| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,§
*
Institute of Immunology, Departments of
Pathology and
Urology, University Hospital Benjamin Franklin, Free University Berlin, Berlin, Germany;
§
Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin, Bochum, Germany; and
¶
Department of Internal Medicine IV, Friedrich-Alexander-University, Erlangen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B sites, which are not active in the
constitutive OX40 expression. Their deletion abrogates Tax
responsiveness in reporter gene analysis. The site-directed mutagenesis
of each NF-B site demonstrates that cooperative NF-B binding is a
prerequisite for Tax-directed activity as neither site alone is
sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax
expression, both sites bind p65 and c-Rel. These data provide new
insight into the direct regulation of OX40 by Tax and add to our
understanding of the possible role of the OX40/OX40 ligand system in
the proliferation of HTLV-I+ T cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HTLV-I infection is associated with the aggressive and lethal
adult T cell leukemia (ATL), as well as chronic inflammatory disorders,
such as the tropical spastic paraparesis/HTLV-I-associated myelopathy
and others (13, 14, 15). T cell transformation requires the
HTLV-I Tax oncoprotein, which is the activator of viral gene
expression. Tax also acts as a transactivator of an increasing number
of host cellular genes, most of which are associated with cell growth.
They include growth factors, like GM-CSF (16) or cytokines
and their receptors such as IL-2 (17), IL-15
(18), or IL-2R
(CD25) (19, 20, 21). This
activation occurs via the interaction of Tax with cellular
transcription factors, which include the serum response factor
(22, 23), members of the activating transcription
factor/cAMP response element binding protein (24, 25), and
NF-B (26).
NF-B defines a family of transcription factors that result from the
combination of its protein members, such as p50, p65 (RelA), c-Rel, and
RelB (27). Inactive NF-B is retained in the cytoplasm,
either by complexing to the IB inhibitor or as a precursor molecule
of the active protein (27). NF-B activation and
subsequent translocation to the nucleus requires phosphorylation and
degradation of IB or proteolytic cleavage of the precursor molecule.
Tax is one of the signals known to cause liberation of NF-B from
IB (26, 28). It can also bind NF-B precursors as
well as active nuclear NF-B proteins (23, 29, 30, 31).
The OX40L promoter was shown to be activated by the Tax oncoprotein
(32) and expression of Tax in the OX40-negative T cell
line, Jurkat, results in OX40 surface expression (33).
These findings suggest that auto or paracrine OX40/OX40L interactions
on HTLV-I-producing cells may provide necessary costimulatory signals
for transformation or survival and proliferation of these cells. Thus
far, however, little is known about the transcriptional regulation of
OX40 gene expression and its modulation by the Tax protein. Therefore,
we have cloned the human OX40 gene, analyzed its promoter region, and
defined basal promoter activity. Moreover, we describe here that Tax
further up-regulates OX40 gene expression in an
OX40+ cell line via two NF-B elements, which
recruit at least three members of the NF-B family: c-Rel, p65, and
p50 in a Tax-dependent manner.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human placental cosmid library pWE15 (Stratagene, Heidelberg, Germany) was screened by standard molecular biology techniques using random-primed radio-labeled human OX40 cDNA (3). The cosmid carrying the OX40 gene was sequenced from both strands of sample DNAs on an automated sequencer (Perkin-Elmer/ABI, Weiterstadt, Germany). The sequence was searched for possible transcription factor binding sites using the programs FACTOR (HUSAR, EMBL, Heidelberg, Germany) and MatInspector (34).
Cell lines and cell culture
The OX40- lymphoid cell lines L363 and DG75 were obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (Braunschweig, Germany). The OX40+ but HTLV-I-negative lymphoid cell line Co (35) was a generous gift from Dr. D. B. Jones (Southhampton, U.K.). The OX40+ but HTLV-I-negative lymphoid cell line CCRF-CEM and the OX40+ and HTLV-I-positive lymphoid cell lines HUT-102 and MT-2 were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were cultured in RPMI 1640, containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated at 37°C and 5% CO2.
