Importin β1 regulates cell growth and survival during adult T cell
leukemia/lymphoma therapy
Chie Ishikawa1,2 & Masachika Senba3 & Naoki Mori1
Received: 17 August 2020 /Accepted: 16 September 2020
# Springer Science+Business Media, LLC, part of Springer Nature 2020
Summary
There is no cure for adult T cell leukemia/lymphoma (ATLL) associated with human T cell leukemia virus type 1 (HTLV-1), and
novel targeted strategies are needed. NF-κB and AP-1 are crucial for ATLL, and both are transported to the nucleus by an
importin (IPO)α/β heterodimeric complex to activate target genes. In this study, we aimed to elucidate the function of IPOβ1 in
ATLL. The expression of IPOβ1 was analyzed by western blotting and RT-PCR. Cell growth, viability, cell cycle, apoptosis and
intracellular signaling cascades were examined by the water-soluble tetrazolium-8 assay, flow cytometry and western blotting.
Xenograft tumors in severe combined immune deficient mice were used to evaluate the growth of ATLL cells in vivo. IPOβ1
was upregulated in HTLV-1-infected T cell lines. Further, IPOβ1 knockdown or the IPOβ1 inhibitor importazole and the IPOα/
β1 inhibitor ivermectin reduced HTLV-1-infected T cell proliferation. However, the effect of inhibitors on uninfected T cells was
less pronounced. Further, in HTLV-1-infected T cell lines, inhibitors suppressed NF-κB and AP-1 nuclear transport and DNA
binding, induced apoptosis and poly (ADP-ribose) polymerase cleavage, and activated caspase-3, caspase-8 and caspase-9.
Inhibitors also mediated G1 cell cycle arrest. Moreover, the expression of NF-κB- and AP-1-target proteins involved in cell
cycle and apoptosis was reduced. In vivo, the IPOα/β1 inhibitor ivermectin decreased ATLL tumor burden without side effects.
IPOβ1 mediated NF-κB and AP-1 translocation into ATLL cell nuclei, thereby regulating cell growth and survival, which
provides new insights for targeted ATLL therapies. Thus, ivermectin, an anti-strongyloidiasis medication, could be a potent
anti-ATLL agent.
Keywords Adult T cell leukemia . Human T cell leukemia virus type 1 . Importin . NF-κB . AP-1
Introduction
Adult T cell leukemia/lymphoma (ATLL) is an intractable T
cell neoplasm caused by latent infection by the retrovirus hu￾man T cell leukemia virus type 1 (HTLV-1) [1]. Despite re￾cent advances in chemotherapy, the 3-year overall survival for
ATLL patients receiving conventional chemotherapy is
approximately 24% due to drug resistance [2]. Although allo￾genic hematopoietic stem cell transplantation is the only cura￾tive therapy, only a fraction of patients benefit because ATLL
develops in the elderly [3]. Therefore, more effective and less
toxic options based on aberrantly activated signaling networks
is highly desired.
Transcription factors such as NF-κB and AP-1 are impor￾tant for ATLL pathogenesis and function by regulating genes
involved in cell proliferation and survival [4, 5]. Further, con￾stitutive NF-κB and AP-1 activation occurs in HTLV-1-
infected T cells and ATLL cells [6, 7], and many studies have
validated these pathways as promising therapeutic targets [8].
The NF-κB family consists of five members, namely RelA
(p65), RelB, c-Rel, p50 and p52. NF-κB can form homo or
heterodimers. Unstimulated cells retain NF-κB in the cyto￾plasm, which is bound to its inhibitor IκBα. Upon activation,
IκBα is phosphorylated, leading to its proteasome-mediated
degradation, causing the release of NF-κB for nuclear import
and subsequent target gene transcription [9]. Another
* Naoki Mori
[email protected]
1 Department of Microbiology and Oncology, Graduate School of
Medicine, University of the Ryukyus, 207 Uehara,
Nishihara, Okinawa 903-0215, Japan
2 Division of Health Sciences, Transdisciplinary Research
Organization for Subtropics and Island Studies, University of the
Ryukyus, Nishihara, Okinawa, Japan
3 Department of Pathology, Institute of Tropical Medicine, Nagasaki
University, Nagasaki, Japan
Investigational New Drugs

https://doi.org/10.1007/s10637-020-01007-z

transcription factor, AP-1, consists primarily of Jun (c-Jun,
JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) di￾mers [5].
Protein translocation from the cytoplasm to the nucleus is a
tightly regulated process. Most proteins require an intrinsic
nuclear localization signal (NLS), which directs their nuclear
transport via the nuclear envelope-localized nuclear pore com￾plexes, which is mediated by importins (IPOs) [10]. During
classical nuclear protein import, IPOα functions as an adaptor,
linking NLS-containing proteins to IPOβ [10, 11]. In the non￾classical pathway, IPOβ directly recognizes and binds to a
specific NLS [12].
IPOβ1 was the first identified and is a major nuclear trans￾port factor [13]. IPOα and IPOβ1 are recruited to facilitate the
nuclear translocation of NF-κB complexes [14, 15]. Further,
AP-1 nuclear import was suggested to be mediated by the
non-classical nuclear import pathway, as AP-1 binds IPOβ1
with higher affinity than IPOα and therefore, was reported to
be transported into the nucleus by IPOβ1 alone [16]. In addi￾tion, IPOβ1 plays a key role in cell cycle transition and the
regulation of mitosis and replication [17–19]. Moreover,
IPOβ1 expression is reportedly upregulated in various can￾cers, including head and neck, cervical, gastric, ovarian and
lung cancer, and correlates with a poor prognosis [20–23].
Therefore, it could be a novel therapeutic target for cancers
[24]. Moreover, IPOβ1 might take part in the pathogenesis of
hepatocellular carcinoma, cervical cancer, diffuse large B cell
lymphoma and multiple myeloma via NF-κB and AP-1 sig￾naling [25–28]. However, the role of IPOs in ATLL is not
clear. Here, we elucidated the roles of IPOβ1 in ATLL￾derived and HTLV-1-transformed T cell lines.
Materials and methods
Cell lines and cell culture
HTLV-1-transformed T cell lines (MT-2 [29], MT-4 [30], C5/
MJ [31], SLB-1 [32] and HUT-102 [33]), ATLL-derived T
cell lines (MT-1 [34], TL-OmI [35] and ED-40515(−) [36])
and an uninfected Jurkat T cell line [37] were cultured in
RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 1% penicillin/streptomycin (Nacalai
Tesque, Inc.) and 10% fetal bovine serum (Biological
Industries, Kibbutz Beit Haemek, Israel) at 37 °C in a 5%
CO2 humidified atmosphere. The C5/MJ, HUT-102, MT-1
and Jurkat cells were obtained from Fujisaki Cell Center,
Hayashibara Biochemical Laboratories, Inc. (Okayama,
Japan). The MT-2 and MT-4 cells were provided by Dr.
