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B
Department of Orthopaedics [V. B. A., J. E. P., R. J. O., R. N. R.] and Immunology/Rheumatology Unit, Department of Medicine [E. M. S.], University of Rochester, Rochester, New York 14642
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B
(NFB) in tumor metastasis, we generated a murine lung alveolar
carcinoma cell line (Line 1) defective in NFB-signaling by
retroviral delivery of a dominant negative inhibitor of NFB. The
NFB signal blockade resulted in the down-regulation of prometastatic
matrix metalloproteinase 9, a urokinase-like plasminogen activator, and
heparanase and reciprocal up-regulation of antimetastatic tissue
inhibitors of matrix metalloproteinases 1 and 2 and plasminogen
activator inhibitor 2. NFB signal blockade did not affect tumor cell
proliferation in vitro or in vivo but
prevented intravasation of tumor cells in an in vivo
chick chorioallantoic membrane model of metastasis as well as
spontaneous metastasis in a murine model. These findings suggest that
NFB plays a central and specific role in the regulation of tumor
metastasis and may be an important therapeutic target for development
of antimetastatic cancer treatments. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B2
is a dimeric transcription factor that is involved in the regulation of
genes associated with pathological processes such as inflammation and
apoptosis (1
, 2
, 3)
. NFB has been reported to
selectively enhance the expression of proinflammatory cytokines such as
tumor necrosis factor 
, IL-1, and IL-6 as well as the expression of
degradative enzymes such as MMPs. Thus NFB has a pivotal role in
sustaining chronic inflammatory reactions (4
, 5)
. Many of
the cellular events that occur during inflammation, such as vascular
penetration by inflammatory cells, tissue degradation, and
neoangiogenesis, also occur during tumor metastasis. Both NFB
(6
, 7)
and proinflammatory cytokine expression are
increased in malignancies (8
, 9)
, supporting the concept
of metastasis as an analogue of inflammation. Whereas the role of
NFB in malignancies has been explored in relation to regulation of
apoptosis, tumor progression, and responses to chemotherapy and
ionizing radiation (2
, 7
, 10)
, its role specifically in
the process of metastasis has not been examined.
MMP9 and its activator, uPA, are essential for basement membrane
transgression by tumor cells and have recently been implicated as
critical requirements for tumor cell intravasation and extravasation
during metastasis (11)
. The fact that NFB is a
transcriptional activator of MMP9 and uPA (12
, 13)
supports a role for the NFB signal transduction pathway in the
metastatic process. Recent data also implicate another degradative
enzyme, heparanase, as a critical element in tumor metastasis, allowing
degradation of the heparan sulfate proteoglycan components of basement
membranes (14
, 15)
. MMP 9 and heparanase are known to
colocalize in neutrophil tertiary granules, suggesting possible
coordinate regulation of their production and extracellular delivery
(16)
.
In resting cells, NFB is sequestered in the cytoplasm by IB
proteins. After a variety of stimuli that activate the NFB pathway,
IB is phosphorylated and degraded, resulting in the release and
nuclear translocation of NFB. A dominant negative mutant form of
IB (mIB) has been engineered that cannot be
phosphorylated, and thus does not dissociate from NFB in response to
stimulation of the IB kinase pathways (17
, 18)
. Thus,
mIB effectively blocks the NFB signal transduction pathway. In
the present study we examined the effect of NFB signal blockade on
tumor metastasis through its regulation of prometastatic and
antimetastatic factors using mIB transfection in a murine
lung alveolar carcinoma cell line (Line 1). These cells were infected
with recombinant retroviruses containing the LacZ (control)
or mIB gene, and stably expressing clones were
obtained (17
, 18)
. This report provides evidence for
NFB as a major controller of the metastatic phenotpye through its
reciprocal regulation of known prometastatic and antimetastatic genes.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Retrovirus was produced using 293 kidney fibroblasts stably expressing
the Moloney gag and pol under control of the cytomegalovirus
promoter-enhancer as the packaging cell line. Twenty µg of
pLXSN retroviral vector containing: (a) either mIB
or the bacterial LacZ gene upstream of the neomycin
resistance gene; (b) 5 µg of plasmid pVSVG,
containing the vesicular stomatitis virus envelope downstream of the
cytomegalovirus promoter-enhancer; and (c) 1 µg of
pRSV ß-gal containing the bacterial LacZ gene
downstream of the Rous sarcoma virus promoter-enhancer to aid in
assessing transfection efficiency (in the case where the retroviral
vector contained mIB) were tranfected into the packaging cell line
by CaPO4 precipitation.
