Human T-Cell Leukemia Virus Type I Tax Transactivates Human Interleukin 8 Gene Through Acting Concurrently on AP-1 and Nuclear Factor-KB-Like Sites

[CANCER RESEARCH 58. 3993-4000. September 1. 1998] Human T-Cell Leukemia Virus Type I Tax Transactivates Human Interleukin 8 Gene through Acting Concurrently on AP-1 and Nuclear Factor-KB-like Sites

Naoki Mori,1 Naofumi Mukaida, Dean W. Ballard, Kouji Matsushima, and Naoki Vaniamolo

Department of Preventive Medicine and AIDS Research. Research Field of Palhogenesis and Clinical Sciences, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan Â¡N.M.. N. YJ; Department of Pharmacology. Cancer Research Institute, Kanazawa University, Kanayiwa 920, Japan Â¡N.M.Â¡:Department of Molecular Preventive Medicine, School of Medicine. University of Tok\o. Tokyo 113, Japan Â¡K.M.I: and Howard Hughes Medical Institute. Vanderhilt University School of Medicine. Nashville, Tennessee 37232-0295 [D. W. BJ

ABSTRACT unclear but could be one of the common pathological findings among diseases associated with HTLV-I infection. The transactivator protein, Tax, from the human T-cell leukemia virus IL-8 has potent chemotactic activity for T lymphocytes, as well as type I (HTLV-I) transactivates both viral and cellular genes. Previously, neutrophils (1, 2). Interestingly, T lymphocytes are 10-fold more we had shown that interleukin 8 (II.-8) is constitutively expressed in sensitive to IL-8 than neutrophils in chemotaxis (3). Thus, IL-8 is an HTLV-I-infected cells and in cells transiently expressing Tax. We show here that the II -X promoter is Tax responsive in Jurkat T cells. Further important mediator for tissue infiltration of various subsets of leuko more, using several deletion and mutated plasmids of the 5'-flanking cytes (7). Previously, we and others have shown that IL-8 is consti regulatory region of the ll.-fi gene linked to the luciferase gene as a tutively expressed in HTLV-I-infected T-cell lines and fresh ATL reporter and mutant tax gene expression vectors, we have established that cells (16, 17). These findings raise the possibility that aberrant pro both AP-1 at -126 to -120 and nuclear factor (NF)-KB-like rÃ´-element at duction of IL-8 by leukemic cells in ATL or HTLV-I-infected T cells -80 to â€”¿71areessential and sufficient for the induction of the IL-8 gene in chronic inflammatory disorders might promote accumulation of T by HTLV-I Tax. In addition, overexpression of the dominant-negative lymphocytes in affected tissues. Yet, the precise mechanism by which mutants of NF-KB inhibitor molecules, In-Hir and Ik-H/i, abolished the HTLV-I induces IL-8 gene expression remains to be defined. Tax-induced activation of IL-8 gene. Gel mobility shift assays detected A possible candidate for the induction of IL-8 gene expression is proteins specifically binding to the AP-1 and NF-KB-like sites in Tax- the tax gene product. HTLV-I regulatory protein Tax is a potent expressing T-cell lines infected with HTLV-I. Similarly, the nuclear trans- transactivator of the HTLV-I long terminal repeat (18, 19) and nu location of proteins specifically bound to these two motifs was shown in JPX-9 cells, a subclone of Jurkat cells, carrying the Tax sequences under merous cellular genes, including IL-2 (20), IL-2Ra (20, 21), IL-la the control of an inducible promoter. Taken together, these results suggest (22), IL-6 (23), IL-10 (24), c-fas (25, 26), and intercellular adhesion that the cooperation of transcription factors NF-KB and AP-1 is essential molecule I (27). Tax does not appear to bind DNA directly (28) but, for transactivation of IL-8 gene by HTLV-I Tax. rather, activates transcription by inducing or modifying the activity of certain host transcription factors, including members of the activating transcription factor/cyclic AMP-responsive element-binding protein INTRODUCTION family, serum responsive factor, AP-1 proteins, and NF-KB (29-32). IL-8,2 a member of the leukocyte chemotactic cytokine (chemo- The KB element is also activated in another manner by Tax. Tax interacts with its inhibitors (plOO. pl05, IÂ«Ba,and IxB-y) and disso kine) family, induces chemotaxis of neutrophils, basophils, and T cells (1-3). Although initially identified in medium from human blood ciates their complexes with NF-KB proteins, inducing the nuclear monocytes stimulated with phorbol myristate acetate or lectins (4, 5), translocation of NF-KB proteins (33-37). Recent studies have dem it was soon realized that many different cell types, including endo- onstrated that Tax induces the phosphorylation and degradation of thelial, epithelial, synovial and T cells, fibroblasts, and some tumor iKBa and IÂ«BÃŸ(38,39). cells have been shown to produce IL-8 (1, 3, 6-8). The production of Previously, we have shown that transient transfection of an expres IL-8 is generally not constitutive but can be induced by a variety of sion vector for Tax led to induction of IL-8 expression at both signals including IL-1, tumor necrosis factor a, endotoxin, lectins, and transcriptional and translational levels in Jurkat T-cell line (16). The phorbol esters in a broad range of cells (1, 2, 4, 7). present study was therefore undertaken to determine the regulatory HTLV-I is the causative agent of ATL, an aggressive and usually elements in the IL-8 promoter that were responsive to Tax. We fatal T-cell malignancy (9, 10). ATL is also characterized by unique demonstrated that the viral transactivator Tax is capable of inducing clinical features such as a remarkable tendency of organ infiltration by the IL-8 promoter in T cells. We determined the regulatory elements leukemic cells into lymph nodes, spleen, liver, lung, and skin (11). in the IL-8 promoter responsive to Tax. HTLV-I infection is also associated with chronic inflammatory dis orders such as tropical spastic paraparesis/HTLV-I-associated mye- MATERIALS AND METHODS lopathy, HTLV-I-arthropathy, and others (12-15). Infiltration of Cell Culture. The human T-cell lines Jurkat, MOLT-4, and the HTLV-I- HTLV-I-infected T cells is also a characteristic commonly found in infected T-cell lines MT-1, HUT-102. C5/MJ. and MT-4 were grown in RPMI these HTLV-I-associated diseases. The mode of prominent tissue 1640 containing lO^r fetal bovine serum. JPX-9 and JPX-9/M (provided by Dr. infiltration of HTLV-I-positive T cells and leukemic cells is still M. Nakamura, Tokyo Medical and Dental University, Tokyo, Japan) are subclones of Jurkat cells, expressing Tax and truncated nonfunctional Tax, Received 2/23/98; accepted 7/1/98. respectively, under the control of the metallothionein promoter (26). The cosls of publication of this article were defrayed in pan by the payment of page Western Blotting. Cellular lysates were fractionated by 10% SDS-PAGE, charges. This article must therefore be hereby marked advertisement in accordance with electrophoretically transferred to polyvinylidine difluoride membranes (Immo- 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Department of Preventive bilon-P; Millipore. Bedford, MA) and then analy/.ed for immunoreactivity with a mouse anti-Tax antibody, Lt-4 (provided by Dr. Y. Tanaka, Kitasato Uni Medicine and AIDS Research. Research Field of Pathogenesis and Clinical Sciences. Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto. Nagasaki 852. versity, Kanagawa, Japan; Ref. 40). and horseradish peroxidase-conjugated Japan. Phone: 81-95-849-7846: Fax: 81-95-849-7805. sheep anti-mouse IgG (Amersham Inc., Arlington Heights, IL) with an en 2 The abbreviations used are: IL. interleukin: HTLV-I, human T-cell leukemia virus type I: ATL, adult T-cell leukemia: IL-2Ra, interleukin 2 receptor a; RT-PCR. reverse hanced chemiluminescence detection system (ECL; Amersham, Inc.). transcription-PCR; EMSA. eleclrophoretic mobility shift assay; CRE, cyclic AMP- RT-PCR. Total RNA samples (each l /tg) were converted to single-strand responsive element. cDNA by reverse transcription using the RNA PCR kit (Takara Shuzo, Kyoto. 3993