RNase protection assay
Total RNA from OX40-positive cells was isolated using the RNA-Clean isolation kit (AGS, Heidelberg, Germany). A PCR fragment carrying the sequence from position -251 to +80 with respect to the transcription start site was generated using 35 cycles of PCR (94°C, 1 min; 60°C, 2 min; 72°C, 2 min, and 5 min final extension) with top-strand primer 5'-GCTCCATGGACCTTTTTGCTGAGGG-3' (-260 to -236) and bottom-strand primer 5'-GAGGAGCAGAGCCGCACACGGC-3' (+59 to +80) and the cosmid containing the 5' region of the OX40 gene as a template. The resulting fragment was cloned into the pAMP1 vector (Life Technologies, Karlsruhe, Germany). For the generation of radioactively labeled antisense-RNA, the construct was linearized with XbaI and in vitro transcribed with the T7 RNA polymerase (Riboprobe In Vitro Transcription System; Promega, Heidelberg, Germany). Then, 15 µg RNA were used in each RNase protection assay according to manufacturer’s instructions (RNase Protection Kit; Boehringer Mannheim, Mannheim, Germany). The resulting fragment was resolved on a 6% polyacrylamide gel.
Flow cytometry
For FACS analysis, 5 x 105 cells
were incubated for 30 min on ice with the human FITC-conjugated OX40 Ab
(Ber-Act35) (3) or with an isotype-matched control Ab,
mouse IgG1, FITC-conjugated (Dako, Hamburg, Germany) in PBS with
10% FCS. After washing, the cells were analyzed by a FACSort (Becton
Dickinson, Heidelberg, Germany).
DNA constructs
For the PCR-based generation of OX40 promoter deletion
constructs, the parental cosmid containing the OX40 5'-flanking region
was used as a template. Top-strand primers were -1259,
5'-GAGGGgtACCAACTTAGGGACC-3' (-1266 to -1245); -1184,
5'-GCCAGCGGtacCCACGGCTGGGAATTTC-3' (-1195 to -1168); -1184 mut1,
5'-GCCAGCGGtacCCACGGCTGcGAATTTC-3' (-1195 to -1168); -1124 mut2,
5'-TTCCTGTGgTaCcGGGAGGAGccGATTTCC-3' (-1137 to -1108); -1021,
5'-CTGGTacCAGTTATTTGAAAGG-3' (-1028 to -1007); -944,
5'-GGGCAGgTacCATCCTCTGGGC-3' (-954 to -933); -649,
5'-ggtaCCACCTCTGCTTTGCAACTTC-3' (-649 to -629); -426,
5'-CGGGAGgtACCGTGCCTGGCCATGGG-3' (-434 to -409); -138,
5'-GGTaCCCAGACCCCGCCTTTG-3' (-142 to -122); -64,
5'-GgTaCCCTCCTCCCCTCTCCC-3' (-68 to -48); and the bottom-strand
primer +19, 5'-CTCGagTCTGCTGTCGCCAGAG-3' (+4 to +25). Nucleotides
that were exchanged to introduce KpnI and XhoI
restriction sites or to mutate the OX40 NF-B sites are given in
lower case letters. The annealing conditions for each PCR standard
reaction (as described above) were modified according to the melting
temperatures of the primers used. The site-directed mutagenesis of the
NF-B site 2 in construct -1184 mut2 was generated according to the
protocol of the QuickChange kit (Stratagene) with the pGL2 basic -1184
reporter construct as the template and a primer pair -1184 mut2
covering 5'-GCTCCTGGGAGGAGccGATTTCCAGCCCC-3' (-1130 to -1102) on
both strands. Promoter fragments were sequenced throughout the
PCR-amplified region. The pSGtax, coding for full-length HTLV-I Tax and
pSGtaxmutant2, coding for a truncated HTLV-I Tax of amino acids 1–284
(36) were a generous gift from Dr. Kazuo Sugamura (Sendai,
Japan).
