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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2360-2368
Constitutive Activation of NF- B in Primary Adult T-Cell Leukemia
Cells
By
Naoki Mori,
Masahiro Fujii,
Shuichi Ikeda,
Yasuaki Yamada,
Masao Tomonaga,
Dean W. Ballard, and
Naoki Yamamoto
From the Department of Preventive Medicine and AIDS Research,
Research Field of Pathogenesis and Clinical Sciences, Institute of
Tropical Medicine, Nagasaki University, the Department of Laboratory
Medicine, Nagasaki University School of Medicine, the Department of
Hematology, Atomic Disease Institute, Nagasaki University School of
Medicine, Nagasaki; the Department of Medicine, City of Sasebo General
Hospital, Sasebo; the Department of Virology, Niigata University School
of Medicine, Niigata, Japan; and Howard Hughes Medical Institute,
Vanderbilt University School of Medicine, Nashville, TN.
 |
ABSTRACT |
Human T-cell leukemia virus type I (HTLV-I) is an
etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax
induces the activation and nuclear translocalization of transcription factor NF- B, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells
from ATL patients. In this study, we examined the basis for NF-B
activation in freshly isolated leukemic cells from ATL patients. We
found that leukemic cells from ATL patients, like HTLV-I-infected
T-cell lines, display constitutive NF-B DNA binding activity and
increased degradation of IB (an inhibitor of NF-B). Whereas
the NF-B binding activity in Tax-expressing T-cell lines consisted
mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65
heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1,
displayed constitutive NF-B activity, as well as enhanced
degradation of IB, despite the lack of detectable Tax expression.
Interestingly, the NF-B in TL-Om1 consists of p50/p50 and p50/p65
like that in fresh primary leukemic cells. Our results suggest that
activation of NF-B occurs through a Tax-independent mechanism in
leukemic cells of ATL patients, possibly due to differential NF-B
subunit activation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ADULT T-CELL leukemia (ATL) is a highly
aggressive T-cell malignancy etiologically linked to a retrovirus,
human T-cell leukemia virus type I (HTLV-I).1,2 As the
viral genome does not encode a known oncogene3 or promote
cis-activation of endogenous proto-oncogenes by
site-specific integration,4 the events leading to the
occurrence and progression of this hematologic disease remain to be
elucidated. A 40-kD nuclear oncoprotein, termed Tax, appears to be
responsible for the transforming features of HTLV-I. HTLV-I Tax is a
potent transcriptional activator of the HTLV-I long terminal
repeat,5,6 as well as numerous cellular genes involved in
T-cell activation and growth, such as those encoding interleukin-2
(IL-2)7,8 and the  chain of IL-2 receptor (IL-2R).8-10 Because Tax does not bind DNA directly, Tax
appears to induce cellular target genes indirectly via host cellular
pathways,11 including activation of the NF-B/Rel family
of transcription factors.12-14
In resting T cells, NF-B/Rel proteins are sequestered in the
cytoplasm as latent precursors by their inhibitor,
IB.15 Subsequent to cellular activation and through
proteolytic degradation of IB, NF-B is released and
translocates to the nucleus where it transactivates several genes,
including IB itself.16 Recent studies have shown that
Tax induces the degradation of IB, which may contribute to
constitutive activation of NF-B in HTLV-I-infected T-cell
lines.17-21
Although NF-B is known to be constitutively activated in
Tax-expressing and HTLV-I-infected T-cell lines, the activation status
of NF-B in leukemic cells from ATL patients remains unclear. Some of
the genes that can be transactivated by Tax are constitutively expressed in leukemic cells from ATL patients. On isolation, ATL cells
spontaneously display surface IL-2R22 and express mRNA for cytokines including IL-1,23,24
IL-1 ,23-25 IL-6,26,27 IL-8,28
IL-9,29 IL-10,30 tumor necrosis factor
,31 transforming growth factor ,32,33 and
parathyroid hormone-related protein.34,35 Among them, genes
for IL-2R,12-14 IL-1,36
IL-6,27 IL-8,28 and tumor necrosis factor
37 harbor the B enhancer element and Tax activates
transcription of these cellular genes through NF-B binding site.
