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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3915-3921
NEOPLASIA
From the Department of Preventive Medicine and AIDS Research,
Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan;
Department of Laboratory Medicine, Nagasaki University School of
Medicine, Nagasaki, Japan; Department of Hematology, Atomic Disease
Institute, Nagasaki University School of Medicine, Nagasaki, Japan;
Department of Internal Medicine, City of Sasebo General Hospital,
Sasebo, Japan; Department of Internal Medicine, Kokura Memorial
Hospital, Kitakyushu, Japan; Department of Infectious Disease and
Immunology, Okinawa-Asia Research Center of Medical Science, Faculty of
Medicine, University of the Ryukyus, Okinawa, Japan; Department of
Virology, Niigata University School of Medicine, Niigata, Japan.
Human T-cell leukemia virus type-I (HTLV-I) is the etiologic agent
of adult T-cell leukemia (ATL). This study examined the status of the
oncogenic transcription factor AP-1 in leukemic cells freshly isolated
from patients with ATL. Leukemic cells from peripheral blood of all
patients with ATL exhibited constitutive AP-1 DNA binding activity,
whereas mononuclear cells from normal individuals did not. In agreement
with previous studies, HTLV-I transforming protein, Tax, was found to
stimulate the DNA binding activity of AP-1 in a T-cell line. However,
HTLV-I genes, including Tax, were not significantly expressed in
leukemic cells freshly obtained from patients with ATL. Moreover, all
T-cell lines derived from leukemic cells of patients with ATL also
displayed constitutive AP-1 DNA binding activity, but expressed little
Tax protein. Thus, leukemic cells of patients with ATL appear to have
Tax-independent mechanisms that induce AP-1 activity, both in vivo and
in vitro. In antibody supershift experiments, AP-1 in fresh leukemic
cells and ATL-derived cell lines were found to contain JunD.
Consistently, all primary ATL cells and ATL-derived cell lines
expressed high levels of JunD messenger RNA. Our results suggest that
AP-1 is activated in leukemic cells of patients with ATL through a
Tax-independent mechanism and this may play a role in the deregulated
phenotypes of ATL leukemic cells.
(Blood. 2000;95:3915-3921)
Adult T-cell leukemia (ATL) is an aggressive and fatal
T-cell malignancy caused by infection with human T-cell leukemia virus type I (HTLV-I).1-3 HTLV-I is also associated with the
development of a variety of chronic inflammatory diseases such as
HTLV-I-associated myelopathy/tropical spastic paraparesis and uveitis.
In addition to structural proteins, the HTLV-I genome encodes several
regulatory proteins, including Tax,4 a 40-kd
transcriptional transactivator of HTLV-I gene expression. There is
increasing evidence that Tax plays a crucial role in cellular
transformation.5 For instance, Tax transforms fibroblast
cell lines and immortalizes primary human T cells in vitro, in the
presence of interleukin (IL)-2. Tax also induces various tumor types,
including neurofibromas and sarcomas, in addition to large granular
lymphocytic leukemia in transgenic animals.
Tax activates a number of cellular genes, including those encoding
cytokines,6-8 their receptors,9 and nuclear
proto-oncogenes.10-12 Some of these genes are thought to
contribute to T-cell immortalization or transformation. Tax activates
the transcription of cellular genes through selective enhancer elements
such as cyclic adenosine monophosphate-responsive element (CRE), the
NF- The development of leukemia in individuals infected with HTLV-I is
preceded by a long clinical latent period of 40 to 50 years. In
addition, only 5% of HTLV-I carriers develop ATL. Thus, it seems that
several events in HTLV-I-infected cells are required for the
development of the full malignant phenotype. Most leukemic cells do not
express significant levels of Tax in vivo. Indeed, the expression of
Tax can only be detected using a highly sensitive reverse
transcriptase-polymerase chain reaction (RT-PCR)
method.24 Thus, the expression of viral proteins, including
Tax, does not seem to be necessary for the proliferation of leukemic
cells in the late stages of the disease. However, the cellular events
that cause deregulated proliferation of leukemic cells in vivo, have not yet been elucidated.
In this study, we investigated AP-1 activity in peripheral blood
mononuclear cells (PBMC), freshly obtained from patients with ATL.
Results showed that AP-1 complexes containing JunD were highly
expressed in PBMC from all patients with ATL, although no viral gene
expression was detected. Furthermore, all T-cell lines derived from
patients with ATL displayed constitutive AP-1 DNA binding activity
involving JunD, but expressed little Tax protein. These findings
suggest that Tax is not the only mechanism for constitutive activation
of AP-1 in HTLV-I-infected T cells. Hence, a Tax-independent mechanism
appears to exist for constitutive AP-1 activation in leukemic cells of
patients with ATL. These findings are discussed in the context of
deregulated proliferation of leukemic cells in the absence of
detectable viral gene expression in vivo.