Transfections and luciferase assays
Transfections were performed using the DMRIE-C reagent protocol (Life Technologies). Briefly, for each transfection 5 x 105 cells were pelleted, resuspended in Opti-MEM media (Life Technologies) and mixed with 1 µg of each plasmid DNA. Cell extract preparation and luciferase assay were performed 48 h later according to the Luciferase assay system kit (Promega). Enzyme activity was measured with a Lumat LB9501 (Berthold, Wildbad, Germany). Transfection efficiency was normalized by cotransfection of a reporter plasmid, pCH101 (Pharmacia Biotech, Freiburg, Germany), containing the ß-galactosidase gene under the control of the SV40 promoter. ß-galactosidase activity was determined using the Galacto-light kit (Tropix, Bedford, MA), following manufacturer’s instructions. For comparison of promoter activities in different cell lines, luciferase activities were referred to luciferase control vector, pGL2 control, containing the SV40 promoter and enhancer. All measurements were conducted in duplicates from three independent transfections.
Nuclear extracts and gel mobility shift assay
Nuclear extracts were prepared as previously described
(37). Five micrograms of extract were incubated with 1 ng
of labeled DNA (20,000 cpm) in a buffer containing 20 mM HEPES (pH
7.9), 60 mM KCl, 0.5 mM dithiotreitol, 4% Ficoll or 1%
polyvinylalcohol, 5% glycerol, and 2 µg poly (dI-dC) (Boehringer
Mannheim) for 30 min at 4°C. The following oligonucleotides with
consensus sites underlined and mutations in lower case
letters were used as probes: NF-B site 1,
5'-ACGGCTGGGAATTTCCCCACGGGTGGG-3' (-1182/-1156);
NF-B site 2, 5'-TCCTGGGAGGAGGGGATTTCCAGCCCC-3'
(-1128/-1102); NF-Bmut,
5'-GATGGATGAGTTGAGGcGACTTTCCCTCTTTACTG-3'. Oligonucleotides
carrying an Sp-1 site, Sp-1cons,
5'-ATTCGATCGGGGCGGGGCGAGC-3' were purchased from Santa
Cruz (Heidelberg, Germany). The reaction mixtures were
electrophoresed through a 4% polyacrylamide gel. In competition
experiments a 50-fold excess of unlabeled oligonucleotides, and in
supershift assays 2 µg of NF-B p50 (sc-1191X), NF-B p65
(sc-372X), or NF-B c-Rel (sc-70-GX) Abs (Santa Cruz), were added to
the binding reaction before the addition of the labeled probe.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine the genomic structure of the OX40 Ag, a cosmid
containing the entire OX40 gene was obtained. The complete coding
region was found to be within only 2.7 kb and to include 7 exons and 6
introns (DDBJ/EMBL/GenBank Nucleotide Sequence Database accession
number AJ277151). To identify the transcription initiation site, we
performed RNase protection assays with RNA isolated from
OX40+ HUT-102 cells. We obtained a protected band
that mapped the start site to an adenosine 26 nt upstream of the ATG
initiation codon (Fig. 1
A).
The specificity of the transcription start site was confirmed using RNA
of a different OX40+ cell line, MT-2 (data not
shown).
|
B depicts the 5'-flanking region and putative
transcription factor binding sites, based on sequence homology.
Although an initiator element (Inr) matching the consensus sequence Py
Py A+1 N T/A Py Py (38) overlapped
the start site for transcription, the sequence analysis additionally
revealed a TATA-like box at position -27 as well as a CCAAT box.
Several elements with homology to Sp-1 binding sites, such as CCCCTCC
(39) or GT box sequences (40), were found
throughout the 5' region. Furthermore, we found two putative E boxes,
as well as potential binding sites for AP-1, AP-2, Yin Yang-1, and
Icaros-2. Finally, at the very 5'-end of the analyzed region two
NF-B binding sites were detected (Fig. 1B). Functional analysis of the human OX40 promoter region
First, to test the promoter activity of the 5'-flanking sequence
of the OX40 gene, a fragment spanning the region between nucleotides
-1259 to +19 was cloned upstream of the luciferase gene of the pGL2
basic vector. This reporter construct was then transiently transfected
into OX40- DG75 and L363 cells, and into Co and
CCRF-CEM cells, which both constitutively express low levels of OX40 as
determined by FACS analysis (Fig. 2). We
compared the ability of the OX40 5' fragment to induce luciferase
activity in these cell lines.