However, primary leukemic cells isolated from ATL patients typically
express Tax mRNA and protein at low levels, if at all.38,39
These findings imply that there is another mechanism independent of Tax
underlying the overexpression of several cellular genes in fresh ATL cells.
In this study, we examined the activation of NF-B and IB in
leukemic cells isolated from ATL patients and in control cells from
healthy subjects. Tax expression in the primary cells isolated from
these patients was hard to estimate quantitatively with Northern and
Western blot analyses. We found evidence for constitutive degradation
of IB protein, increased nuclear expression of NF-B, and
increased expression of IB mRNA in ATL cells. Also, TL-Om1, which
does not express Tax at all, still exhibits constitutive NF-B
binding activity, composed of p50 and p65, as seen in ATL cells. These
observations suggest Tax-independent mechanisms operating for
constitutive NF-B activation in leukemic cells from ATL patients.
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MATERIALS AND METHODS |
Cells.
MT-2, HUT-102, SLB-1, C5/MJ, and TL-Om1 are human T-cell lines infected
with HTLV-I. Jurkat, MOLT-4, and H-9 are human T-cell lines. These
T-cell lines were maintained in RPMI 1640 supplemented with 10% fetal
bovine serum, glutamine, and antibiotics. JPX-9 is a derivative of
Jurkat cells carrying the transfected Tax gene under the control of
metallothionein promoter.40 Expression of Tax was induced
by adding 20 µmol/L CdCl2 into the medium.
Peripheral blood mononuclear cells (PBMC) from 7 patients, 5 with
acute-type ATL (patients 3 to 7) and 2 with chronic type ATL (patients
1 and 2),41 were analyzed. The diagnosis of ATL was based
on clinical features, hematologic characteristics, the presence of
serum antibodies to ATL-associated antigens, and the presence of the
HTLV-I proviral genome in DNA from leukemic cells. PBMC were isolated
by Ficoll/Hypaque (Pharmacia LKB, Uppsala, Sweden) by density gradient
centrifugation. Each patient had more than 90% leukemic cells in the
blood at the time of analysis.
Detection of IL-2R-positive cells.
The cells were washed once with phosphate-buffered saline (PBS)
containing 0.1% NaN3 and mixed with fluorescein
isothiocyanate-conjugated murine monoclonal antibody to the chain
of the human IL-2R. After incubation for 30 minutes at 4°C, they
were washed with PBS containing 0.1% NaN3 and then
analyzed using a FACScan (Becton Dickinson, San Jose, CA).
Northern blot analysis.
Total RNA was extracted from cells using the Trizol method as described
by the manufacturer (GIBCO-BRL, Gaithersburg, MD). Twenty micrograms of
RNA was electrophoresed through a formaldehyde-agarose gel and
transferred to a nylon membrane. Membranes were prehybridized (0.5 mol/L sodium phosphate, 0.1% bovine serum albumin, 7% sodium dodecyl
sulfate [SDS], 100 µg/mL salmon testis DNA, and 100 µg/mL yeast
RNA) for 2 hours at 65°C and then hybridized overnight with the
following [-32P] radiolabeled probes: cDNA of human
IB,42 IL-2R (kindly provided by Dr T. Taniguchi,
Tokyo University, Tokyo, Japan),43 glyceraldehyde-3-phosphate dehydrogenase (GAPDH),44 and
HTLV-I Tax.45 Radiolabeled probes were generated by using a
Megaprime DNA Labeling system (Amersham, Arlington Heights, IL).
Western blotting.
