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Department of Microbiology, Immunology, and Molecular Genetics, and Jonsson Comprehensive Cancer Center, University of California School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent NO
generation increases the trans-activation of the Fas
promoter, and this increase was blocked by the NOS inhibitor
(NG-monomethyl-L-arginine), but
could be restored by the addition of the NO donor
S-nitroso-N-acetylpenicillamine.
Systematic deletion of the Fas promoter revealed that the functional
region responsible for the NO-mediated effect was located at the
silencer region, suggesting that NO may be responsible for the
disruption of a repressor mechanism. We demonstrate that NO
up-regulates the expression of the Fas receptor on AD10 cells via the
specific inactivation of the transcription repressor yin-yang 1 DNA
binding activity to the silencer region of the Fas promoter. These
findings reveal a new mechanism of NO-mediated gene regulation by
interfering with a repressor transcription factor at the silencer
region of the Fas promoter. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
and by the activation of lymphocytes (2, 4, 5). We have demonstrated that one of the cellular mechanisms by
which IFN- sensitizes the human ovarian carcinoma cell line AD10 to
Fas-mediated apoptosis is mediated by the induction of inducible NO
synthase (iNOS)3 and
subsequent generation of NO in the tumor cells. The generation of NO
correlated with an increased expression of the Fas gene at both the
transcriptional level and the surface protein level (6).
It has been shown that NO increases expression of the Fas receptor in
aortic vascular smooth muscle cells and correlates with increased
sensitivity to Fas-mediated apoptosis. Further, up-regulation of Fas
expression by NO was shown to be a cGMP-independent mechanism
(7). However, the molecular mechanism of the interaction
of NO with the Fas transcriptional machinery remains elusive. Thus, we
hypothesized that NO, directly or indirectly, might modify the
transcriptional machinery toward increased expression of the Fas
gene.
Functional analysis of the 5'-flanking region of the Fas gene has
revealed three major regions within the 
2000 bp upstream of the
translational initiation site. The Fas promoter exhibits silencer
activity residing between nucleotide positions -1781 and -1007 and a
strong enhancer region between -1007 and -425. A basal promoter
activity resided in the region between -425 and -1 (8).
Several putative transcription factor binding sites have been
identified by sequence comparison (8, 9, 10), but the
functional analysis of the Fas promoter is not completely
understood.
In this study we aimed to determine 1) whether NO is implicated in the
molecular mechanism of the IFN--mediated up-regulation of Fas
expression on AD10 cells, 2) the specific functional region of the Fas
promoter that is responsible for the stimulatory effect of NO, and 3)
the identity of the putative NO-sensitive transcription factor(s) and
its mechanism(s) of action toward the regulation of Fas expression in
AD10 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The AD10 cell line is an adriamycin-resistant, multidrug
resistant phenotype-expressing, subline derived from the human
ovarian carcinoma cell line A2780 and was obtained from Dr. R. Ozols
(Fox Chase Cancer Center, Philadelphia, PA). The PC-3 cell line is a
metastatic bone-derived human prostatic adenocarcinoma obtained from
American Type Culture Collection (Manassas, VA), CRL-1435. Cell
cultures were maintained as previously described (6). For
every experimental condition cells were cultured in 1% FBS 24 h
before treatments.
S-nitroso-N-acetylpenicillamine (SNAP) was
provided and synthesized by Dr. J. Fukuto (University of California,
Los Angeles, CA). DEA-NONOate (diethylamine NONOate) and DETA-NONOate
(diethylenetriamine NONOate) were purchased from Cayman Chemical Co.
(Ann Arbor, MI). For iNOS induction, cultured cells were stimulated
18 h with 100 U human recombinant IFN- (PeproTech, Rocky Hill,
NJ). NOS activity was blocked by incubation with 1 mM
NG-monomethyl-L-arginine
(L-NMA; Alexis, San Diego, CA) 18 h before
IFN- treatment. DTT (Life Technologies, Gaithersburg, MD) was added
directly to the nuclear extracts where indicated.
Flow cytometry
Surface Fas Ag expression on tumor cells was determined by flow cytometry as previously reported (6).