Naoki Yamamoto (Tokyo Medical and Dental University,
Tokyo, Japan). The SLB-1 and ED-40515 (−) cells were ob￾tained from Dr. Diane Prager (UCLA School of Medicine,
Los Angeles, CA, USA) and Dr. Michiyuki Maeda (Kyoto
University, Kyoto, Japan), respectively. The TL-OmI cells
were obtained from Dr. Masahiro Fujii (Niigata University,
Niigata, Japan). Human peripheral blood mononuclear cells
(PBMCs) obtained from Lifeline Cell Technology (Frederick,
MD, USA) were cultured with 20 μg/ml of phytohemagglu￾tinin (PHA; Sigma-Aldrich Co., St. Louis, MO, USA) stimu￾lation for 72 h.
Reagents
Importazole was purchased from Merck Millipore
(Burlington, MA, USA) and Abcam (Cambridge, UK).
Ivermectin and z-VAD-FMK were obtained from Wako
Pure Chemical Industries (Osaka, Japan) and Promega Corp.
(Madison, WI, USA), respectively. Antibodies against
IPOβ1, Bcl-xL, Bax, Bak, survivin, cellular inhibitor of apo￾ptosis (c-IAP)1, RelA, cleaved poly (ADP-ribose) polymerase
(PARP), and cleaved caspase-8, caspase-9 and caspase-3 were
purchased from Cell Signaling Technology, Inc. (Beverly,
MA, USA). Antibodies against cyclin-dependent kinase
(CDK)2, CDK4, CDK6, cyclin E and actin were obtained
from Neomarkers, Inc. (Fremont, CA, USA). Antibodies
against X-linked inhibitor of apoptosis protein (XIAP) and
cyclin D1 were purchased from Medical & Biological
Laboratories, Co. (Aichi, Japan). Antibodies against c-IAP2,
cyclin D2, JunB, JunD, lamin B, and NF-κB subunits p50,
p52, RelA, c-Rel and RelB, and AP-1 subunits c-Fos, FosB,
Fra-1, Fra-2, c-Jun, JunB and JunD for supershift assays were
obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX,
USA). An antibody recognizing c-Myc was purchased from
Wako Pure Chemical Industries.
Co-cultivation of PBMCs and HTLV-1-infected T cells
MT-2 cells were used as the HTLV-1-infected T cell line, and
these cells produce viral particles. MT-2 cells were treated
with 200 μg/ml of mitomycin C (MMC; Sigma-Aldrich Co.)
for 1 h. After pipetting vigorously and washing three times
with phosphate-buffered saline, MMC-treated MT-2 cells
were co-cultured with PBMCs from a healthy donor
(Lifeline Cell Technology) in RPMI-1640 supplemented with
10 ng/ml of interleukin-2 (IL-2; kindly provided by Takeda
Pharmaceutical Company Ltd., Osaka, Japan). Half of the
culture medium was changed with fresh medium containing
IL-2 every 3 days. Because MT-2 cells were treated extensive￾ly with MMC, no discernible contamination of MT-2 cells
was found in this system.
Small interfering RNA (siRNA)
To repress IPOβ1, a pre-designed double-stranded siRNA
(ON-TARGET plus SMART pool; Dharmacon Inc.,
Lafayette, CO, USA) was used. The siCONTROL non￾Invest New Drugs
targeting siRNA pool (Dharmacon Inc.) was used as a nega￾tive control. All siRNA transfections were performed using a
Microporator MP-100 (Digital Bio Technology, Seoul,
Korea) by pulsing twice at 1000 V for 20 ms.
Cell proliferation and cytotoxicity assays
Cell proliferation and toxicity were evaluated using the water￾soluble tetrazolium (WST)-8 uptake method according to the
supplier’s instructions (Nacalai Tesque, Inc.). Cells were seed￾ed on 96-well plates and treated as indicated for up to 72 h.
After WST-8 reagent was added to each well, the absorbance
at 450 nm was determined using a Wallac 1420 Multilabel
Counter (PerkinElmer, Inc., Waltham, MA, USA). Triplicate
wells were used for each experimental condition. The optical
density of each sample was compared to that of the control.
Cell cycle analysis
The cell cycle was assessed using propidium iodide (PI),
which can label cellular nuclear DNA. Cells were stained
using the CycleTEST Plus DNA Reagent kit (Becton￾Dickinson Immunocytometry Systems, San Jose, CA, USA)
according to instructions of the kit. The cell cycle distribution
was analyzed using an Epics XL flow cytometry (Beckman
Coulter, Inc., Brea, CA, USA) and MultiCycle software (ver￾sion 3.0; Phoenix Flow Systems, San Diego, CA, USA).
Histograms of PI signal intensity were generated and the per￾centage of cells in each phase of the cell cycle was determined.
Analysis of apoptosis
Cells were treated with importazole and ivermectin for up to
72 h and then permeabilized by incubating them with digito￾nin, after which, they were treated with a phycoerythrin￾conjugated APO2.7 antibody (1:10; Beckman Coulter, Inc.,
Marseille, France). Rates of apoptosis were quantified imme￾diately after staining using an Epics XL flow cytometry. In
addition, to evaluate nuclear morphological changes in the
nuclei, cells were stained with Hoechst 33342 (Dojindo
Molecular Technologies, Inc., Kumamoto, Japan) and ob￾served under a DMI6000 microscope (Leica Microsystems,
Wetzlar, Germany).
Analysis of caspase activity
For the detection of caspase-3, caspase-8 and caspase-9 acti￾vation, Colorimetric Caspase Assay kits (Medical &
Biological Laboratories, Co.) were used according to the man￾ufacturer’s instructions. Briefly, cells were lysed in the cell
lysis buffer supplied with the kit, and cell lysates were incu￾bated with respective caspase-specific labeled substrates. The
release of the chromophore ρ-nitroanilide after cleavage from
substrates was measured using a Wallac 1420 Multilabel
Counter. Caspase activity was assessed as the ratio of the
colorimetric output in the treated sample relative to that in
the control, which was set to 1.
Reverse transcriptase (RT)-PCR
Total RNA was isolated from cells using TRIzol reagent
(Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA
was generated from a total of 1 μg of RNA using a
PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan).
PCR was performed using a combination of individual
sequence-specific primer sets (Table 1).
Electrophoretic mobility shift assay (EMSA)
To examine NF-κB and AP-1 activation, nuclear extracts from
cells were prepared, and EMSAs were performed with 5 μg of
protein, as described previously [38]. Nuclear extracts were
incubated with 32P-labeled EMSA probes. The oligonucleo￾tide probe sequences were as follows: for a typical NF-κB
element of the IL-2 receptor (IL-2R) α gene, 5′-GATC
CGGCAGGGGAATCTCCCTCTC-3′ and for the consensus
AP-1 element of the IL-8 gene, 5′-GATCGTGATGAC
TCAGGTT-3′. The underlined sequences are the NF-κB￾and AP-1-binding elements, respectively.
