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J. Biol. Chem., Vol. 278, Issue 32, 29776-29782, August 8, 2003
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B Complex in P19 Mouse Embryonal Carcinoma Cells*
From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Received for publication, March 31, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B. SNP treatment reduces the DNA
binding ability of the constitutively active NF-B heterodimer, p65/p50,
and its consequent transactivation of the c-myc promoter. Repression
can be blocked by the peroxynitrite scavenger, deferoxamine, but not by
dithiothreitol, which triggers reduction of S-nitrosylated residues.
In HEK293 cells, where tumor necrosis factor-
can activate NF-B,
SNP likewise suppresses the binding of the active NF-B complex,
restoring the binding of the repressive p50/p50 homodimer complex. This effect
of SNP in HEK293 cells is also blocked by deferoxamine. Chromatin
immunoprecipitation analysis of SNP-treated P19 cells reveals reduced
association of p65, but not of p50, with the promoter region of the endogenous
c-myc gene. SNP-induced p65 dissociation was associated with the
recruitment of histone deacetylase 1 and 2 to the endogenous c-myc
gene promoter and the subsequent deacetylation of its chromatin histone. This
study is the first to demonstrate that NO modulates the transcriptional
activity of the c-myc gene promoter by dissociating the active form
of NF-B and replacing it with a repressive NF-B complex,
correlated with the recruitment of gene-silencing histone deacetylases. In
light of findings that NF-B stimulates Myc oncoprotein expression in
cancers, our findings suggest that NO should be investigated as a prospective
therapeutic cancer agent. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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c-Myc is a transcription factor of the basic helix-loop-helix leucine zipper family. It forms a heterodimer with Max on the E-box of target gene promoters to activate transcription. In contrast, a heterodimer of Max and Mad binds to the E-box to suppress transcription. A functional role for c-Myc has been established in a wide variety of cellular processes including proliferation, metabolism, apoptosis, differentiation, genomic stability (11–14), and cell cycle progression from quiescent to synthetic phases (15, 16). Our recent study of P19 stem cells presented evidence that c-Myc functions as an activator for the mouse KOR gene by binding to its promoter. We also showed that NO suppressed KOR expression by rapidly down-regulating c-Myc protein synthesis in P19 cells (10). This result prompted us to investigate the mechanisms underlying rapid down-regulation of c-Myc by NO in P19 cells.
Regulation of c-myc gene expression has been shown to occur at
multiple levels, including gene transcription, premature termination of
translation (17,
18), and translocation
(19,
20). In Burkitt's lymphoma the
c-myc gene is translocated to the immunoglobulin heavy chain gene
loci on either chromosome 2, 14, or 22. Transcription of the mouse gene can be
initiated from two promoters, P1 and P2, separated by 
160 bp. P2 is the
major promoter where several transcription factors and regulatory DNA elements
have been identified. For instance, three cis-acting elements for
ME1a2, E2F, and ME1a1 have been found in P2
(21,
22). The pituitary
tumor-transforming gene product can bind to a region near the P2 initiation
site as a protein complex containing the upstream stimulatory factor-1
(23). A Smad-responsive
element has been shown to be responsible for transforming growth
factor- mediated suppression of c-myc transcription
(24). An intron IX box is
known to be involved in leukemia HL-60 differentiation
(25,
26). In addition, two
functional NF-B elements have been identified in both the human and the
murine c-myc genes
(27–29),
one located from —1261 to —1251 in the upstream region of P2 and
the other located from +280 to +289. Importantly, transcription of the
translocated c-myc gene in Burkitt's lymphoma is stimulated by
constitutively expressed NF-B
(19). Our finding of rapid
repression of c-Myc by NO in P19 cells
(10) and the identification of
two NF-B binding sites in the c-myc promoter prompted us to
examine the potential role of NF-B in mediating NO regulation of
c-myc in P19 cells.
NF-B is an inducible transcription factor. Five members of
NF-B are known, including p50 (NF-B1), p52 (NF-B2),
c-Rel, RelB, and p65 (RelA). The major active form of this transcription
factor is the p65/p50 heterodimer
(30–32).