Cells
Northern blot analysis
Western blotting
Oligonucleotides The sequence of the oligonucleotide corresponding to the AP-1 motif, derived from IL-8 gene was 5'-gatcGTGATGACTCAGGTT-3'. Underlined sequences represent the AP-1 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 a mutated AP-1 binding site, a consensus AP-1 binding site, and a TPA (12-O-tetradecanoylphorbol 13-acetate)-responsive element (TRE) sequence, derived from the human metallothionein gene promoter,39 were used. The sequences were 5'-gatcGaaACTCAGCGCG-3', 5'-gatcCGCTTGATGAGTCAGCCGGAA-3', and 5'-gatcGTGACTCAGCGCG-3', respectively. For preparation of the probe used in the electrophoretic mobility shift assay (EMSA), a radiolabeled double-stranded oligonucleotide was prepared by annealing and filling the overhang using the Klenow fragment of DNA polymerase I in the presence of -32P-deoxycytidine triphosphate and
-32P-deoxyadenosine triphosphate.
Preparation of nuclear extracts Nuclear extracts were prepared as described by Antalis et al,40 with some modifications. Cells (107) were washed twice with cold phosphate-buffered saline and the cell pellet was suspended in 400 µL 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-benzene sulfonyl 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 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. The protein concentration was measured using the Bradford assay (Bio-Rad, Richmond, CA).EMSA As previously described,7 nuclear extracts (5 µg of protein) were preincubated in 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 poly-deoxy-inosinic-deoxy-cytidilic 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. Samples were analyzed by electrophoresis using 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. Supershifts were performed by adding antibodies to the incubating mixture of nuclear extracts and the labeled DNA probe. One microgram of anti-c-Fos, Fra-1, c-Jun, JunB, and JunD antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and 2 µg of anti-FosB and Fra-2 antibodies (Santa Cruz Biotechnology) were used per lane.Plasmids and transfection A reporter plasmid 2× AP-1 site LUC (a kind gift from Dr N. Mukaida, Kanazawa University, Kanazawa, Japan) is a luciferase expression plasmid controlled by 2 copies of the AP-1 binding site from the IL-8 promoter.41 The Tax expression plasmid, pH2R40M,42 was a gift from Dr M. Hatanaka (Shionogi Institute for Medical Science, Osaka, Japan). The JunD expression plasmid, pSG-JunD, was prepared by subcloning a cDNA fragment of human JunD into the EcoRI site of pSG5. Transfections were performed by electroporation.7 In all cases, the reference plasmid, pRL-TK, an internal control renilla luciferase expression vector (Toyo Ink Co, Tokyo, Japan), was cotransfected to correct for transfection efficiency. For the luciferase assay, transfected cells were lysed in lysis reagent (Toyo Ink Co), and luciferase activity was measured according to the protocol provided by the manufacturer. Each assay was independently repeated at least 3 times.
ATL-derived cell lines exhibit little expression of viral genes, including Tax Three HTLV-I-negative and 10 HTLV-I-positive cell lines were used for these experiments. HTLV-I-positive cell lines can be classified into 2 groups (Table 1). The 4 cell lines (MT-2, MT-4, C5/MJ, and SLB-1) were established by the in vitro transformation of normal T cells with HTLV-I. Five cell lines (MT-1, TL-OmI, SO-4, ST-1, and KK-1) originated from leukemic cells of patients with ATL. The leukemic cell origin of these cell lines was previously confirmed by having the same provirus integration sites or T cell receptor -chain gene
rearrangement profiles as those of their original leukemic cells, by
Southern blot25 or chromosomal analysis. HUT-102 could not
be classified by this criterion because its clonal origin had not been examined.
Constitutive AP-1 binding activities containing JunD in
HTLV-I-infected cell lines
Activation of JunD in HTLV-I-infected T-cell lines We next examined components of the AP-1 complex in the ATL-derived cell line, MT-1. The antibody supershift assay showed that the AP-1 complex in MT-1 contained JunD, because the anti-JunD antibody induced a slowly migrating (supershift) complex when added to the assay reaction (Figure 2A). No obvious changes were found when antibodies against c-Fos, FosB, Fra-1, Fra-2, c-Jun, and JunB were used (Figure 2A). The complex was found to contain JunD in the remaining HTLV-I-infected cell lines (data not shown). Therefore, we examined by Northern blot analysis the expression of JunD mRNA in these T-cell lines, which either did or did not express Tax. All HTLV-I-infected T-cell lines, including the 5 ATL-derived cell lines, expressed high levels of JunD mRNA, whereas JunD mRNA was not detectable in 3 HTLV-I-negative cell lines (Figure 2B). Thus, ATL-derived cell lines overexpressed JunD mRNA, even though they expressed little Tax.