Line 1 tumor cells were transformed by retroviral infection with
recombinant virus-bearing mutant IB or the
bacterial LacZ gene and selected for 2 weeks with 0.5 mg of
G418 (Life Technologies, Inc., Gaithersburg, MD) as we have described
previously (18)
. G418-resistant clones were pooled to
constitute a heterogeneous population of mIB-expressing cells. This
measure excludes the confounding possibility of a random mutagenic
effect of transgene insertion.
NFB DNA-binding and Functional Activity.
EMSA was used to assess NFB DNA-binding by a method described
previously. Briefly, 5 µg of nuclear extracts from cells were mixed
with 2 µg of poly(dI-dC) and DNA-binding buffer [50 mM
NaCl, 5 mM HEPES (pH 7.5), 5 mM EDTA,
%mM EGTA, 30% Glycerol, and 1.25 µg BSA] in a total
volume of 10 µl and incubated on ice for 20 min. NFB and Oct1
oligonucleotides (Santa Cruz Biotech) were end-labeled by use of T4
polynucleotide kinase and [32P] cytosine
triphosphate (DuPont NEN) and 20,000 cpm of 32P-
labeled oligonucleotide added to the binding reaction and incubated for
30 min at room temperature. The complexes were then separated on 6%
polyacrylamide gel under nondenaturing conditions at 125 V for 3 h. Gels were dried on 3-M Whatman papers
and the DNA-protein complexes visualized by autoradiography. To
identify NFB subunits of the heterodimeric complex, EMSA supershifts
were done by preincubating nuclear extracts with antibodies specific
for each subunit (p50, p52, p65, c-rel antibody; Santa Cruz Biotech)
for 10 min on ice.
Functional NFB activity was assessed by cotransfecting 1.5 µg
pNFB-Luc (firefly luciferase driven by a TATA box with 5 NFB
sites in the enhancer element) and 2 ng of pRL-SV40 (renilla luciferase
driven by SV-40) reporter constructs (Promega) complexed to 3 µl of
Fugene transfection reagent (Boehringer Mannheim) in 100 µl of
serum-free culture medium. Three hundred thousand cells seeded in
six-well plates (Falcon) in 3 ml of culture medium were transfected by
adding 100 µl of the Fugene- plasmid complex and assaying for
luciferase activity 24 h after using the Dual Luciferase Reporter
Assay System (Promega) following the manufacturer’s instructions. The
EMSA and heterologous promoter/reporter assays were carried out on
cells with and without a 30-min stimulation with 20 nm PMA (Sigma) and
20 µg/ml ionomycin A23187 (Calbiochem), a commonly used regimen for
the induction of NFB in cultured cells through activation of the
protein kinase C/mitogen-activated protein kinase pathway
(3)
.
Assessment of Cell Proliferation.
Thymidine incorporation assays were used to assess cell proliferation
by a method described previously (19)
. Briefly, cell
cultures of 20,000 cells in 24-well plates (Falcon) were labeled with
8mCi/ml [3H]thymidine (New England Nuclear,
Boston, MA) in the presence of 5 mM of unlabeled thymidine
in culture medium. After 4 h of incubation, the DNA was
precipitated and centrifuged and the pellet redissolved in NaOH.
Radioactivity was determined by liquid scintillation spectrometry. All
samples were assayed in quadruplicate.
