HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1α in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1α protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1α by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1α protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1α protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics.
HTLV-1 (human T-cell leukaemia virus type 1) is a human retrovirus that is the causative agent of ATL (adult T-cell leukaemia), which is an aggressive and fatal T-cell malignancy characterized by dysregulated proliferation of CD4-positive T-cells [1–3]. HTLV-1 causes ATL in 3–5% of infected individuals after a long latent period of 40–60 years . The prognosis of patients with aggressive ATL remains poor with a median survival time of less than 1 year despite advances in both chemotherapy and supportive care [5,6]. Currently, the molecular mechanism of malignant transformation by HTLV-1 remains undefined. However, it has been reported that the 40-kDa transactivator protein Tax encoded by HTLV-1 plays a crucial role in T-cell transformation and leukaemogenesis. Tax triggers viral transcription as well as induction of cellular genes involved in cell proliferation and anti-apoptotic signalling. Tax can immortalize primary human T-cells derived from peripheral blood or cord blood [7,8] and induce tumours and leukaemia in transgenic mice [9,10] through: (i) activation of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway and transcription factors such as CREB (cAMP-response-element-binding protein), serum-responsive factor, activator protein 1, JAK (Janus kinase)/STAT (signal transducer and activator of transcription) and NF-κB (nuclear factor κB); (ii) repression of tumour suppressor p53; and (iii) interference with cell-cycle regulators, such as cyclins and cyclin-dependent kinase inhibitors .
HIF-1 (hypoxia-inducible factor 1) has been identified as a transcription factor that is induced in cells exposed to decreased oxygen concentrations (hypoxia) and bound to the cis-acting HRE (hypoxia-response element) located in the 3′-flanking region of the human EPO gene, which encodes erythropoietin . HIF-1 activates the transcription of over 60 genes that encode proteins which are required for critical aspects of tumour progression such as cell survival, angiogenesis, metabolism, invasion and metastasis . HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits . Although HIF-1β is not regulated by oxygen, HIF-1α levels increase exponentially as the cellular oxygen concentration decreases and HIF-1α protein undergoes rapid ubiquitination and degradation by the proteasome in the presence of oxygen . Thus HIF-1α appears to determine many responses to hypoxic conditions.
Expression of HIF-1α protein is up-regulated in common human cancers . Overexpression of HIF-1α in many cancers is associated with a poor response to treatment and increased patient mortality [15,16]. High HIF-1α levels in tumours reflect not only the frequent presence of intratumoural hypoxia, but also many common genetic alterations in oncogenes and tumour suppressor genes, such as PTEN (phosphatase and tensin homologue deleted on chromosome 10) , p53 , von Hippel–Lindau  and Src . HIF-1α protein expression is also regulated by the PI3K/Akt pathway in response to growth factors and cytokines . H-Ras has been reported to increase the expression of HIF-1 target genes in both normoxia and hypoxia via PI3K/Akt signalling . Although the PI3K/Akt signalling pathway is implicated in regulating HIF-1α expression in breast, colon and prostate cancer [13,21,23], cell-type-specific mechanisms may be involved . Previously, we and others have demonstrated that PI3K/Akt signalling was activated by Tax in HTLV-1-infected T-cells. PI3K/Akt signalling is required for malignant growth of HTLV-1-infected T-cells [25–30]. However, neither the activity of HIF-1 nor the relationship between activation of the PI3K/Akt pathway and HIF-1 function in HTLV-1-infected T-cells has been elucidated. In the present study, we examined whether HIF-1 was activated in HTLV-1-infected T-cell lines and primary ATL cells and investigated the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway.
