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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2002-01-0151.
NEOPLASIA
From the Department of Preventive Medicine and AIDS
Research, Institute of Tropical Medicine, Nagasaki University, Japan;
the Department of Hematology, Molecular Medicine Unit, Atomic Bomb
Disease Institute, and the Department of Laboratory Medicine, Nagasaki
University School of Medicine, Japan; the Department of Internal
Medicine, Sasebo City General Hospital, Japan; the Department of
Internal Medicine, Kokura Memorial Hospital, Kitakyushu, Japan; the
Department of Infectious Disease and Immunology, Okinawa-Asia Research
Center of Medical Science, Faculty of Medicine, University of the
Ryukyus, Nishihara, Okinawa, Japan; and the Division of Virology,
Niigata University Graduate School of Medicine and Dental Sciences,
Japan.
Human T-cell leukemia virus type I (HTLV-I) is the causative agent
of an aggressive form of leukemia designated adult T-cell leukemia
(ATL). We have previously demonstrated that all T-cell lines infected
with HTLV-I and primary leukemic cells from ATL patients display
constitutively high activity of transcription factor NF- Adult T-cell leukemia (ATL) is an aggressive
malignancy of CD4+ T cells, and its development is
closely associated with human T-cell leukemia virus type I (HTLV-I)
infection.1-3 In vitro, HTLV-I transforms primary human
CD4+ T cells in interleukin-2 (IL-2)-dependent and
IL-2-independent manners. Although the mechanism of transformation and
leukemogenesis is not fully elucidated, there is evidence to suggest
that the viral Tax protein plays a crucial role in these processes. For instance, like HTLV-I, Tax immortalizes primary human CD4+
T cells in vitro and transforms rat fibroblast cell
lines.4,5 In addition, Tax inhibits apoptosis induced by
various stimuli in T-cell lines.6
Tax activates viral gene expression through the binding sequence
for cAMP response element binding protein (CREB)/activating transcription factor (ATF) in the 21-bp repeats of the HTLV-I long
terminal repeat (LTR). Tax has been shown to activate the expression of
a number of cellular genes through several distinct transcription
factors, such as NF- Accumulating evidence suggests that activation of cellular genes by Tax
through NF- In resting T cells, NF- ATL continues to have a poor prognosis, mainly because of its
resistance to conventional as well as high-dose chemotherapy. Therefore, the establishment of new therapeutic strategies for ATL is
important. Tax is a candidate for such strategies because it is
necessary for the transformation of HTLV-I-infected T cells. However,
the expression level of Tax in leukemic cells of ATL patients is
extremely low; expression can be detected only by reverse
transcriptase-polymerase chain reaction.26 Furthermore, leukemic cells from several ATL patients possess mutation and truncation of the Tax coding region, which inactivates its
functions.27,28 Thus, Tax may not be essential in the
maintenance of leukemic phenotype in the last stage of the
leukemogenesis, indicating that Tax may not be a good therapeutic
target for ATL.
In a previous study, we showed that the NF- Cell lines and human specimens
Growth inhibition assay
Apo2.7 immunostaining Quantification of apoptosis was performed by immunostaining cells with Apo2.7, which specifically detects the 38-kDa mitochondrial membrane antigen 7A6, which is present only on the mitochondrial membrane of apoptotic cells and can be used as a late apoptotic marker in nonpermeabilized cells.36,37 ATL cells cultured for 48 hours with Bay 11-7082 or media were labeled with the Apo2.7-phycoerythrin-conjugated monoclonal antibody (Beckman-Coulter/Immunotech, Miami, FL) or mouse IgG1 isotype control (Beckman-Coulter/Immunotech) and subsequently analyzed by flow cytometry.Plasmids and transfections The B-LUC construct, which was kindly provided by Dr J. Fujisawa (Kansai Medical University, Osaka, Japan),38
contains 5 tandem repeats of an NF-B binding site from the IL-2R
 -chain gene. HTLV-I LTR-LUC and IL-8 AP-1-LUC39
constructs were generously provided by Dr I. Futsuki (Nagasaki
University School of Medicine, Japan) and Dr N. Mukaida (Kanazawa
University, Kanazawa, Japan), respectively. Transient transfections
were performed in JPX-9 cells by electroporation, using 5 × 106 cells and 5 µg of appropriate reporter plasmids. To
normalize transfection efficiencies, a thymidine kinase (TK)
promoter-driven Renilla luciferase plasmid
(pRL-TK, 1 µg; Promega, Madison, WI) was cotransfected as an internal
control plasmid. Then, 16 hours after transfection, CdCl2
was added to the cultures at a concentration of 20 µM to induce Tax
expression and the cells were further cultured for 48 hours for assay
of luciferase activity. Transfected cells were collected by
centrifugation, washed with phosphate-buffered saline, and lysed in
reporter lysis buffer (Promega). Lysates were assayed for reporter gene
activity with the dual luciferase reporter assay system (Promega).