Zymography.
Zymography was used for the analysis of MMP and uPA activity secreted
into the culture medium of cell lines as described previously. Briefly,
a confluent 10-cm dish was maintained in 2 ml of serum-free medium for
24 h, after which the medium was concentrated 50-fold by
ultrafiltration (Centricon 10; Amicon, Stonehouse, Gloucester, United
Kingdom) before analysis. Aliquots of conditioned medium were then
separated on a 6% SDS-polyacrylamide gel copolymerized with 1 mg/ml
type-1 gelatin from porcine skin (Sigma) +/- 600 units of human
plasminogen (Calbiochem) under nondenaturing conditions. After
electrophoresis, the gels were incubated in 1% Triton x-100 for 1 h (to remove SDS from the gels) and subsequently incubated for 24 h in substrate buffer [50 µM Tris (pH 7.5) and 10
µM CaCl2] at 37°C.
After incubation, gels were stained in a solution containing 0.1% Coomassie Blue R250, 50% methanol, and 10% acetic acid for 1 h and destained in a solution of 30% methanol and 10% acetic acid to put into evidence clear bands of gelatinolytic activity on a blue background. Proteases were identified on the basis of their molecular weights using high molecular weight protein standards (Sigma) run in parallel with samples.
Western Blot Analysis.
Analysis of expression of uPA in culture medium was done by separating
10 µg of protein concentrate on a 10% SDS-polyacrylamide gel, with
subsequent transfer onto Immobilon-P membrane (Millipore, Bedford, MA).
Membranes were blocked and probed with polyclonal antibodies (Santa
Cruz Biotech) in 5% nonfat dried milk in a solution of PBS containing
0.1% Tween 20. After two 30-min washes in PBS-0.1% Tween 20, blots
were incubated with horseradish peroxidase-conjugated secondary
antibodies for 1 h followed by two 30-min washes in PBS/0.1%Tween
20. Immunoreactive bands were detected using the enhanced
chemiluminescence system (Amersham) after exposure of the membrane to
Hyperfilm ECL (Amersham) for 30–60 s.
RNA Isolation, RT-PCR Northern Blot Analysis.
RNA was isolated using Quaigen RNAeasy miniprep columns following the
manufacturer’s protocol and quantitated by UV absorption (Genequant).
Using oligonucleotide primers pair (5'-TTCGATCCCAAGAAGGAATCAAC-3' and
5'-GTAGTGATGCCATGTAACTGAATC-3') for murine heparanase, 1 µg of total
cellular RNA from Line 1 cells was reverse-transcribed and
PCR-amplified using Acces RT-PCR (Promega) following the
manufacturer’s instructions. PCR conditions for amplification were an
initial denaturation of 4 min at 94°C and subsequent denaturation for
45 s at 94°C, annealing for 1 min at 60°C and an extension for
1 min at 72°C (50 cycles). An aliquot of the PCR product (3 µl) was
then ligated into a PCR II plasmid vector (Invitrogen) following the
manufacturer’s protocol and subcloned into DH5 competent cells.
Probes for heparanase (generated from the PCRII vector), MMP 9, and
TIMP 1 and TIMP 2 (generous gifts from Dr. Dylan Edwards) were used for
Northern blot analysis. Briefly, total RNA isolated from cells was
subjected to 1% agarose gel electrophoresis with formaldehyde and
transferred to nylon membranes (Hybond N; Amersham); blots were
hybridized overnight with 32P-labeled cDNA probes
using the Quikhyb hybridization solution (Stratagene) at a temperature
of 68°C, washed twice for 15 min at room temperature with 2 x SSC buffer and 0.1% (w/v) SDS wash solution and once for 30
min at 60°C with 0.1 x SSC buffer and 0.1% (w/v) SDS
wash solution , and then overnight exposure to Biomax scientific
imaging films (Kodak) Oligonucleotide primer pairs
(5'-ACAGCAGATGGCCAAAGTGCT-3' and 5'-AGAACCTTCGGGTAGCAGGTT-3') for
plasminogen activator inhibitor 2 and (5'-ATGGATGACGATATCGCTGC-3' and
5'-GCTGGAAGGTGGACAGTGAG-3') for ß-actin were used for the
reverse transcription of 100 µg of total cellular RNA and PCR
amplification under the following conditions: (a) initial
denaturation at 90°C for 30 s; (b) annealing at
60°C (PAI 2) and 55°C (ß-actin) for 30 s ; and
(c) extension at 72°C for 1.30 min for 26 cycles.