MATERIALS AND METHODS
The HTLV-1-uninfected T-cell leukaemia cell lines MOLT-4 and CCRF-CEM, and the HTLV-1-infected T-cell lines MT-2 , MT-4 , SLB-1  and HUT-102  (a gift from the Fujisaki Cell Center, Hayashibara Biomedical Laboratories, Okayama, Japan) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), 50 units/ml penicillin and 50 μg/ml streptomycin (Sigma–Aldrich) at 37 °C in 5% CO2. MT-2, MT-4 and SLB-1 are HTLV-1-transformed T-cell lines, established by an in vitro co-culture protocol. HUT-102 was established from a patient with ATL, but it is unclear whether HUT-102 cells represent the tumour clone from the donor ATL patient. HeLa (a human cervix adenocarcinoma cell line) cells were maintained in DMEM (Dulbecco's modified Eagle's medium), supplemented with 10% heat-inactivated FBS, 50 units/ml penicillin and 50 μg/ml streptomycin at 37 °C in 5% CO2.
The diagnosis of ATL was based on clinical features, haematological findings and the presence of anti-HTLV-1 antibodies in the sera. Monoclonal HTLV-1 provirus integration into the DNA of leukaemia cells was confirmed by Southern blot hybridization in all patients (results not shown). PBMCs (peripheral blood mononuclear cells) from healthy volunteers and patients with ATL were isolated by Ficoll–Paque density gradient centrifugation (GE Healthcare). The study protocol was approved by the Human Ethics Review Committee of the University of the Ryukyus, Nishihara, Japan, and all samples were obtained after written informed consent.
Proteasome inhibitor LLnL (N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) and the PI3K inhibitor LY294002 were purchased from Calbiochem. CoCl2 and cycloheximide were obtained from Sigma–Aldrich.
Western blot analysis
Western blot analysis was performed as described previously . We used primary antibodies against Tax (1:3000; Lt-4) , HIF-1α (1:1000; BD Biosciences), Akt (1:1000; Cell Signaling Technology), phosphorylated Akt (phosphorylated at Ser473; 1:1000; Cell Signaling Technology) and actin (1:3000; Lab Vision). Horseradish-peroxidase-conjugated secondary antibodies were purchased from GE Healthcare.
RT (reverse transcriptase)-PCR
Total cellular RNA was extracted from cells using TRIzol® as described in the manufacturer's protocol (Life Technologies). First-strand cDNA was synthesized in a 10 μl reaction volume using an RNA-PCR kit (Takara Bio) with random primers. Thereafter cDNA was amplified for HIF-1α, VEGF (vascular endothelial growth factor) and β-actin. The oligonucleotide primers used were as follows: for HIF-1α sense, 5′-CCTGCACTCAATCAAGAAGTTGC-3′ and antisense, 5′-TTCCTGCTCTGTTTGGTGAGGCT-3′; for VEGF sense, 5′-GAAGTGGTGAAGTTCATGGAT-3′ and antisense, 5′-CGATCGTTCTGTATCAGTCTT-3′; for β-actin sense, 5′-GTGGGGCGCCCCAGGCACCA-3′ and antisense, 5′-CTCCTTAATGTCACGCACGATTTC-3′. Product sizes were 619 bp for HIF-1α, 408 bp and 541 bp for VEGF, and 548 bp for β-actin. The amplification programs were as follows: for HIF-1α, denaturion at 94 °C for 1 min, an annealing step at 60 °C for 1 min, and an extension step at 72 °C for 1 min for 27 cycles; for VEGF, denaturion at 94 °C for 2 min, an annealing step at 64 °C for 1.5 min, and an extension step at 72 °C for 2 min for 30 cycles; for β-actin, denaturion at 94 °C for 30 s, an annealing step at 60 °C for 30 s, and an extension step at 72 °C for 90 s for 28 cycles. The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining.
siRNA (small interfering RNA)
To repress HIF-1α expression, a predesigned double-stranded siRNA (siGENOME SMART pool Human HIF1A; Dharmacon) was used. An siCONTROL non-targeting siRNA pool (Dharmacon) was used as a negative control. siRNAs were transfected into HUT-102 cells by electroporation with a microporator (Degital Bio Technology). Transfected cells were incubated for 12 h, seeded into 24-well plates at 1×105 viable cells per well, and incubated for the times indicated. The number of viable cells was determined every 24 h by counting Trypan-Blue-excluding cells in a haemocytometer.