Plasmids constructs and reporter system
The human Fas promoter containing 1781 bp upstream of the translation initiation site was amplified by PCR using the forward primer AAG CTT TTT TGG CTA CAT TTT T and the reverse primer GGT TGT TGA GCA ATC CTC CGA A. The genomic DNA extracted from cultured AD10 cells was used as a template. PCR were conducted using the Advantage-HF 2 PCR kit (Clontech, Palo Alto, CA) following the manufacturer’s recommendations. The PCR product was gel-purified and incubated 30 min with Taq polymerase (Life Technologies) in the presence of 50 µM dATP for adenosine tailing. The resultant fragment was ligated to pCR2.1 TA vector (Invitrogen, Carlsbad, CA), resulting in generation of the pCR-FasP construct. The cloned Fas promoter was released from the pCR-FasP construct by digestion with SpeI and XbaI and ligated to pGL3/Basic (Promega, Madison, WI) that had been digested with NheI and BglII, yielding the pGL3-FasP construct. We further generated two constructs deleting two functional region upstream of the Fas gene core promoter, pGL3-FasEC (silencer deleted) and pGL3-FasC (silencer and enhancer deleted). Two forward primers with the sequences pAGT AAT GAT GTC ATT ATC CAA ACA TAC C (flanking the enhancer region of the Fas gene promoter -1007) and pCTG CAG GAA CGC CCC GGG ACA GGA ATG C (flanking the Fas gene core promoter region -425) and a universal reverse primer with the sequence pACG CGT AAG AGC TCG GTA CC (located in the pGL3 vector) were used in PCR as described above. The resulting amplified products were circularized and replicated after being transformed into competent TOP10-F' Escherichia coli strain. The truncations were confirmed by automated sequencing and restriction digestion.
Transcriptional elements search
Specific DNA sequences from GenBank comprising the Fas/CD95 5'-flanking region (accession no. X87625 and D31968) and functional regions derived from them (8) were analyzed for the presence of potential trans-acting elements using Transcription Element Search software on the Internet (Computational Biology and Informatics Laboratory, University of Pennsylvania School of Medicine, Philadelphia, PA; http://www.cbil.upenn.edu/tess/).
RT-PCR
Total RNA was extracted and purified from 1 x
106 cells for each experimental condition by a
single-step monophasic solution of phenol and guanidine
isothiocyanate-chloroform using TRIzol reagent (Life Technologies). One
microgram of total RNA was reverse transcribed to first-strand cDNA for
1 h at 42°C with 200 U SuperScript II reverse transcriptase and
20 µM random hexamer primers (Life Technologies). Amplification of
1/10th of these cDNA by PCR was performed using the gene-specific
primers, yin-yang 1 (YY1) forward (5'-GGC CAC CAC CAC CAC CAC
CA-3') and YY1 reverse (5'-TTC TTG TTG CCC GGG TCG GC-3'; 407-bp
expected product). Internal control for equal cDNA loading in each
reaction was assessed using the following gene-specific GAPDH primers:
GAPDH sense, 5'-GAA CAT CAT CCC TGC CTC TAC TG-3'; and GAPDH antisense,
5'-GTT GCT GTA GCC AAA TTC GTT G-3' (355-bp expected product). PCR
amplifications of each specific DNA sequence were conducted using the
hot start method with Platinum Taq polymerase (Life
Technologies), followed by a two-step thermal cycling incubation
(95°C for 15 s and 60°C for 30 s for 25 cycles, and a
final extension at 72°C for 10 min). The numbers of PCR cycles were
established based on preliminary titration of the relative amount of
amplified product for each gene representing the linear phase of the
amplification process. The amplified products were resolved on 1.5%
agarose gel electrophoresis, and their relative concentrations were
assessed by densitometric analysis of the digitized ethidium
bromide-stained image, performed on a Macintosh computer (Apple
Computer, Cupertino, CA) using the public domain National Institutes of
Health Image program (developed at the U.S. National Institutes of
Health and available on the Internet at
http://rsb.info.nih.gov/nih-image/).
Nuclear extracts preparation
Cultured cells (1 x 106) treated under different experimental conditions were washed twice with ice-cold Dulbecco’s PBS (MediaTech, Herndon, VA). P-40 lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, and 0.1 mM EDTA) was added to the top of the washed cells and incubated on ice for 5 min. Lysed cells were collected by gentle pipetting three or four times and transferred to a microcentrifuge tube. Nuclear pellets for each experimental condition were generated by two consecutive centrifugation and washing steps at 1200 rpm. Nuclear pellets were lysed in buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, and 0.5 mM PMSF). Total nuclear protein concentrations were determined using the method of Bradford (11).