The antibody used in these experiments was rabbit polyclonal antibody
to IB, C-21 (Santa Cruz Biotechnology, Santa Cruz, CA). Cells
were lysed by incubation in radio-immuno-protein-assay (RIPA) buffer (0.5% sodiumdeoxycholate, 1% Nonidet
P-40, 0.1% SDS, 66 µg/mL aprotinin, 100 µg/mL phenylmethylsulfonyl
fluoride, and 1 mmol/L sodium orthovanadate) for 30 minutes at 4°C.
Equal amounts (50 µg) of protein from cell lysates were
electrophoresed on 10% SDS-polyacrylamide gel electrophoresis gels and
transferred to polyvinylidine difluoride membranes. The membranes were
washed with Tris-buffered saline-Tween (0.05%) with 3% nonfat dried
milk overnight at 4°C and then blotted for 45 minutes with the
antibody. The membranes were washed with Tris-buffered saline-Tween and incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated antirabbit immunoglobulin (Amersham). Thereafter, membranes were developed with enhanced chemiluminescence reagents (Amersham) and
exposed to film.
Oligonucleotides.
The sequence of the oligonucleotide corresponding to the B element
from IL-2R gene was
5'-gatcCGGCAGGGGAATCTCCCTCTC-3'.46 Underlined sequences represent the B motif. The oligonucleotide, 5'-gatcTGTCGAATGCAAATCACTAGAA-3', containing the
consensus sequence of the octamer binding motif (underlined) was used
to identify specific binding of the transcription factor Oct-1. This
transcription factor regulates transcription of a number of so-called
housekeeping genes. For competition studies, oligonucleotides
containing mutant B and two B motifs derived from the human
immunodeficiency virus type 1 long terminal repeat (LTR) were used, and
the sequences are 5'-gatcCGGCAGatctATCTCCCTCTC-3'
and
5'-gatcACAAGGGACTTTCCGCTGGGGACTTTCCAG-3', respectively. For the preparation of a probe in electrophoretic mobility shift assay (EMSA), a radiolabeled double-stranded
oligonucleotide was prepared by annealing and filling in the overhang
with the Klenow fragment of DNA polymerase I in the presence of
[-32P]deoxycytidine triphosphate
(dCTP) and [-32P]deoxyadenosine
triphosphate (dATP).
Preparation of nuclear extracts.
Nuclear extracts were prepared as described by Antalis et
al47 with modifications. Cells (107) were
washed twice with cold PBS and the cell pellet was suspended in 400 µL of hypotonic buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol [DTT], 2 mmol/L aminoethyl-benzenesulfonyl fluoride
[AEBSF], and 0.2% Nonidet P-40) for 10 minutes at
4°C. Nuclei were prepared by microcentrifugation for 5 minutes at
4°C. The nuclear pellet was suspended in 75 µL of buffer C (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 2 mmol/L AEBSF, 33 µg/mL aprotinin, 10 µg/mL leupeptin,
10 µg/mL E-64, and 10 µg/mL pepstatin A) and incubated for 30 minutes at 4°C with brief mixing. The mixture was microcentrifuged
(15,000 cpm) for 15 minutes at 4°C. Protein concentration was
measured by using the Bradford assay (Bio-Rad, Richmond, CA).
EMSA.
As previously described,36 nuclear extracts (5 µg of
protein) were preincubated in a 20-µL total reaction volume
containing 10 mmol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 5% glycerol, and 1 µg of
polydeoxyinosinic-deoxycytidylic acid (Pharmacia, Piscataway, NJ) for
15 minutes at room temperature. The reaction mixture was then incubated
with the radiolabeled oligonucleotide (50,000 cpm) for 15 minutes at
room temperature. The samples were analyzed by electrophoresis in a 4%
nondenaturing polyacrylamide gel with 0.25 × TBE buffer (22.3 mmol/L Tris, 22.2 mmol/L boric acid, and 0.5 mmol/L EDTA). The gels
were dried and analyzed by autoradiography.
Plasmids and transfection.