Electrophoretic mobility shift assay Cells were placed in culture at 1 × 106 cells/mL (cell lines) or 5 × 106 cells/mL (PBMCs) and examined for inhibition of NF-B after exposure to Bay 11-7082 (0, 1.25, 2.5, and 5 µM) for 1, 3, and 24 hours at 37°C . Nuclear proteins were extracted and NF-B and AP-1 binding activities
to B or AP-1 elements were examined by electrophoretic mobility
shift assay (EMSA) as described previously.29,40 In brief,
5 µg nuclear extracts were preincubated in a binding buffer containing 1 µg poly-deoxy-inosinic-deoxy-cytidylic acid (Pharmacia, Piscataway, NJ) followed by addition of 32P-labeled
oligonucleotide probes containing B or AP-1 elements (approximately
50 000 cpm). These mixtures were incubated for 15 minutes at room
temperature. The DNA-protein complexes were separated on a 4%
polyacrylamide gel and visualized by autoradiography. To examine the
specificity of the B element probe, unlabeled competitor
oligonucleotides were preincubated with nuclear extracts for 15 minutes
before incubation with probes. The probes or competitors used were
prepared by annealing the sense and antisense synthetic oligonucleotides as follows: a typical B element from the IL-2R -chain gene, 5'-gatcCGGCAGGGGAATCTCCCTCTC-3'; B
mutant, 5'-gatcCGGCAGatctATCTCCCTCTC-3'; and AP-1 element
of the IL-8 gene, 5'-gatcGTGATGACTCAGGTT-3'. (Underlined
sequences represent the NF-B or AP-1 binding sites, and mutations
are indicated in lowercase.) To identify NF-B proteins in the DNA
protein complex revealed by EMSA, we used antibodies specific for
various NF-B family proteins, including p50, p65, c-Rel, and p52
(Santa Cruz Biotechnology, Santa Cruz, CA), to elicit a supershift DNA
protein complex formation. These antibodies were incubated with the
nuclear extracts for 45 minutes at room temperature before incubation
with radiolabeled probes.
Western blot analysis Cells from treatment were solubilized at 4°C in lysis buffer (0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 66 µg/mL aprotinin, 100 µg/mL phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Cell lysates (50 µg) were resolved by electrophoresis on a 10% polyacrylamide gel and transferred to polyvinylidine difluoride membrane. After blocking of the membrane in 3% skimmed milk and 0.05% Tween 20 in Tris-buffered saline, the blots were incubated with the mouse monoclonal antibody to Tax, Lt-4,41 p53 (NeoMarkers, Fremont, CA), or Bcl-xL (Transduction Laboratories, Lexington, KY) or the rabbit polyclonal antibody to cyclin D1 or cyclin D2 (Santa Cruz Biotechnology). Phosphospecific antibodies to P-Ser15 and P-Ser392 (Oncogene Research Products, Boston, MA) were used in the detection of phosphorylated residues of p53. After several washes, the protein bands recognized by the antibodies were visualized by means of the enhanced chemiluminescence Western blotting detection system (Amersham, Arlington Heights, IL).Northern blot analysis Total RNA (20 µg) was subjected to electrophoresis through a formaldehyde-agarose gel and transferred to a nylon filter. Filters were prehybridized (in 0.5 M sodium phosphate, 0.1% bovine serum albumin, 7% sodium dodecyl sulfate, 100 µg/mL salmon testis DNA, and 100 µg/mL yeast RNA) for 2 hours at 65°C. Hybridization was then carried out overnight in a prehybridization buffer containing the following32P-radiolabeled probes: cDNA of cyclin D1,
cyclin D2, Bcl-xL, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Radiolabeled probes were generated by means of a
Megaprime DNA labeling system (Amersham).