EMSA
Nuclear protein extracts (2 µg) were assayed for DNA
interaction by EMSA as described previously with modifications
(12, 13). The double-stranded YY1 consensus binding
sequence 5'-GGG GAT CAG GGT CT C CAT TTT G AA GCG GGA TCT CCC-3'
(Geneka, Montreal, Canada) oligonucleotide was radiolabeled with
[-32P]ATP (ICN Pharmaceuticals, Costa Mesa,
CA) by incubation with 10 U T4 polynucleotide kinase (New England
Biolabs, Beverly, MA) and further purified using the QIAquick
nucleotide removal kit (Qiagen, Valencia, CA). After the DNA-binding
reaction the samples were resolved on 4–15% Tris-HCl-polyacrylamide
minigels (Bio-Rad, Richmond, CA), and the gels were dried and
autoradiographed. The specificity of the DNA-binding reaction was
determined by competition assays performed with a 100-fold excess of
unlabeled YY1 oligonucleotide and by supershifting using a rabbit YY1
polyclonal Ab (Geneka Biotechnology, Montreal, Canada). Relative
concentrations of specific YY1-shifted bands were assessed by
densitometric analysis of the digitized autoradiographic images using
the National Institutes of Health Image program described above.
Transfections and reporter gene system
AD10 cells (7 x 106/vector) were
transfected with 10 µg DNA using 60 µl Lipofectamine reagent (Life
Technologies) according to the manufacturer’s recommendations.
Transfected cells were then distributed onto a six-well culture plate
and incubated under different experimental conditions. After incubation
for each experimental condition, the cells were harvested, and
luciferase activity was assessed using the enhanced luciferase assay
kit (Analytical Luminescence Laboratory, Ann Arbor, MI) and reading in
an automatic luminometer (Analytical Luminescence Laboratory) following
the manufacturer’s recommendations. For internal transfection
efficiency assessment cells were cotransfected with a reporter vector
containing the 
-galactosidase gene driven by the CMV promoter
(Clontech, Palo Alto, CA).
Statistical analysis
The experimental values were expressed as the mean ± SEM for the number of separate experiments indicated in each case. One-way ANOVA was used to compare variances within groups and among them. Bartlett’s tests were used to establish the homogeneity of variance on the basis of the differences among SD values. Whenever needed, post hoc unpaired multiple comparison tests (Bonferroni’s test) and Student’s t test were used for comparison between two groups. Significant differences were considered for probabilities <5% (p < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent activation of the human Fas
promoter in AD10 cells
We have reported that the IFN--induced sensitization of the
human ovarian carcinoma AD10 cell line to Fas-mediated apoptosis is due
in part to the generation of NO by the induction of iNOS in these cells
and subsequently by the up-regulation of Fas gene expression (Fig. 1
A) (6). We
examined whether IFN--dependent induction of iNOS and subsequent
generation of NO mediate activation of the human Fas promoter in AD10
cells. Transient transfection of AD10 cells with the luciferase-based
reporter vector pGL3-FasP and further treatment with IFN- (100 U/ml)
for 18 h revealed a significant increase in functional activation
of the Fas promoter, as determined by an increase in luciferase
activity (Fig. 1B, lane 2). In contrast,
functional activation of the Fas promoter was completely abrogated when
pGL3-FasP-transfected AD10 cells were treated with IFN- in the
presence of 1 mM of the NOS inhibitor L-NMA (Fig. 1B, lane 3). To confirm the specific role of NO
in the control of human Fas gene expression, we assessed Fas promoter
activity in the presence of the NO donor SNAP. Treatment with SNAP (100
µM) of pGL3-FasP-transfected AD10 cells that were incubated in the
presence of 1 mM L-NMA and IFN- completely
restored the activity of the Fas promoter (Fig. 1B,
lane 4). These results strongly suggest that the
IFN--mediated up-regulation of Fas gene expression in AD10 cells
could be due in part to generation of NO. Furthermore, using the
prostate adenocarcinoma cell line PC-3, we confirmed the same
phenomenon of sensitization by IFN- and its correlation with the
induction of iNOS and subsequent generation of NO by these tumor cells,
including the up-regulation of Fas gene expression (data not shown).
These findings suggest the broad distribution of this type of mechanism
in solid tumor systems.