Chick Embryo Metastasis Model.
The CAM of 9-day-old chick embryos were exposed by creating a window on
their eggshells. Tumor cells (1 x 106) were then seeded on the upper CAM and the
eggs maintained at 37°C in an egg incubator for 48–50 h. The upper
pole of the egg was then discarded and genomic DNA extracted from the
chorioallantoic membrane of the lower CAM to detect metastatic tumor
cells. Oligonucleotide primers pairs (5'-GGCGAGGTGGCACACGCCTTTATCCC-3')
and (5'-AGGGCTACACAGAAACCCTGTCTC-3') for mouse ALU were used to
detect tumor cell DNA in the genomic DNA extract from the lower CAM.
One µg of genomic DNA was PCR-amplified under the following
conditions: (a) 95°C for 10 min; (Ib) 95°C for 30 s; (c) 58°C for 45 s; (c) 72°C for
45 s; and (d) 72°C for 10 min. Samples were then
separated on a 2% agarose gel and the bands visualized by ethidium
bromide staining.
In Vivo Pulmonary Metastasis Model.
Thirty-six syngeneic BALB/CbyJ mice (Jackson Laboratories, Bar Harbor,
ME) were used to create 3 study groups of 12 each. All mice
received a single i.m. injection of 0.05 ml of suspension containing
2 x 105 Line 1 cells (Parental,
mIB, and LacZ control). At 25 days, the mice were euthanized by
CO2 asphyxiation. All mice then had their
tracheas canulated with a 21-gauge i.v. catheter under loupe
magnification and their lungs insufflated with 2.5 ml of India ink. The
lungs were dissected from the mice and fixed in Fekete’s solution
(61% ethanol, 32% formaldehyde, and 4.3% acetic acid) and the
primary tumors excised and fixed in 10% formalin. Lung metastasis,
evident as white nodules on a black background, were counted and
measured using a dissecting microscope and graduated measuring
reticule.
Immunohistochemistry.
Fixed and paraffin-embedded tissue specimens were deparaffinized,
rehydrated, denatured for 3 min in a microwave oven and treated to
block nonspecific staining. For this, sections were incubated for 30
min at 25°C with 1% H2O2
in methanol, and then by blocking for 30 min with 5% normal goat serum
in PBS. Polyclonal antibodies to MMP 9 and p65 were added (1:500
dilution) overnight at 25°C, and then by incubation for 30 min at
room temperature with biotinylated secondary goat-antirabbit IgG
antibodies and 30 min with avidin-biotin peroxidase conjugate (1:50
dilution). Color was developed using Sigma Fast 3,3-diaminobenzidine
tablet sets (Sigma) for 10 min and then counterstained with Mayer’s
hematoxylin.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B Signal Blockade in Tumor Cell Lines.B in blocking NFB signaling, we
analyzed the expression of IB and NFB activity in these cells
with and without stimulation by PMA and ionomycin (Fig. 1)
. Immunoblot analysis of cellular protein extracts showed that the
mIB clones contain elevated levels of IB protein, which are
insensitive to induced proteolysis that degrades the IB in the
parental cells (Fig. 1A)
. Extracts of nuclear proteins from
the transfectant and wild-type cells were analyzed by EMSA for NFB
binding activity with and without stimulation. As shown in Fig. 1B
, NFB-binding activity under basal conditions was
decreased in the mutant (Lane 3) in comparison with the wild
type (Lane 1), and the stimulation of NFB-binding
activity by PMA/ionomycin was absent (Lane 4). Oct-1
DNA binding, which was used as a constitutively expressed control, was
equivalent in all of these samples (Lanes 5–8). Supershift
assays revealed that the proteins in the shifted complex are the p50
and p65 isoforms of NFB in the wild-type cells (Lanes 9
and 11). The shift in p65 (Lane 11) appears as
decreased NFB-DNA binding complexes because of conformational
changes in the p65 subunit induced by antibody binding, that preclude
DNA binding. Additionally, we confirmed the differential
transactivational activity of NFB in the wild-type and mutant cell
lines by heterologous promoter-reporter assays. The basal NFB
activity was 7-fold lower in the mIB transfected cells, and there
was no inducible luciferase activity in these cells after PMA/ionomycin
stimulation (Fig. 1C)
. These data confirm the specific
suppression of NFB in the mIB-expressing Line 1 cells.
