HTLV-1 (human T-cell leukemia virus type 1) is a human retrovirus and the causative agent of ATL (adult T-cell leukemia), 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]. Infiltration of leukemic cells into various organs, such as lymph nodes, liver, spleen, lung, skin and intestinal tract, is a frequent manifestation of ATL. This type of cell infiltration often poses serious clinical problems for ATL patients, affecting the disease profile and prognosis. Because tumor cell survival and growth are maintained by nutrients, especially glucose and oxygen supplied by blood vessels, angiogenesis is considered to be essential for tumor malignancy .
Currently, the molecular mechanism of malignant transformation by HTLV-1 remains undefined. However, Tax, the 40-kDa transactivator protein encoded by HTLV-1, plays a crucial role in T cell transformation and leukemogenesis. Tax triggers viral transcription as well as induction of cellular genes involved in cell proliferation and anti-apoptotic signaling. In addition to activation of transcription, Tax transforms the infected cells by some mechanisms due to protein-protein interaction between Tax and other proteins [8, 9]. Moreover, one key feature of ATL is aneuploidy and chromosomal instability. Tax also contributes transformation of the cells by inducing aneuploidy and inactivating chromosomal instability checkpoint . Indeed, it immortalizes primary human T cells derived from peripheral blood or cord blood [11, 12] and induces tumors and leukemia in transgenic mice [13, 14].
NF-κB (nuclear factor κB) is a major survival signaling pathway activated by HTLV-1. This pathway is constitutively active in HTLV-1-transformed T-cells and primary ATL cells [15, 16].Tax can activate NF-κB pathway by associating with various signaling molecules in this pathway. For example, Tax binds IKKγ (also known as NEMO) and triggers the phosphorylation of IKKα and IKKβ, which form a complex with IKKγ . Subsequently the IKK complex phosphorylates IκBα, leading to its proteasome-mediated degradation, which frees IκBα-sequestered cytoplasmic NF-κB to migrate into the nucleus where it activates the transcription of NF-κB-responsive genes . Tax can also stimulate an alternative NF-κB pathway through the IKKα-dependent processing of the NF-κB p100 precursor protein to its active p52 form . The NF-κB signaling pathways are activated in ATL cells that do not express Tax, although the mechanism of activation remains unknown . One of the potential mechanisms by which ATL cells could develop resistance to apoptosis is through the activation of NF-κB. From this point of view, NF-κB has become an attractive target for therapeutic intervention.
AMPK (AMP-activated protein kinases) are a class of serine/threonine kinases that are activated by increased intracellular concentrations of AMP. ARK5 is a fifth member of the AMPK catalytic subunit family [18-20], and involved in tumor invasion and metastasis , and also known to induce cell survival during nutrient starvation or death receptor activation [22, 23]. ARK5 promoter contains two putative MARE (Maf-recognition element) sequences . The
2. Materials and methods
Bay 11-7082 and LY294002 were purchased from Calbiochem. D-(+)-glucose was purchased from Nakalaitesque.
2.2. Cell lines
The HTLV-1-uninfected T-cell leukemia cell lines MOLT-4 and CCRF-CEM, the HTLV-1-infected T-cell lines MT-2 , MT-4 ,C5/MJ , SLB-1 , HUT-102 , MT-1  and TL-OmI were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin (Sigma-Aldrich) at 37˚C in 5% CO2. MT-2, MT-4, C5/MJ and SLB-1 are HTLV-1-transformed T-cell lines which were established by an
2.3. RT (reverse transcriptase)-PCR
Total cellular RNA was extracted from cells using Trizol reagent as described by the supplier (Invitrogen). First-strand cDNA was synthesized in a 10-μl reaction volume using RNA-PCR kit (TAKARA BIO) with random primers. Thereafter, cDNA was amplified for ARK5 and c-Maf. The oligonucleotide primers used were as follows: for ARK5; sense, 5’- GAGTCCACTCTATGCATC-3’ and antisense, 5’- ATGTCCTCAATAGTGGCC-3’; for c-Maf; sense, 5’- TGCACTTCGACGACCGCTTCT C-3’ and antisense, 5’- CGCTGCTCGAGCCGTTTTCTC-3’. Product sizes were 256-bp for ARK5 and 327-bp for c-Maf. The amplification programs were follows: denaturing at 94˚C for 2 min, an annealing step at 55˚C for 30 s and an extension step at 72˚C for 30 s. Amplification cycles were 35 cycles for ARK5 and c-Maf, 25 cycles for β-actin. The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining.