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In an effort to determine which region of the human Fas promoter
is responsible for the responsiveness to NO, we performed a systematic
deletion of the Fas promoter based on previous functional analysis of
the human Fas promoter (8). pGL3-FasP was used to generate
two other reporter constructs, pGL3-FasEC and pGL3-C, by selective PCR
amplification; these constructs contain -1007 and -425 bp upstream of
the translational initiation of the Fas gene, respectively (Fig. 2A). pGL3-FasEC comprises the
previously identified enhancer and core promoter region in which the
silencer region has been removed. pGL3-FasC consists of only the core
promoter region in which the whole silencer and enhancer regions have
been deleted. Transient transfection of these three Fas promoter-driven
reporter vectors in AD10 revealed that the Fas promoter responds to NO,
but lacks its responsiveness to NO upon deletion of the silencer
region. Although removal of the silencer region resulted in an expected
increase in luciferase activity compared with that of the full
promoter, the sensitivity to NO was minimal and not significant.
Removal of both silencer and enhancer regions resulted in a basal
promoter activity that was not responsive to SNAP treatment (Fig. 2B). Therefore, these results suggest that NO, or a
derivative of it, is likely to act in the disruption of a repressor
mechanism located at the silencer region of the Fas promoter.
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The finding that the main region that retained responsiveness to
NO on the Fas promoter was located at the silencer suggested the
repressive nature of a possible trans-acting element. This
trans-acting element could be inactivated in the presence of
NO, resulting in release of the Fas promoter transcriptional machinery.
To identify the presence of a putative repressor
trans-acting element in the silencer region of the Fas
promoter (-1781/-1007 bp from the translational initiation site), we
analyzed this region using the Transcription Element Search software,
searching for potential recognized repressor trans-acting
elements. This analysis revealed a noticeable stretch of sequences that
matched the consensus transcription element (CCWTNTTNNNW) for the
transcription factor YY1, a recognized transcriptional repressor
(14). Three potential YY1-responsive elements were found
to cluster in a very narrow segment within the Fas promoter silencer
region, between -1619 and -1533 bp from the translational initiation
site (Fig. 3). Repeated IFN- silencer
B motifs also colocalized within this same region, and some of them
overlapped with the YY1 silencer cluster. However, no functional
relevance of the IFN- silencer B motifs has been shown in the
control of Fas gene expression in several cell types (15).
YY1 is a DNA-binding zinc finger transcription repressor that is highly
conserved in mammalian cells. Moreover, zinc finger proteins have been
suggested to be primary targets of NO-induced disruption of their
functional structure (16). Thus, we hypothesized that YY1
is a potential candidate to function as a repressor of Fas gene
expression and can be affected by NO.
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To investigate the specific role of NO in the activity of the
transcription factor YY1, we first examined the relative mRNA
expression of YY1 in quiescent AD10 cells cultured for 18 h in the
presence of 100 and 500 µM SNAP. Semiquantitative RT-PCR reflected no
significant changes in levels of YY1 mRNA expression upon treatment
with SNAP (Fig. 4). Similarly, treatment
of these cells with NO donors had no effect on the constitutive levels
of YY1 protein levels (data not shown). Further, we analyzed by EMSA
the DNA binding activity of YY1 in AD10 cells using the double-stranded
YY1 consensus binding oligonucleotide. As shown in Fig. 5, nuclear extracts from AD10 cells
treated for 6 h in the presence of 100 and 500 µM SNAP exhibited
a negligible DNA binding activity of YY1 (Fig. 5, lanes 5
and 6) compared with the untreated control (Fig. 5, lane 1). As a control for potential secondary metabolites
other than NO derived from SNAP, we used 500 µM of a SNAP solution
allowed to fully decompose for 1 wk (Fig. 5, lane 4) with no
significant effect on the YY1 DNA binding activity. The impaired YY1
binding activity caused by NO was completely restored upon addition
1 h before the DNA-binding reaction of 1 mM of the reducing agent
DTT to the SNAP-treated nuclear extract (Fig. 5, lane 7).