In most cells, NF-B complexes are predominantly cytoplasmic and are
usually associated with an inhibitory protein, the IB family. Upon
stimulation by pro-inflammatory cytokines such as tumor necrosis
factor- (TNF-) or interleukin-1, NF-B dimer dissociates
from IB and is translocated into nuclei to act on the target
gene (33,
34). Translocation into nuclei
is considered to be a general, underlying mechanism for the activation of
NF-B in gene regulation. It has been shown that NO can inhibit
NF-B by modifying the p50 subunit via redox-based
S-nitrosylation of a cysteine residue in its N-terminal region. This
effect of NO is sensitive to dithiothreitol (DTT)
(4,
35,
36). In our previous study,
the effect of NO on c-Myc in P19 cells was not blocked by DTT
(10), indicating the
involvement of other NO-triggered mechanisms in the regulation of NF-B
activity.
In this study we found that a NO donor sodium nitroprusside (SNP) repressed
the DNA binding and activating function of the constitutively active p65/p50
form of NF-B in P19 cells. Although not affected by DTT, it was blocked
by deferoxamine (DFO), a peroxynitrite scavenger. Similarly, the NO donor also
repressed TNF--activated NF-B in cells where NF-B
normally existed as an inactive complex. The effect of NO in these cells was
likewise blocked by DFO. We then confirmed the effect of NO on NF-B and
the c-myc promoter by examining the dynamic behavior of the
NF-B complex and histone deacetylases (HDACs), and chromatin
acetylation on the endogenous c-myc promoter. This study
demonstrated, for the first time, that NO modulates the transcription of the
c-myc gene by inducing the dissociation of p65/p50 NF-B from
the c-myc promoter region and the recruitment of the repressive p50
homodimer complex, in correlation with increased recruitment of HDACs and
decreased histone acetylation on the endogenous c-myc promoter.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B specific
reporter construct, p(Igk)4-LUC, that contains four tandem repeats
of NF-B binding sites was a gift from Dr. N. Mackman (Scripps
Institute) (39). Cell Culture and Transient Transfection—P19 embryonal carcinoma cell and COS-1 cell lines were maintained as described previously (10). HEK293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% nonessential amino acids. Transient transfection was performed in P19 and COS-1 cells using the calcium chloride method, and reporter assays were conducted as described previously (40).
Analyses of c-myc mRNA—The endogenous c-myc mRNA was analyzed by a reverse transcriptase-coupled polymerase chain reaction (RT-PCR) as described (10). Total RNA was isolated (by using a Trizol® kit (Invitrogen)) from P19 cells which were pretreated with 2 µg/ml actinomycin D (ActD) or 2 µg/ml cycloheximide. Five mM DTT or 0.1 mM DFO was added for 30 min prior to the addition of 0.5 mM SNP for 6 h. To determine c-myc mRNA stability, P19 cells were treated with 2 µg/ml ActD for different durations in the presence of 1 mM SNP for 6 h. Primers specific to c-myc mRNA for the amplification procedure are 5'-CCATATGCCCCTCAACGTGAAC-3' and 5'-GGGATCCTTATGCACCAGAGTTT-3', which span a 1350-bp coding region.
Electrophoretic Mobility Shift Assay (EMSA) and Western
Blot— Nuclear extracts and whole cell lysates were isolated from
P19 cells treated as above. HEK293 cells were treated with 10 ng/ml
TNF- for 8 h to activate NF-B, followed by the addition of SNP
for 6 h. EMSA was performed as described
(41). Four types of
double-stranded oligonucleotides (upstream responsive element (URE), internal
responsive element (IRE), mt-URE, and nonspecific KOR gene promoter sequences)
(see Fig. 2) were each used as
probes that were labeled with [-32P]dCTP or used as
competitors without labeling. Whole cell lysates were analyzed on a Western
blot by using anti-c-Myc antibody as described
(10).