2.4. Real-time RT-PCR
Total RNA was extracted from cells with Trizol reagent. Total RNA was reverse transcribed to obtain single-strand cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was carried out in a total volume of 25 μl of reaction mixture containing 1 μl of diluted cDNA, 12.5 μl of Brilliant SYBR® Green QPCR Master Mix (Stratagene), and 100 nM of each primer with a Mx3000P® Real-Time PCR System (Stratagene). For precise quantitative determination of the transcripts, we assessed the expression levels of GAPDH as an internal control. PCR conditions were set according to the instructions supplied by the manufacturer. The real-time PCR assay of each sample was conducted in triplicate, and the mean value was used as the mRNA level. The PCR primer pairs used in this study for ARK5 and c-Maf are listed above and those for Tax and GAPDH were as follow: for Tax; sense, 5’- CCCACTTCCCAGGGTTTGGACAGA-3’ and antisense, 5’- CTGTAGAGCTGAGCCGATAACGCG-3’; for GAPDH; sense, 5’-GAGTCAACGGATTTGGTCGT-3’ and antisense, 5’- GACAAGCTTCCCGTTCTCAG-3’.
2.5. Western blot analysis
Western blot analysis was performed as described previously . In brief, cells were lysed in sodium dodecyl sulfate (SDS) sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 10% glycerol, 6% 2-mercaptoethanol and 0.01% bromophenol blue. The lysates were resolved by electrophoresis on polyacrylamide gels and then electroblotted onto polyvinylidene difluoride membranes (Millipore). The membranes were incubated overnight with the appropriate primary antibody, as indicated, at 4°C. After washing, the blots were exposed to the appropriate secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The reaction products were visualized using enhanced chemiluminescence reagent (GE Healthcare) according to the instructions provided by the manufacturer. We used primary antibodies against Tax (Lt-4) , phosphorylated IκBα (Ser32/36), phosphorylated AKT(Ser473), AKT, NF-κB (p65) (Cell Signaling Technology), IκBα, (Santa Cruz Biotechnology) and actin (Lab Vision). Horseradish-peroxidase-conjugated secondary antibodies were purchased from GE Healthcare.
The reporter assay construct for ARK5 promoter was described previously . In brief, based on the results of a Genomic BLAST Search, primers with the NheI (upstream primer) or XhoI (downstream primer) site were synthesized, and PCR was then performed with the primers for genomic DNA extracted from PANC-1 cells. The PCR fragment digested with NheI and XhoI was ligated into pGL2-basic.A series of expression vectors for Tax (Tax WT) and mutants thereof (Tax M22 and Tax 703) were described previously [40, 41]. IκBα ΔN and IκBβ ΔN are deletion mutants of IκBα and IκBβ lacking the N-terminal 36 amino acids and 23 amino acids, respectively. IKKβ K44A and NEMOΔC are the dominant negative mutants of IKKβ and NEMO, respectively [42, 43]. The expression vector for mouse c-Maf was described previously . NF-κB (p65) expression plasmid was described previously .
2.7. Transfection and luciferase assay
Transfections were performed in CCRF-CEM cells by electroporation with Microporator MP-100® (Digital Bio Technology) according to the instructions supplied by the manufacturer for optimization and use. In all cases, the reference plasmid phRL-TK, which contains the
2.8. siRNA (small interfering RNA)
To knockdown ARK5 and c-Maf expression, predesigned double-stranded siRNAs (siGENOME SMART pool Human ARK5 and Human MAF;Dharmacon) were used. The siCONTROL non-targeting siRNA pool (Dharmacon) was used as a negative control. siRNAs were transfected into MT-2 cells by electroporation with MicroporatorMP-100®.