The specificity of the DNA-binding reaction was determined by
competition assays performed with a 100-fold excess of unlabeled YY1
oligonucleotide and by supershifting using a rabbit YY1 polyclonal Ab
(Fig. 5, lane 2). These results suggest that a potential
mechanism by which YY1 binding activity is inhibited is via redox
regulation by NO and occurs through thiol modification with consequent
disruption of the YY1 activity. Additional experiments performed in the
presence of the short half-life (2.5 min) NO donor DEA-NONOate in a
cell-free EMSA (treatment of the nuclear extracts) and the long
half-life (20 h) DETA-NONOate on cultured cells revealed results
similar to those of the SNAP-treated groups (data not shown).
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To determine whether the transcription factor YY1 binds
specifically to its putative cluster at the silencer region of the Fas
promoter, the YY1 cluster identified above was PCR-amplified and used
as a probe in the EMSA. Using the forward primer 5'-ATT TTG TCA ATT GTC
CTT TC-3' and the reverse primer 5'-AGT TCA TTT AAA TAA AAA AA-3'
flanking the putative YY1 cluster at the Fas promoter silencer region
(Fig. 3
, underlined sequence), we amplified using the pGL3-FasP vector
as a template. The PCR product was gel-purified and radiolabeled as
described in the previous section. Specific YY1 DNA binding activity
was observed in untreated AD10 cells. Although other shifted complexes
were formed that reflect the multiplicity of overlapping binding sites
that can be present in the YY1 cluster used as a probe, the prominent
shifted band (Fig. 6, lane 1)
was supershifted in the presence of the rabbit YY1 polyclonal Ab (Fig. 6, lane 2). These results demonstrate that YY1 is able to
bind the putative binding sites for YY1 on the silencer region of the
Fas promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- and by activation of lymphocytes (2, 4).
Therefore, the expression of the Fas receptor marks a critical point in
the decision of the fate of the cell in terms of its survival or death.
We have shown that IFN--mediated sensitization to Fas-induced
apoptosis of the resistant human carcinoma cell line AD10 is partially
due to the induction of iNOS and the subsequent generation of NO. In
addition, we have demonstrated the presence of a strong correlation
between the generation of NO and the up-regulation of Fas gene
expression in AD10 cells (6). In the present study
evidence is presented for the first time of a novel molecular mechanism
that demonstrates that NO is disrupting a repressor mechanism located
primarily at the silencer region of the Fas gene promoter, thereby
increasing its expression. Moreover, the NO-mediated effect is due
specifically to the disruption of the DNA binding activity of the
transcription factor YY1, which normally represses Fas expression by
binding to a cis-element clustered at the silencer region of
the Fas promoter. Hence, NO promotes the removal of YY1 repressor
activity and frees the Fas promoter to initiate or increase
transcriptional activation of the Fas gene. Several lines of evidence support the hypothesis that NO regulates the expression of some genes that are implicated in the signal pathway involving regulatory cytokines that modify the cellular response to apoptotic stimuli (18). However, the regulation of apoptosis-related genes by NO is not completely understood. The biological effect of NO has always been shown to be paradoxical. NO appears to inhibit Fas-induced apoptosis in transformed cells derived from the hemopoietic lineage, whereas it mediates sensitization to Fas apoptosis, as observed here in ovarian tumor cells and several other solid tumor cells. This dichotomy represents a crucial point of divergence between the biological natures of these two cell types, and it needs to be considered when making strategies toward the use of NO as an anti-tumor agent. Several reports addressed the question of the inhibition of caspases by NO and further interruption of the programmed cell death. Hence, under the light of recent reports, the final outcome of NO-mediated effects would be determined by many factors, including the local concentration and sources of NO and the presence of reactive molecules that might redirect the redox status in the tumor cell.
Previous studies suggested the regulation of Fas gene expression on
normal or tumor cells by NO (6, 7), although the
underlying molecular mechanism of this effect is not known.
Characterization of the human Fas gene promoter has revealed three
major regions within the 2000-bp 5'-flanking region. Functional
analysis identified a silencer activity residing between nucleotide
position -1781 and -1007 and a strong enhancer region between -1007
and -425 in the human Fas gene. The region between -425 and -1
retained a basal promoter activity (8).
Transiently transfected human ovarian carcinoma cells AD10 with the Fas
promoter-driven luciferase expression reporter vector (pGL3-FasP)
responded to NO by an increase of luciferase activity. We observed that
IFN--mediated up-regulation of luciferase activity was blocked by
use of the specific NOS inhibitor L-NMA. Furthermore,
treatment with the NO donor SNAP rescued the
L-NMA-inhibited luciferase activity in these cells (Fig. 1). These results strongly suggested the participation of NO in the
regulation of Fas promoter activity in AD10 cells.