EMSA (electrophoretic mobility-shift assay)
Nuclear extracts were prepared from cells and HIF-1 DNA-binding activity was analysed by EMSA as described previously . The probes or competitors used were prepared by annealing the following sense and antisense synthetic oligonucleotides: HIF-1 consensus binding motif (W18) derived from the EPO promoter 5′-gatcGCCCTACGTGCTGTCTCA-3′ containing a putative HRE (underlined), mutant HIF-1 consensus binding motif (M18) 5′-gatcGCCCTAAAAGCTGTCTCA-3′ containing a mutated HRE (underlined), IL-2R κB 5′-gatc CGGCAGGGGAATCTCCCTCTC-3′ containing a typical NF-κB element from the IL2RA (interleukin 2 receptor α-chain gene) (underlined), or Oct-1 element 5′-gatc TGTCGAATGCAAATCACTAGAA-3′ containing an Oct-1-binding site (underlined). To identify HIF-1α proteins in the DNA–protein complex revealed by EMSA, we used a specific antibody for HIF-1α protein (Santa Cruz Biotechnology) to elicit a supershift DNA–protein complex formation.
pCEP4/HIF-1α is an HIF-1α expression plasmid . pGL3-6×HRE-Luc contains six tandem copies of the HRE recognition sequence for HIF-1 . A series of expression vectors for Tax [Tax WT (wild-type)] and mutants thereof (Tax M22, Tax 703 and Tax K88A) have been described previously [39,40]. The dominant-negative Akt (AKT-DN) expression plasmid (pCMV5-K169A, T308A, S473A-AKT) has the Lys169, Thr308 and Ser473 to an alanine residue mutations.
Transfection and luciferase assay
HeLa cells were transfected using Lipofectamine™ (Invitrogen). Cells were transiently transfected with the indicated effector plasmids and luciferase reporter constructs. In all cases, the reference plasmid phRL-TK (Promega), which contains the Renilla luciferase gene under the control of the TK (thymidine kinase) promoter, was co-transfected to correct for transfection efficiency. Luciferase assays were performed using the Dual-Luciferase Reporter System (Promega), in which relative luciferase activities were calculated by normalizing transfection efficiency according to the Renilla luciferase activities.
RESULTS AND DISCUSSION
HIF-1α protein is overexpressed in HTLV-1-infected T-cell lines
Western blot analysis of the HTLV-1-infected T-cell lines showed that they all contained elevated levels of HIF-1α protein compared with barely detectable levels in HTLV-1-uninfected T-cell lines (Figure 1A). The expression levels of HIF-1α in HTLV-1-infected T-cell lines were almost the same as those in CoCl2-treated HeLa cells (Figure 1A). The cell lysates from MT-2 cells showed two Tax-immunoreactive bands, consistent with the 40-kDa Tax protein and a 69-kDa fusion between the envelope and the Tax coding sequence, because MT-2 cells have a defective provirus as reported previously . HIF-1α mRNA was detectable by RT-PCR in all HTLV-1-infected and uninfected T-cell lines, but no significant difference was observed in the HIF-1α mRNA level between HTLV-1-infected and uninfected T-cell lines (Figure 1B). These results suggest that expression of HIF-1α is regulated by post-transcriptional mechanisms in these cell lines. Then, to examine whether there were any differences in degradation of HIF-1α protein between HTLV-1-infected and uninfected T-cell lines, HTLV-1-infected HUT-102 cells and uninfected MOLT-4 cells were treated with the proteasome inhibitor LLnL. The accumulation of HIF-1α protein was detected in the presence of LLnL in both cell lines (Figure 1C). We obtained similar results in HTLV-1-infected MT-2 cells and uninfected CCRF-CEM cells (results not shown). We also examined the half-life of the HIF-1α protein in these cell lines. Cycloheximide was added to the cell culture medium to block new protein synthesis. Western blot analysis showed that HIF-1α protein was degraded in both cell lines (Figure 1D). Therefore the difference in HIF-1α protein levels between HTLV-1-infected and -uninfected T-cell lines was attributable not to differences in stabilization of this protein, but rather to differences in protein synthesis.
