNA probe (Fig. 1D,
lane 2), not observed with the nonspecific (
22/+9) DNA probe.
DNA from normal breast nuclear matrix failed to show any affinity with
either probes (Fig. 1D, lane 1). These results (Fig.
1B-D) demonstrate the presence of a MAR-like DNA element
within the immediate upstream of c-erbB-2 promoter. These
sequences are rich in GGA and not in AT, which traditionally is part
of, but not a prerequisite for, MAR. This GGA-rich region (
79/
22)
has also been mentioned as an activator of gene promoter and an
alternate transcription start site region of the c-erbB-2
gene (35, 36). Other groups have reported the presence of MAR in
chicken oviduct, chicken lysozyme gene, avian-globin gene, mouse
immunoglobulin K chain, and 5
-upstream regulatory region of human H4
histone gene promoter (25, 31, 32, 37, 38).
Fig. 1.
Breast tumor NM detects a MAR in
c-erbB-2 gene promoter and a NMP present in the nuclear
matrices of human breast tumor tissues and cell line only and not in
benign breast tissues that bind to this MAR-DNA sequence. A,
a schematic representation of the c-erbB-2 gene promoter's
regulatory region (34). Distinct regions are shown, where +1
corresponds to the transcription start site. A+T-rich region (-450 to
-390), GGA-rich region (-73 to -22), and B and SP1 binding positions
are indicated. Underneath are the B recognition motifs from different
genes, including c-erbB-2. B, the nuclear matrices of human
breast tissues and breast tumor cell line, BT-20, identify a MAR in the
c-erbB-2 gene enhancer-promoter. Nuclear matrices
(5A260nm) were mixed with 50,000 cpm of
5-end-labeled oligonucleotide probe mixture corresponding to different
regions of c-erbB-2 enhancer-promoter in the presence of 150 µg/ml Escherichia coli DNA. The bound DNA was processed
and resolved on a 8% acrylamide gel in 1 × TBE (0.045 M Tris borate, 0.001 M EDTA) (33). Lane 1, c-erbB-2 DNA probes mixture mixed with human breast tumor
nuclear matrix; lane 2, with human benign (normal) breast
nuclear matrix; lane 3, with breast tumor cell line BT-20
nuclear matrix; and lane 4, A+T-rich DNA probe alone with
human breast tumor nuclear matrix. The extreme left lane is
the mixture of input c-erbB-2 DNA probes. The
arrow indicates the position of nuclear matrix bound (matrix
attachment) GGA-rich region (
79/
22) that is observed only due to
binding with tumor tissue (lane 1) and tumor cell line BT-20
(lane 3) and not with benign tissue (lane 2),
while a less specific breast tumor nuclear matrix-bound A+T sequence is
seen in lane 4. C, specificity of DNA attachment onto the
nuclear matrices of tumor tissue and tumor cell line BT-20 in the
presence of increasing amounts of competitor E. coli DNA.
Lanes 1 and 2, DNA probes mixture with nuclear
matrices from breast tumor tissue and cell line BT-20, respectively,
with 100 µg/ml E. coli competitor DNA; lanes 3 and 4, in the presence of 200 µg/ml E. coli
competitor DNA. In both cases GGA-rich DNA (
79/
22) is the dominant
bound element observed. D, GGA-rich element of
erbB-2 is preferentially associated with the nuclear matrix
of breast tumor. DNAs from normal and tumor breast nuclear matrix were
resolved on a 1.2% agarose gel and then transferred to nitrocellulose
paper. Filters were hybridized with GGA-specific (
79/
22) and
nonspecific (
22/+9) DNA probes. Lane 1, in both panels,
DNA from normal breast nuclear matrix; lane 2, DNA from
breast tumor nuclear matrix.
[View Larger Version of this Image (34K GIF file)]
Fig. 2.
Breast tumor NMPs contain a unique
sequence-specific DNA-binding protein. The MAR-like region binds
to breast tumor NMPs, but not benign breast tissue NMPs, in a
sequence-specific manner. DNA-protein binding EMSA and SouthWestern
blot assay were performed (8, 9) with 5-end-labeled GGA-rich
(
79/
22, specific) and
22/+9 (nonspecific) oligonucleotide probes
and solubilized NMP from breast tumor, benign tissues, and breast tumor
cell line BT-20 (see "Materials and Methods"). A, EMSA
with three sets (panels a-c) of breast biopsy tissue NMPs,
one set (panel d) of breast cell line NMPs and with the MAR
(
79/
22) probe. Lanes 1, 3, and 5 are
DNA-protein complexes from benign (normal) tissues, while lanes
2, 4, and 6 are complexes from tumor tissues.