A reporter plasmid B-LUC (a kind gift from Dr J. Fujisawa, Kansai
Medical University, Osaka, Japan) was constructed by inserting five
repeats of the NF-B binding sequence of the IL-2R gene fused to
an enhancerless promoter of HTLV-I into pGL2-Basic containing the
luciferase gene, dN-LUC.21 The NF-B expression plasmids, human p50, p65, and c-Rel, were the generous gifts of Dr K. Yamamoto (Kanazawa University, Kanazawa, Japan). Transfections were performed by
electroporation.36 In all cases, the reference plasmid
pRL-CMV, which contains the Renilla luciferase gene under the control
of the cytomegalovirus immediate early enhancer/promoter, was
cotransfected to correct for transfection efficiency. For luciferase
assay, the transfected cells were lysed in a lysis reagent (Toyo Ink Co, Tokyo, Japan), and luciferase activities were measured according to
the manufacturer's procedures. Each assay was independently repeated
at least three times.
| |
RESULTS |
HTLV-I-infected T-cell lines constitutively express a factor mediating
activation of NF-B element.
As an initial test of the activity of NF-B in HTLV-I-infected
T-cell lines, a transfection analysis was performed using a reporter
construct, B-LUC, containing a luciferase gene under the control of
the NF-B binding site from the IL-2R gene. The level of
luciferase activity detected in uninfected cells transfected with
B-LUC was similar to that of cells transfected with dN-LUC (Fig 1A). On the other hand, in all of the
five HTLV-I-infected cell lines, the B-LUC reporter gene was 9 to
13 times more active than dN-LUC, indicating enhanced NF-B activity
in HTLV-I-infected cell lines.
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| Fig 1.
(A) HTLV-I-infected T-cell lines express a constitutive
NF-B-related activity. Various cell lines were transfected with 10 µg of a reporter plasmid containing the luciferase gene fused to five
repeats of the B motif of the IL-2R gene and enhancerless
promoter of HTLV-I (B-LUC). After 24 hours of incubation, lysates
were prepared and assayed for luciferase activity. Ratio of luciferase
activity in extracts of B-LUC-transfected cells is expressed to
that for dN-LUC-transfected cells. The results represent the mean of
three experiments. (B) Northern blot analysis of Tax in various cell
lines. Total RNA (20 µg of each) was isolated from various cell
lines. Jurkat, MOLT-4, and H-9 are HTLV-I-uninfected T-cell lines.
MT-2, HUT-102, SLB-1, TL-Om1, and C5/MJ are HTLV-I-infected T-cell
lines. Blots were sequentially hybridized, exposed, stripped, and
rehybridized with Tax and GAPDH probes.
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Among five HTLV-I-infected cell lines, four (MT-2, HUT-102, SLB-1,
C5/MJ) expressed high levels of HTLV-I Tax transcript. In contrast, the
TL-Om1 cell line48 failed to express significant amounts of
Tax at the mRNA level (Fig 1B). TL-Om1 did not express Tax mRNA at all
by a highly sensitive polymerase chain reaction method coupled with
reverse transcription.49 These results suggest that Tax is
not required to maintain NF-B-dependent transcription in
HTLV-I-infected T-cell lines.
Potential alterations in cellular gene activation associated with the
Tax-independent activation of NF-B in TL-Om1 were next studied by
measuring expression of the IL-2R protein and its mRNA. Like the
other four Tax-expressing cell lines, TL-Om1 expressed IL-2R on its
surface (Fig 2A). In addition, constitutive
expression of the 25S and 16S forms of IL-2R mRNA50 was
detected in TL-Om1, as well as the other four HTLV-I-infected cell
lines, whereas it was not in uninfected cell lines (Fig 2B). These
results showed that TL-Om1 has a Tax-independent constitutive
expression of the IL-2R gene, possibly due to the activation of
NF-B.
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| Fig 2.
Expression of IL-2R at protein (A) and mRNA (B) levels
in various cell lines. (A) The percentages of positive cells for
IL-2R are shown. (B) Total RNA (20 µg of each) was isolated from
various cell lines and assessed for IL-2R or GAPDH mRNA
expression.