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Chromatin Immunoprecipitation (ChIP) Assays—P19 cells were treated with SNP or SNP plus DFO for 6 h (or in a time-dependent manner), followed by cross-linking with 1% formaldehyde. ChIP assay was as described previously (10) by using 2 µg of anti-acetylated histone H4, anti-p65, anti-p50, anti-HDAC1, or anti-HDAC2 antibodies. Precipitated DNA was amplified with primers specific to the URE flanking sequences in the endogenous c-myc promoter and to its 3'-untranslated region (UTR), followed by Southern blot analyses of amplified endogenous c-myc sequences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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20
min in both control cells (lanes 1–5) and cells exposed to NO
(lanes 6–10). Therefore, c-myc mRNA stability is not
affected by NO, whereas its gene transcription is directly repressed by NO
signal.
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Repression of c-myc Gene Transcription by NO through Inactivation of
NF-B Activity—To define the target of NO-mediated
repression of c-myc gene transcription, the regulatory regions of
c-myc gene were carefully examined. This gene utilizes two promoters
located 160 bp apart, with the second promoter residing in exon 1.
Interestingly, two putative NF-B binding sites, an URE (GGGTTTCCCC)
located at —1261 to —1251 bp in the 5'-flanking region of
promoter 1 and an IRE (GGGAATTTTT) located at +280 to +289 relative to the
promoter P2 (27,
28), were found to be relevant
(Fig. 2A). NF-B
has been shown to be a target protein of NO signal
(3,
42). We examined how the
endogenous NF-B activity in P19 cells might be affected by NO and
augment c-myc gene expression. P19 cells were treated with SNP,
followed by analyzing its nuclear extract for DNA binding ability to the
B binding sites of the c-myc promoter in EMSA. As shown in
Fig. 2B, the
32P-labeled URE probe revealed a major retarded band and several
minor bands (lane 1), which could be competed out specifically by the
wild type cold probe (lane 2) but not by the mutated cold probe
(mt-URE, lane 3) or nonspecific oligonucleotides (lane 4).
The anti-p65 antibody was able to block the major binding species that is the
heterodimeric p65/p50 NF-B complex (lane 5), and the anti-p50
antibody was able to supershift the minor species, the p50 complex (lane
6). The control, a nonspecific antibody such as the anti-Sp1, had no
effect (lane 7). Therefore, the URE consensus sequences of the
c-myc gene indeed can be bound by p65/p50 heterodimer and p50
homodimer of NF-B, but the major complex in P19 cells is the
transcriptionally active p65/p50 heterodimer. The second putative NF-B
binding site, IRE, was also tested in EMSA as shown in
Fig. 2C, which
appeared to share a very similar binding pattern as that of the URE.
Furthermore, SNP decreased the intensity of the p65/p50 NF-B band but
increased the p50 complex for both NF-B sites (lanes
1–4), revealing a decreased activator p65/p50-DNA interaction of
P19 nuclear extract as a result of SNP treatment.
To determine whether the transcriptional activity of endogenous NF-B
was affected by NO, transient transfection assays were conducted in P19 stem
cells and fully differentiated COS-1 cells by using a specific NF-B
reporter, p(Igk)4-LUC
(39), without introducing any
other expression vectors. As shown in Fig.
3A, SNP effectively suppressed the specific reporter
activity in a dose-dependent manner in P19 cells but not in COS-1 cells, which
is consistent with our previous conclusion that NO suppressed KOR gene
transcription only in P19 cells but not in COS-1 cells
(10). This suppressive effect
of NO on NF-B reporter plasmid in P19 cells agreed with the reduced DNA
binding activity of NF-B from SNP-treated P19 cells
(Fig. 2C). The
resistance of the same reporter activity to SNP in COS-1 cells was because of
the absence of p65/p50 species binding to the URE in COS-1 cells that
contained primarily the p50 species binding to the URE site (data not shown).
To further confirm that the effect of SNP was indeed mediated by p65/p50
NF-B, the specific reporter and a pMT2T-p65 expression vector
(37) were simultaneously
introduced into COS-1 cells (Fig.
3A). Indeed, ectopic expression of p65 increased the
specific reporter activity in COS-1 cells, which was again suppressed by SNP
in a dose-dependent manner (Fig.