1 23456 Site-directed mutagenesis of the IL-8 AP-1, NF-IL-6-like. and Â«B-likesites was carried out using â€”¿133-lucplasmid as the wild-type template (46), which converted the AP-1 site TGACTCA (-126 to -120) to TatCTCA, the NF- IL-6-like site CAGTTGCAAATCGT (-94 to -81 ) to agcTTGCAAATCGT. and the icB-like site GGAATTTCCT (-80 to -71 ) to taAcTTTCCT (sites of imitation are indicated in lowercase). Â«B-luc, WT-luc (provided by Dr. J. Fujisawa. Kansai Medical University. Osaka. Japan), and pCArG-luc (provid ed by Dr. K. Shimotohno, Kyoto University. Kyoto, Japan) are luciferase expression plasmids regulated by five copies of the KB element from the IL-8 Â¡L-2Ragene, five copies of the 21-bp viral enhancer (CRE), a monomer of the 302 bp CArG box from the c-fos gene, respectively. Cell Transfection and Luciferase Assays. Transient transfection of cells was performed by electroporation as described previously (22). In all cases, the reference plasmid pRL-CMV, which contains the Renilla luciferase gene under the control of the CMV immediate early enhancer/promoter, was cotransfected to correct for transfection efficiency. Twenty-four h after electroporation, cell lysates were prepared using PicaGene Dual (Toyo Ink Co., Tokyo, Japan). The luciferase activity was normali/ed according to the Renilla luciferase control. Results were confirmed by at least three independent transfections. EMSA. Nuclear extracts were obtained as described (47). Binding reac tions were performed essentially as described previously (22, 24). In some Tax experiments, nuclear extracts were incubated with 100-fold molar excess 203 bp

JPX-9 Cd(uM) 0 1 5 10 20

0-actm Tax 548 bp Â°^ '* B JPX 9 JPX 9/M

HTLV-I (-) HTLV-I (4) Cd

Fig. I. RT-PCR analysis for expression of Ihe Il.-X and lax genes in the HTLV-I- infected cells. Jurkal and MOLT-4 are human T-eell lines free from HTLV-I infection. HUT-102. C.VMJ. MT-1. and MT-4 are human T-cell lines harboring HTLV-I provirus genomes. The internal control, ÃŸ-actinmRNA, was also analyzed.

Japan), and the resulting cDNA was amplified by PCR using Taq DNA polymerase (Takara Shu/ol with three different sets of gene-specific primers. All PCR amplification was carried out on a DNA thermal cycler by 35. 28. and 28 cycles of IL-8, Tax, and ÃŸ-actin.respectively, consisting of 94Â°Cfor 30 s IL 8 (120 s for the first one), 60Â°Cfor 30 s, and 72Â°Cfor 90 s (5 min for the last 302 bp one). After amplification, 10 ;u,lof each reaction solution were electrophoresed in a gel of 2% agarose containing ethidium bromide. Sequences of the oligonucleotides used as primers have been described previously (16, 24). Plasmids. pH2Rneo (-Tax) and pH2R40M ( + Tax) plasmids (kindly pro vided by Dr. M. Hatanaka. Shionogi Institute for Medical Science, Osaka, Japan) have been described previously (41). Both plasmids contain the SV40 promoter. Tax wild-type (TaxMT-2) and mutant expression vectors (TaxM22 and Tax703) were provided by Dr. K. Matsumoto (Osaka Red Cross Blood Center. Osaka, Japan). These genes were cloned into pHÃŸPrf).I-neo, which has ÃŸ-actin a ÃŸ-actinpromoter for protein expression. The Tax mutants TaxM22 and 548 bp Tax703 have been characterized previously (42). The NF-icB p65 expression vector was obtained from Dr. H. Shimi/u (Kana/awa University. Kanazawa. Japan). Deletion mutants of IÂ«Ba (IÂ«BaAN; Ref. 38) and IxBÃŸ(IxBÃŸAN: Fig. 2. Tax induces expression of the endogenous 1L-R gene. JPX-9 and JPX-9/M are Ref. 39) lacking the NH2-terminal 36 amino acids and 23 amino acids, Jurkat subclones stably transformed with the HTLV-l-encoded transactivator Tax or respectively, were cloned into the pCMV4 polylinker. A series of 5' deletion truncated nonfunctional Tax. respectively, under the control of the metallothionein pro of the IL-8 promoter was generated and inserted into a firefly luciferase moter. A. JPX-9 cells were incubated with various concentrations of CdCI2 for 48 h and then collected for preparation of whole-cell extracts. Whole-cell extracts isolated from expression vector, and the protocols used for the generation of the different JPX-9 cells were subjected to immunoblotting with anti-Tax antibody. B. total cellular deletion constructs were essentially the same as in the case of the chloram- RNA isolated from either nontreated or CdCU (20 Â¿iMl-trealedJPX-9 or JPX-9/M cells phenicol acetyltransferase expression vector described previously (43-45). was subjected to RT-PCR. 3994