Lane 7 is MCF10A (normal) and lane 8 is BT-20
(tumor cell line). B, criteria for sequence-specific binding
of breast tumor NMPs, EMSA with specific MAR (-79/-22) probe
(panel a) and nonspecific (
22/+9) probe (panel
b). Two more breast tissue sets and cell line NMPs were used to
test the specificity of breast tumor NMPs. Panel a, specific
binding. Lanes 1 and 3 of panel a are
DNA-protein complexes due to normal (benign) tissues NMPs, while
lanes 2 and 4 are their tumor counterparts.
Lane 5 is due to control MCF10A line, while lane
6 is due to BT-20 cell line NMPs. Panel b, nonspecific
binding. Similar experiment with c-erbB-2 other region
(
22/+9) DNA sequences are unable to form any DNA-protein complex.
C, a SouthWestern blot assay of NMPs from breast tumor tissues and BT-20 cell line with the same two oligonucleotide probes as
used above recognizes a dominant 68-kDa NMP in the breast tumor tissues
and BT-20 cell line only with specific probe,
79/
22. 10 µg of NMP
were resolved on SDS-polyacrylamide gel electrophoresis, electrotransferred, and hybridized with 1 × 106
cpm/ml of specific GGA-rich (
79/
22) and nonspecific (
22/+9) oligonucleotide probes for 15 h at room temperature (9).
Lanes 1 and 3 are NMPs from normal (benign)
breast tissues, whereas lanes 2 and 4 are NMPs
from tumor breast tissues. Lane 5 is NMP from the BT-20 cell
line. The arrow indicates the position of the protein
observed, while no such protein was recognized with other DNA (
22/+9)
probe. D, maximum DNA binding activity is localized in the
nuclear matrix compartment. EMSA was performed with 10 µg of
cytoplasmic extract (lane 1), nuclei (lane 2),
nuclear extract (lane 3), NM (lane 4), and
soluble NMP pool (lane 5) of breast tumor tissues and with
specific GGA-rich (
79/
22) probe. The arrow indicates the
position of the DNA-protein complex formation, with maximum DNA binding
activity shown in lanes 5 (NMP) and 4 (NM) and
the least DNA binding activity shown in lanes 1, 2, and 3 (cytoplasmic, nuclei, and nuclear extracts,
respectively).
[View Larger Version of this Image (66K GIF file)]
22/+9). Again the gel shift assay from
two more sets of breast tumor, but not breast normal NMPs clearly
demonstrates the formation of a specific DNA-protein complex only with
the GGA-rich,
79/
22, DNA probe (Fig. 2B, panel a, lanes
2 and 4 versus lanes 1 and 3; BT-20 NMP,
lane 6 versus MCF10A NMP, lane 5). No binding is
observed in these NMP extracts with other DNA (
22/+9) probe (Fig.
2B, panel b). These results were supported by an
SouthWestern blot assay (Fig. 2C), probed with the same two
DNA probes. The MAR-like GGA probe (
79/
22) identifies a dominant
nuclear matrix protein of 68 kDa, seen only in the breast tumor NMPs
and not in their normal counterparts (Fig. 2C, lanes 2 and
4 over lanes 1 and 3 as well as in
BT-20 NMP, lane 5). The second DNA probe (
22/+9) fails to
demonstrate any binding. Furthermore, we investigated whether this
binding activity of GGA with breast tumor NMPs is localized in the
nuclear matrix domain only or is a general phenomenon, found in other
compartments of the cell. Results (Fig. 2D) of a DNA binding
assay (EMSA) with GGA-rich probe and breast tumor extracts show an
optimized binding activity with the nuclear matrix (lane 4)
and solubilized nuclear matrix proteins (lane 5) in
comparison with cytoplasmic extract (lane 1), nuclei
(lane 2) and nuclear extract (lane 3). A slower migrating intense DNA-protein complex seen only in the nuclei (lane 2) and nuclear extract (lane 3) could
probably be due to CAAT and TATA (as well as some other
unknown)-binding proteins, since the specific probe (
79/
22) also
contains CAAT and TATA binding sequences. This complex is totally
absent from the nuclear matrix compartment (lanes 4 and
5), affirming the fact that the nuclear matrix is devoid of
these high-salt-sensitive nuclear factors.
79/
22) affinity column. The purified NMP from breast
tumors displays a dominant polypeptide of 68 kDa (Fig.