Protein extraction and immunoblot analysis
Immunoblot analysis was performed on whole cell lysates and
nuclear fractions. The cultured cells were lysed with lysis
buffer containing 62.5 mM Tris-HCl (pH 6.8) (Nacalai
Tesque, Inc.), 2% sodium dodecyl sulfate (SDS; Nacalai
Tesque, Inc.), 10% glycerol (Nacalai Tesque, Inc.), 6% 2-
mercaptoethanol (Nacalai Tesque, Inc.) and 0.01%
bromophenol blue (Wako Pure Chemical Industries). Protein
concentrations were determined using the DC Protein Assay
kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Samples
containing 20 μg of total protein were separated using SDS￾polyacrylamide gels, transferred to polyvinylidene difluoride
membranes (Merck KGaA, Darmstadt, Germany), and blotted
with primary antibodies (1:1000). Following incubation with
horseradish peroxidase-conjugated secondary anti-mouse
(1:1000; Cell Signaling Technology, Inc.) or anti-rabbit
(1:1000; Cell Signaling Technology, Inc.) IgG antibodies, im￾munoreactivity was visualized using an enhanced chemilumi￾nescence reagent (Amersham Biosciences Corp., Piscataway,
NJ, USA).
Xenograft tumor model
All animal experiments were conducted in strict accordance
with the Guidelines for Animal Experimentation of University
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of the Ryukyus and approved by the Animal Care and Use
Committee of University of the Ryukyus (A2016149). HUT-
102 cell suspensions (1 × 107
/0.2 ml of RPMI-1640 medium)
were inoculated subcutaneously into 5-week-old female C.B-
17/Icr-severe combined immune deficient (SCID) mice
(Kyudo, Co., Tosu, Japan) on day 0. The mice were random￾ized into two groups (n = 4, each) and then treated with vehi￾cle or ivermectin (4 mg/kg) between days 1 and 28.
Ivermectin was solubilized in soybean oil (Wako Pure
Chemical Industries) and administered via oral gavage five
times per week. The three dimensions, height (h), length (l)
and width (w), of each tumor were measured weekly using a
shifting caliper, and tumor volume was calculated according
to the following formula: π/6 × h × l × w [39]. Body weights
were also measured weekly. The xenograft tumors and blood
samples were collected immediately after the mice were
sacrificed on day 28. Tumor weights were measured, and the
sera were stored at −80 °C until they were assayed for human
soluble IL-2Rα (sIL-2Rα) and sCD30.
Biomarker analysis
The concentrations of sIL-2Rα and sCD30 were determined
in the sera of mice treated or untreated with ivermectin by
enzyme-linked immunosorbent assays (ELISAs) for human
sIL-2Rα (Diaclone SAS, Besançon, France) and sCD30
(Affymetrix eBioscience, San Diego, CA, USA), according
to the manufacturers’ instructions.
Hematoxylin and eosin (HE) staining and terminal
deoxynucleotidyl transferase deoxyuridine triphos￾phate nick-end labeling (TUNEL) assay
Tumor specimens were collected from mice and fixed with
10% formalin (Wako Pure Chemical Industries). After dehy￾dration through a graded ethanol series (Japan Alcohol Selling
Co., Tokyo, Japan), the samples were embedded in paraffin
(Sakura Finetek Japan Co., Tokyo, Japan). The paraffin￾embedded tissue sections of ATLL tumors were stained with
HE (Merck KGaA), and pathological changes were evaluated.
DNA fragmentation was analyzed by TUNEL assays using a
commercial kit (Roche Applied Science, Penzberg, Germany)
according to the manufacturer’s instructions. Cells were ex￾amined under a light microscope (Axioskop 2 Plus) with an
Achroplan 40×/0.65 lens (both from Zeiss, Hallbergmoos,
Germany). Images were acquired with an AxioCam 503 color
camera and AxioVision LE64 software (Zeiss GmbH, Jena,
Germany).
Statistical analysis
The results are expressed as the mean ± standard deviation
(SD). Experimental data analysis was performed with a
Student’s t test or ANOVA with the Tukey-Kramer test.
Differences were considered significant at P < 0.05.
Results
Expression of IPOβ1 in HTLV-1-infected T cells
Expression levels of IPOβ1 were evaluated by western blot
analysis in eight HTLV-1-infected T cell lines and PBMCs
from two healthy donors. IPOβ1 was upregulated in all
HTLV-1-infected T cell lines compared to the expression in
normal PBMCs (Fig. 1a). Notably, PHA stimulation induced
the expression of IPOβ1.
To investigate the ability of HTLV-1 infection to induce
IPOβ1 expression in vitro, PBMCs were co-cultured with
MMC-treated MT-2 cells. After co-cultivation for 7 days,
the PBMCs were harvested for the assessment of HTLV-1
viral gene expression by RT-PCR. As shown in Fig. 1b,
Table 1 Primer sequences used
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PBMCs co-cultured with MT-2 cells expressed Tax mRNA.
Furthermore, the expression levels of IPOβ1 and IL-2Rα, a
known target gene of Tax [40], were increased in these cells
following the induction of Tax expression. These results sug￾gest that HTLV-1 infection can induce the expression of
IPOβ1 in PBMCs.
Knockdown of IPOβ1 suppresses the growth of HTLV-
1-infected T cell lines
To investigate the cellular function of IPOβ1, siRNA was
utilized to inhibit its expression. MT-2 cells were transfected
with siRNA against IPOβ1, and the effect on IPOβ1 expres￾sion was examined by RT-PCR and western blotting. The
mRNA and protein levels of IPOβ1 were reduced by trans￾fection with this siRNA (Figs. 1c, d). A scrambled siRNA
served as a negative control. To determine whether the inhi￾bition of IPOβ1 expression had biological relevance, cell
growth was analyzed. The results showed that inhibition of
IPOβ1 expression significantly reduced MT-2 cell prolifera￾tion (Fig. 1e). We then analyzed the expression of cell cycle￾associated genes such as cyclin D1, cyclin D2 and c-myc in
cells by RT-PCR. Knockdown of IPOβ1 decreased the ex￾pression of these genes (Fig. 1c).
IPOβ1 inhibitors reduce the viability of HTLV-1-
infected T cell lines
Importazole and ivermectin have been shown to be potent
inhibitors of IPOβ1 and IPOα/β1 activities, respectively
[41, 42]. Both inhibitors were investigated for their potential
to inhibit cell viability using HTLV-1-infected T cell lines
(MT-2, MT-4, HUT-102 and TL-OmI), an uninfected T cell
line (Jurkat) and PBMCs from a healthy volunteer by
performing WST-8 assays. As shown in Figs. 2a, b (left
panels), exposing HTLV-1-infected T cell lines to importazole
and ivermectin resulted in a dose-dependent cytotoxic effect.