Overexpression of HIF-1α protein in HTLV-1-infected T-cell lines
HIF-1 DNA-binding activity is enhanced in HTLV-1-infected T-cell lines
HIF-1 DNA-binding activity was detected by EMSA in all HTLV-1-infected T-cell lines which had enhanced expression of HIF-1α protein. In contrast, no significant HIF-1 DNA-binding activity was detected in extracts of HTLV-1-uninfected T-cell lines (Figure 2A). Competition and supershift assays showed that the observed DNA–protein complexes were specific for the HRE site and included HIF-1α protein (Figure 2B).
HIF-1 DNA-binding activity is enhanced in HTLV-1-infected T-cell lines
Suppression of HIF-1α expression inhibited cell growth of an HTLV-1-infected T-cell line
To examine the role of HIF-1α in HTLV-1-infected T-cell growth, HUT-102 cells were transfected with HIF-1α siRNA. Cell growth was inhibited in HUT-102 cells transfected with HIF-1α siRNA compared with the cells transfected with non-targeting siRNA (Figure 3A). The suppression of HIF-1α and its target gene (VEGF) mRNA expression was confirmed by RT-PCR (Figure 3B).These results indicate that the expression of HIF-1α is needed for the growth of HTLV-1-infected T-cells. Tax activates HIF-1 transcriptional activity through the PI3K/Akt signalling pathway
Knockdown of HIF-1α suppresses cell growth of a HTLV-1-infected T-cell line
In a transient transfection assay using pGL3-6×HRE-Luc as a reporter, the expression of Tax WT or HIF-1α alone in HeLa cells activated the luciferase reporter. The combination of HIF-1α and Tax WT had an even greater effect on the activation of the luciferase reporter (Figure 4A). We confirmed that Tax induced HIF-1α protein expression in HeLa cells (Figure 4B) and co-expression of Tax with HIF-1α enhanced HIF-1α protein accumulation (Figure 4C). In previous studies, the activation of PI3K/Akt signalling was associated with an accumulation of HIF-1α [21,23]. Moreover, we have shown previously that Tax induced phosphorylation of Akt and Akt kinase activity via activation of CREB . In the present study, we tested expression plasmids encoding three mutant forms of Tax (Tax M22, Tax 703 and Tax K88A) which have been described previously [39,40]. Tax M22 activates CREB but does not affect NF-κB; Tax 703 and Tax K88A activate NF-κB but do not affect CREB. In a previous study, we have shown that WT Tax and the mutant Tax M22 enhanced Akt activity but Tax 703 and Tax K88A did not affect its activity . In the present study, Tax M22, but not Tax 703 or Tax K88A mutant forms, activated the pGL3-6×HRE-Luc reporter (Figure 4D). The transcriptional activity induced by the combination of HIF-1α and either Tax WT or Tax M22 was inhibited by the PI3K inhibitor LY294002 in a dose-dependent manner (Figure 4E). In contrast, the combination of HIF-1α and either Tax 703 or Tax K88A did not increase luciferase reporter activity and no inhibitory effect of LY294002 was observed (Figure 4E). We used a dominant-negative mutant form of Akt (AKT-DN) to directly examine the involvement of Akt in Tax-induced HIF-1 transcriptional activity. AKT-DN suppressed the effect of Tax alone or Tax plus HIF-1α on reporter gene transcription in a dose-dependent manner (Figure 4F). These results indicate that HIF-1 transcriptional activation by Tax is mediated via the PI3K/Akt signalling pathway.