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Lower protein levels of IB in HTLV-I-infected T-cell lines.
In a variety of cell lines, NF-B is localized in the cytoplasm
through binding to specific cytoplasmic molecules called IB. After cellular stimulation by multiple inducers, activation of signal
transduction cascades leads to the degradation of IB. In
agreement with prior studies,21 the protein level of
IB was significantly lower in HTLV-I-infected cell lines
(Fig 3A). More importantly, TL-Om1 cells,
which do not express Tax, have reduced IB protein at a similar
level to HTLV-I-infected cell lines expressing Tax. These results
suggest that IB protein expression is downregulated in TL-Om1 by
a Tax-independent mechanism.
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| Fig 3.
Expression of IB at protein (A) and mRNA (B) levels
in various cell lines. (A) Whole-cell extracts were isolated from the
cells and analyzed by Western blotting using anti-IB antibody.
(B) Total RNA (20 µg of each) was isolated from various cell lines
and assessed for IB or GAPDH mRNA expression.
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Overexpression of IB mRNA in HTLV-I-infected T-cell lines.
Next, we performed Northern blot analysis of IB mRNA in the
T-cell lines characterized above. As shown in Fig 3B, the expression of
IB mRNA in TL-Om1, as well as Tax-expressing cell lines, was
strikingly higher than in uninfected ones. Thus, decreased IB
protein level detected in HTLV-I-infected cell lines, including TL-Om1, is not due to impaired IB gene activity, but due to the
rapid turnover of IB. It has been reported previously that Tax
disrupts feedback regulation of NF-B through destabilization of
IB,17-21 thus establishing constitutive activation of
NF-B. JPX-9 cells are derived from a human T-cell line Jurkat, and
they are permanently transfected with a metallothionein promoter-driven Tax expression vector.40 As a result of the expression of
Tax in JPX-9 cells by CdCl2 treatment, IB mRNA
expression was markedly upregulated (Fig
4A). However, the total protein levels for IB remained constant
(Fig 4B), possibly indicative of a more rapid turnover of IB.
Taken together, these results show that Tax-mediated destabilization of
IB is the mechanism for reduction of IB protein in
HTLV-I-infected cell lines expressing Tax. These data also suggest
that rapid degradation of IB is maintained by mechanisms other
than Tax in TL-Om1 cells.
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| Fig 4.
Tax-induced proteolytic degradation of IB. JPX-9
cells were incubated with CdCl2 (20 µmol/L) for the
indicated time periods and then collected for preparation of either
total cellular RNA or whole-cell extracts. (A) Total RNA isolated from
either nontreated or CdCl2-treated JPX-9 cells was
subjected to Northern blot analysis with Tax, IB, and GAPDH
probes. (B) Whole-cell extracts isolated from CdCl2-treated
JPX-9 cells were subjected to Western blotting using anti-IB
antibody.
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Enhanced IB degradation in primary ATL cells.
We had postulated that the IB/NF-B pathways may already be
activated in ATL leukemic cells in vivo. Therefore, we examined whether
the expression of IB in freshly obtained PBMC of ATL patients is
upregulated. PBMC were isolated from peripheral blood of seven ATL
patients and RNA was prepared for Northern blot analysis. As shown in
Fig 5A, IB mRNA levels in freshly
isolated PBMC of ATL patients were much higher than those in PBMC from
healthy control subjects. Furthermore, IL-2R expression was stronger in primary ATL cells than in normal PBMC (Fig 5A). These results are
similar to the high expression of IB and IL-2R mRNA observed in HTLV-I-infected T-cell lines. In addition, Tax expression in the
primary cells isolated from these patients was hard to estimate quantitatively with Northern and Western blot analyses (data not shown).
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| Fig 5.