In contrast, the effects of these agents on the viability of nor￾mal PBMCs and Jurkat cells were less pronounced compared
to those on HTLV-1-infected T cell lines (Fig. 2a, right panel
and Fig. 2b, middle and right panels).
Fig. 1 Overexpressed IPOβ1 regulates cell growth of HTLV-1-
infected T cells. a. The expression of IPOβ1 in HTLV-1-infected T cell
lines and normal PBMCs was analyzed by western blotting. PBMCs from
a healthy volunteer were stimulated with PHA for 72 h (PHA-PBMC).
Actin served as an internal control. b. IPOβ1 expression in HTLV-1-
infected PBMCs. PBMCs from a healthy donor were co-cultured with
MMC-treated MT-2 cells. After co-cultivation for the indicated periods,
PBMCs were harvested and the expression of the indicated genes was
examined by RT-PCR. GAPDH was used as the control. c. The knock￾down of IPOβ1 in cells via siRNA treatment inhibited the expression of
cyclin D1, cyclin D2 and c-myc. MT-2 cells were transfected with control
siRNA or IPOβ1-targeting siRNA. Then, 48 and 72 h after transfection,
cells were harvested and subjected to RT-PCR. d. Protein expression of
IPOβ1 was assessed using western blotting. MT-2 cells were transfected
with control siRNA or IPOβ1-targeting siRNA. After 48 h, cells were
harvested and subjected to western blotting. e. A WST-8 assay showed
that knockdown of IPOβ1 inhibited cellular proliferation. MT-2 cells
were transfected with control siRNA or IPOβ1-targeting siRNA, and
then passaged into a 96-well plate. The relative cell growth was deter￾mined by performing WST-8 assays. *P < 0.001 compared to the control
siRNA-transfected cells
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Induction of apoptosis in HTLV-1-infected T cell lines
treated with IPOβ1 inhibitors
We next investigated whether the inhibition of IPOβ1 could
induce apoptosis in HTLV-1-infected T cell lines. First, mor￾phological changes induced by importazole and ivermectin
were examined by microscopy. Characteristic morphological
features of apoptosis such as nuclear fragmentation and chro￾matin condensation were observed in these cells upon treat￾ment with importazole and ivermectin (Fig. 3a). Next, the
induction of apoptosis by these IPOβ1 inhibitors was ana￾lyzed by APO2.7 staining [43]. As expected, importazole
(Fig. 3b) and ivermectin (Fig. 3c) both induced apoptosis in
HTLV-1-infected T cell lines in a dose- and time-dependent
manner. In contrast, the effects of these agents on the apopto￾sis of Jurkat cells were less pronounced (Fig. 3d).
IPOβ1 inhibitors induce caspase activation
We then measured the cleavage of a known caspase-3 sub￾strate, cleaved PARP, as well as caspase-3, caspase-8 and
caspase-9. In MT-2 and HUT-102 cells treated with
importazole and ivermectin, cleaved PARP, caspase-3,
caspase-8 and caspase-9 were increased in a dose-dependent
manner after 48 h of treatment (Figs. 4a, b). Caspase-3,
caspase-8 and caspase-9 activities were also induced after
importazole and ivermectin treatment (Fig. 4c). To explore
whether caspases are involved in IPOβ1 inhibitor-induced
cell death in HTLV-1-infected T cell lines, we performed
WST-8 assays, assessing the effect of pre-treatment with the
pan-caspase inhibitor z-VAD-FMK on importazole- and
ivermectin-mediated cytotoxicity. We found that pre￾treatment with this pan-caspase inhibitor partly inhibited
IPOβ1 inhibitor-induced cytotoxicity (Fig. 4d). These results
suggest that importazole and ivermectin induce caspase￾mediated cytotoxicity in HTLV-1-infected T cell lines.
Effect of IPOβ1 inhibitors on the cell cycle in HTLV-1-
infected T cell lines
To determine whether the inhibition of cell proliferation in￾duced by IPOβ1 inhibitors was associated with changes in
cell cycle progression, cell cycle analysis was performed on
HTLV-1-infected T cell lines treated with importazole and
ivermectin. As shown in Fig. 5, flow cytometric analysis
showed an increase in the number of cells arrested at G1 phase
and a decrease in the proportion of cells in S phase upon
treatment with IPOβ1 inhibitors, as compared to the propor￾tions in untreated cells. These results suggest that IPOβ1 can
modulate cell proliferation via G1–S progression.
IPOβ1 inhibitors downregulate the nuclear
localization of RelA, JunB and JunD
We next speculated that the high expression of IPOβ1
might be related to the persistent hyperactivation of
NF-κB and AP-1 signaling pathways in ATLL. As shown
in Fig. 6a, constitutive DNA binding by NF-κB and AP-1
was observed in HUT-102 and MT-2 cells but not in
Jurkat cells. Competition EMSA demonstrated the
Fig. 2 Importazole and
ivermectin inhibit cell viability
of HTLV-1-infected T cell lines.
Cell viability was evaluated by
WST-8 assays after importazole
(a) and ivermectin (b) treatment
using HTLV-1-infected T cell
lines, an uninfected T cell line and
PBMCs from a healthy volunteer
for 24–72 h. The values were
compared to those obtained with
medium control (100%). Data are
presented as the mean ± SD of
triplicate cultures. *P < 0.001
compared to PBMCs.
**P < 0.001 compared to Jurkat
cells
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specificity of these binding reactions. Specifically, cold
competitors, but not unrelated oligonucleotides, competed
for binding (Fig. 6b, lanes 2 and 3). The components
present in nuclear extracts were then analyzed by the ad￾dition of specific antibodies against NF-κB and AP-1
family members. Supershift analysis indicated that p50/
RelA/c-Rel/RelB (Fig. 6b, left panels, lanes 4–6 and 8)
and JunB/JunD (Fig. 6b, right panels, lanes 9 and 10)
were the major NF-κB and AP-1 subunits, respectively.
To determine the effect of IPOβ1 inhibitors on NF-κB
and AP-1 signaling in ATLL, we examined whether
importazole and ivermectin could alter nuclear NF-κB
and AP-1 DNA binding in HTLV-1-infected T cell lines.
EMSA results indicated that the specific shifted band was
reduced when cells were incubated with both inhibitors
(Figs. 6c, d). Next, we conducted western blot analysis
to determine nuclear levels of RelA, JunB and JunD in
HUT-102 and MT-2 cells after treatment with IPOβ1 in￾hibitors. As shown in Figs. 6e, f, nuclear RelA, JunB and
JunD were decreased in cells treated with both inhibitors.
Together, these results confirm that IPOβ1 is necessary
for the nuclear localization of NF-κB and AP-1.