2.9. EMSA (electrophoretic mobility-shift assay)
Nuclear extracts were prepared from cells and DNA-binding activity was analyzed by EMSA, as described previously . Briefly, 5 μg of nuclear extracts were pre-incubated in a binding buffer containing 1 μg poly-deoxy-inosinic-deoxy-cytidylic acid (Amersham Biosciences), followed by addition of [α-32P]-labeled oligonucleotide probe. These mixtures were incubated for 15 min at room temperature. The DNA-protein complexes were separated on 4% polyacrylamide gels and visualized by autoradiography. The probes or competitors used were prepared by annealing the following sense and antisense synthetic oligonucleotides: NF-κB binding sites ARK5 κB A and ARK5 κB B derived from the ARK5 gene promoter 5’- gatcCTCTTGGGGTTCTCCTGGAC-3’ and 5’-gatcAGGTGGGGGAAGCCCTGGCT-3’, respectively. Mutants ARK5 κB A and ARK5 κB B are 5’-gatcCTCTTGGCCACGAGCTGGAC-3’and 5’-gatcAGGTGGGCCTCCAGCTGGCT-3’, respectively. To identify NF-κB proteins in the DNA-protein complex identified by EMSA, we used antibodies specific for various NF-κB family proteins, including p50, p65, c-Rel, RelB and p52 (Santa Cruz Biotechnology), to elicit a supershift DNA-protein complex formation. These antibodies were incubated with the nuclear extracts for 45 min at room temperature before incubation with radiolabeled probes.
2.10. Cell proliferation assay
The cells transfected with siRNA were incubated for 12 h, then seeded into 24-well plates at 1×105 viable cells per well, and incubated in glucose-containing or non-containing medium for the indicated time periods. The number of viable cells was determined every 24 h by counting trypan blue-excluding cells in a hemocytometer.
2.11. Statistical analysis
Data were expressed as mean ± SD. Differences between groups were analyzed by the unpaired Student’s
3.1. ARK5 and c-Maf are highly expressed in HTLV-1-infected T-cell lines
Expression of ARK5 and c-Maf mRNA was examined in 6 HTLV-1-infected (MT-2, MT-4, C5/MJ, SLB-1, HUT-102, MT-1 and TL-OmI) and 2 HTLV-1-uninfected (MOLT-4 and CCRF-CEM) T-cell lines. ARK5 mRNAs were detectable in all HTLV-1-infected T-cell lines, but not in uninfected T-cell lines (Figure 1A, left panel). c-Maf expression was relatively higher in HTLV-1-infected T-cell lines than in HTLV-1-uninfected T-cell lines (Figure 1A, right panel). The high expression of MafB was detected only in HTLV-1-infected MT-2 cells, but no differences in expression were noted between other infected and uninfected T-cell lines (results not shown). Although Tax protein was not detectable in ATL-derived T-cell lines (Figure 1C), Tax mRNA was expressed in all HTLV-1-infected T-cell lines by real time RT-PCR, which is more sensitive method than Western blot (Figure 1B). These results suggest a close association between HTLV-1 infection and induction of ARK5 and c-Maf mRNA expression.
3.2. HTLV-1 Tax induces ARK5 and c-Maf expression in T cells
To examine the direct association between ARK5 or c-Maf mRNAs induction and HTLV-1 infection, we used HTLV-1-infected TY8-3/MT-2 cells, which were established from TY8-3 cells by cocultivation with HTLV-1-infected MT-2 cells . Although MafB expression level was slightly increased in TY8-3/MT-2 cells (results not shown), the expression of ARK5 and c-Maf mRNAs was clearly higher in TY8-3/MT-2 cells than parental TY8-3 cells (Figure 2A). Because Tax induces various cellular genes, we next examined whether this includes the expression of ARK5 and c-Maf mRNAs in T cells. We used JPX-9 cells, which stably carry Tax expression plasmid, in which Tax expression is induced by the addition of CdCl2 . The expression of ARK5, c-Maf, and Tax mRNAs was analyzed by real time RT-PCR (Figure 2B). The addition of CdCl2 to the culture medium of JPX-9 cells induced the expression of Tax within 2 h, which persisted until 72 h after treatment. A concomitant increase of ARK5 mRNA within 10 h of treatment with CdCl2 was observed in JPX-9 cells. Rapid expression of c-Maf mRNA was also observed within 2 h, and peaked after 10 h of treatment with CdCl2. The induction of ARK5 or c-Maf could not be attributed to CdCl2 treatment, since ARK5 or c-Maf expression was not induced in JPX/M cells, which express Tax mutant protein, after treatment with CdCl2 (results not shown). These results indicate that Tax can increase the expression of ARK5 and c-Maf in T cells.