Expression of IB at mRNA (A) and protein (B) levels
in primary leukemic cells from ATL patients and normal healthy
subjects. (A) Total RNA was extracted from PBMC and assessed for
IB, IL-2R, or GAPDH mRNA expression. A representative Northern
blot is shown from patients with ATL and healthy subjects (normal). (B)
PBMC obtained from patients with ATL or healthy subjects (normal) were
assessed for IB by Western blot analysis.
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We next determined the protein levels of IB in blood mononuclear
cells from patients with ATL and from healthy subjects. Whole-cell
extracts were prepared from PBMC and subjected to Western blot analysis
(Fig 5B). The IB reactivity band was strongly visible in PBMC
from healthy subjects. In contrast, IB reactivity was weak in
PBMC derived from two cases (ATL patients 5 and 6). The remaining five
samples did not show apparent reduction of the bands. However, these
results were not influenced by the contents of leukemic cells in these
samples. While IB mRNA levels were dramatically upregulated in
ATL cells, the total protein levels did not increase, possibly
indicative of protein degradation.
Constitutive NF-B binding activity in HTLV-I-infected
T-cell lines and primary ATL cells.
To determine whether degradation of IB protein in
HTLV-I-infected T-cell lines was associated with NF-B activation,
nuclear extracts of cells were subjected to EMSA. In uninfected T-cell lines, no NF-B-specific protein DNA complexes were detected in nuclear extracts by EMSA (Fig 6A, lanes 1 through 3). In contrast, enhanced NF-B activity was present in each
of the HTLV-I-infected T-cell lines tested (Fig 6A, lanes 4 through
8). The specificity of the binding activities was shown by competition
with excess wild-type and mutant oligonucleotides (data not shown).
Constitutive NF-B binding activity in MT-2 cells was composed
predominantly of c-Rel and p50, as antibodies against c-Rel and p50
partially inhibited formation of the NF-B DNA complex and also
produced a shifted complex (Fig 6B, left panel). Similar analyses
showed that c-Rel and p50 subunits represented the main DNA binding
components in HUT-102, SLB-1, and C5/MJ cells (data not shown). In
TL-Om1 cells, two forms of complexes were detected (Fig 6A). Incubation of the nuclear extracts with various antibodies represented that the
upper band contains p50 and p65, and the lower band contains only the
p50 (Fig 6B, right panel).
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| Fig 6.
NF-B binding activity in various cell lines. Nuclear
extracts were prepared from cells. Nuclear extracts (5 µg) were
incubated with a [32P]-radiolabeled oligonucleotide that
corresponds to the B sequence of the IL-2R gene and analyzed by
EMSA. (B) In super-shift assays, antibody specific for each NF-B
subunit (indicated above the lanes) was incubated with MT-2 and TL-Om1
extracts before probe addition.
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We determined whether degradation of IB protein in primary ATL
cells involved abnormal NF-B-specific DNA binding activity. To
address this question, nuclear extracts of PBMC obtained from seven
patients with ATL and healthy subjects, the same samples as analyzed in
Fig 5, were subjected to EMSA. In extracts from healthy subjects,
NF-B-specific complex was detected (Fig
7A, left panel). This protein DNA complex was composed primarily of p50; interaction with anti-p65, c-Rel, and p52 antibodies was negligible (Fig 7D, right panel). On the other hand, two forms of
complexes were detected, an upper band and a lower band in extracts
from primary ATL cells (Fig 7A, left panel). Although the observed
complexes formed broad bands in patients 2, 5, 6, and 7, lighter
exposure of the gel showed two bands in these cases (Fig 7A, right
panel). The specificity of these complexes was verified by competition
with an excess of wild-type and mutant oligonucleotides (Fig 7C).
Incubation of the protein DNA complex with anti-p50 resulted in almost
complete supershift of the upper and lower migrating bands (Fig 7D,
left panel). Incubation with anti-p65 reduced only the upper band
completely. Further, anti-c-Rel and p52 antibodies failed to shift any
bands (Fig 7D, left panel). This result suggests that the upper band
contains p50 and p65, and the lower band contains the p50 homodimers.