3A, panel a, lane 2). This purified NMP binds
the GGA-rich (specific) probe in a sequence-specific manner, as
demonstrated by SouthWestern (Fig. 3A, panel b) and EMSA
(Fig. 3B) assays. GGA-rich (specific) probe observes a
strong band with 50 ng of purified NMP and 10 µg of breast tumor NMP
pools (Fig. 3A, panel b, lanes 2 and 1,
respectively), while the nonspecific (
22/+9) DNA probe fails to bind
these NMPs. The EMSA result (Fig. 3B) with 50 ng of purified
NMP displays the same DNA-protein complex (Fig. 3B, lane 3)
as in breast tumor NMP pools (10 µg, lane 2), which is
competed out with specific (GGA-rich,
79/
22) cold DNA (lane
4), but not with nonspecific (
22/+9) cold DNA (lane
5).
Fig. 3.
DNA affinity purification of the MAR-specific
NMP from the human breast tumor NMP pools. Large NMP pools from
breast tumor tissues were purified on the GGA-rich DNA affinity column (see "Materials and Methods"). Purified NMP was tested by
SDS-polyacrylamide gel electrophoresis and SouthWestern and EMSA
assays. A: panel a, a silver stain of protein gel.
Lane 1, 5 µg of breast tumor NMP pool; lane 2,
30-40 ng of purified NMP. Panel b, SouthWestern blot assay
of the same samples with specific (GGA-rich, MAR, 79/
22) and
nonspecific (
22/+9) oligonucleotide probes. The arrow
indicates the position of the protein (68 kDa) in panels a
and b. B, EMSA with the purified NMP. Lane
1, GGA,
79/
22, probe alone; lane 2, 10 µg of
breast tumor NMP pool; lane 3, 50 ng of purified NMP; lane 4, 50 ng of purified NMP with 50-fold cold specific
probe (
79/
22); and lane 5, 50 ng of purified NMP with
50-fold cold nonspecific (
22/+9) probe. The arrow
indicates the DNA-protein complex formation with NMP pools (lane
2) and purified NMP (lane 3), which is abolished in the
presence of specific (lane 4), but not with nonspecific
(lane 5), probe.
[View Larger Version of this Image (33K GIF file)]
B binding sequences
(see "Materials and Methods," "Oligonucleotide Probes"). Addition of increasing amounts of purified NMP into MDA-MB231 and BT-20
nuclear extracts and with DNA probe (
22/+9) of c-erbB-2 does not have any effect on the DNA-protein complex formation (Fig.
4A, lanes 2-4 versus lane 1). Similarly
addition of NMP into these extracts and with DNA probe (
78/
42) of
BRCA1 also does not influence the DNA-protein complex (Fig.
4B, lanes 2-4 versus lane 1). Interestingly, addition of
purified NMP to these nuclear extracts and with NF-
B binding DNA
probe appears to have a selective stimulatory effect on the DNA binding
activity of nuclear factor NF-
B (Fig. 4C, lanes 2-4 versus
lane 1 and lanes 6-8 versus lane 5). Cold competition
with
B DNA (lane 9), with mutant
B DNA (lane
10), and with anti-p65 antibody (lane 11) confirm the
specificity of the NF-
B complex. This increase in NF-
B binding
activity appears to be highly selective, which is not observed with
other two probes (of
22/+9 of c-erbB-2 and
78/
42 of
BRCA1) tested (Fig. 4, A and B).
Purified NMP factor alone does not bind to
B or any other probe
except its specific GGA-rich recognition sequence (
79/
22).
Fig. 4.
Purified NMP from breast tumor tissues
stimulates NF-B DNA binding activity in breast cell nuclear extracts
in a specific manner. EMSA was performed with three labeled
oligonucleotide probes:
22/+9 of c-erbB-2 promoter
(A),
78/
42 of exon 1 of BRCA1 (16)
(B), and NF-
B sequences (see "Materials and Methods") (C) and with two breast cell lines, MDA-MB231 and BT-20,
nuclear extracts. Increasing amounts (50, 100, and 150 ng) of purified NMP were added to 10 µg of nuclear extracts of MDA-MB231 and BT-20, together with 15,000 cpm of 5
-labeled probes, and the EMSA reactions were resolved on 6% native acrylamide gels in 1 × TBE (8, 9). A, with
22/+9, c-erbB-2 probe. Lanes
1 of MDA-MB231 and BT-20 without NMP; lanes 2-4, with
increasing amounts (50, 100, and 150 ng, respectively) of purified NMP.