3B). Thus, NO not only suppresses the DNA binding
activity of p65/p50 NF-B, but also efficiently reduces NF-B
activity in transcriptional control. The effect is primarily at the p65
subunit.
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Repression of c-myc Gene Transcription by NO through
Peroxynitrite—Previously we have suggested that
S-nitrosylation was not involved in c-myc repression by SNP,
a NO donor, in P19 cells because this suppressive effect could not be reversed
by DTT, an agent that blocks S-nitrosylation of Cys residues by NO
(10). Although it is accepted
that NF-B activity can be inhibited by NO through
S-nitrosylation on a Cys residue of its p50 subunit
(35,
36), the failure of DTT to
reverse the repressive effects of SNP in P19 cells raised the question as to
what other pathway could be involved in this novel suppressive effect of SNP
in P19 cells. The alternative modification elicited by NO is Tyr nitration
triggered by the highly reactive peroxynitrite (ONOO—)
generated from NO and superoxide
(4). To test this possibility,
we treated P19 cells with DTT or DFO that is known to scavenge peroxynitrite,
followed by examining the expression of c-myc mRNA as shown in
Fig. 4. DTT failed to reverse
the repressive effect of SNP on c-myc transcription
(Fig. 4A, lane
3), whereas DFO efficiently blocked the repressive effect of SNP
(lane 4). Consistently, c-Myc protein expression
(Fig. 4B) was
repressed by SNP (lane 2), which was effectively reversed by DFO
(lane 4) but not by DTT (lane 3). The potent cGMP analog
8-bromo-cGMP and guanylate cyclase inhibitor
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one affected neither
c-myc mRNA nor its protein level (data not shown). To confirm whether
peroxynitrite was indeed responsible for inactivating NF-B activity,
transient transfection assays were performed using the specific reporter,
p(Igk)4-LUC, in P19 cells. As shown in
Fig. 4C, DTT failed to
reverse the repressive effect of SNP on the NF-B reporter, but DFO very
efficiently reversed the repression to 75% of the control level. These
results have ruled out the S-nitrosylation and cGMP-dependent
pathways and suggested that peroxynitrite generated from NO could play a role
in the repression of c-myc transcription in P19 cells, which may
involve nitration of NF-B.
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It was noted that changes had occurred in the DNA binding pattern of
endogenous NF-B and p50 homodimer in P19 cells following SNP treatment
(Fig. 1C). To further
confirm this point, we made use of HEK293 cells where the endogenous
NF-B existed primarily as the inactive complexes and could be activated
by TNF-. By testing DNA binding patterns of HEK293 extract as shown in
Fig. 5, the portion of
heterodimeric NF-B increased in response to TNF- stimulation as
predicted (lane 2), which was blocked by SNP and replaced with the
p50/p50 homodimer complex after SNP treatment (lane 3). Furthermore,
the SNP-triggered blockage of NF-B heterodimer formation was again
reversed by DFO (lane 4). The identity of p65/p50 heterodimer and
p50/p50 homodimer was again confirmed by using specific antibodies (lanes
5–8) that either blocked (for p65/p50 heterodimer) or supershifted
(for p50/p50 homodimer) the complexes. This result confirms that indeed SNP
triggers the dissociation of the p65/p50 complex from, and the association of
p50 complex with, the target DNA, consistent with the effects of SNP and DFO
in the regulation of c-myc gene, as well as its reporter, in P19
cells (Fig. 4).
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The results of the two cell lines have demonstrated that peroxynitrite
indeed disrupts DNA binding of the activated NF-B, i.e. the
p65/p50 heterodimer, which can be blocked by DFO, a scavenger of
peroxynitrite.
Recruitment of HDACs 1 and 2 to the Endogenous c-myc Promoter by SNP
Treatment—The activity of transcription factors can be modulated by
altering the recruited coregulators in addition to changes in DNA binding
ability. For the repressive activity of p50 homodimer, the recruitment of
HDACs has been shown to play a major role
(43,
44). To confirm the effect of
SNP and thus replacement of p65/p50 with p50/p50 on its target gene, the role
of HDAC activity was examined. P19 cells were transfected with the specific
NF-B reporter p(Igk)4-LUC and treated with SNP, trichostatin
A (TSA), or both. As shown in Fig.