No differences between ATL patients and healthy subjects in binding to
the octamer motif on DNA were found (Fig 7B).
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| Fig 7.
Activation of NF-B in primary ATL cells. (A) Nuclear
extracts (5 µg) from PBMC from patients with ATL or from healthy
subjects (normal) were assessed for activation of NF-B by EMSA. The
right panel represents a lighter exposure of patients 2, 5, 6, and 7. (B) Nuclear extracts from patients with ATL or from healthy subjects
(normal) were analyzed for protein binding to the octamer consensus
sequence by EMSA. (C) Nuclear extracts from patient 3 sample were
coincubated with 100-fold excess of unlabeled oligonucleotides and
assessed in parallel. (D) Nuclear extracts were preincubated with
NF-B subunit-specific antibody, as indicated above each lane, before
the addition of radiolabeled probe (as in Fig 6).
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Transactivational activities of different members of the NF-B/Rel
family at the IL-2R B site.
To determine the transcription activity of different subunit members of
the NF-B/Rel family, expression plasmids for three B-related
proteins, p50, p65, and c-Rel, were cotransfected in Jurkat cells with
a reporter plasmid, B-LUC. As shown in
Fig 8, expression of p65 and c-Rel, by
themselves or in combination with p50, led to an increase in luciferase
activity, while expression of p50 was devoid of effect on the reporter
gene transcription. These results recapitulate the observed EMSA and
IL-2R expression data, indicating that p65 or c-Rel binding can
activate NF-B-dependent transcription. However, p50 homodimers can
bind to the B element in the IL-2R, but are unable to support
transcriptional activation.
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| Fig 8.
Effect of NF-B subunit on NF-B transcriptional
activity. Jurkat cells were transfected by electroporation with a
reporter plasmid (5 µg) containing the luciferase gene fused to five
repeats of the B motif of the IL-2R gene and enhancerless
promoter of HTLV-I (B-LUC) along with combinations of the cDNA
expression plasmids encoding p50 (5 µg), p65 (5 µg), and c-Rel (5 µg) indicated at the bottom of the panel. After 24 hours of growth,
cells were harvested and assayed for luciferase activity. Increase of
the luciferase activity in cell extracts is shown relative to that
after parental plasmid transfection. The mean ± standard error of
mean (SEM) for three independent assays is presented.
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| |
DISCUSSION |
We analyzed for the first time the functional status of the
NF-B/IB pathway in primary leukemic cells from ATL patients. In all patients, NF-B protein's DNA binding activity was detected by EMSA in the leukemic cell extracts. Furthermore, this study shows
that in contrast to healthy subjects, IB in freshly isolated leukemic cells from patients with ATL has already undergone proteolytic degradation. IB mRNA was upregulated in ATL cells of patients. However, IB protein levels did not increase in concert with the
highly elevated expression of the mRNA. Furthermore, IB protein
levels were significantly lower in two samples. Thus, increased
turnover of the IB protein in leukemic cell extracts of patients
with ATL was associated with activation of NF-B. These results were
similar to those observed in HTLV-I-infected and Tax-expressing T-cell
lines. Recently, like IB, Tax-mediated breakdown of IB was
reported.51,52 In preliminary experiments, little or no
detectable amount of IB was found in primary ATL cells. Together,
these results suggest that the degradation of IB and IB may
play a role in the constitutive activation of NF-B in ATL.
Importantly, the identity of the shifted bands was confirmed by
supershifting with NF-B-specific antibodies. We observed that the
dynamic alterations in NF-B binding activity occurred in ATL
patients. In all patients' extracts, the DNA protein complex consists
of p50/p65 heterodimers and p50 homodimers. In contrast, NF-B
binding activity in healthy subjects was composed of p50 homodimers
alone. Interestingly, cotransfection with various NF-B subunits
indicated that p65 and c-Rel, but not p50, was able to activate
transcription at the IL-2R B site. The observation that
constitutive binding of heterodimeric p50/p65 correlated with
expression of IL-2R in ATL patients supports the notion that in vivo
p50/p65 heterodimers and p50 homodimers can bind to the B element in
the IL-2R promoter, but only p50/p65 heterodimers can activate transcription.
