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Materials and Methods (details)
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Chemicals cells and cell-culture conditions
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were prepared from blood bank of the Transfusion Department, Oita University Hospital
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Proteasome inhibitor MG-132, caspase inhibitor zVAD-fmk, autophagy inhibitors
3-methyladenine (3MA) and 5-amino-4-imidazole carboxamide riboside (AICAR) were
were purchased from Sigma. Geldanamycine and its water-soluble and less-toxic
derivative 17-DMAG25 were purchased from Invivogen. ATL transformed cell lines C8166
and MT4, Jurkat and HEK293 cell lines were obtained from the ATCC. ATL cell lines
established from ATL patient’s PBL (HUT-102, ED, S1T, SU9T, ATL4 and ATL9) and
Non-ATL leukemic cell lines (HL-60, MOLT-4 and HUT-78) are laboratory stocks of KM
and HI. PBLs from ATL patents (Pat1 to Pat4), or healthy donors (PBL1, PBL2 and PBL3)
in accordance with the guidelines of the Ethical Committee of the Oita University Faculty
of Medicine. Suspension cells were cultured in complete RPMI-1640 supplemented with
10% FBS. Each medium contains the appropriate amount of antibiotics. Tumor cells from
ATL patients were maintained in RPMI 1640 medium supplemented with 15% FBS
(Equitech-Bio), penicillin G (50 U/mL), and streptomycin (50 μg/mL; Sigma-Aldrich).
Ethical permission for use of patient-derived cells and pathologic materials was approved
by the Ethical Committee of the Oita University Faculty of Medicine, and informed
consent was obtained in accordance with the Declaration of Helsinki. Adherent cells were
cultured in complete DMEM/high glucose with 10% fetal bovine serum (FBS).
Co-Immunoprecipitation (Co-IP) and Immunoblot (IB)
One million cells of MT-4, C8166 treated with or without 17-DMAG and HEK293 cells
transfected with each plasmid (maximum 1 μg) by FugeneHD (Roche) for 40 hours were
lysed
with
Co-IP
buffer
[0.5%
Nonidet
P-40,
20mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.3), 150mM NaCl,
2mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.1mM sodium orthovanadate
(Na3VO4), 1mM sodium fluoride (NaF), 10% glycerol and protease inhibitor mix (Roche)].
In all, 200 μg of lysates were precleared with 30 μl of protein G agarose (CalBiochem) for
2 hours and incubated with 2 μg of rabbit polyclonal anti-Hsp90 (Stressgen) or rabbit
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anti-FLAG antibody (Sigma) and 30 μl of protein G agarose for at least 3 hours at 4
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Real-time quantitative reverse transcriptase-polymerase chain reaction
degree C. Antibody–agarose complexes were washed four times with 500μl of Co-IP
buffer, resolved by 12 or 15% SDS–poly acrylamide gel electrophoresis (SDS–PAGE)
and transferred onto a PVDF membrane (Millipore) using semi-dry TRANS-BLOT SD
(BioRad) under the manufacture’s instructions. Specific proteins were detected by
specific monoclonal Tax, Hsp90 (Stressgen), Flag and Tubulin (Sigma) or polyclonal
IKKβ (Cell Signaling Technology), antibodies respectively.
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(RT-qPCR) by LightCycler system
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(Wako) and contaminated DNA was removed. cDNA was constructed by the
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to the level of glucose-6-phosphate 1-dehydrogenase (G6PD) measured in the same
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Total RNA from MT-4 cells treated with or without 17-DMAG was isolated using ISOGEN
Thermoscript reverse transcriptase-polymerase chain reaction system (Invitrogen)
according to the manufacturer’s instructions. Real-time quantitative PCRs (RT-qPCR) for
Tax and glyceraldehyde glucose-6-phosphate 1-dehydrogenase (G6PD) were performed
on a Roche LC480 (Roche) set-up according to the directions. The set of primers and
universal probe for Tax were as follows: Left primer: 5’–TTCCGAAATG GATACATGGA
A-3’; Right primer: 5’–TCTGGAAAAG ACAGGGTTGG-3’; and LightCycler Universal
Probe Library (UPL) #44: 6FAM- TTCCGAAATG GATACATGGA ACCCACCCTT
GGGCAGCACC TCCCAACCCT GTCTTTTCCA GA-TA MRA. All data were normalized
sample with Left primer: 5’– AACAGAGTGA GCCCTTCTTC A-3’; Right primer:
5’–GGAGGCTGCA TCATCGTACT-3’; and LightCycler UPL #6: 6FAM- AACAGAGTGA
GCCCTTCTTC
AAGGCCACCC
CAGAGGAGAA
GCTCAAGCTG
GAGGACTTCT
TTGCCCGCAA CTCCTATGTG GCTGGCCAGT ACGATGATGC AGCCTCC-TA MRA
and plotted the averages of three independent results.