B, same as in A but with
78/
42 of exon 1 of
BRCA 1. C, same as in A but with NF-
B probe; lanes 1-4 and lanes 5-8 are exactly similar as
in A and B; lane 9, with 10 µg of
BT-20 nuclear extract and 50-fold cold NF-
B oligonucleotide;
lane 10, same as in lane 9 except with 50-fold cold mutant NF-
B oligonucleotide; and lane 11, with
anti-p65 antibody. The top band in C is the specific NF-
B
complex. Unlike in A and B, a gradual increase in
NF-
B DNA binding activity in C is observed due to the
addition of the purified NMP.
[View Larger Version of this Image (55K GIF file)]
B is present in the nuclear matrix of
breast tissues, we performed a Western blot analysis of the nuclear
matrix preparations from normal and tumor breast tissues, which were
subsequently probed with anti-p65 and anti-p50 antibodies of NF-
B.
As per our prediction, reasonable amounts of NF-
B (p65 as well as
p50) subunits were found preferentially associated with the breast
tumor nuclear matrix (Fig. 5A, panel a; lane
2 in both panels) and not with the normal breast nuclear matrix
(Fig. 5A, panel a; lane 3 in both panels). Lane 1 in both panels are standard control p65 and p50 peptides. To
demonstrate that equal amounts of proteins were used, we silver-stained
a parallel gel with these samples and also performed a Western blot assay with a control anti-human nuclear matrix antibody. The results of
the silver stain (Fig. 5A, panle b) and Western blot
analysis (Fig. 5A, panel c) confirm that equivalent amounts
of protein were used from the tumor (lane 2) and normal
(lane 3) breast tissue nuclear matrices. Additionally, we
also performed a NF-
B-specific Western blot analysis with the total
nuclear content along with the nuclear matrix from the breast tumor
tissue. The results of Fig. 5B clearly demonstrate that a
much smaller yet a reasonable fraction of total nuclear NF-
B is
associated with the nuclear matrix (Fig. 5B, panel a, lane 3 versus lane 2). A silver stain of the nuclear extract (lane
2) and nuclear matrix (lane 3) are shown underneath
(Fig. 5B, panel b) to show that equivalent amounts of
protein were used for the analysis.
Fig. 5.
NF-B protein is preferentially found on
the nuclear matrix of breast tumors. A: panel a, Western
blot analysis was performed with 20 µg of nuclear matrix from normal
and tumor breast tissues. Nitrocellulose-bound nuclear matrix samples
were probed with anti-p65 and anti-p50 antibodies of NF-
B.
Lane 1, left and right panels, control p65 and
p50 peptides, respectively; lane 2, left and right
panels, nuclear matrix from breast tumor; and lane 3, left
and right panels, nuclear matrix from normal breast tissue. The
arrow indicates the position of NF-
B protein. Panel b, a silver stain of nuclear matrix from breast tumor (lane
2) and nuclear matrix from normal breast tissue (lane
3), indicating equal amounts of samples. Panel c, a
Western blot analysis of tumor (lane 2) and normal
(lane 3) nuclear matrices (as in panels a and
b) with anti-human nuclear matrix protein 45, confirming equal amounts of samples used in the assay. B: panel a, a
smaller fraction of total nuclear NF-
B is preferentially associated
with the breast tumor nuclear matrix. A Western blot analysis of 20 µg of total nuclear extract (lane 2) and nuclear matrix
(lane 3) from breast tumor tissue was probed with anti-p65
antibody of NF-
B. Lane 1 is control p65 peptide.
Panel b, a silver stain of total nuclear extract (lane
2) and nuclear matrix (lane 3) from breast tumor tissue
(as in B, panel a), indicating equal amounts of samples used
in the assay.
[View Larger Version of this Image (27K GIF file)]
) of actively transcribed genes (31,
32).
B
at those sites. It can thereby rationalize the increased NF-
B DNA
binding activity observed in malignant breast and other solid tumors
that express c-erbB-2 (and BRCA1) genes (44,
45).2 Increased transcription of this
tumorigenic protein could then be a factor in tumor progression; but
one must now approach the difficult question of how the specific NMP is
expressed and regulated in breast tumors.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: IRSP,
SAIC/Frederick, NCI-FCRDC, Bldg. 567, Rm. 152, Frederick, MD
21702-1201. Tel.: 301-846-5745; Fax: 301-846-6863.
1
The abbreviations used are: NM, nuclear matrix;
NMP, nuclear matrix proteins; EMSA, electrophoretic mobility shift
assay; MAR, matrix-attachment region.
2
A. Raziuddin, D. Court, F. H. Sarkar, Y.-L. Liu,
H.-f. Kung, and R. Raziuddin, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.