Tax induces HRE reporter activity through the PI3K/Akt signalling pathway
PI3K/Akt activation is required for overexpression of HIF-1α protein and HIF-1 DNA-binding activity in HTLV-1-infected T-cell lines
We next examined the effect of PI3K inhibition on Akt activity and HIF-1α protein expression. As shown in Figure 5(A), LY294002 markedly decreased HIF-1α protein levels and inhibited phosphorylation of Akt in the HTLV-1-infected T-cell line HUT-102. To examine whether HIF-1 DNA-binding activity was also mediated via PI3K/Akt signalling in HTLV-1-infected T-cell lines, cells were treated with LY294002 and the HIF-1 DNA-binding activity was analysed by EMSA. HIF-1 DNA-binding activity was decreased by LY294002 treatment in MT-2 and HUT-102 cells (Figure 5B). These results suggest that Tax induced HIF-1 DNA-binding and transcriptional activity by increasing HIF-1α protein expression through activation of the PI3K/Akt signalling pathway. Activated Akt phosphorylates mTOR (mammalian target of rapamycin), which increases the activity of eukaryotic translation initiation factor 4E and thereby increases cap-dependent translation of mRNA into protein . Previously, it has been shown that PI3K/Akt signalling can activate cap-dependent translational machinery through mTOR, which results in enhanced HIF-1α protein synthesis . HIF-1α protein is overexpressed in primary ATL cells. Finally, we examined the expression of HIF-1α protein in primary ATL cells. Overexpression of HIF-1α protein was observed in the majority of primary ATL cells compared with PBMCs from healthy donors (Figure 6). Although Tax protein was hardly detectable (results not shown), HIF-1α expression was strongly correlated with levels of phosphorylated Akt in ATL cells (Figure 6). These results are consistent with the hypothesis that HIF-1 is activated via PI3K/Akt signalling in primary ATL cells as was demonstrated in the HTLV-1-infected T-cell lines. Consistent with our results, Fukuda et al.  recently reported that the levels of phosphorylated Akt were increased in ATL cells and that expression of the inositol phosphatases PTEN and SHIP-1 (SH2-containing inositol phosphatase-1), which antagonize the PI3K/Akt signalling pathway, was down-regulated in these cells.
A PI3K inhibitor represses HIF-1α protein expression and HIF-1 DNA-binding activity in HTLV-I-infected T-cell lines
HIF-1α and phosphorylated Akt are highly expressed in primary ATL cells
In the present study, we report the novel observation that HIF-1α protein is highly expressed in HTLV-1-infected T-cell lines and is associated with HIF-1 DNA-binding and transcriptional activity. Increased HIF-1α expression in these cells is dependent on Tax and the PI3K/Akt signalling pathway. Other viral oncogenes use a variety of molecular strategies to increase HIF-1α expression or activity, including Epstein–Barr virus latent membrane protein 1 , Hepatitis B virus X protein  and Kaposi's sarcoma herpes virus G-protein-coupled receptor .
As HIF-1 plays a major role in activating the transcription of genes whose protein products promote tumour progression, it is an obvious target for the development of novel cancer therapeutics [13,47,48]. Although interventions using dominant-negative and RNA interference technology are under development, the effects of small molecule inhibitors of the HIF-1 signalling pathway are of particular interest [49,50]. The role of HIF-1 in promoting angiogenesis and invasion in ATL is under further investigation in our laboratory using small molecule inhibitors of HIF-1.
This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Foundation for Promotion of Cancer Research in Japan, Uehara Memorial Foundation, Public Trust Haraguchi Memorial Cancer Research Fund, Takeda Science Foundation, and Japan Leukemia Research Fund. We thank the Fujisaki Cell Center, Hayashibara Biomedical Laboratories (Okayama, Japan) for providing the HUT-102 cell line, Dr K. Matsumoto (Osaka Red Cross Blood Center, Osaka, Japan) for providing Tax WT, Tax M22 and Tax 703, Dr C.-Z. Giam (Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, U.S.A.) for providing Tax K88A, and Dr D. Alessi (MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, U.K.) for providing the dominant-negative Akt expression plasmids. We also acknowledge all members of our laboratories for the helpful comments and collaborations.
adult T-cell leukaemia
Dulbecco's modified Eagle's medium
electrophoretic mobility-shift assay
fetal bovine serum
human T-cell leukaemia virus type 1
hypoxia-inducible factor 1
nuclear factor κB
peripheral blood mononuclear cell
phosphatase and tensin homologue deleted on chromosome 10
mammalian target of rapamycin
small interfering RNA
vascular endothelial growth factor