Cell viability assay
One million cells from each ATL cell lines established from ATL patient’s PBL (HUT-102,
ED, S1T, SU9T, ATL4 and ATL9) or ATL cell lines established by co-culture of chord
blood and ATL patient’s PBL (C8166 and MT4), PBLs from ATL patent (Pat1 to Pat4),
Non-ATL leukemic cell lines (Jurkat, HL-60, MOLT-4 and HUT-78) or healthy donors
(PBL1 to PBL3) were incubated in 2 ml of RPMI1640 and treated with 2.5 μM of
17-DMAG for 1 to 4 days. After every 24 hours’ incubation, 5 x 103 of cells were
transferred to each well of a 96 well plate, and 1/10 volume of Cell Counting Kit solution
(Dojindo) was added to each fraction. Thirty minutes after incubation, the absorbance at
465 nm was measured using an E-max precision microplate reader (Molecular Divices).
Caspase3/7 assay
Same cells used in the ‘Cell viability assay’ were also utilised for apoptosis activity
measurement. After every 24 hours’ incubation, 5 x 103 of cells were transferred to each
well of 96 well plate and an 1/2 volume of Cappase3/7 assay solution (Promega) was
added to each fraction. 60 minutes after incubation, the chemical luminescense was
measured by GLOMAX 96.
Plasmids
Plasmid pSG5-Tax is kind gift from Dr. Claudine Pique at Université Paris-Descartes,
Institut Cochin, Paris, France24 and Hsp90 cDNA was a kind gift from Dr. Kyosuke Nagata
at University of Tsukuba.25 Hsp90 and Cdc3726 were amplified from templates previously
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described, and cloned into pcDNA3 (Invitrogen). CMV-Tax was constructed by cloning of
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recognition sites at both ends and the Kozak sequence (-GCCACC-) was placed prier to
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upon request. All the plasmid constructs used in this study were verified by nucleotide
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the PCR amplified Tax open reading flame (ORF) with HindIII-EcoRI restriction enzyme
the first ATG codon of Tax. Purified HindIII-Kozak-Tax-EcoRI DNA fragment was cloned
into the same sites of pcDNA3. LTR-Tax which expresses Tax under the control of the
Tax response element in the U3 region of HTLV-1 proviral DNA was constructed by
replacing BglII-HindIII fragment of CMV-Tax with the HTLV-1 LTR amplified from HpX.11
Tax and Cdc37 cDNA were amplified by PCR and cloned into CoralHue vectors (MBL,
Japan) phmKGN-MC and phmKGC-MN respectively to evaluate their physical interaction
by Kusabira GFP-two hybrid system.27 Hsp90 and NEMO containing plasmids were also
constructed for validation of this system. The details of plasmid vectors will be provided
sequencing analysis.
Luciferase assay
Five hundred thousand HEK293 cells were transfected with plasmid DNA mixture
containing the reporter plasmids (50 ng of NF-κB-Luc or HTLV1-LTR-Luc11 and 50 ng of
RSV-b-galactosidase as a transfection indicator) and 250 ng of Tax expression vectors
(pSG5-Tax, CMV-Tax or LTR-Tax) by HugeneHD. The same amount of pcDNA3 was
used for transfection control. After 24 hours incubation of the transfection, where
indicated, 17-DMAG at concentrations listed in figures were added and further incubated
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for 16 hour. Cells were removed from 12 well dishes with 1mM-EDTA/PBS and
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Microscopic observation of cells
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transfered to 1.5 ml microtube and washed once with PBS then cells were lysed with 100
μl of Lysis Buffer (Promega) and the luciferase activity was measured with a luciferase
assay kit (Promega) in GLOMAX 96 microplate luminometer (Promega). The
beta-galactosidase activity of the same cell extracts used for the luciferase assays was
quantitated by Galacto-Star (Tropix) for normalization. Protein concentration of cell
extracts was measured by Protein Assay reagents (BioRad) and used for SDS-PAGE
and IB analysis.
Five hundred thousand HEK293 cells seeded on the six-well plates were transfected with
phmKGN-MC-Tax
and
phmKGC-MN-Cdc37
or
its
mutant-Cdc37(N200)
and-Cdc37(N180) by FugeneHD as described in the ‘Luciferase assay’. After 48 hours
incubation, the transfected HEK293 cells were treated with Hoecst34442 (Sigma) at the
final concentration of 1 μM. Light and fluorescent (GFP and Hoecst34442) microscopic
observation and photography was performed by BZ-9000 Biorevo all-in-one fluorescence
microscope (Keyence).
Preparation of leukemic Lck-Tax cells and injection to SCID mice
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SCID mice were obtained from Clea Japan Inc, Japan. Tumor cells from spleens of
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lymphomatous cells, were isolated using a Lymphoprep kit (Axis-Shield ProC As), and
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(Miltenyi Biotec).