6, trichostatin A alone induced this reporter (lanes 4
and 5) and rescued the repressive effect of SNP (lanes
1–3 and 6–8), confirming the involvement of HDACs in
the repressive activity of NO on the transcription of the NF-B target
gene.
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To further examine the association pattern of HDACs with the affected
promoter in cells treated with SNP, ChIP assay was performed. This experiment
was conducted to examine histone acetylation
(Fig. 7A) status and
the association of p65, p50, and HDACs
(Fig. 7B) with the
endogenous c-myc gene promoter in P19 cells, mediated through the
NF-B binding site URE. The top panel in
Fig. 7A shows the
relative primer positions used to amplify the endogenous c-myc
sequences surrounding the URE site, as well as its 3'-UTR, which was
precipitated in the immune complexes. As shown in
Fig. 7A, middle
panel, treatment of P19 cells with SNP caused a dramatic decrease in the
level of histone acetylation on the endogenous c-myc sequences
flanking the URE site (lane 3), and the addition of DFO completely
recovered histone acetylation in this region (lane 4). As a control,
histone acetylation was not detected in its 3'-UTR (bottom
panel). Fig. 7B
showed that in control cells, p65 and p50 were associated with the endogenous
c-myc promoter regions flanking the URE site (top left
panel), but not the 3'-UTR (top right panel). In
SNP-treated cells, p65 but not p50 association was dramatically reduced. By
using anti-HDAC1 and anti-HDAC2 antibodies in ChIP assays, it was apparent
that both HDAC1 and HDAC2 were recruited to the endogenous c-myc
promoter regions, but HDAC1 appeared to be preferentially recruited
(bottom panels). Thus, SNP treatment triggers dissociation of p65,
but not p50, from the endogenous c-myc promoter correlated with
increased HDAC recruitment to and decreased histone acetylation of this
promoter.
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To follow the dynamic behavior of transcription complexes on the endogenous c-myc promoter immediately after exposure to SNP, ChIP assays were conducted to examine p65 and acetylated histones of the endogenous c-myc promoter every 30 min following SNP treatment. The dissociation of p65 subunit from, and histone deacetylation of, the endogenous c-myc promoter started between 90 and 120 min following SNP treatment (Fig. 7C). Thus, SNP treatment triggers early cofactor exchange on the endogenous c-myc promoter, in particular the dissociation of p65 and recruitment of HDACs, resulting in rapid histone deacetylation of this promoter.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B is an inducible transcription factor that has attracted
considerable attention because of its role in a wide variety of cellular
processes. It is the major player in immune and stress responses and, in
particular, NO-elicited pathophysiological effects
(32,
33). It is generally believed
that S-nitrosylation of the p50 subunit is responsible for the effect
of NO on NF-B activity
(3,
35,
36,
42). Our study presents the
first evidence for a potentially important role for NO-induced modification of
p65/p50 NF-B activity, which is shown here to modulate c-myc
gene transcription. We propose that SNP exerts at least two important effects
on NF-B, i.e. reduced DNA binding of the p65/p50 heterodimer
and replacement of the p65/p50 with the repressive p50/p50 homodimer, leading
to enhanced recruitment of HDACs. Our study also reveals, for the first time,
that the proto-oncogene c-myc is an endogenous NF-B target
gene, as demonstrated by ChIP analyses of the endogenous c-myc gene
promoter.
The effect of NO to inhibit NF-B activity and subsequently repress
c-myc transcription in P19 cells, but not COS-1 cells, suggests that
NF-B is constitutively active in P19 stem cells but is inactive in
differentiated COS-1 cells. Consistent with this assumption, P19 cells show
constitutive histone hyperacetylation of the c-myc gene promoter
regions surrounding the B sites
(Fig. 7). As such, neither
TNF- nor lipopolysaccharide could further stimulate NF-B
activity in P19 cells (data not shown). EMSA studies reveal that in P19 stem
cells where the c-myc gene is constitutively active, the p65/p50
heterodimer is the major species of NF-B complex that binds to the
B binding sites on the c-myc promoter
(Fig. 2). This observation may
be physiologically relevant as elevated NF-B activity is detected in
numerous solid tumor cell lines and human cancers
(45–48).