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B Signal Blockade on Cell Proliferation and the
Expression of Prometastatic and Antimetastatic Genes.B suppression affected cell
proliferation as determined by measurement of thymidine incorporation
into DNA (Average ± SE of four experiments:
Wild-type, 19,700 cpm ± 270; LacZ, 19,000
cpm ± 630; and mIB, 19,373 cpm ± 385). Northern blots for MMP9 and heparanase demonstrated markedly
lower levels of these transcripts in the mIB transfectants as
compared with the wild-type cells and the control LacZ transfectants.
Inhibition of uPA protein expression by mIB was also demonstrated by
immunoblot analysis of conditioned medium from the cell lines using an
antibody that recognizes the ß-chain of uPA, common to the inactive
and active uPA. (Fig. 2A)
. In contrast, Northern blot analysis of TIMPs 1 and 2,
known antimetastatic regulators of MMP activity (20)
,
demonstrated significantly higher levels of these transcripts in the
mIB-transfected cells compared with the wild-type. In the case of
TIMP 2, only the 3.5 kb of mRNA was regulated in this manner, whereas
the expression of the 1.0-kb transcript was not regulated. Similarly
PAI 2, a known antimetastatic regulator of uPA and the MMP activation
cascade (21)
was elevated in the mIB Line 1 cells as
determined by RT-PCR (Fig. 2B)
.
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B
transfectants (Fig. 3A)
. NFB inhibition thus reciprocally regulates
matrix-degrading enzymes and their inhibitors resulting in a
significant suppression of functional degradative enzymatic activity
known to be essential for tumor metastasis.
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B
expression on tumor cell intravasation in vivo using a chick
CAM model of metastasis (11)
. Wild type-, control LacZ-,
or mIB-transfected Line 1 cells were seeded on the CAM of day 9 old
chick embryos, and metastasis to the opposite pole of the CAM was
determined by PCR amplification of murine specific Alu sequences as
described in "Materials and Methods." The method demonstrated high
sensitivity to detect small numbers of metastatic cells in control
experiments (Fig. 3A)
. The wild-type and LacZ cell lines all
exhibited metastasis; in contrast there was no detectable metastasis in
any of the mIB-expressing transfectants (Fig. 3B)
.
Finally, the effect of NFB signal blockade was evaluated in an
in vivo murine model more closely simulating natural cancer
metastasis. In this model, the low class I HLA-DR expression in the
Line 1 cells allows transplantation in syngeneic, immunologically
normal BALB/c ByJ mice. After inoculation of tumor cells, all mice
developed large primary tumors at the injection site.