|
A). The OX40 promoter in the
OX40+ cells was three times less active than the
SV40 promoter and enhancer of the pGL2 control vector. This showed that
the sequence up to -1259 serves as a promoter of OX40 gene expression
in the OX40+ cell lines.
|
B, all constructs displayed a reduced luciferase activity
compared with the -944 construct, which was the most potent one and
therefore set as 100%. The longest construct -1259 displayed a
>2.5-fold lower activity than the -944 construct. Deleting the region
to -1021 resulted in an increased promoter activity being only
1.4-fold lower than the -944 construct. However, as neither Co (see
Fig. 5B) nor CCRF-CEM cells (data not shown) exhibit nuclear
NF-B activity the NF-B sites within this region do not play a
role in maintaining the constitutive OX40 promoter expression in these
cells.
|
B).
Remarkably, the promoter activity of the -138 construct was only
1.4-fold lower than the -944 construct. Indeed, this sequence lacks
the CCAAT element but contains the TATA element and several Sp-1
binding sites, which were found to bind both Sp-1 and Sp-3 in EMSA
(data not shown). A further 5' truncation to nucleotide -64 that
deleted the Sp-1 site located at position -118, diminished the
promoter activity 5-fold compared with the -944 construct, but still
maintaining it above background levels. Thus these data demonstrate
that the region between -138 and +19 can serve as a minimal promoter
whose activity seems to be dependent on the number of Sp-1 sites
present. Tax modulates OX40 promoter activity
To determine whether Tax is able to further up-regulate OX40 Ag
expression, we transfected Co cells with Tax expression plasmids:
wild-type Tax (pSGtax) or, as a negative control, a truncated Tax
(pSGtaxmutant2) lacking amino acids 285–353, which was shown to have
lost its transactivation ability (36, 41). FACS analysis
demonstrated that OX40 expression could be up-regulated only upon
wild-type Tax and not upon transfection with the pSGtaxmutant2 plasmid,
as shown in Fig. 4A. Thus,
functional Tax expression increases OX40 surface expression.
|
B). However, cotransfection of the -1259 construct
with pSGtax was associated with a 6-fold increase over control in
luciferase activity. Thus Tax expression indeed induced OX40 promoter
activity.
To localize the promoter region responsive to Tax, the 5'-end deletion
constructs, -1021, -944, and -138, were employed for cotransfection.
In fact, the deletion of the region between -1259 and -1021,
including two Sp-1 sites and NF-B sites 1 and 2, fully abolished
Tax-induced promoter activity (Fig. 4B). In addition, Tax
expression did not alter the activity of the minimal promoter as
evidenced with the -138 construct. These results indicate that the
minimal promoter of the OX40 gene is Tax independent and that the
upstream region containing Sp-1 and two NF-B sites accounts for the
promoter responsiveness upon Tax.
Two NF-B sites are required for Tax inducibility
We wondered whether the two NF-B sites found within the -1259
and -1021 region might mediate OX40 promoter inducibility upon Tax.
Therefore, a series of reporter constructs carrying mutations within
either NF-B site was generated (Fig. 5A). To exclude that any
relevant binding to the sequence upstream of both NF-B sites could
influence Tax modulation, we generated construct -1184 deleting the
region between nucleotides -1259 and -1184. To this construct we then
introduced point mutations either to the NF-B site 1, -1184 mut1,
or to the NF-B site 2, -1184 mut2. Furthermore, construct -1124
mut2 lacked the NF-B site 1 and contained a mutated NF-B site 2.