Involvement of Tax in induction of AP-1 binding activity In the next series of experiments, we used the JPX-9 cell line to obtain direct evidence for the induction of AP-1 activity by Tax. JPX-9 is derived from the Jurkat T-cell line and has an inducible Tax gene under the control of a metallothionein promoter. Treatment of JPX-9 cells with CdCl2 (Figure 3A, lanes 2, 4, and 6) resulted in a significant increase in IL-8 gene complex formation with the AP-1 site. The level of expression of Tax mRNA and protein in these cells was determined by Northern and Western blot analyses (Figure 3B and C). The specificity of the complex was confirmed by performing competition assays with the autologous oligonucleotide (Figure 3D, lane 3), TRE oligonucleotide (lane 4), the consensus AP-1 site (lane 5), and the mutated AP-1 site oligonucleotide (lane 6). We further analyzed the composition of the AP-1 complex using the antibody supershift assay. As shown in Figure 3E, the complex in JPX-9 stimulated with CdCl2 contained JunD, because antisera specific for JunD induced a supershift complex (lane 9). Thus, Tax can lead to activation of AP-1, containing the JunD protein, in a T-cell line.
Constitutive activation of AP-1 in freshly prepared primary leukemic cells from patients with ATL The above data showed that cell lines originating from ATL cells express high levels of AP-1 activity. We next examined whether AP-1 is indeed activated in vivo in primary leukemic cells of patients with ATL. Nuclear extracts were prepared from PBMC of 11 patients, including 7 with acute-type ATL and 4 with chronic-type ATL. High levels of binding to the AP-1 site of the IL-8 gene were detected in nuclear extracts prepared from all patients (Figure 4A, lanes 3-13). However, no such activity was found in PBMC extracts from 2 healthy volunteers (lanes 1 and 2). The specificity of this activity was verified by competition with an excess of wild-type and mutant oligonucleotides (Figure 4B). However, no differences between patients with ATL and healthy volunteers were observed when the octamer motif sequence was used as a probe for EMSA.44 Furthermore, no quantitative or qualitative differences in AP-1 binding activity were observed between patients with acute and chronic disease (Figure 4A). It should be noted that Tax expression was not detectable by Northern and Western blot analyses of primary cells isolated from these patients, although it could be detected by RT-PCR (references 24 and 45, and data not shown). Thus, these results indicate a high level of AP-1 activity is present in primary leukemic cells of ATL patients in vivo and that this activity is likely to be independent of Tax.
Activation of JunD in leukemic cells of patients with ATL We next examined the components of the AP-1 complex in fresh primary leukemic cells obtained from patients with ATL, by supershift experiments using appropriate antibodies. The AP-1 complex in all 11 ATL samples contained JunD (Figure 4C and data not shown). No obvious supershift complex was found when antibodies against c-Fos, FosB, Fra-1, Fra-2, c-Jun, and JunB were used (Figure 4C). Thus, AP-1 in primary leukemic cells of patients with ATL contains the JunD protein. Northern blot experiments showed that JunD mRNA was highly expressed in leukemic cells of all patients, but only in trace amounts in normal PBMC (Figure 5). Thus, high expression of JunD mRNA is likely to be involved in the activation of AP-1 in primary ATL cells in vivo.
JunD transactivates AP-1 element and can potentially work with Tax To investigate whether JunD is involved in AP-1 site-mediated transcription in T cells, we performed a cotransfection experiment with a luciferase reporter plasmid containing 2 AP-1 elements (2× AP-1 site LUC), using the human T-cell line, Jurkat. Jurkat exhibited a low level of endogenous AP-1 activity (Figure 1C). Cotransfection experiments showed that Tax stimulated the expression of the luciferase gene regulated by IL-8 AP-1 sites, by 8.7-fold. JunD stimulated expression 3.2-fold; and JunD and Tax together stimulated expression 14.1-fold (Figure 6). These results indicate that JunD as well as Tax can stimulate the transcription of cellular genes regulated by the AP-1 site in T cells, both alone and synergistically.
AP-1 is a pleiotropic regulator of inducible expression of several genes. These genes encode proteins involved in the modulation of inflammatory and host defense processes in eucaryotic cells. The protein components of AP-1 are encoded by a set of genes called "immediate early genes" whose transcription is rapidly induced following cell stimulation, independent of de novo protein synthesis. AP-1 has been shown to alter gene expression in response to growth factors, cytokines, tumor promoters, and carcinogens. It is believed that deregulation of these early genes or an imbalance between oncoproteins of the AP-1 complex may contribute in part to the malignant transformation.
We thank Drs N. Mukaida and M. Hatanaka 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, MT-1, and C5/MJ. Recombinant human IL-2 was kindly provided by Takeda Chemical Industries (Osaka, Japan). We also thank M. Yamamoto and M. Sasaki for excellent technical assistance.
Submitted October 4, 1999; accepted February 3, 2000.
Supported in part by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan, and a Cooperative Research Grant No. 1999-10-A-14 from the Institute of Tropical Medicine, Nagasaki University.
Reprints: Naoki Mori, Department of Preventive Medicine and AIDS Research, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan; e-mail: n-mori{at}net.nagasaki-u.ac.jp.
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.
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Top
Abstract
Introduction
B element, and the CArG box. Tax does not directly interact with
these enhancers, but with cellular transcription factors or
transcriptional modulators such as CRE-binding
protein,