Immunohistochemical evaluation of the primary tumors using antibodies
that recognize the nuclear localization sequence of the p65 subunit of
NFB demonstrated suppression of activated (nuclear) p65 NFB (Fig. 4A
and 4B)
and MMP9 expression (Fig. 4C
and 4D)
in the mIB Line 1 tumors, confirming maintenance
of the mIB effect in vivo. The primary tumor weights were
not statistically different among the wild type-, LacZ-, and
mIB-transfected Line 1 cells (Fig. 4E)
. Thus, as expected
from the in vitro thymidine assays, there was no effect of
the transfection or mIB expression on cell proliferation in
vivo. However, we observed a marked suppression of pulmonary
metastatic nodules in the mice inoculated with the mIB cells (Fig. 4E
and 4F)
. Metastatic nodules, when present in
the mIB group, were smaller, and thus the total lung tumor burden
was also dramatically decreased in this group (data not shown). The
in vivo metastasis experiments have been reproduced with
similar findings for several different pooled populations of different
mIB clones, minimizing the possibility of clonal variation
accounting for the diminished metastatic capability of the mIB
cells.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Another enzyme critical for metastasis, the mammalian heparanase, has
been recently cloned and may represent the dominant endoglucuronidase
activity in mammalian tissues (14
, 15)
. Enhanced
expression is found in metastatic malignant tumors, and transfection of
heparanase into nonmetastatic cell lines enhances metastatic
capability. Furthermore, pharmacological inhibition of heparanase has
been shown to have a dramatic effect on reducing metastasis in a rodent
model (23)
. In the present study, we found that heparanase
was expressed in the Line 1 cells, and its expression was significantly
inhibited by blockade of the NFB signal transduction pathway. This
is the first demonstration of heparanase regulation by NFB, which
may contribute to the efficacy of the mIB transfection in
suppressing metastasis. The reciprocal up-regulation of antimetastatic
TIMPs 1 and 2 and PAI 2 by NFB, may further enhance the
antimetastatic effects of NFB signal blockade. In a previous report,
NFB up-regulated MMPs and proinflammatory cytokines in inflammatory
cells but did not up-regulate anti-inflammatory cytokines or MMP
inhibitors (4)
. However, the present study represents the
first identification of a reciprocal regulation of MMPs and TIMPs by
NFB in tumor cells. The NFB p50/p65 heterodimer identified as the
DNA binding complex in the Line 1 tumor cells is a transcriptional
activator, although the effect exerted on antimetastatic factors
appears to be inhibitory. Therefore, the reciprocal suppression of
antimetastatic genes may be an indirect effect mediated by an as-yet
unidentified other factor(s). This is further supported by the absence
of classic NFB-DNA binding sites in the promoters of TIMPs 1, 2 and
PAI 2.
NFB proteins have been implicated as playing a role in cellular
transformation by either providing continued positive growth stimuli
such as that mediated by cytokines, or through inhibition of apoptotic
pathways (24)
. The role of NFB in regulating tumor
growth remains unclear because of inconsistent findings among different
cell types, and may be cell-specific. In the present study, inhibition
of NFB did not influence tumor cell growth in vitro or
in vivo with the Line 1 cell model, and the observed effects
on phenotype were more specific to genes involved in metastasis. NFB
is known to stimulate the expression of a number of proinflammatory
cytokines such as tumor necrosis factor, IL-6, and IL-1, as well as the
degradative enzymes MMP 9 and uPA that may enhance tumor metastasis.
Because proinflammatory cytokines can, in turn, stimulate NFB
activation, a positive feedback loop could occur to sustain enhanced
levels of NFB activity, cytokine production, and tumor cell
degradative activity, thus sustaining the metastatic phenotype
(25)
. Through local cytokine production, tumor cells may
also recruit host stromal cells to participate in the metastatic
cascade. The fact that NFB inhibition markedly suppressed, but did
not eliminate, metastasis in the in vivo murine model may
reflect production of some of the essential proteins for metastasis by
host stromal cells stimulated by unknown mediators from the tumor cells
or local inflammatory processes. Alternately, it is possible that the
mIB expression in some of the tumor cells could be eliminated by
"in vivo promoter shut-off" of the long terminal
repeat. Consequently, these cells could revert to their
metastatic phenotype. Nonetheless, the lack of an effect of NFB
suppression on cell proliferation in vitro, and of tumor
growth in vivo, indicates a relatively specific role of
NFB in the metastatic aspects of malignancy rather than at the level
of growth regulation. Data from the chick CAM model of metastasis
suggest that tumor cells defective in NFB signaling are incapable of
intravasation, consistent with the observed suppression of metastatic
nodule development in the lungs of the mice inoculated with the
mIB-expressing cells.