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. Tax-INDUCED IL-8 EXPRESSION unlabeled oligomers for 15 min before radiolabeled probe was added. The sequences of the DNA fragments were as follows; the AP-1 binding site of the IL-8 gene (-130 to -116: 5'-gatcGTGATGACTCAGGTT-3'). mutation of AP-1 binding site of the IL-S gene (5'-gatcGTGATatCTCAGGTT-3'). the Control NF-KB-like site of the IL-8 gene (-83 to -68; 5'-galcCGTGGAATTTC- CTCTG-3'). mutation of NF-KB binding site of the IL-8 gene (5'-gatcCGT- taAcTTTCCTCTG-3'). the NF-KB-like site of the IL-2Ra gene (5'-gatcCG- Tax GCAGGGGAATCTCCCTCTC-3' ). and the consensus binding site for AP-1 (5'-gatcCGCTTGATGAGTCAGCCGGAA-3') (the underlined sequence is the binding site). After another 15-min incubation at room temperature, the samples were electrophoresed in a 4% polyacrylamide gel. For the supershift M22 studies, antibodies (Santa Cruz Biotechnology. Santa Cruz. CA) to different transcription factors were added to the preincubation reaction. 703

RESULTS 50 100 150 IL-8 Transcript in Human T Cells Infected with HTLV-I. To of wild-type activity determine whether IL-8 expression in T cells correlates with HTLV-I infection, RNA was extracted from HTLV-I-infected or uninfected B human T-cell lines, and expression of the IL-8 gene was analyzed by RT-PCR. Three HTLV-I-infected cell lines expressed the viral trans- Control â€¢¿ activator Tax (Fig. 1, Lane 3, HUT-102; Lane 4, C5/MJ; Lane 5, A/7"-/). The IL-8 transcript was also detectable in these three cell lines, whereas it was undetectable in the uninfected lines, Jurkat and MOLT-4 (Lanes I and 2). The other infected cell line MT-4, without Tax Tax transcript, also did not express IL-8 (Lane 6). In the MT-4 cells, G -1481-luC a dramatic reduction in the expression of viral proteins is linked to E3 -133-luc methylation of the HTLV-I provirus (48). These results show a M22 plausible correlation between IL-8 expression and the viral transactivator Tax expression, suggesting that Tax is responsible for the stimulated transcription of IL-S gene like other Tax-responsive cellu 703 lar genes.

25 50 75 100 125

of wild-type activity â€¢¿126/-120 AP-1 Fig. 4. Tax transactivation of the IL-8 promoter is dependent of the KB-like element. B, Jurkat cells were transfected with IL-K-luc constructs, containing the ll.-H gene Octamer AP-3 fragment â€”¿1481/+44or â€”¿13.V+44.together with expression plasmids encoding Tux. its â€¢¿91/-83 -821-77 indicated mutants, or the parental expression vector with no insert iCtwtrttl) as well as the reference plasmid pRL-CMV. A, reporter luciferase plasmids with the KB (KB-luc). the CRE element (WT-luc). or the CArG box (pCArG-luc) were used as control promoters for Tax mutants. After a 24-h incubation, the lucifcra.se activity was determined. Luciferase B activity was normalized for Renillu luciferase activity. The activities of Tax mutants. M22 and Tax703. relative to wild-type Tax. were calculated by comparing the luciferase activity in the presence of M22 or Tax703 with the luciferase activity in the presence of wild-type Tax. The results are shown as means of three experiments; burs. SD.

D -Tax Tax Induces Expression of IL-8 Gene. To examine the effect of

ED +Tax Tax on IL-S gene expression, we used JPX-9 cells that were derived from the human T-cell line Jurkat currying the transfected Tax cDNA under the control of metallothionein promoter (26). Western blot analysis demonstrated that CdCl2 added to the culture medium in duced Tax protein in JPX-9 cells (Fig. 14). IL-8 mRNA was also induced (Fig. 2B). Thus, we concluded that Tax was sufficient to induce expression of the IL-8 gene at the transcriptional level. CdCU Relative luciferase activity did not induce IL-8 gene expression in the parental Jurkat and JPX- Fig. 3. A. potential binding sites for ci.Ã¯-actingelements in the .V-regulatory region of 9/M. a Jurkat cell line permanently transfected with a nonfunctional the Ã•L-Xgene.B, identification of Tax-responsive elements required for the transcriptional activation of the IL-8 gene. Various lengths of 5'-flanking region of the IL-ti gene, linked Tax protein. The 5'-Flanking Sequence Is Responsible for the Induced Ex to lucÃ/erase gene (5 u.g), were transfected into Jurkat cells together with the expression plasmid for HTLV-I Tax. pH2R40M ( -I-Tax. 5 (ig) or a control plasmid. pH2Rnco (-Tax, pression by Tax. To examine whether the 5'-flanking sequence of 5 Â¿ig).The transfection efficiency was monitored by cotransfecting the reference plasmid human IL-8 gene is responsible for the expression stimulated by Tax, pRL-CMV (O.I ng). After 24 h of incubation, cells were harvested and assayed for luciferase activity as described in "Materials and Methods." Luciferase activity was the luciferase gene was used to monitor IL-8 promoter activity (Fig. normalized for Renilla luciferase activity. The results are shown as means of three 3). The reporter plasmid, -1481-luc, which has the IL-8 promoter independent experiments; htirs. SD. Relative luciferase activity was calculated from the luciferase activity of each reporter plasmid relative to the value of -50-luc, cotransfected fragment between -1481 and +44, was transfected into recipient with pH2Rneo and assigned a value of 1. cells with or without the Tax plasmid, and transient expression of the 3995