These constructs were transfected into Co cells in the presence or
absence of pSGtax cotransfection. In the absence of pSGtax
cotransfection, all constructs exhibited promoter activities equivalent
to the -1021 construct. As expected, point mutations in either NF-B
site did not affect the constitutive promoter activity. This is
consistent with our findings in EMSAs where nuclear NF-B activity is
absent in Co cells (Fig. 5B). Furthermore the point
mutations within NF-B site 2 mutated the overlapping Sp-1 site as
well, with no effect on the resulting promoter activity. However,
when we cotransfected pSGtax we detected a >5-fold
increase of the activity in the nonmutated -1184 construct. The
respective point mutations of either NF-B site severely reduced the
inducibility of luciferase activity by Tax to <2-fold (Fig. 5A). Finally, the deletion of NF-B site 1 and a mutation
of NF-B site 2 (-1124 mut2) completely abolished OX40 promoter
inducibility upon Tax coexpression. Therefore, the NF-B sites,
although not required for a constitutive OX40 expression, play a
central role in OX40 promoter regulation upon Tax expression.
Furthermore, both sites are required for optimal Tax-induced promoter
modulation.
Tax induces NF-B binding to its motifs in the OX40 promoter
To determine whether NF-B complexes bind to NF-B site 1 and
2 upon Tax transactivation, we performed EMSAs using the two NF-B
sites as synthetic probes. The binding activities of nuclear extracts
from HTLV-I+ untransfected HUT-102 and Co cells,
transiently transfected with the Tax-expressing plasmid pSGtax (Co
tax), were compared with the binding activities of nuclear extracts
obtained from untransfected Co cells, which served as controls.
As shown in Fig. 5B, nuclear extracts from either HUT-102
and Co tax cells formed two major NF-B complexes, defined as I and
II, with NF-B site 1 and NF-B site 2. Because both complexes were
formed only with nuclear extracts from HUT-102 and Co tax-transfected
but not in the nontransfected extracts, this indicated that they were
Tax induced. Binding specificity was shown by complete inhibition of
complex formation upon addition of the unlabeled probe but not by
mutated NF-B site oligonucleotides. Additional binding complexes
were observed with NF-B site 2 in all nuclear extracts (Fig. 5B). These could likely result from Sp-1 proteins binding to
the NF-B site 2 probe, because an Sp-1 binding site overlaps the
NF-B site on the complementary strand. In fact, Sp-1 binding could
be specifically competed by Sp-1 consensus oligonucleotides (Fig. 5B). However, as these Sp-1 complexes were also detectable
with nuclear extracts from untransfected Co cells, they were not
considered to be of functional relevance for OX40 promoter induction
upon Tax expression.
Next, we wished to identify protein components of the NF-B complexes
formed by nuclear extracts from Tax-expressing cells. We focused on p65
or c-Rel, because they were both shown to bind Tax and activate
transcription (29) and because c-Rel is preferentially
induced upon HTLV-I infection or in Tax-expressing cells (42, 43). Supershift experiments with extracts from HUT-102 cells
were performed using Abs against the two NF-B protein family
members, c-Rel and p65 (Fig. 5C).
Indeed, NF-B complexes I and II, formed with both NF-B sites,
were supershifted by anti-p65 and anti-c-Rel Abs, respectively
(Fig. 5C). Supershifted bands were also observed upon
addition of anti-p50 Abs but did not result in any detectable
disappearance of either complex I or II (Fig. 5C). However,
because the simultaneous addition of anti-p65 and anti-c-Rel
Abs did not fully supershift complexes I and II, one is tempted to
speculate that p50 is contained within the binding complex. The Sp-1
associated complexes detected with NF-B site 2 (Fig. 5B,
NF-B site 2) were weakly competed with unlabeled Sp-1
oligonucleotides, as they overlaid the anti-p50 supershift (Fig. 5C, NF-B site 2). Taken together, these results suggest
that p65, c-Rel, and, to a lesser extent, p50-binding to the NF-B
site 1 and site 2 are responsible for mediating Tax-induced OX40
promoter up-regulation.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We define a 157-bp DNA fragment as a minimal promoter for constitutive
expression with several Sp-1 binding sites modulating its activity.
Furthermore, we show that Tax can enhance OX40 surface expression and
that it can transcriptionally up-regulate the promoter activity.