3.3. c-Maf does not alter ARK5 expression in T cells
3.4. Tax activates ARK5 transcriptional activity through NF-κB pathway
Next, we investigated whether Tax could directly enhance the activity of
3.5. NF-κB inhibitor suppresses ARK5 expression in an HTLV-1-infected T-cell line
NF-κB is constitutively activated not only in HTLV-1 transformed T-cell lines but also in ATL-derived T-cell lines and primary ATL cells . We analyzed the effects of an NF-κB inhibitor Bay11-7082, an inhibitor of phosphorylation of IκBα, on the expression of ARK5 in an HTLV-1-infected T-cell line. The expression of ARK5 mRNA in MT-2 cells was reduced by treatment with Bay11-7082 (Figure 5A, left panels). Inhibition of phosphorylation of IκBα and stabilization of IκBα protein were confirmed by Western blotting (Figure 5A, upper right panels). LY249002, a PI3K (phosphatidyl inositol3-kinase)/AKT inhibitor, did not affect the expression of ARK5 (Figure 5A, left panels). Using Western blotting, we also confirmed inhibition of phosphorylation of AKT by LY294002 (Figure 5A, lower right panels). Inhibition of NF-κB DNA-binding activity by Bay11-7082 was also detected by EMSA using oligonucleotide probes of ARK5 κB A and B sites (Figure 5B). These results support out findings in Figure 4 that indicate the contribution of NF-κB signaling to induction of
3.6. ARK5 maintains tolerance to glucose starvation in HTLV-1-infected T-cells
Finally, we investigated the role of ARK5 on the growth of HTLV-1-infected T-cells. Knockdown of ARK5 expression in MT-2 (Figure 6A, upper panels) and HUT-102 (Figure 6A, lower panels) cells did not affect growth of cells in the complete medium, which contained 2000 mg/mL glucose (Figure 6A, left panels). In contrast, knockdown of ARK5 expression reduced the cell growth in the glucose-free medium (Figure 6A, right panels). The knockdown efficiency was analyzed by real-time RT-PCR and almost equal knockdown efficiency was detected between with and without glucose conditions in both cell lines (Figure 6B). These results suggest that ARK5 maintains tolerance to glucose starvation in HTLV-1-infected T-cells.
Some tumor cells have a strong tolerance to nutrient starvation; tolerance to glucose starvation can be induced by hypoxia. AKT and AMPK appear to be involved closely in the mechanism of tolerance [47-49]. ATL cells often invade the lung, liver, bone, intestine and nerves. Invading leukemia cells might be under nutrient-starvation condition. Therefore, we investigated the roles of ARK5, which is a member of the AMPK family and downstream target of AKT in leukemogenesis by HTLV-1. The results of this study showed high expression of ARK5 and c-Maf in HTLV-1-infected T-cells and that such expression was induced by HTLV-1 Tax (Figure 1 and 2). The promoter region of
NF-κB signaling pathway is not only activated by Tax but also constitutively activated in primary ATL cells which express little amount of Tax . Therefore, NF-κB inhibitors are promising therapeutic agents for ATL. At present, several trials are being conducted using the Bay11-7082  and the proteasome inhibitor PS-341  for treatment of ATL. Recently, a new NF-κB inhibitor, dehydroxy-methyle poxy-quinomicin, has been found to inhibit NF-κB signaling pathway induced by Tax as well as the constitutive NF-κB activation in primary ATL cells, without affecting normal peripheral blood mononuclear cells [52, 53]. In the present study, we demonstrated that Bay11-7082 reduced ARK5 expression in an HTLV-1-infected T-cell line (Figure 5), suggesting that NF-κB inhibitors may modulate ATL cells invasion into multiple organs.
Another important finding in this study is that ARK5 is necessary for the growth of HTLV-1-infected T-cells during glucose starvation (Figure 6). Previously, we and others have demonstrated activation of PI3K/AKT signaling in HTLV-1-infected T-cells and Tax-expressing cells . These findings are important because PI3K/AKT signaling is required for malignant growth of HTLV-1-infected T-cells [55, 56]. However, there are numerous other downstream targets of PI3K/AKT . ARK5, one of the downstream targets of PI3K/AKT signaling, contains the consensus sequence of the AKT phosphorylation at amino acids 595-600, and is directly activated by AKT [21, 23]. We propose that Tax has dual roles as an accelerator to induce glucose tolerance in HTLV-1-infected T-cells (Figure 7); 1) induction of ARK5 expression through NF-κB activation (present study), and 2) activation of PI3K/AKT signaling pathway [55, 56].