relative to wild-type Tax, was similarly efficient in transactivating the

AP-1 NF-IL-6 NF-KB IL-8 promoter fragments â€”¿1481-lueand â€”¿133-luc.In contrast, TaxM22, which fails to induce through the NF-KB transcription factor pathway, induced only low levels of luciferase activity. Controls for these experiments demonstrated that Tax703, but not TaxM22, trans- activated xB-luc (Fig. 4/1). Conversely, Tax mutants M22. but not 703, induced luciferase expression from WT-luc and pCArG-luc (Fig. 4A). These results suggest that, for the Tax responsiveness of the IL-8 NF-KBmut-luc promoter, an NF-KB element is required. AP-1 and NF-KB but not NF-IL-6-like Sites Are Essential for B Activation by Tax. The regulatory role of the AP-1, NF-IL-6-like, and KB-like sites was further studied by site-directed mutagenesis of each element in the -133-luc plasmid (Fig. 5A). Among these lucif erase expression plasmids, the -133 (wild-type)-luc construct and the NF-IL-6 site mutant caused inducibility of luciferase activity by Tax (Fig. 5ÃŸ).However, the mutation of either the AP-1 site or the KB-like site severely reduced inducibility of luciferase activity by Tax. Col lectively, two elements (AP-1 and KB-like sites) were involved in the NF IL-6 mut-luc IL-8 gene activation in Jurkat cells in response to Tax stimulation. Complex Formation of the IL-8 AP-1 and KB-like Sites in the HTLV-I-infected T Cells. To characterize the specific nuclear pro NF.Â«Bmut-luc teins binding to the AP-1 and KB-like sites in Tax-expressing T-cell lines, we carried out EMSA. With the IL-8 - 130/- 116 probe, con 0 25 50 75 100 125 taining the AP-1 site (TGACTCA; - 126 to - 120), one complex was % of wild-type activity

Fig. 5. The relative importance of the AP-1, NF-IL-6-like, and KB-like sites in the IL-8 promoter for responsiveness to Tax. A, schematic representation of wild-type and mutant IL-8 reporter constructs. The AP-1 site (-126 to -120: TGACTCA). NF-IL-6-like site (-94 to -81; CAGTTGCAAATCGT). or KB-like site (-80 to -71; GGAATTTCCT) in Cell: the IL-8 promoter (- 133 to +44). linked to lucifertue gene, were mutated to TalCTCA. agcTTGCAAATCGT. and laAcTTTCCT, respectively. B. effect of point mutations on the inducibility of luciferase activity. The wild-type and mutated plasmids (5 fig) were transfected into Jurkat cells with ( + ) or without (-) Tax expression plasmid (5 /j,g) as well as the reference plasmid pRL-CMV (0.1 fig). Luciferase activity was normalized for Renillu luciferase activity. Tax-dependent increases in reporter gene activity are expressed as a percentage of the value of - 133-luc colransfected with pH2R40M. The results are shown as means of three experiments; him, SD. luciferase gene was analyzed. As shown in Fig. 3ÃŸ,the promoter activity was activated by Tax in Jurkat cells. To localize the Tax-responsive element in the S'-tlanking sequence of the IL-8 gene, deletion constructs from the 5'-end were prepared. As shown in Fig. 3/J, the transfection with either â€”¿272-lucorâ€”¿133- luc constructs gave the highest induction of luciferase activity when stimulated with Tax, suggesting that negative regulatory element(s) may exist in the 5'-flanking region from â€”¿1481toâ€”¿212.Further B more, deletion from -133 to -98 drastically decreased the inducibil ity of luciferase activity by Tax. Furthermore, deletion up to -50 Competitor: â€”¿ abolished the inducibility of luciferase activity by Tax. These results suggest that the minimally essential elements for Tax activation in the IL-8 gene arc present between â€”¿133and â€”¿50.Theseare three known c/i-elements, AP-1 (-126 to - 120), NF-IL-6-like (-94 to -81), and KB-like ( â€”¿80to â€”¿71)siteswithin the region downstream of 133 bp (Fig. 3/4; Refs. 43-45). Tax up-regulates a number of cellular genes via effects on several transcription factor pathways. Each pathway is independent of the other and can be differentially activated by various mutants of HTLV-I Tax (49). The roles of NF-KB in regulation of the IL-8 gene can, therefore, be studied by cotransfection of the appropriate HTLV-I Tax expression vector and the IL-8 promoter coupled to a luciferase Fig. 6. Nuclear factors binding to the AP-1 site in the HTLV-I-infected T cells. A, nuclear extracts were prepared from HTLV-I-infected (HUT-102. C5/MJ. MT-1, and reporter gene. Tax protein mutants that are incapable of activating MT-4) and uninfected (Jurkal and MOLT-4) T cells and incubated with the labeled IL-8 either NF-KB (M22) or activating transcription factor/cyclic AMP- promoter ( - 130 to - 116). containing AP-1 site ( - 126 to - 120; TGACTCA ). B. nuclear extracts from HUT-102 cells were mixed with the labeled IL-8 AP-1 site probe in the responsive element-binding protein and serum-responsive factor absence (Lane lÃ¬or presence of IL-8 AP-1 site (Lane 2). consensus AP-1 site (Lane 3), (Tax703) were used. As shown in Fig. 4B, a Tax mutant Tax703, or mutated IL-8 AP-1 site (TatCTCA) competitors (Lane 4). 3996

activity in HUT-102 cells is composed predominantly of p50 and c-Rel. Collectively, the increased nuclear expression of the AP-1 and NF-KB-like activities clearly correlated with the increased transcrip Antibody: - tion of the IL-8 gene in the HTLV-I-infected T-cells. Activation of AP-1 and NF-KB by Tax in JPX-9 Cells. We further examined nuclear extracts from JPX-9 without or with CdCU treatment for 48 h. With the IL-8 -130/- 116 probe, containing the AP-1 site, the complex was strongly induced in JPX-9 after induction of Tax by CdCl2 treatment (Fig. 9A,Lanes I and 2). This complex was inhibited by the autologous oligonucleotide and the consensus AP-1 sequence but not by the mutated AP-1 site oligonucleotide (Fig. 9A, Lanes 3-5). In addition, treatment of JPX-9 cells with CdCl2 in creased the retardation of the labeled IL-8 â€”¿837â€”68oligomer,con 12345678 taining the KB-like site (Fig. 9B, Lanes I and 2). The formation of D complex is due to specific binding of NF-KB-like protein, because the appearance of complex was inhibited by the unlabeled KB-likesite of Antibody: -