IPOβ1 inhibitor-induced apoptosis is associated with
the modulation of apoptotic regulatory proteins
The activation of NF-κB and AP-1 initiates the expression
of various target genes that contribute to cancer progres￾sion by enhancing proliferation and mediating the evasion
of apoptosis. Since it was established that NF-κB and AP-
1 require IPOβ1 for their transport into the nucleus, we
postulated that inhibiting IPOβ1 should alter the expres￾sion of NF-κB- and AP-1-target genes. Apoptosis is reg￾ulated by a balance between pro-apoptotic and anti￾apoptotic genes. As shown in Fig. 7, levels of the anti￾apoptotic proteins survivin, c-IAP1/2 and XIAP were re￾duced by treatment with importazole and ivermectin in
HUT-102 and MT-2 cells in a dose-dependent manner.
In addition, Bcl-xL was suppressed by ivermectin treat￾ment in HUT-102 cells. In contrast, Bak protein levels
Fig. 3 Importazole and ivermectin induce apoptosis in HTLV-1-
infected T cell lines. HTLV-1-infected T cell lines and uninfected
Jurkat cells were treated with importazole and ivermectin and assayed
for apoptosis. a. Morphological changes in MT-2 and HUT-102 cells
treated with importazole and ivermectin were analyzed by microscopy
after Hoechst 33342 staining. b, c. Apoptosis was evaluated by APO2.7
staining after treating HTLV-1-infected T cell lines with various concen￾trations of importazole (b) or ivermectin (c) for 24–72 h. d. Apoptosis
was evaluated by APO2.7 staining after treating Jurkat cells with various
concentrations of ivermectin or importazole for 24 h. Data are presented
as the mean ± SD of triplicate cultures. *P < 0.005 and **P < 0.001, com￾pared to the vehicle-treated control
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Fig. 4 Importazole and ivermectin induce caspase activation in
HTLV-1-infected T cell lines. a, b. Importazole and ivermectin
increased the cleavage of PARP and caspases in HTLV-1-infected T cell
lines. HUT-102 and MT-2 cells were treated with importazole (a) and
ivermectin (b) for 48 h, and cell extracts were analyzed for the cleavage of
PARP and caspases by western blotting. c. MT-2 and HUT-102 cells
were treated with importazole and ivermectin for 48 h, and cell extracts
were analyzed for caspase activities. Data are the mean ± SD of triplicate
cultures. *P < 0.001 compared to the vehicle-treated cells. d. Pre￾treatment with a pan-caspase inhibitor (z-VAD-FMK) reduced
importazole- and ivermectin-induced cytotoxicity in cells, as assessed
by WST-8 assays. Cells were pre-incubated with 20 μM of z-VAD￾FMK for 2 h before importazole and ivermectin treatment for 24 h.
Data are the mean ± SD of triplicate cultures. *P < 0.01 and
**P < 0.005, compared to the importazole-alone or ivermectin-alone
group
Fig. 5 Cell cycle analysis of
HTLV-1-infected T cell lines
after treatment with various
concentrations of importazole
(a) and ivermectin (b) for 24 h.
Data are the mean ± SD of
triplicate cultures. *P < 0.005 and
**P < 0.001, compared to the
vehicle-treated control
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Fig. 6 IPOβ1 inhibitors suppress the nuclear import and DNA
binding of NF-κB and AP-1. a. NF-κB- and AP-1-binding activity in
various cell lines. EMSAs with NF-κB and AP-1 DNA oligonucleotides.
b. Nuclear extracts were incubated with an excess of unlabeled NF-κB or
AP-1 elements (lanes 2 and 3) and assessed by EMSA. The specificities
of the antibodies (Ab) added to the DNA–protein complexes are indicated
on the top of each panel. Arrows indicate the migrational location of each
non-supershifted NF-κB and AP-1 DNA complex. Arrowheads indicate
the mobility of the supershifted complexes incubated with Ab. c, d. The
treatment of cells with importazole (c) and ivermectin (d) inhibited NF-
κB and AP-1 DNA binding. Cells were treated with the indicated con￾centrations of importazole (c) and ivermectin (d) for 24 h and then har￾vested. Nuclear extracts were incubated with oligonucleotide probes. e, f.
Cells were treated with the indicated concentrations of importazole (e)
and ivermectin (f) for 24 h and then harvested. The nuclear protein frac￾tion was extracted, and RelA, JunB, JunD and lamin B levels were
assessed by western blot analysis. Lamin B was used as a control to asses
nuclear fraction purity and loading levels
Fig. 7 Levels of apoptotic
regulatory proteins are
modulated by IPOβ1 inhibitors
in HTLV-1-infected T cell lines.
Cells were treated with the
indicated concentrations of
importazole (a) and ivermectin
(b) for 48 h and then harvested.
The levels of anti-apoptotic and
pro-apoptotic proteins were
assessed by western blot analysis
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were increased in response to importazole in HUT-102
and MT-2 cells and also in response to ivermectin in
HUT-102 cells.
IPOβ1 inhibitors decrease the expression of proteins
required for cell cycle transition from G1 to S phase
We next determined which of proteins that are involved in
mediating the transition from G1 to S phase are regulated by
IPOβ1. As shown in Fig. 8, importazole and ivermectin de￾creased the protein levels of cyclin D1/D2/E, CDK2/4/6 and
c-Myc in a dose-dependent manner. These NF-κB- and AP-1-
target proteins are required for the cell cycle transition from
G1 to S phase, which determine cell division.
Ivermectin treatment suppresses tumor growth in an
ATLL xenograft model
Because ivermectin resulted in cytotoxicity in vitro, we further
investigated its efficacy towards ATLL in vivo using a mouse
xenograft model. Ivermectin potently suppressed HUT-102
tumor growth throughout the duration of treatment (Fig. 9a).
Tumor weight was also significantly reduced in the ivermectin
treatment group, as compared to that in the control group (Fig.
9c). Furthermore, the serum concentrations of human sIL-
2Rα [44] and sCD30 [45] in HUT-102 tumor-bearing mice
were decreased in the treatment group compared to those in
the control group, although these changes were not statistical￾ly significant (Fig. 9c). Signs of apoptosis such as chromatin
condensation, cell shrinkage and uniformly dense nuclei were
also observed in ivermectin-treated group, as shown in Fig.
9d. Furthermore, tumor sections from the ivermectin treatment
group showed numerous TUNEL-positive cells. Moreover, it
was determined that 4 weeks of ivermectin administration was
not toxic to mice, as determined by mouse body weights (Fig.
9b), appearance and behavior. These results suggest that the
IPOβ1 inhibitor exerts an anti-tumor effect in an ATLL
model.
Discussion
The intracellular localization of proteins is crucial for their
function in cells. Targeting nuclear transport is suggested to
be a novel anti-cancer approach. Increased expression of
IPOβ1 was shown to increase nuclear import efficiency in
transformed cells [46]. Moreover, progress has been made in
targeting the activity of several nuclear import receptors [47].