Tax-induced promoter modulation requires a 79-bp sequence located 1.1
kb upstream in the OX40 enhancer region. The cooperative function of
two distinct NF-B sites within this sequence is shown here to be the
prerequisite to confer Tax responsiveness by binding nuclear p50, p65,
and c-Rel.
Examining the OX40 gene promoter, we have determined that transcription could be initiated by an Inr element and a TATA box. Inr elements are often found in TATA-less promoters because they can replace the TATA box to direct the precise transcription start (44). However, when Inr and a TATA box are present in the same core promoter with the TATA box 25–30 bp upstream of the Inr, they can cooperate to enhance promoter activity, even if the TATA sequence only has a weak TATA box homology (38, 45). Such a weak TATA box-like element is indeed found at position -27. Our functional studies revealed that the sequence between -138 and +19 displays a remarkable promoter activity to function as a minimal promoter. Sp-1 binding sites (46) within this region together with the TATA box element might control the strength of the minimal promoter.
Previously, it has been reported that OX40 Ag expression is induced by
the viral transcriptional activator HTLV-I Tax (33). Here
we show that Tax enhances OX40 expression in an
OX40+ cell line. In fact, this suggests that Tax
is not only capable to induce de novo gene expression, but can also
modify cellular Ag expression to meet the requirements for viral
persistence. On the promoter regulatory level, this modified OX40 gene
expression upon Tax relies on the presence of two NF-B sites
spanning a region of 79 bp in the enhancer region. These sites are
sufficient for conferring the Tax responsiveness of the whole OX40
5'-flanking region, examined in Fig. 5A. They are
dispensable in the constitutive expression of the OX40 gene, as
untransfected Co cells do not exhibit any nuclear NF-B binding
activity (Fig. 5B).
Upon Tax expression, both OX40 NF-B sites bind the same set of
NF-B complexes with equal strength (Fig. 5A). These
complexes consist of mainly p65 and c-Rel (Fig. 5B). Other
cellular promoters have also been shown to be up-regulated by Tax
through NF-B proteins, such as the IL-2R (19) or
OX40L (32). The NF-B site in the IL-2R promoter,
GGGAATCTCC, differs from the OX40 NF-B site 1 in one and from site 2
in only two positions of the decameric motif. Like OX40, the IL-2R
NF-B site has also been shown to bind p65 and c-Rel upon Tax
expression, with c-Rel serving as the major protein responsible for Tax
transactivation (19). Interestingly, one NF-B site in
the IL-2R promoter is sufficient to mediate Tax-directed
transcriptional up-regulation, whereas in the OX40 promoter two
cooperatively acting NF-B sites are required (Fig. 5A).
However, two NF-B elements, i.e., GGGAAATTCA and GGGAACTTCT, have
also been described to be required for Tax transactivation in the OX40L
promoter. Upon Tax expression, no binding of c-Rel but rather of
RelB was detected on these sites (32). The respective
NF-B motifs in the OX40 and OX40L promoters differ in at least two
or three positions from each other. Thus, the exact NF-B motif in a
given promoter may account for altered binding affinities for NF-B
proteins, and how NF-B proteins regulate a cellular promoter in
response to Tax may depend on both the sequence of the NF-B motif
and the number of sites present.
The fact that OX40 is constitutively expressed on HTLV-I-expressing cell lines and can be detected in ATL cells could account for some of the observations reported for ATL or its related diseases (14, 15). For example, OX40 expressed on leukemic cells isolated from peripheral blood of ATL patients was shown to mediate cell adhesion to OX40L-expressing vascular endothelial cells (11), suggesting the involvement of OX40 for leukemic cell migration into tissues. Studies on normal T cells have demonstrated that OX40 is a potent costimulatory molecule, which leads to a sustained proliferation of differentiated effector T cells (8). In addition, OX40 has not been shown so far to promote cell death. Recently, reported data rather suggest that signaling through OX40 may serve to inhibit activation-induced T cell death (47). This could be important for supporting the survival of HTLV-I-expressing T cells as Tax not only guarantees for constitutive OX40 expression, but also of its ligand, OX40L (4, 32). Therefore, auto or paracrine OX40/OX40L interactions might contribute to the survival, proliferation, and clonal expansion of ATL cells and may substitute the IL-2/IL-2R pathway when the cell proliferation becomes IL-2 independent (48). Thus, inhibition of OX40 expression is a promising target for therapeutic intervention in ATL.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Silvia Bulfone-Paus, Institute of Immunology, University Hospital Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany.