DISCUSSION Previously, we showed that IL-8 is constitutively expressed in AN human T-cell lines carrying HTLV-I and fresh ATL cells derived from patients (16). We also demonstrated that Tax was capable of Competitor: - inducing the IL-8 expression (16). In this report, we have further extended our study to investigate the mechanism of the aberrant IL-8 Induction: - gene regulation in HTLV-I-infected T cells. Our data show that HTLV-I Tax can transactivate the IL-8 promoter in T cells, the natural target cells of HTLV-I. Transactivation of the IL-8 promoter appears to require induction and binding of multiple transcription factors. One Tax-responsive region includes an AP-1 site at â€”¿126to â€”¿120.Mu tation in the AP-1 site of the IL-8 promoter reduces Tax activation. Several reports have pointed out an AP-1-like site as a responsive element for Tax (50-52). Indeed, the expression levels of the AP-1 genes (c-Fos, Fra-l, c-Jun, JunB, and JunD) induced by Tax were 12345 constitutively high in the HTLV-I-infected T cells as reported previ ously (53). In EMSA, nuclear extracts of HTLV-I-infected T cells had an increased ability to form the IL-8 AP-1 binding site complex. Furthermore, CdCU-treated JPX-9 cells demonstrated that induction B of Tax expression led to the nuclear expression of AP-1 DNA binding factors. The second Tax-responsive region, NF-KB-like site, resides within Competitor: _ <Â¿ *Ã ^ positions â€”¿80toâ€”¿71ofthe IL-8 promoter. Our study revealed that Induction: this region is absolutely required for IL-8 gene expression. Loss of the NF-KB activation pathway of Tax eliminates transactivation through the promoter. The observation that binding of NF-KB transcription M tt factors to the KB-like site and reduction of activity of the IL-8 p65 pee

12345 Fig. 9. Induction of nuclear factors binding to the IL-8 AP-1 and KB-likc sites in JPX-9 cells afler treatment with CdCK. Nuclear extracts prepared from JPX-9 cells without ( â€”¿) or with ( + ) induction of Tax with 20 (Â¿MCdCl, for 48 h were analyzed by EMSA with the IL-8 AP-1 site probe (Ai or Â«B-likesite probe (ÃŸ)in the absence (Litnes I and 2) or presence of the indicated cold competitors ILttnex 3-5). nuclear localization signals. Previously, it was demonstrated that both IÂ«Ba and IÂ«BÃŸmutantsin their NH2-terminal region escape from proteolytic breakdown and prevent NF-KB activation in Tax-express B '*.Ba AN(ug) ing cells (38, 39). As an additional means of determining the involve ment of NF-KB in IL-8 activation with Tax. transient cotransfections Tax Tax were done with a mutant of IÂ«Ba (UBaAN) or IÂ«BÃŸ(IxBÃŸAN) lacking the NH-,-terminal amino acids. As shown in Fig. 10A. both IxBaAN and IicB/SAN abolished p65-dependent transcription of Â«B- luc, containing a lucifemse gene under the control of the NF-KB binding site of the IL-2Ra gene. Furthermore, both IxBaAN and IxBÃŸANalso inhibited Tax-induced transcription from Â«B-luc(Fig. 10#). To evaluate the specificity of this interaction, either IxBaAN or IxBÃŸANeffector plasmid was cotransfected with Tax and a distinct Tax-responsive reporter construct. In sharp contrast to Â«B-luc,both IÂ«BaANand IxBÃŸANfailed to significantly down-regulate transcrip tion directed from WT-luc, which responds to Tax by five repeats of the 21-bp viral enhancer (CRE). Tax-induced transcription from the IL-8 promoter was also abolished in cells expressing both IÂ«BaAN Fig. 10. Repression of Tax-dependent transcription from the IL-8 promoter. Jurkat cells and IxBÃŸAN(Fig. 10S). These observations provide additional sup were transfected with 5 /xg of a p65 (A) or Tax (B) expression vector, graded amounts of iKBaAN or InBÃŸANeffector plasmid, and 5 /xg of a luciferase reporter construct under port for the involvement of NF-KB in inducible IL-8 gene expression transcriptional control of either the KB-luc, WT-luc, or â€”¿133-luc.Input DNA for all and indicate that modulation of active NF-KB complex by 1KB is transfections was normalized by addition of blank pCMV4 expression vector. For each titration point (n - 3), the increase in reporter gene activity over basal levels is expressed likely play a role in the regulation of IL-8 gene expression mediated as the mean percentage of luciferase activity measured in control cells lacking the by HTLV-I Tax. iKBorAN or I/iBÃŸANexpression vector. 3998