IPOβ1 and IPOα isoforms work together to transport cargo
into the nucleus, although IPOβ1 can also transport cargo
independently. Therefore, targeting IPOβ1 might result in
broader-spectrum inhibition of nuclear import [48].
Mounting evidence suggests that NF-κB and AP-1 signaling
pathways, as cell cycle progression and apoptosis regulators,
can play a key role in the occurrence and development of
ATLL [6–8]. Previous studies have shown that IPOβ1 medi￾ates NF-κB and AP-1 signal transduction in the nuclei of cells
[14–16]. Therefore, we hypothesized that IPOβ1 might be
Fig. 8 Expression of proteins required for cell cycle transition from
G1 to S phase in HTLV-1-infected T cell lines in response to IPOβ1
inhibitors. Cells were treated with the indicated concentrations of
importazole (a) and ivermectin (b) for 48 h and then harvested. The
levels of proteins required for cell cycle transition from G1 to S phase
were assessed by western blot analysis
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indispensable for ATLL cell growth and survival. In this
study, we found that the expression of IPOβ1 in HTLV-1-
infected T cells was increased compared to that in normal
PBMCs. Further, knockdown of IPOβ1 in these cells led to
cell growth inhibition. Taken together, these results showed
that IPOβ1 might be a novel therapeutic target for ATLL. We
further showed that IPOβ1 inhibitors, namely importazole
and ivermectin, display anti-ATLL activity in vitro and
in vivo. Treatment with importazole and ivermectin was high￾ly cytotoxic to HTLV-1-infected T cells, whereas normal
PBMCs were less sensitive. These results indicate that normal
PBMCs and HTLV-1-infected T cells respond differently to
IPOβ1 inhibition, which is advantageous and promising in
terms of anti-ATLL drug development.
The effects of importazole and ivermectin on cell growth
and killing were analyzed, and these inhibitors were found to
induce G1 cell cycle arrest and apoptosis in HTLV-1-infected
T cell lines. NF-κB and AP-1 play a primary role in cell cycle
progression and survival in ATLL cells [6–8]. As both require
access to the nucleus to be functional, we hypothesized that
inhibiting nuclear import via IPOβ1 might lead to their inac￾tivity and ultimately various downstream effects on ATLL cell
growth. Indeed, IPOβ1 inhibitors interfered with the nuclear
localization of cargo, namely the transcription factors NF-κB
and AP-1. Consequently, the expression levels of their target
genes such as cyclin D1/D2/E, CDK2/4/6, c-myc, survivin, c￾IAP1/2, XIAP and Bcl-xL [49–54] were decreased. NF-κB and
AP-1 are known to act synergistically to increase expression
of the same target genes. Therefore, inhibition of more than
one transcription factor that acts synergistically might be a
more appropriate approach to inhibit these processes. Thus,
inhibiting IPOβ1 could be a way to specifically target multi￾ple overactive transcription factors that are required for ATLL
cell biology. In addition, the NF-κB pathway is considered
one of the important mechanisms underlying the development
of resistance to chemotherapy [55], suggesting that the inhibi￾tion of this signaling pathway through IPOβ1 suppression is a
promising approach to the enhance efficacy of and prevent
acquired resistance to ATLL treatment.
The development of nuclear import inhibitors lags behind
that of nuclear export inhibitors, and nuclear import inhibitors
have not yet entered clinical trials [47]. The anti-parasitic
agent ivermectin is licensed for the treatment of strongyloidi￾asis, and Strongyloides stercoralis affects the development of
ATLL [56]. Ivermectin has been reported to cure patients with
strongyloidiasis, particularly those positive for anti-HTLV-1
antibodies [57]. We showed that ivermectin could induce cell
death in HTLV-1-infected T cell lines in vitro and delay tumor
growth in vivo. Although no prior clinical studies have direct￾ly evaluated ivermectin as an anti-ATLL agent, the measure￾ment of viral DNA load after ivermectin treatment in patients
co-infected with HTLV-1 and S. stercoralis might be useful to
assess the anti-ATLL activity of ivermectin. The doses of
ivermectin used in this study were higher than the maximum
obtained plasma concentrations of ivermectin that have been
reported after a single oral dose of 150 μg/kg of ivermectin in
patients with onchocerciasis [58]. However, higher concentra￾tions of ivermectin were well tolerated in our in vivo study.
An efficient dose for ATLL treatment could be clarified in the
future by performing clinical studies.
Fig. 9 Ivermectin decreases
tumor growth in vivo. HUT-102
tumor-bearing mice were treated
orally with vehicle (control) or
ivermectin for 4 weeks. a. Tumor
volume curve after treatment. b.
Ivermectin did not affect mouse
body weight. c. Tumor weight
and serum concentrations of sIL-
2Rα and sCD30 in mice. Data are
presented as the mean ± SD (n =
4). *P < 0.05, **P < 0.01 and
***P < 0.005, compared to con￾trols. d. Histological observations
of tumor tissues in mice treated
with ivermectin based on HE and
TUNEL staining (magnification,
400×)
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In conclusion, our results proved that IPOβ1 expression
was higher in HTLV-1-infected T cells, as compared to that
in normal PBMCs. The knockdown of IPOβ1 or inhibitors of
IPOβ1 could inhibit cell growth and induce cell apoptosis
in vitro and in vivo via the inhibition of NF-κB and AP-1
activities. Thus, we propose that IPOβ1 is critical for ATLL
cell survival and an attractive target for future ATLL therapy.
Acknowledgments The authors would like to thank Fujisaki Cell Center,
Hayashibara Biochemical Laboratories, Inc. for providing C5/MJ, HUT-
102 and MT-1 cells, Dr. Naoki Yamamoto (Tokyo Medical and Dental
University) for providing MT-2 and MT-4 cells, Dr. Diane Prager (UCLA
School of Medicine) for providing SLB-1 cells, Dr. Michiyuki Maeda
(Kyoto University) for providing ED-40515(−) cells, and Dr. Masahiro
Fujii (Niigata University) for providing TL-OmI cells. Recombinant hu￾man IL-2 was kindly provided by Takeda Pharmaceutical Company Ltd.
The measurement of protein concentrations was performed at the
University of the Ryukyus Center for Research Advancement and
Collaboration. We would like to thank Editage (www.editage.jp) for
English language editing.
Funding This study was supported partially by JSPS KAKENHI
(17 K07175).
Compliance with ethical standards
Conflict of interest No potential conflict of interest is reported by the
authors.
Ethical approval All animal experiments were approved by the Animal
Care and Use Committee of University of the Ryukyus (A2016149).