In conclusion, the finding that a single factor (NFB) can
reciprocally regulate the expression of several different prometastatic
and antimetastatic genes implicates NFB as an important regulator of
the metastatic phenotype and implies that dysregulation of NFB in
tumor cells can have a dramatic impact on metastatic potential. In
other experiments, we have found similar effects of NFB signal
blockade on prometastatic and antimetastatic gene expression in
multiple clones of human prostatic carcinoma (PC3) and breast carcinoma
cells (MDA-MB 231; data not shown), suggesting that our findings
are not specific to either particular clones or cell types, and that
this regulatory pathway may play a more widespread role in the control
of metastasis. Our findings suggest that NFB may be an important
therapeutic target for the development of future antimetastatic cancer
treatments.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at University of Rochester Medical Center, 601 Elmwood
Avenue, Box 665, Rochester, NY 14642. Phone: (716) 275-3100; Fax:
(716) 756-4727; E-mail: Randy Rosier{at}urmc.rochester.edu 
2 The abbreviations used are: NFB, nuclear
factor B; IL, interleukin; MMP, matrix metalloproteinases; uPA,
urokinase-like plasminogen activator; EMSA, electrophoretic mobility
shift assay; RT-PCR, reverse transcription-PCR; CAM, chorioallantoic
membrane; PMA, phorbol 12-myristate13-acetate; EMSA, electrophoretic
mobility shift assay; TIMP, antimetastatic tissue inhibitors of matrix
metalloproteinases; PAI, plasminogen activator inhibitor.
Received 5/ 1/00. Accepted 10/16/00.
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma. Cancer Res., 59: 3495-3504, 1999.This article has been cited by other articles:
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 M. Jorda, D. Olmeda, A. Vinyals, E. Valero, E. Cubillo, A. Llorens, A. Cano, and A. Fabra Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor J. Cell Sci., August 1, 2005; 118(15): 3371 - 3385. [Abstract] [Full Text] [PDF]
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A. H. Gordon, R. J. O'Keefe, E. M. Schwarz, R. N. Rosier, and J. E. Puzas Nuclear Factor-{kappa}B-Dependent Mechanisms in Breast Cancer Cells Regulate Tumor Burden and Osteolysis in Bone Cancer Res., April 15, 2005; 65(8): 3209 - 3217. [Abstract] [Full Text] [PDF]
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 V B Andela, F Siddiqui, A Groman, and R N Rosier An immunohistochemical analysis to evaluate an inverse correlation between Runx2/Cbfa1 and NF{kappa}B in human osteosarcoma J. Clin. Pathol., March 1, 2005; 58(3): 328 - 330. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, H. Seino-Noda, et al. Inhibition of Inhibitor of Nuclear Factor-{kappa}B Phosphorylation Increases the Efficacy of Paclitaxel in in Vitro and in Vivo Ovarian Cancer Models Clin. Cancer Res., November 15, 2004; 10(22): 7645 - 7654. [Abstract] [Full Text] [PDF]
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 S. Yamamoto, Y. Tomita, Y. Hoshida, S. Toyosawa, H. Inohara, M. Kishino, M. Kogo, M. Nakazawa, S. Murakami, N. Iizuka, et al. Expression level of valosin-containing protein (VCP) as a prognostic marker for gingival squamous cell carcinoma Ann. Onc., September 1, 2004; 15(9): 1432 - 1438. [Abstract] [Full Text] [PDF]