promoter by mutation in the Â«B-likesite correlates with the require ACKNOWLEDGMENTS ment for the NF-KB pathway of Tax activation. Taken together, the IL-8 promoter response to Tax requires induction of both NF-KB and We thank Drs. W. C. Greene, M. Hatanaka, H. Shimizu. J. Fujisawa, K. AP-1 transcription factors. This is in contrast to Tax activation of Shimotohno. and K. Matsumoto for providing plasmids: Y. Tanaka for pro viding anti-Tax monoclonal antibody; M. Nakamura tor providing JPX-9 and other genes such as IL-2, IL-2Ra, IL-la, IL-6, and the human immu JPX-9/M: and Fujisaki Cell Center. Hayashibara Biochemical Laboratories, nodeficiency virus long terminal repeat that require only NF-KB Inc. (Okayama, Japan) for providing Jurkat. HUT-102. C5/MJ. and MT-1. We activation (21-23,49). are very grateful to Dr. M. Fujii for helpful discussions and advice. We also It has been shown that a variety of agents, such as IL-1, phorbol thank M. Yamamoto, M. Sasaki, and Y. Honda for excellent technical assist- esters, tumor necrosis factor a, hepatitis B virus X protein, and respiratory syncytial virus, can activate IL-8 gene expression (43, 44, 54). These analyses reveal that the sequence from -94 to -71, REFERENCES consisting of NF-IL-6 and NF-KB-like sites, is required for respon Matsushima. K.. Baldwin. E. T., and Mukaida. N. lnlcrleukin-8 and MCAF: novel siveness to these inducers. In contrast to previous reports, we now leukocyte recruitment and activating eytokines. Chem. Immunol.. 5/: 236-265. 1992. report that Tax induction of IL-8 is independent of an NF-IL-6-like Mukaida. N.. Harada. A., Yasumoto. K.. and Matsushima. K. Properties of pro- site. Rather, IL-8 gene activation by Tax appears to be mediated by the inflammatory cell type-specific leukocyte chemotactic cytokines. interleukin 8 (IL-8) and monocyte chemotactic and activating factor (MCAF). Microbio!. Immunol.. 36: AP-1 site in conjunction with the NF-KB-like site. The requirement 773-789. 1992. for a similar region for IL-8 transcription by cytokines. cytomegalo- Larsen. C. G.. Anderson. A. O.. Appella. E.. Oppenheim. J. J.. and Matsushima. K. The neutrophil-activating protein (NAP-l) is also chemotactic for T lymphocytes. virus, and paclitaxel has been observed in a human gastric cancer cell Science (Washington DC). 243: 1464-1466. 1989. line (45), a human monocytic cell line (55), and a human ovarian Baggiolini. M.. Walz. A., and Kunkel. S. L. Neutrophil-activating peptide-l/interleukin 8, a novel cytokine thai activates neutrophils. J. Clin. Invest.. 84: 1045-1049. cancer cell line (56), respectively. These results suggest that different 1989. sets of nuclear transcription factors may be responsible for the regu Yoshimura. T.. Matsushima. K.. Tanaka. S.. Robinson. E. A.. Appella. E.. lation of IL-8 gene transcription in a cell type- and inducer-specific Oppenheim. J. J.. and Leonard. E. J. Purification of a human monocyle-derived neutrophil chemotactic factor that has peptide sequence similarity to other host manner. defence cytokines. Proc. Nati. Acad. Sci. USA. 84: 9233-9237. 1987. In spite of the apparent expression of IL-8 mRNA and protein in Baggiolini. M.. and Clark-Lewis. 1. Interleukin-8. a chemotactic and inflammatory cytokine. FEBS Lett.. 307: 97-101. 1992. fresh ATL cells. Tax expression in all of the ATL samples exam Oppenheim. J. J.. Zachariae. C. O.. Mukaida. N.. and Matsushima. K. Properties of ined was extremely low, and could be detected only by RT-PCR the novel proinflammatory supergene "intercrine" cytokine family. Annu. Rev. Im munol.. 9: 617-648. 1991. (16). These results indicated that the transactivation by Tax may Stricter. R. M.. Kunkel. S. L.. Showell. H. J.. Remick. D. G.. Phan. S. H.. Ward. P. A., not be the only mechanism for overexpression of IL-8 gene in and Marks. R. M. Endothetial celi gene expression of a neutrophil chemotaetic factor HTLV-I-infected T cells. The similar paradox, i.e., strong up- by TNF-n, LPS. and IL-I0. Science (Washington DC). 243: 1467-1469, 1989. Poiesz. B. J.. Ruscelli. F. W.. Gazdar. A. F.. Bunn. P. A.. Minna. J. D.. and Gallo. regulation of a Tax-responsive gene in fresh ATL cells in the R. C. Detection and isolation of type C retrovirus particles from fresh and cultured absence of Tax, could be applied to other genes such as IL-2Ra, lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Nati. Acad. Sci. USA. 77: 7415-7419. 1980. IL-la, IL-6, and IL-10. It is possible that Tax production is to. Yoshida, M., Miyoshi. I., and Hinuma. Y. Isolation and characterization of retrovirus necessary for the observed overexpression of cellular genes but from cell lines of human adult T-cell leukemia and its implication in the disease. Proc. that it could occur early in the course of HTLV-I infection. By the Nati. Acad. Sci. USA. 79: 2031-2035. 1982. 11.Takatsuki. K.. Yamaguchi. K.. Watanabe. T.. Mochizuki. M.. Kiyokawa. T.. Mori. S.. time ATL cells appear in peripheral blood. Tax is gone. The and Miyata, N. Adult T-cell leukemia and HTLV-I related diseases, in: K. Takatsuki. Y. Hinuma, and M. Yoshida (eds.). Gann Monograph on Cancer Research No. 39. circulating ATL cells would be in a terminally differentiated, Advances in Aduli T-Ccll Leukemia and HTLV-I Research, pp. 1-15. Boca Raton: autonomous state and may no longer require Tax production for CRC. 1992. expression of cellular genes. Tax might still be functional in other 12. Gassain. A.. Barin. F.. Vernanl. J. C.. Gout. O.. Maurs. L.. Calender, A., and de The. G. Antibodies to human T-lymphotropic virus lype-I in patients with tropical spastic tissues such as lymph node and skin, although Tax expression in paraparesis. Lancet. 2: 407-410. 1985. leukemic cells in peripheral blood is extremely low. 13. Osarne. M.. Usuku. K.. Izumo. S.. Ijichi. N.. Amilani. H.. Igata. A.. Matsumoto. M.. and Tara. M. HTLV-I associated myelopathy. a new clinical entity. Lancet. I: In the present study, we found that the deletion mutants of iKBa 1031-1032. 1986. (iKBaAN) and iKBÃŸ(iKBÃŸAN)that lacked these NH2-terminal reg 14. Nishioka. K.. Maruyama. I.. Sato. K.. Kitajima. !.. Nakajima. Y.. and Osarne. M. ulatory region, did inhibit Tax-induced transcription from the IL-8 Chronic inflammatory arthropathy associated wilh HTLV-1. Lancet. /: 441. 1989. 15. Mochizuki. M.. Watanabe. T.. Yamaguchi. K.. Takatsuki. K.. Yoshimura. K.. Shirao. promoter. Furthermore, overexpression of both IKBaAN and iKBÃŸAN M.. Nakasnima. S.. Mori. S.. Araki. S., and Miyala. N. HTLV-I uveitis: a distinct significantly reduced Tax-induced NF-KB DNA binding in CdCU- clinical entity caused by HTLV-I. Jpn. J. Cancer Res.. 83: 236-239. 1992. 16. Mori. N.. Murakami. S., Oda. S.. Prager. D., and Eto. S. Production of interleukin X treated JPX-9 cells and constitutive NF-KB DNA binding in HUT-102 in adult T-cell leukemia cells: possible transactivation of the inierleitkin H gene by cells (data not shown). In contrast, no significant changes were human T-cell leukemia virus type 1 tax. Cancer Res.. 35: 3592-3597, 1995. observed in the levels of AP-1 binding with the same extracts (data 17. Yamada. Y.. Ohmoto. Y.. Hala. T.. Yamamura. M.. Murata. K.. Tsukasaki. K.. Kohno, T.. Chen, Y., Kamihira. S., and Tomonaga. M. Features of the cytokines not shown). These results indicate that these dominant-negative mu secreted by adult T cell leukemia (ATL) cells. Leuk. Lymphoma. 21: 443-447. 1996. tants of 1KBare constitutive repressors of NF-KB with specific defects 18. Fujisawa, J.. Seiki. M.. Kiyokawa, T., and Yoshida, M. Functional activation of the long terminal repeal of human T-cell leukemia virus type I by a iniru-acting factor. in their ability to transduce Tax-dependent signals that uncouple Proc. Nati. Acad. Sci. USA. 82: 2277-2281, 1985. NF-KB/lKB complexes. Inhibition of NF-KB expression by antisense 19. Sodroski. J. G.. Rosen, C. A., and Haseltine, W. A. TVwi.v-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected oligodeoxynucleotides was shown to inhibit the growth of Tax-trans cells. Science (Washington DC). 225: 381-385. 1984. formed fibroblasts as well as an HTLV-I-transformed T-cell line, thus 20. Siekevitz. M.. Feinberg. M. B.. Holbrook. N.. Wong-Staal. F.. and Greene. W. C. demonstrating that NF-KB expression was necessary for the mainte Activation of inlerleukin 2 and interleukin 2 receptor (Tac I promoter expression by the trans-activator (;<Â»)gene product of human T-cell leukemia virus type I. Proc. nance of the malignant phenotype (57). HTLV-I Tax deregulates the Nati. Acad. Sci. USA. 84: 5389-5393, 1987. function of NF-KB, resulting in the aberrant expression of a subset of 21. Ballard, D. W.. Bohnlein. E.. Lowenlhal. J. W.. Wano. Y.. Pranza. B. R., and Greene, W. C. HTLV-I tax induces cellular proteins that activate the KB element in the IL-2 growth-related cellular genes including IL-2 and IL-2Ra. Our findings receptor a gene. Science (Washington DC), 241: 1652-1655. 1988. demonstrate that dominant-negative mutants of IKB. in combination 22. Mori. N.. and Prager. D. Transactivation of the interleukin-la promoter by human T-cell leukemia virus type I and type II Tax proteins. Blood. 87: 3410-3417. 1996. with an appropriate delivery system, are potential therapeutic agents 23. Mori. N.. Shirakawa. F.. Shimizu. H.. Murakami. S.. Oda. S., Yamamoto. K.. and Elo. for the treatment of HTLV-I-associated diseases. S. Transcriptional regulation of the human inlerleukin-6 gene promoter in human 3999