Bay 11-7082 specifically inhibits NF- B
phosphorylation inhibitor, Bay 11-7082, on NF-B activity in
HTLV-I-infected T-cell lines. As shown in Figure
1A, all 4 HTLV-I-infected T-cell lines
(MT-4, SLB-1, C5/MJ, and HUT-102) displayed significantly greater
NF-B/DNA binding than the uninfected T-cell lines (Jurkat and
CCRF-CEM). The observed protein/DNA binding was specific for NF-B,
since the binding was effectively competed and abrogated by excess
unlabeled NF-B oligonucleotide, but not by AP-1 and mutated NF-B
oligonuleotides. Bay 11-7082 inhibited NF-B/DNA binding in all 4 HTLV-I-infected T-cell lines (Figure 1B). To determine the subunit
composition of NF-B complex, we used p50-, p65-, c-Rel-, or
p52-specific antibody to induce a supershift. In HUT-102 cells, p50 and
c-Rel led to supershifting of a fraction of the NF-B complex.
Complex containing p50 and c-Rel was decreased by Bay 11-7082 (Figure
1C). NF-B/DNA binding in SLB-1 cells was reduced within 1 hour after
the addition of Bay 11-7082, and the inhibition was maintained until 24 hours (Figure 1D). To assess the specificity of NF-B inhibition by
Bay 11-7082, the effect of Bay 11-7082 on another transcription factor,
AP-1, was evaluated in SLB-1 cells. Bay 11-7082 at concentrations of
1.25 µM to 5 µM did not significantly affect the binding of AP-1 to
DNA in SLB-1 cells, whereas it reduced that of NF-B, especially at
the 5 µM concentration, suggesting that the inhibitory effects of Bay
11-7082 are specific to NF-B (Figure 1E). Therefore, we used a
5-µM concentration of Bay 11-7082 in subsequent experiments to
evaluate the activity of Bay 11-7082.
Bay 11-7082 specifically inhibits Tax-induced NF- B. JPX-9 is a derivative of the human
HTLV-I-negative T-cell line Jurkat, and has an inducible
tax gene under the control of metallothionein
promoter.33,34 Using JPX-9, we next examined whether Bay
11-7082 inhibits Tax-induced activation of NF-B. Luciferase
expression plasmids regulated by 3 Tax-inducible enhancers were
transfected into JPX-9 cells, and the cells were then treated with
CdCl2. Treatment with CdCl2 efficiently
activated 3 distinct transcription factor pathways NF-B, CREB/ATF,
and AP-1in JPX-9 (Figure 2A), whereas
the same treatment failed to activate the same pathways in JPX/M (data
not shown), as reported previously.12,33 Activation of
NF-B by Tax in JPX-9 cells was significantly inhibited by culture
with 5 µM Bay 11-7082 for 48 hours, whereas the compound had little
if any effect on activation of CREB/ATF and AP-1. We confirmed NF-B
inactivation by the use of EMSA. EMSA showed that the culture of
CdCl2-treated JPX-9 cells with Bay 11-7082 for 48 hours
mostly abrogated NF-B/DNA binding. However, EMSA with the AP-1
binding site as a probe did not reveal any change of the binding of
AP-1 to DNA in CdCl2-treated JPX-9 cells (Figure 2B). These
results indicate that Bay 11-7082 selectively inhibits Tax-induced
NF-B activity in a human T-cell line.
Bay 11-7082 selectively induces apoptosis of HTLV-I-infected T-cell lines To evaluate the role of NF-B in the cell growth of
HTLV-I-infected T-cell lines, we cultured these cells in the presence of Bay 11-7082 for 48 hours. WST-1 cell growth assay showed that Bay
11-7082 reduced cell growth of 4 HTLV-I-infected T-cell lines in a
dose-dependent manner, whereas it only weakly affected the cell growth
of uninfected T-cell lines (Jurkat and CCRF-CEM) even when used at high
concentration (Figure 3A). To examine
whether the induction of apoptosis accounts for the cell growth
inhibition observed in HTLV-I-infected T-cell lines, we used anti-7A6
antibody (Apo2.7, a mitochondrial membrane antigen expressed in
late-stage apoptosis) conjugated with phycoerythrin to stain cells
treated with Bay 11-7082 and then analyzed the stained cells by flow
cytometry (Figure 3B). Significant apoptosis of HTLV-I-infected T-cell
lines, compared with uninfected cells, was noted. DNA extracted from HTLV-I-infected cells treated with Bay 11-7082 revealed marked DNA
fragmentation, another marker of apoptosis, compared with uninfected
and untreated T-cell lines (data not shown). Considered together, these
data suggest that constitutively high NF-B activity in
HTLV-I-infected T cells is required for their survival, and that
specific inhibition of this activity results in the induction of
apoptosis.