References
1. Iwanaga M, Watanabe T, Yamaguchi K (2012) Adult T-cell leuke￾mia: a review of epidemiological evidence. Front Microbiol 3:322
2. Ishitsuka K, Tamura K (2014) Human T-cell leukaemia virus type I
and adult T-cell leukaemia-lymphoma. Lancet Oncol 15:e517–
e526
3. Hishizawa M, Kanda J, Utsunomiya A, Taniguchi S, Eto T,
Moriuchi Y, Tanosaki R, Kawano F, Miyazaki Y, Masuda M,
Nagafuji K, Hara M, Takanashi M, Kai S, Atsuta Y, Suzuki R,
Kawase T, Matsuo K, Nagamura-Inoue T, Kato S, Sakamaki H,
Morishima Y, Okamura J, Ichinohe T, Uchiyama T (2010)
Transplantation of allogeneic hematopoietic stem cells for adult
T-cell leukemia: a nationwide retrospective study. Blood 116:
1369–1376
4. Qu Z, Xiao G (2011) Human T-cell lymphotropic virus: a model of
NF-κB-associated tumorigenesis. Viruses 3:714–749
5. Gazon H, Barbeau B, Mesnard J-M, Peloponese J-M Jr (2018)
Hijacking of the AP-1 signaling pathway during development of
ATL. Front Microbiol 8:2686
6. Mori N, Fujii M, Ikeda S, Yamada Y, Tomonaga M, Ballard DW,
Yamamoto N (1999) Constitutive activation of NF-κB in primary
adult T-cell leukemia cells. Blood 93:2360–2368
7. Fujii M, Iwai K, Oie M, Fukushi M, Yamamoto N, Kannagi M,
Mori N (2000) Activation of oncogenic transcription factor AP-1 in
T cells infected with human T cell leukemia virus type 1. AIDS Res
Hum Retrovir 16:1603–1606
8. Mori N (2009) Cell signaling modifiers for molecular targeted ther￾apy in ATLL. Front Biosci 14:1479–1489
9. Hayden MS, Ghosh S (2004) Signaling to NF-κB. Genes Dev 18:
2195–2224
10. Aggarwal A, Agrawal DK (2014) Importins and exportins regulat￾ing allergic immune responses. Mediat Inflamm 2014:476357
11. Stewart M (2007) Molecular mechanism of the nuclear protein
import cycle. Nat Rev Mol Cell Biol 8:195–208
12. Lee SJ, Sekimoto T, Yamashita E, Nagoshi E, Nakagawa A,
Imamoto N, Yoshimura M, Sakai H, Chong KT, Tsukihara T,
Yoneda Y (2003) The structure of importin-β bound to SREBP-
2: nuclear import of a transcription factor. Science 302:1571–1575
13. Kutay U, Izaurralde E, Bischoff FR, Mattaj IW, Görlich D (1997)
Dominant-negative mutants of importin-β block multiple pathways
of import and export through the nuclear pore complex. EMBO J
16:1153–1163
14. Fagerlund R, Kinnunen L, Köhler M, Julkunen I, Melén K (2005)
NF-κB is transported into the nucleus by importin α3 and importin
α4. J Biol Chem 280:15942–15951
15. Liang P, Zhang H, Wang G, Li S, Cong S, Luo Y, Zhang B (2013)
KPNB1, XPO7 and IPO8 mediate the translocation of NF-κB/p65
into the nucleus. Traffic 14:1132–1143
16. Forwood JK, Lam MHC, Jans DA (2001) Nuclear import of Creb
and AP-1 transcription factors requires importin-β1 and ran but is
independent of importin-α. Biochemistry 40:5208–5217
17. Harel A, Forbes DJ (2004) Importin beta: conducting a much larger
cellular symphony. Mol Cell 16:319–330
18. Mosammaparast N, Pemberton LF (2004) Karyopherins: from
nuclear-transport mediators to nuclear-function regulators. Trends
Cell Biol 14:547–556
19. Angus L, van der Watt PJ, Leaner VD (2014) Inhibition of the
nuclear transporter, Kpnβ1, results in prolonged mitotic arrest and
activation of the intrinsic apoptotic pathway in cervical cancer cells.
Carcinogenesis 35:1121–1131
20. Martens-de Kemp SR, Nagel R, Stigter-van Walsum M, van der
Meulen IH, van Beusechem VW, Braakhuis BJM, Brakenhoff RH
(2013) Functional genetic screens identify genes essential for tumor
cell survival in head and neck and lung cancer. Clin Cancer Res 19:
1994–2003
21. van der Watt PJ, Maske CP, Hendricks DT, Parker MI, Denny L,
Govender D, Birrer MJ, Leaner VD (2009) The Karyopherin pro￾teins, Crm1 and Karyopherin β1, are overexpressed in cervical
cancer and are critical for cancer cell survival and proliferation.
Int J Cancer 124:1829–1840
22. Zhu J, Wang Y, Huang H, Yang Q, Cai J, Wang Q, Gu X, Xu P,
Zhang S, Li M, Ding H, Yang L (2016) Upregulation of KPNβ1 in
gastric cancer cell promotes tumor cell proliferation and predicts
poor prognosis. Tumor Biol 37:661–672
23. Smith ER, Cai KQ, Smedberg JL, Ribeiro MM, Rula ME, Slater C,
Godwin AK, Xu X-X (2010) Nuclear entry of activated MAPK is
restricted in primary ovarian and mammary epithelial cells. PLoS
One 5:e9295
24. van der Watt PJ, Stowell CL, Leaner VD (2013) The nuclear import
receptor Kpnβ1 and its potential as an anticancer therapeutic target.