T-cell leukemia virus type l-infected T-cell lines: evidence for the involvement of 41. Tanaka. A., Takahashi. C., Yamaoka, S.. Nosaka. T., Maki. M., and Hatanaka. M. NF-KB. Blood, 84: 2904-2911. 1994. Oncogenic transformation by the lax gene of human T-cell leukemia virus type I in 24. Mori, N.. Gill, P. S., Mougdil. T., Murakami. S., Elo. S., and Prager. D. Interleukin-10 vitro. Proc. Nati. Acad. Sci. USA. 87: 1071-1075, 1990. gene expression in adult T-cell leukemia. Blood. 88: 1035-1045. 1996. 42. Matsumoto, K.. Shibata, H., Fujisawa, J., Inoue, H.. Hakura, A., Tsukahara, T., and 25. Fujii, M.. Sassone-Corsi. P.. and Verma. I. M. c-/u.v promoter Irans-activation by the Fujii, M. Human T-cell leukemia virus type 1 Tax protein transforms rat fibroblasts laxl protein of human T-cell leukemia virus type 1. Proc. Nati. Acad. Sci. USA, 85: via two distinct pathways. J. Virol., 71: 4445-4451. 1997. 8526-8530, 1988. 43. Mukaida. N.. Mahe. Y.. and Matsushima, K. Cooperative interaction of nuclear 26. Nagata, K.. Ohtani, K.. Nakumura. M.. and Sugamura. K. Activation of endogenous factor-KB and c/.v-regulatory enhancer binding protein-like factor binding elements in c-fia proto-oncogene expression by human T-cell leukemia virus type 1-encoded activating the interleukin-8 gene by pro-inflammatory cytokines. J. Biol. Chem., 265: p40"" protein in the human T-cell line. Jurkat. J. Virol.. 63: 3220-3226. 1989. 21128-21133. 1990. 27. Mori. N.. Murakami. S.. Oda. S.. and Elo, S. Human T-cell leukemia virus type I tax 44. Mahe. Y.. Mukaida. N., Kuno, K., Akiyama. M., Ikeda, N., Matsushima, K.. and induces intracellular adhesion molecule-1 expression in T cells. Blood. 84: 350-351, Murakami. S. Hepatitis B virus X protein transactivates human interleukin-8 gene 1994. through acting on nuclear factor KB and CCAAT/enhancer-binding protein-like 28. Armstrong, A. P., Franklin, A. A., Uillenbogaard. M. N.. Giebler. H. A., and Nyborg, (â€¢Â«-elements.J.Biol. Chem.. 266: 13759-13763. 1991. J. K. Pleiolropic effect of the human T-cell leukemia virus Tax protein on the DNA 45. Yasumolo, K.. Okamoto, S.. Mukaida, N.. Murakami. S., Mai. M.. and Matsushima. binding activity of eukaryotic transcription factors. Proc. Nati. Acad. Sci. USA. 90: K. Tumor necrosis factor a and interferon y synergistically induce interleukin 8 7303-7307. 1993. production in a human gastric cancer cell line through acting concurrently on AP-I 29. Suzuki. T., Fujisawa. J.. Toita. M.. and Yoshida. M. The trans-activator tax of human and NF-KB-like binding sites of the interleukin 8 gene. J. Biol. Chem.. 267: 22506- T-cell leukemia virus lype 1 (HTLV-1) interacts with cAMP-responsive element 22511, 1992. (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of 46. Ishikawa. Y.. Mukaida. N.. Kuno. K.. Rice. N.. Okamoto. S.. and Matsushima, K. HTLV-1. Proc. Nail. Acad. Sci. USA. 90: 610-6I4. 1993. Establishment of lipopolysaccharide-dependent nuclear factor KB activation in a 30. Wagner. S.. and Green. M. R. HTLV-I Tax protein stimulation of DNA binding of cell-free system. J. Biol. Chem., 270: 4158-4164. 1995. bZIP proteins by enhancing dimeri/alion. Science (Washington EX?). 262: 395-399. 47. Antalis. T. M.. and Godbolt. D. Isolation nuclei from hematopoietic cell types. NucÃ. 1993. Acids Res., 19: 4301. 1991. 31. Zhao, L-J., and Giam. C-Z. Human T-cell lymphotropic virus type I (HTLV-1) 48. Saggioro. D.. Panozzo. M., and Chicco-Bianchi. L. Human T-lympholropic virus type (ranscriptional activator. Tax. enhances CREB binding to HTLV-1 21-base-pair I transcriptional regulation by methylation. Cancer Res., 50: 4968-4973, 1990. repeals by protein-protein interaction. Proc. Nail. Acad. Sci. USA, 89: 7070-7074, 49. Smith. M. R.. and Greene, W. C. Identification of HTLV-1 tax trans-activator mutants 1992. exhibiting novel transcriplional phenotypes. Genes Dev.. 4: 1875-1885. 1990. 32. Fujii, M., Tsuchiya, H., Chuhjo. T.. Akizawa, T., and Seiki, M. Interaction of 50. Kim, S-J.. Kehrl, J. H.. Burton. J.. Tendier, C. L.. Jeang. H-T.. Danielpour. D.. HTLV-1 Taxi with p67SRI' causes the aberrant induction of cellular immediate early Thevenin. C.. Kim. K. Y.. Sporn. M. B., and Roberts. A. B. Transactivation of the genes through CArG boxes. Genes Dev.. f>: 2066-2076. 