HTLV-I Tax has been shown to induce a degradation of IB, which
may contribute to induce the nuclear expression of a number of
NF-B/Rel species.17-21 NF-B is known to mediate the
Tax-dependent transactivation of IL-2R,12-14
IL-1,36 IL-6,27 IL-8,28 and
tumor necrosis factor genes.37 Considering the
previously reported enhanced production of these inflammatory
cytokines23,24,26-28,31 and the increased expression of
surface molecules such as IL-2R22 in leukemic cells of
ATL patients, it is not surprising that activated NF-B/IB
pathway was seen in ATL cells. However, consistent with previous
studies,38,39 primary leukemic cells isolated from ATL
patients did not express Tax at a significant level. Because viral
expression in vivo differs significantly from that observed in cell
lines in vitro, it is of interest to understand the differences of
NF-B activation between leukemic cells in vivo and cell lines
established in vitro. Tax induces predominantly the c-Rel-containing
complexes17,53 and transcriptionally activates the c-Rel
gene.53 Consistent with these studies, in HTLV-I-infected T cells that constitutively express high levels of Tax, p50 and c-Rel
were the major DNA binding components; similar c-Rel containing complexes were also observed previously,19,53,54 suggesting Tax-independent mechanisms by which primary ATL cells exhibit persistent nuclear expression of p50/p65 heterodimers.
In this study, we have shown that the ATL-derived TL-Om1 cell line
fails to express detectable levels of Tax. However, TL-Om1 was shown to
carry full-length proviral genome of HTLV-I by Southern blot
analysis.48 It was also found that the
HTLV-I LTR function was not enhanced in TL-Om1, as the LTR luciferase
activity in TL-Om1 was one fourteenth of that in MT-2. Furthermore, Tax
induced transcription from the LTR in TL-Om1 (data not shown). We have also shown the rapid degradation of IB in TL-Om1 cells, which may
be directly involved in constitutive NF-B activation. This line
still expresses IL-2R mRNA and protein at high levels. In addition,
TL-Om1 cells express chemokine genes such as IP-10 and I309, and
intercellular adhesion molecule 1 at the same levels as those in
Tax-expressing T-cell lines infected with HTLV-I.49,55 These results suggest that Tax is not the only mechanism for
constitutive expression of certain cellular genes in HTLV-I-infected T
cells. We believe in the possibility of Tax-independent mechanisms
operating for constitutive NF-B activation in leukemic cells of ATL
patients. TL-Om1 should help to identify new targets of NF-B by
comparing gene expression between this cell line and other
Tax-expressing cell lines.
| |
ACKNOWLEDGMENT |
We thank Drs T. Taniguchi, J. Fujisawa, and K. Yamamoto for providing
plasmids, Dr M. Nakamura for providing JPX-9, and Fujisaki Cell Center,
Hayashibara Biochemical Laboratories Inc (Okayama, Japan) for providing
Jurkat, HUT-102, and C5/MJ. We also thank Dr Y. Yamasaki for providing
blood of ATL patients and M. Yamamoto and M. Sasaki for excellent
technical assistance.
| |
FOOTNOTES |
Submitted July 29, 1998; accepted November 22, 1998.
Supported in part by a Grant in Aid for Scientific Research from the
Ministry of Education, Science and Culture, Japan, and a Cooperative
Research Grant No. 1998-10-A-14 from the Institute of Tropical
Medicine, Nagasaki University.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Naoki Mori, MD, Department of Preventive
Medicine and AIDS Research, Research Field of Pathogenesis and Clinical
Sciences, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
| |
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TOP
ABSTRACT
INTRODUCTION