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C. N. Papandreou and C. J. Logothetis Bortezomib as a Potential Treatment for Prostate Cancer Cancer Res., August 1, 2004; 64(15): 5036 - 5043. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, M. Saito, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, et al. Inhibition of NF{kappa}B Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models J. Biol. Chem., May 28, 2004; 279(22): 23477 - 23485. [Abstract] [Full Text] [PDF]
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 S. Bien, C. A. Ritter, M. Gratz, B. Sperker, J. Sonnemann, J. F. Beck, and H. K. Kroemer Nuclear Factor-{kappa}B Mediates Up-Regulation of Cathepsin B by Doxorubicin in Tumor Cells Mol. Pharmacol., May 1, 2004; 65(5): 1092 - 1102. [Abstract] [Full Text] [PDF]
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 S. Yamamoto, Y. Tomita, Y. Hoshida, S. Takiguchi, Y. Fujiwara, T. Yasuda, M. Yano, S. Nakamori, M. Sakon, M. Monden, et al. Expression Level of Valosin-Containing Protein Is Strongly Associated With Progression and Prognosis of Gastric Carcinoma J. Clin. Oncol., July 1, 2003; 21(13): 2537 - 2544. [Abstract] [Full Text] [PDF]
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T. Karashima, P. Sweeney, A. Kamat, S. Huang, S. J. Kim, M. Bar-Eli, D. J. McConkey, and C. P. N. Dinney Nuclear Factor-{kappa}B Mediates Angiogenesis and Metastasis of Human Bladder Cancer through the Regulation of Interleukin-8 Clin. Cancer Res., July 1, 2003; 9(7): 2786 - 2797. [Abstract] [Full Text] [PDF]
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G. Helbig, K. W. Christopherson II, P. Bhat-Nakshatri, S. Kumar, H. Kishimoto, K. D. Miller, H. E. Broxmeyer, and H. Nakshatri NF-{kappa} B Promotes Breast Cancer Cell Migration and Metastasis by Inducing the Expression of the Chemokine Receptor CXCR4 J. Biol. Chem., June 6, 2003; 278(24): 21631 - 21638. [Abstract] [Full Text] [PDF]
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C. Belluco, F. Olivieri, M. Bonafe, S. Giovagnetti, E. Mammano, R. Scalerta, A. Ambrosi, C. Franceschi, D. Nitti, and M. Lise -174G>C Polymorphism of Interleukin 6 Gene Promoter Affects Interleukin 6 Serum Level in Patients with Colorectal Cancer Clin. Cancer Res., June 1, 2003; 9(6): 2173 - 2176. [Abstract] [Full Text] [PDF]
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J. C. Hodge, J. Bub, S. Kaul, A. Kajdacsy-Balla, and P. F. Lindholm Requirement of RhoA Activity for Increased Nuclear Factor {kappa}B Activity and PC-3 Human Prostate Cancer Cell Invasion Cancer Res., March 15, 2003; 63(6): 1359 - 1364. [Abstract] [Full Text] [PDF]
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M. H. Ma, H. H. Yang, K. Parker, S. Manyak, J. M. Friedman, C. Altamirano, Z.-q. Wu, M. J. Borad, M. Frantzen, E. Roussos, et al. The Proteasome Inhibitor PS-341 Markedly Enhances Sensitivity of Multiple Myeloma Tumor Cells to Chemotherapeutic Agents Clin. Cancer Res., March 1, 2003; 9(3): 1136 - 1144. [Abstract] [Full Text] [PDF]
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J. H. Wang, B. J. Manning, Q. D. Wu, S. Blankson, D. Bouchier-Hayes, and H. P. Redmond Endotoxin/Lipopolysaccharide Activates NF-{kappa}B and Enhances Tumor Cell Adhesion and Invasion Through a {beta}1 Integrin-Dependent Mechanism J. Immunol., January 15, 2003; 170(2): 795 - 804. [Abstract] [Full Text] [PDF]
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