1992. transforming growth factor ÃŸl(TGF-ÃŸl)gene by human T lymphotropic virus type 33. Hirai. H.. Fujisawa. J.. Su/uki. T.. Ueda. K.. Murumalsu. M.. Tsuboi. A.. Arai. N.. and 1 Tax: a potential mechanism for the increased production of TGF-ÃŸl in adult T cell Yoshida. M. Transcriptional activator Tax of HTLV-1 hinds to the NF-KB precursor leukemia. J. Exp. Med.. 772: 121-129, 1990. pl05. Oncogene, 7: 1737-1742, 1992. 51. Uchijima, M.. Sato. H., Fujii. M.. and Seiki. M. Tax proleins of human T-cell 34. Hirai. H.. Su/uki. T.. Fujisawa, J., Inoue. J., and Yoshida. M. Tax protein of human leukemia virus type I and 2 induce expression of the gene encoding erythroid- T-cell leukemia virus type I hinds to the ankyrin motifs of inhibitory factor KB and polentialing activity (tissue inhibitor of metalloproteinases-1. T1MP-I). J. Biol. induces nuclear translocation of transcription factor NF-KB proteins for transcrip- Chem.. 269: 14946-14950. 1994. tional activation. Proc. Nati. Acad. Sci. USA. 91: 3584-3588. 1994. 52. Tsuchiya. H.. Fujii. M.. Niki. T.. Tokuhara. M.. Matsui. M., and Seiki, M. Human 35. Kanno. T.. Fran/oso. G., and Siebenlist. U. Human T-cell leukemia virus type I T-cell leukemia virus lype 1 Tax activates transcription of the human /m-/ gene Tax-protein-mediated activation of NF-KB from pl()0 (NF-KB2)-inhibited cytoplas- through multiple cis elements responsive to transmembrane signals. J. Virol.. 67: mic reservoirs. Proc. Nati. Acad. Sci. USA. 91: 12634-12638. 1994. 7001-7007. 1993. 36. Kanno. T.. Brown. K.. Fran/oso, G., and Siebenlist. U. Kinetic analysis of human 53. Fujii. M.. Niki. T.. Mori. T.. Matsuda, T.. Matsui. M.. Nomura, N., and Seiki. M. T-cell leukemia virus type I Tax-medialed activation of NF-KB. Mol. Cell. Biol.. 14: HTLV-1 Tax induces expression of various immediate early serum responsive genes. 6443-6451, 1994. Oncogene. 6: 1023-1029. 1991. 37. Suzuki, T., Hirai, H.. Murakami, T., and Yoshida. M. Tax protein of HTLV-1 54. Mastronarde. J. G.. He. B.. Monick. M. M.. Mukaida. N.. Matsushima. K.. and destabili/es the complexes of NF-KB and lKB-a and induces nuclear translocalion of Hunninghake. G. W. Induction of interleukin (IL)-8 gene expression by respiratory NF-KB for transcriptional activation. Oncogene. 10: 1199-1207. 1995. syncytial virus involves activation of nuclear factor (NF)-KB and NF-IL-6. J. Infect. 38. Brockman. J. A.. Scherer. D. C.. McKinsey, T. A.. Hall, S. M., Qi, X.. Lee, W. Y.. Dis., 174: 262-267, 1996. and Ballard. D. W. Coupling of a signal response domain in 1KBÂ«to multiple 55. Murayama. T.. Ohara. Y., Obuchi, M.. Khabar, K. S. A., Higashi, H., Mukaida, N.. pathways for NF-KB activation. Mol. Cell. Biol.. /5: 2809-2818. 1995. and Matsushima, K. Human cytomegalovirus induces interleukin-8 production by a 39. McKinsey. T. A.. Brockman. J. A.. Scherer, D. C.. Al-Murrani, S. W., Green. P. L.. human monocytic cell line. THP-I. through acting concurrently on AP-I-and NF- and Ballard. D. W. Inaclivation of iKBÃŸbythe Tax protein of human T-cell leukemia xB-binding sites of the interleukin-8 gene. J. Virol., 71: 5692-5695. 1997. virus type 1: a potential mechanism for constitutive induction of NF-KB. Mol. Cell. 56. Lee, L-F.. Haskill, J. S., Mukaida, N., Matsushima. K.. and Ting. J. P-Y. Identifica Biol.. 16: 2083-2090. 1996. tion of tumor-specific paclitaxel (Taxol)-responsive regulatory elements in the inter 40. Tanaka. Y.. Yoshida. A.. Takayama. Y., Tsujimoto. H., Tsujimoto. A., Hayami, M.. leukin-8 promoter. Mol. Cell. Biol.. 17: 5097-5105. 1997. and To/awa. H. Heterogeneity of antigen molecules recognized by anti-taxi mono- 57. Kitajima. !.. Shinohara. T.. Bitakovics. J.. Brown. D. A., Xu, X.. and Nerenberg, M. clona] antibody Lt-4 in cell lines bearing human T cell leukemia virus type 1 and Ablation of transplanted HTLV-I Tax-transformed tumors in mice by antisense related rclroviruses. Jpn. J. Cancer Res.. 81: 225-231. 1990. inhibition of NF-KB. Science (Washington DC). 25Â«:1792-1795, 1992.

4000

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. Human T-Cell Leukemia Virus Type I Tax Transactivates Human Interleukin 8 Gene through Acting Concurrently on AP-1 and Nuclear Factor- κB-like Sites

Naoki Mori, Naofumi Mukaida, Dean W. Ballard, et al.

Cancer Res 1998;58:3993-4000.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/17/3993

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/58/17/3993. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.