To clarify the mechanisms of induced apoptosis of HTLV-I-infected T
cell lines, we examined the expression level of viral Tax and cellular
proapoptotic protein p53 by Western blot analysis. Tax was detected in
all 4 HTLV-I-infected cell lines (MT-4, SLB-1, C5/MJ, and HUT-102) as
a specific band of about 40 kDa (Figure 4), but it was not detected in
HTLV-I-negative cells (Jurkat and CCRF-CEM; data not shown). The
expression of Tax in HTLV-I-infected T-cell lines was not affected by
treatment with Bay 11-7082 for 48 hours (Figure 4). Northern blot
analysis also showed that Bay 11-7082 did not induce any significant
change in the levels of the 3 major HTLV-I mRNAs in infected T-cell
lines (data not shown). In addition, p53 expression in cell lines was
not altered by treatment with Bay 11-7082. It has been shown that the
activation of NF-
Bay 11-7082 down-regulates the expression of cyclin D1, cyclin D2, and Bcl-xL in HTLV-I-infected cells To investigate the molecular mechanism through which Bay 11-7082 inhibits cell growth of HTLV-I-infected T-cell lines, we examined the expression of cell cycle-associated genes cyclin D1 and cyclin D2 and antiapoptotic gene Bcl-xL by Northern blot analysis. These 3 genes contain NF-B enhancer elements and are undetectable in
uninfected T-cell lines Jurkat and CCRF-CEM (data not shown). We and
others have previously shown that Tax induces the expression of these
genes through NF-B binding sites.19-21,24 High
expression levels of cyclin D1 and cyclin D2 were detected in 3 exponentially growing HTLV-I-infected T-cell lines (C5/MJ, HUT-102,
and SLB-1), but the expression of cyclin D3 was not detected in these
cell lines (partial data are shown in Figure
5A,B). Signals were quantitated by
densitometry, and values were normalized to those for GAPDH. On the
basis of quantitations from the Northern blots, we deduced that cyclin
D1, cyclin D2, and Bcl-xL mRNA levels in these 3 HTLV-I-infected T-cell lines were down-regulated by exposure
to Bay 11-7082 (Figure 5D). Western blots with antibodies specific for
cyclin D1, cyclin D2, and Bcl-xL confirmed that Bay 11-7082 treatment of SLB-1 cells had a markedly suppressed expression of these
proteins (Figure 5C).
Effect of NF- B/DNA complex in the
nucleus.29 Thus, we examined whether Bay 11-7082 inhibits the cell growth of primary ATL cells such as HTLV-I-infected T-cell lines. As described in our previous study,29 high
NF-B/DNA binding was detected in nuclear extracts from all primary
ATL cells, but treatment with 5 µM of Bay 11-7082 severely reduced their NF-B/DNA binding activity (Figure
6A). Supershift assay showed a dramatic
decrease in NF-B/DNA binding, previously shown to correspond to
p50-p65 heterodimers (Figure 6B).29 Concomitantly, the
growth of these ATL cells was significantly inhibited compared with
that of normal PBMCs treated with Bay 11-7082 (Figure 6C). Apo2.7
staining detected a high level of apoptosis in 7 ATL specimens, although the degree of apoptosis varied among the specimens (Figure 6D). These data further indicate that NF-B inhibition effectively and preferentially targeted primary ATL cells to induce
apoptosis.
Kitajima et al43 showed that NF- NF- To determine the exact mechanisms by which NF- The other major finding of the present study was that Bay 11-7082 induced apoptosis of fresh primary leukemic cells from ATL patients.
Like HTLV-I-infected T-cell lines, primary ATL cells display the
constitutive NF- NF-
We are deeply indebted to the many patients with ATL and the
control subjects who donated blood for these studies. We thank Drs J. Fujisawa, I. Futsuki, and N. Mukaida for providing luciferase reporter
constructs
Submitted January 17, 2002; accepted April 25, 2002.
Prepublished online as Blood First Edition Paper, May 13, 2002; DOI 10.1182/blood-2002-01-0151.
Supported in part by a grant-in-aid for scientific research (category C) from the Japan Society for the Promotion of Science and the Japan Leukemia Research Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Naoki Mori, Department of Virology, Faculty of Medicine, University of the Ryukyus, 207, Uehara, Nishihara, Okinawa 903-0215, Japan; e-mail: n-mori{at}med.u-ryukyu.ac.jp.
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Human T cell lymphotropic virus type I Tax activates IL-15R | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B and induces
apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell
leukemia cells
Top
Abstract
Introduction
,
monocyte chemoattractant protein-1, granulocyte macrophage-colony
stimulating factor, and granulocyte colony-stimulating factor), their
receptors (the 
/NEMO. Tax interacts with
IKK