The molecular mechanisms of induction of tolerance to glucose starvation by ARK5 in HTLV-1-infected T-cells are not elucidated in this study. Previous studies showed that during glucose starvation, survival of human hepatoma HepG2 cells is induced by ARK5 and activation of ARK5 by AKT is necessary for this effect [22, 23]. Glucose tolerance induced by ARK5 in HTLV-1-infected T-cells may also require phosphorylation and activation of ARK5 by AKT. However, we did not analyze the phosphorylation levels or activity of ARK5 in HTLV-1-infected T-cell lines, because a suitable antibody that can recognize phosphorylated ARK5 is not available commercially at present time. ARK5 also negatively regulates death receptors, such as Fas ligand-, TNF-and TRAIL-mediated cell death [22, 58]. When Fas is activated by the ligation of Fas ligand, intracellular interaction of the Fas-death domain, FADD and caspase-8 (death-inducing signaling complex (DISC) recruitment) is initiated for the activation of executioner caspase , and c-FLIP is the inhibitor of DISC recruitment. ARK5 directly inactivates caspase-6 through the phosphorylation at Ser257, resulting in c-FLIP preservation, which in turn suppresses DISC formation . Although cell death during glucose starvation is independent of death receptor, DISC recruitment is needed to induce cell death . In this way, ARK5 may prevent cell death during glucose starvation.
The results showed that c-Maf is highly expressed in HTLV-1-infected T-cells and induced by Tax in T cells (Figure 1 and 2). A previous study showed that c-Maf is expressed in ATL cells in lymph nodes of patients . c-Maf transgenic mice develop T-cell lymphoma and ARK5 is upregulated in c-Maf transgenic thymocytes and T lymphoma cells . In contrast, we found that c-Maf did not activate ARK5 promoter transcription in T-cells. However, c-Maf encodes a Th2-specific transcription factor that activates the expression of IL-4 and IL-10 in T cells . In this regard, a subpopulation of ATL cells produces Th2-associated cytokines . Taken together, it is of interest to identify other downstream target genes responsible for the actions of c-Maf that might contribute to malignant transformation of T cells. For example, some of the target genes of c-Maf, such as those that encode cyclin D2 and integrin β7, have deregulated expression in c-Maf transgenic mice . It might be interesting to investigate the role of c-Maf in the regulation of expression of these genes in ATL cells.
We demonstrated overexpression of ARK5 in HTLV-1-infected T-cells and that Tax induced
We thank the Fujisaki Cell Center, Hayashibara Biomedical Laboratories (Okayama, Japan) for providing HUT-102 cell line, M. Nakamura for providing JPX-9 and JPX/M cells, K. Matsumoto for providing Tax WT, Tax M22 and Tax 703, D.W. Ballard for providing the dominant-negative IκBα and IκBβ (IκBαΔN and IκBβΔN), K.-T. Jeang for providing the dominant-negative NEMO (NEMOΔC) and R. Geleziunas for providing the dominant-negative IKKβ (IKKβK44A) plasmid. We also thank Drs. Kohei Taniguchi, Atsushi Suzuki, Tetsuro Nakazato, Taeko Okudaira, Chie Ishikawa, Yuetsu Tanaka, Satoru Takahashi, Hiroyasu Esumi and Naoki Mori for providing materials, useful comments and discussions. We also acknowledge all members of our laboratories for the helpful comments and collaborations.
This work was supported in part by grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Scientific Research (C) from Japan Society for the Promotion of Science.
No potential conflicts of interest were disclosed.
ATL, adult T-cell leukemia;
AMPK, AMP-activated protein kinases;
ARK5, AMP-activated protein kinase-related kinase 5;
CREB, cyclic AMP response element-binding protein;
DISC, death-inducing signaling complex;
HTLV-1, human T-cell leukemia virus type 1;
EMSA, electrophoretic mobility-shift assay;
MARE, Maf-recognition element;
NF-κB, nuclear factor-kappa B;
PI3K, phosphatidyl inositol 3-kinase;
RT, reverse transcriptase;
SDS, sodium dodecyl sulfate;
siRNA, small interfering RNA;
WT, wild type.