Systematic deletion of the previously identified functional regions on
the Fas promoter revealed that the responsiveness to NO observed using
the luciferase-based reporter system is present within the silencer
region (Fig. 2). This result suggested that one possible mechanism by
which NO increases the expression of the Fas gene is by inactivation of
a potential repressive transcription factor located on the silencer
region of the Fas promoter. Thus, we anticipated that by removing
negative regulatory trans-acting signals on the Fas
promoter, NO would increase Fas gene expression in AD10 cells. Indeed,
our findings concur with this hypothesis.
Silencers have been reported to act synergistically to increase or
modify repressor function and to play a definitive role in determining
eukaryote gene expression (19). The transcription factor
YY1 has been identified as a potential repressor factor in the human
IFN- gene (20, 21), the IL-3 gene promoter
(22), and the GM-CSF gene promoter (23, 24).
Although many putative repressor motifs have been localized by sequence
comparison in the 5'-flanking region of the Fas gene, no functional
implications have been assigned to these trans-regulatory
elements (8, 15). Significantly, we have identified a very
relevant putative repressor cluster at the silencer region that matched
the consensus sequence that binds the transcription factor YY1. Three
binding sites for YY1 are located in a very narrow sequence stretch at
-1619, -1590, and -1543 bp from the translational initiation site of
the Fas gene (Fig. 3). YY1 is a zinc finger transcription factor
involved in the negative regulation of many mammalian genes
(25). NO is known to interfere in the DNA binding activity
of many zinc finger transcription factors via
S-nitrosylation of cysteine thiols groups and subsequent
S-nitrosothiol formation (16, 26). Exposure of
AD10 cells to various concentrations of the NO donor SNAP and other NO
donors significantly inhibited the specific DNA binding activity of the
transcription factor YY1 (Fig. 5). Furthermore, NO-based inhibition of
YY1 DNA binding activity was completely restored by incubation with 1
mM of the reducing agent DTT. It is noteworthy that NO was not able to
modify YY1 gene expression in AD10 cells, as assessed by RT-PCR (Fig. 4). Our findings are consistent with the role of NO in blocking YY1
binding activity. Hence, our results suggest that a potential mechanism
by which NO inhibits the YY1 DNA binding activity is through redox
regulation by NO and is likely to occur through thiol modification with
consequent disruption of the YY1 functional structure.
We confirmed the specific binding of the transcription factor YY1 to
the silencer region by EMSA and supershift analysis using the sequence
comprising the identified putative YY1 binding sites (Fig. 6, underlined sequence) on the Fas silencer region. Multiple shifted
complexes were formed using the YY1 cluster probe, which revealed the
possible interaction of overlapping transcription factors within this
region with a prominent concentration of the specific band
corresponding to YY1. It has been shown that YY1 repression activity
probably reflects its ability to interfere with the communication
between transcription activators and their targets within the general
transcription machinery. The simplest mechanism of repression by YY1 is
through the transcription activator displacement from their
cis-acting element within the promoter region and/or the
recruitment of corepressor molecules (14).
In conclusion, our findings demonstrate that the mechanism by which NO up-regulates the expression of the Fas receptor on AD10 cells is probably due to specific inactivation of the transcription repressor YY1 DNA binding activity to the silencer region of the Fas promoter. The identification of NO sensitive trans-regulatory elements provides the molecular basis to explain how such a pleiotropic and reactive molecule could modulate the expression of specific genes involved in the sensitivity of normal and tumor cell to apoptosis. Therefore, a new role for NO-mediated regulation of gene expression is revealed.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Benjamin Bonavida, Department of Microbiology, Immunology, and Molecular Genetics, University of California School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1747. E-mail address: bbonavida{at}mednet.ucla.edu
3 Abbreviations used in this paper: iNOS, inducible NO synthase; SNAP, S-nitroso-N-acetylpenicillamine; L-NMA, NG-monomethyl-L-arginine; YY1, yin-yang 1.
Received for publication January 24, 2001. Accepted for publication April 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B: role in sensitization of human tumor cells to TNF--induced cytotoxicity. J. Biol. Chem. 276:8918.B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58:227.[Medline]
promoter. J. Biol. Chem. 269:25728. interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element. Mol. Cell. Biol. 16:4744.[Abstract]
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