In contrast, the p50 homodimer appears to be the major complex binding to the
target promoter in fully differentiated HEK293 cells
(44) where NF-B is
normally inactive and where TNF- is able to trigger the formation of
the active p65/p50 heterodimer (Fig.
5). In COS-1 cells where most endogenous NF-B subunits are
also of the p50/p50 species, ectopic expression of p65 dramatically elevated
NF-B reporter gene activity, indicating that additional p65 activates
NF-B (Fig. 3).
Furthermore, this activation was suppressed by the NO donor SNP. Therefore,
SNP exerts a similar repressive effect on both constitutive and
TNF--stimulated active p65/p50 NF-B complex, leading to the
dissociation of NF-B from its target DNA and the replacement with the
repressive p50/p50 complex.
NF-B activity can be modulated by a number of events, including
changes in the status of the inhibitory protein IB, its shuttling
between the cytoplasm and the nucleus, and several types of protein
modification such as phosphorylation
(49–51),
acetylation (52,
53), and
S-nitrosylation (35,
36). It is generally believed
that the primary mode of NF-B modification by NO is through
S-nitrosylation of the p50 subunit and subsequent reduction in its
DNA binding ability. However, because SNP exerted no effects on COS-1 cells,
where p50/p50 homodimers represent the major species of NF-B (data not
shown), it is tentative to speculate that the p65/p50 heterodimer, not the
p50/p50 homodimer, may be the preferred target of SNP-elicited suppression.
Moreover, DTT could not reverse SNP repression of c-myc, reporter
gene activities, or DNA binding activity of NF-B (Figs.
4 and
5), but a peroxynitrite
scavenger, DFO, was able to do so (Fig.
4). These results rule out S-nitrosylation as the
underlying mechanism and suggest that Tyr nitration could be the actual
underlying mechanism for this novel effect of NO. It is proposed that an
alternative pathway of protein modification by NO, possibly through Tyr
nitration of the p65 subunit of NF-B, is able to rapidly inactivate
NF-B activity. We are in the process of determining specific Tyr
residue(s) of NF-B that could be NO targets.
NO is known to be involved in numerous pathophysiological processes. In
particular, Tyr nitration of various proteins by peroxynitrite has been
detected in a number of inflammatory or degenerative diseases
(54,
55) and tumors
(9,
56). As such, it has generated
enthusiasm for managing diseases with agents that modulate NO signaling
pathways. NF-B is an important transcription factor that coordinates
the expression of a wide variety of genes
(32) and is known to be an
inhibitor of apoptosis because of its ability to induce anti-apoptotic factors
such as cellular inhibitors of apoptosis and the members of the BCL2 family
(58,
59). The c-Myc protein is
involved in numerous important cellular processes, including proliferation,
metabolism, cell cycle control, apoptosis, differentiation, genomic stability,
and tumor formation
(11–13,
57,
60). The ability of a NO
donor, SNP, to directly and effectively repress NF-B activity provides
a potentially important route for therapeutic intervention of numerous disease
conditions. In particular, its rapid transcriptional repression of the
c-myc gene via inactivating NF-B could provide an efficacious
strategy for the use of NO donors in managing tumors.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Pharmacology, University
of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St. SE,
Minneapolis, MN 55455. Tel.: 612-625-9402; Fax: 612-625-8408, E-mail:
weixx009{at}tc.umn.edu.
1 The abbreviations used are: NO, nitric oxide; SNP, sodium nitroprusside;
DTT, dithiothreitol; DFO, deferoxamine; HDAC, histone deacetylase;
TNF-, tumor necrosis factor-; URE, upstream responsive element;
IRE, internal responsive element; ActD, actinomycin D; RT-PCR, reverse
transcriptase-coupled polymerase chain reaction; ChIP, chromatin
immunoprecipitation; UTR, untranslated region; KOR, kappa opioid receptor;
EMSA, electrophoretic mobility shift assay.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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