3 Abbreviations used in this paper: OX40L, OX40 ligand; ATL, adult T cell leukemia; HTLV-I, human T cell leukemia virus type I; Inr, initiator element.
Received for publication December 22, 1999. Accepted for publication April 11, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B with other transcription factor families. Oncogene 8:3189.[Medline]
B site. Proc. Natl. Acad. Sci. USA 95:2452. B enhancer include the product of the c-rel proto-oncogene. Oncogene 8:867.[Medline]
B element in the IL-2 receptor gene. Science 241:1652.B-like factor. Nature 333:776.[Medline]
B p50 and serum response factor (SRF) and associates with enhancer DNAs of the NF-B site and CArG box. Oncogene 8:2391.[Medline]
B and induces nuclear translocation of transcription factor NF-B proteins for transcriptional activation. Proc. Natl. Acad. Sci. USA 91:3584.B. Annu. Rev. Cell. Biol. 10:405.
B kinase activity and NF-B activation. Cell 93:875.[Medline]
B p65 and c-Rel proteins bound to the NF-B binding site and activates transcription. Oncogene 9:3099.[Medline]
B precursor p105 enhances nuclear localization of NF-B p50 and p65 in transfected cells. Oncogene 8:2949.[Medline]
B proteins in viral oncogenesis and the regulation of viral transcription. Semin. Cancer Biol. 8:121.[Medline]
B signalling pathway. Oncogene 9:841.[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. Pichler, T. Kattan, J. Gentzsch, A. K. Kress, G. P. Taylor, C. R. M. Bangham, and R. Grassmann Strong induction of 4-1BB, a growth and survival promoting costimulatory receptor, in HTLV-1-infected cultured and patients' T cells by the viral Tax oncoprotein Blood, May 1, 2008; 111(9): 4741 - 4751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Komori, A. Hasegawa, K. Kurihara, T. Honda, H. Yokozeki, T. Masuda, and M. Kannagi Reduction of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Proviral Loads in Rats Orally Infected with HTLV-1 by Reimmunization with HTLV-1-Infected Cells. J. Virol., August 1, 2006; 80(15): 7375 - 7381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mori, Y. Yamada, S. Ikeda, Y. Yamasaki, K. Tsukasaki, Y. Tanaka, M. Tomonaga, N. Yamamoto, and M. Fujii Bay 11-7082 inhibits transcription factor NF-kappa B and induces apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell leukemia cells Blood, August 13, 2002; 100(5): 1828 - 1834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takahashi, Y. Tanaka, A. Yamashita, Y. Koyanagi, M. Nakamura, and N. Yamamoto OX40 Stimulation by gp34/OX40 Ligand Enhances Productive Human Immunodeficiency Virus Type 1 Infection J. Virol., August 1, 2001; 75(15): 6748 - 6757. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
X174 DNA served as a size marker
(lane 4). B, Sequence of the human OX40
5'-upstream region. The transcription initiation site is marked as +1.
Sequences matching Inr, TATA, and CCAAT elements are boxed. Putative
transcription factor binding sites are underlined.
) and CCRF-CEM (
) cells were transfected
with a series of pGL2 basic constructs containing 5'-terminus promoter
deletions, as indicated on the abscissa. Luciferase activities were
measured in duplicates and normalized to ß-galactosidase activity.
The mean of three independent experiments is shown as a percentage of
the activity of the -944 construct (100%) with SDs given on top of
each bar.
GGCAATTTCC), a
mutated NF-
). Luciferase
activities were measured in duplicates and normalized to
ß-galactosidase activity. The mean of three independent experiments
is shown as a percentage of the activity of the -944 construct (100%)
with SDs given on top of each bar.