Crit Rev Eukaryot Gene Expr 23:1–10
25. Yang L, Hu B, Zhang Y, Qiang S, Cai J, Huang W, Gong C, Zhang
T, Zhang S, Xu P, Wu X, Liu J (2015) Suppression of the nuclear
transporter-KPNβ1 expression inhibits tumor proliferation in hepa￾tocellular carcinoma. Med Oncol 32:128
26. Stelma T, Leaner VD (2017) KPNB1-mediated nuclear import is
required for motility and inflammatory transcription factor activity
in cervical cancer cells. Oncotarget 8:32833–32847
27. He S, Miao X, Wu Y, Zhu X, Miao X, Yin H, He Y, Li C, Liu Y, Lu
X, Chen Y, Wang Y, Xu X (2016) Upregulation of nuclear trans￾porter, Kpnβ1, contributes to accelerated cell proliferation- and cell
Invest New Drugs
adhesion-mediated drug resistance (CAM-DR) in diffuse large B￾cell lymphoma. J Cancer Res Clin Oncol 142:561–572
28. Yan W, Li R, He J, Du J, Hou J (2015) Importin β1 mediates
nuclear factor-κB signal transduction into the nuclei of myeloma
cells and affects their proliferation and apoptosis. Cell Signal 27:
851–859
29. Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y,
Shiraishi Y, Nagata K, Hinuma Y (1981) Type C virus particles
in a cord T-cell line derived by co-cultivating normal human cord
leukocytes and human leukaemic T cells. Nature 294:770–771
30. Yamamoto N, Okada M, Koyanagi Y, Kannagi M, Hinuma Y
(1982) Transformation of human leukocytes by cocultivation with
an adult T cell leukemia virus producer cell line. Science 217:737–
739
31. Popovic M, Sarin PS, Robert-Gurroff M, Kalyanaraman VS, Mann
D, Minowada J, Gallo RC (1983) Isolation and transmission of
human retrovirus (human T-cell leukemia virus). Science 219:
856–859
32. Koeffler HP, Chen IS, Golde DW (1984) Characterization of a
novel HTLV-infected cell line. Blood 64:482–490
33. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo
RC (1980) Detection and isolation of type C retrovirus particles
from fresh and cultured lymphocytes of a patient with cutaneous
T-cell lymphoma. Proc Natl Acad Sci U S A 77:7415–7419
34. Miyoshi I, Kubonishi I, Sumida M, Hiraki S, Tsubota T, Kimura I,
Miyamoto K, Sato J (1980) A novel T-cell line derived from adult
T-cell leukemia. Gan 71:155–156
35. Sugamura K, Fujii M, Kannagi M, Sakitani M, Takeuchi M,
Hinuma Y (1984) Cell surface phenotypes and expression of viral
antigens of various human cell lines carrying human T-cell leuke￾mia virus. Int J Cancer 34:221–228
36. Maeda M, Shimizu A, Ikuta K, Okamoto H, Kashihara M,
Uchiyama T, Honjo T, Yodoi J (1985) Origin of human T￾lymphotrophic virus I-positive T cell lines in adult T cell leukemia:
analysis of T cell receptor gene rearrangement. J Exp Med 162:
2169–2174
37. Kaplan J, Tilton J, Peterson WD Jr (1976) Identification of T cell
lymphoma tumor antigens on human T cell lines. Am J Hematol 1:
219–223
38. Mori N, Prager D (1996) Transactivation of the interleukin-1α pro￾moter by human T-cell leukemia virus type I and type II tax pro￾teins. Blood 87:3410–3417
39. Tomayko MM, Reynolds CP (1989) Determination of subcutane￾ous tumor size in athymic (nude) mice. Cancer Chemother
Pharmacol 24:148–154
40. Inoue J, Seiki M, Taniguchi T, Tsuru S, Yoshida M (1986)
Induction of interleukin 2 receptor gene expression by p40x
encoded by human T-cell leukemia virus type 1. EMBO J 5:
2883–2888
41. Soderholm JF, Bird SL, Kalab P, Sampathkumar Y, Hasegawa K,
Uehara-Bingen M, Weis K, Heald R (2011) Importazole, a small
molecule inhibitor of the transport receptor importin-β. ACS Chem
Biol 6:700–708
42. Wagstaff KM, Sivakumaran H, Heaton SM, Harrich D, Jans DA
(2012) Ivermectin is a specific inhibitor of importin α/β-mediated
nuclear import able to inhibit replication of HIV-1 and dengue
virus. Biochem J 443:851–856
43. Zhang C, Ao Z, Seth A, Schlossman SF (1996) A mitochondrial
membrane protein defined by a novel monoclonal antibody is pref￾erentially detected in apoptotic cells. J Immunol 157:3980–3987
44. Kamihira S, Atogami S, Sohda H, Momita S, Yamada Y,
Tomonaga M (1994) Significance of soluble interleukin-2 receptor
levels for evaluation of the progression of adult T-cell leukemia.
Cancer 73:2753–2758
45. Nishioka C, Takemoto S, Kataoka S, Yamanaka S, Moriki T, Shoda
M, Watanabe T, Taguchi H (2005) Serum level of soluble CD30
correlates with the aggressiveness of adult T-cell leukemia/lympho￾ma. Cancer Sci 96:810–815
46. Kuusisto HV, Wagstaff KM, Alvisi G, Roth DM, Jans DA (2012)
Global enhancement of nuclear localization-dependent nuclear
transport in transformed cells. FASEB J 26:1181–1193
47. Kosyna FK, Depping R (2018) Controlling the gatekeeper: thera￾peutic targeting of nuclear transport. Cells 7:221
48. Stelma T, Chi A, van der Watt PJ, Verrico A, Lavia P, Leaner VD
(2016) Targeting nuclear transporters in cancer: diagnostic, prog￾nostic and therapeutic potential. IUBMB Life 68:268–280
49. Iwanaga R, Ohtani K, Hayashi T, Nakamura M (2001) Molecular
mechanism of cell cycle progression induced by the oncogene prod￾uct tax of human T-cell leukemia virus type I. Oncogene 20:2055–
2067
50. Iwanaga R, Ozono E, Fujisawa J, Ikeda MA, Okamura N, Huang Y,
Ohtani K (2008) Activation of the cyclin D2 and cdk6 genes
through NF-κB is critical for cell-cycle progression induced by
HTLV-I tax. Oncogene 27:5635–5642
51. Kawakami H, Tomita M, Matsuda T, Ohta T, Tanaka Y, Fujii M,
Hatano M, Tokuhisa T, Mori N (2005) Transcriptional activation of
survivin through the NF-κB pathway by human T-cell leukemia
virus type I tax. Int J Cancer 115:967–974
52. Wang C-Y, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS
Jr (1998) NF-κB antiapoptosis: induction of TRAF1 and TRAF2
and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science
281:1680–1683
53. Pahl HL (1999) Activators and target genes of Rel/NF-κB tran￾scription factors. Oncogene 18:6853–6866
54. Hess J, Angel P, Schorpp-Kistner M (2004) AP-1 subunits: quarrel
and harmony among siblings. J Cell Sci 117:5965–5973
55. de Castro Barbosa ML, da Conceicao RA, Fraga AGM, Camarinha
BD, de Carvalho Silva GC, Lima AGF, Cardoso EA, de Oliveira
Freitas Lione V (2017) NF-κB signaling pathway inhibitors as an￾ticancer drug candidates. Anti Cancer Agents Med Chem 17:483–
490
56. Yamaguchi K, Matutes E, Catovsky D, Galton DAG, Nakada K,
Takatsuki K (1987) Strongyloides stercoralis as candidate co-factor
for HTLV-I-induced leukaemogenesis. Lancet 2:94–95
57. Zaha O, Hirata T, Uchima N, Kinjo F, Saito A (2004) Comparison
of anthelmintic effects of two doses of ivermectin on intestinal
strongyloidiasis in patients negative or positive for anti-HTLV-1
antibody. J Infect Chemother 10:348–351
58. Baraka OZ, Mahmoud BM, Marschke CK, Geary TG, Homeida
MMA, Williams JF (1996) Ivermectin distribution in the plasma
and tissues of patients infected with Onchocerca volvulus. Eur J
Clin Pharmacol 50:407–410
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