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HTLV-I Tax-transgenic mice, in which normal splenocytes were replaced with
suspended in RPMI 1640 medium. Thereafter, lymphomatous cells (106/mL) were
intraperitoneally injected into SCID mice. At 28 days after injection, tumor cells were
again isolated from ascites and spleens of the injected SCID mice. The isolated tumor
cells from SCID mice (primary murine lymphoblastoid [pML] cells) were harvested and
kept frozen until use. The pML cells were cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS; Hyclone), antibiotics, and 2 mM L-glutamine, and
maintained at 37°C in 5% CO2 and investigated after 1 or 2 days in culture. As a control,
T cells were isolated from spleens of C57BL/6 mice with a Pan T-cell Isolation Kit
NOG mice
Female 6-weeks-old NOD/Shi-scid/IL-2Rgncull (NOG) mice were purchased from the
Central Institute of Experimental Animals (Kawasaki, Japan). All mice were handled
under sterile conditions and were maintained in germ-free isolators. The animal
experiments were approved by the Animal Care Committees of Kansai Medical
University.
Purification of human CD133+ cells from cord blood
Cord blood samples from full-term human deliveries with signed informed consent were
obtained from Keihan Cord Blood Bank (Osaka, Japan) for research use due to the
shortage of stem cell number for transplantation to human patients. Mononuclear cells
(MNCs) were isolated using Ficoll-Conray (Lymphosepar I, Fujioka, Japan) density
gradient centrifugation. After collecting MNCs, CD133 MicroBead Kit (Miltenyi Biotec Inc,
Auburn, CA) was used to isolate human CD133+ cells according to the manufacturer's
instructions. The purity of collected cells was evaluated by FACS analysis using
anti-human CD133-PE (Miltenyi Biotec) and CD34-FITC (BD Biosciences) antibodies
(≧95%).
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Generation of huNOG
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HTLV-1 infection to huNOG
NOG mice were transplanted with human CD133+ cells by intra-bone marrow injection.
Briefly, 7-weeks-old mice were sublethally irradiated with 250 cGy from a 137Cs source
(GAMMACELL). After irradiation within 24 hours, 5Å~104 human CD133+ cells were
injected to each mouse.
The HTLV-1 producing Jurkat cell line, JEX, was irradiated with 10 Gy from a 137Cs
source (GAMMACELL). Suspension of irradiated 1Å~106 JEX cells intraperitoneally into
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huNOG mice at age of between 24 and 28 weeks. The body weights of mice were
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their maximum weight. Peripheral blood smears were prepared by May-Grunwald
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routinely measured, and the mice were euthanized when the weights decreased <70% of
Giemsa staining and examined with light microscopy. Infection was performed in a
Biosafety Level P2A laboratory in accordance with the guidelines of Kansai Medical
University.
Flow cytometry
Peripheral blood cells were routinely collected every 2 weeks after infection. When mice
sacrificed, the spleen, bone marrow and lymph node were collected and gently
homogenized using a 70μm Nylon Cell Strainer (BD Falcon) to generate single cell
suspensions in PBS with 2.5% FCS. To stain surface markers, anti-human CD45-PerCP
or APC-Cy7, CD3-FITC or PE-Cy7, CD4-PE, CD8-PerCP-Cy5.5, CD19-PE, CD25-FITC,
CCR4-APC antibodies and mouse IgG1-FITC for isotypecontrol were used (all BD
Biosciences). For PB analysis, AccuCount Ultra Rainbow Fluorescent Particles
(Spherotech) was employed to determine absolute cell number according to
manufacturer’s protocol. Flow cytometric analysis was performed on BD FACSCan for
3-color staining and BD FACSCant II (BD Biosciences) for 7-color staining.23 Data
acquisition used with CellQuest and Diva software respectively (BD Biosciences).
Collected data was analysed by FCS express 3 (De Novo software).
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DNA isolation and quantification of proviral load (PVL)
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TGGTCTCCTTAAACCTGTCTTG-3’,
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Genomic DNA was extracted from single cell suspension of tissue or PB followed by
conventional phenol extraction method. PVL were measured by quantitative PCR using
MyiQ or CFX96 real-time PCR system (Bio-Rad). The primers and probe for HTLV-1 pX
region were as follows: forward primer 5 ’ - ACAAAGTTAACCATGCTTATTATCAGC-3 ’ ,
reverse primer 5 ’ - TCTCCAAACACGTAGACTGGGT-3 ’ , and FAM-labeled probe 5 ’
-FAM-TTCCCAGGGTTTGGACAGAGTCTTCT-BHQ-3’. A target region for human
β-globin (HBB) was used to measure the copy number of human genome. The
primers
and
probe
for
HBB
region
TGAGGAGAAGTCTGCCGTTAC-3’,
were
as
reverse
and
follows:
forward
primer
FAM-labeled
primer
5’5’-
probe
5’-FAM-AAGGTGAACGTGGATGAAGTTGGTGG-BHQ-3’. A plasmid containing PCR
products for HTLV-1 pX region and HBB was constructed using T-Vector pMD20
(TaKaRa) and was used as quantified control template for real-time PCR24. The PVL was
calculated as: [(copy number of pX) / (copy number of β-globin / 2)] × 100.
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Supplement Figures
Supplement 1. Tax degradation and I-κBα stabilization by 17-DMAG but not by
Dexamethasone treatment of ATL cells. Two million C8166 cells were treated with 2.5
μM of 17-DMAG or 2.5 μM of Dexamethasone for 48 hours and photographed (lower
panels a, b: untreated controls; c, d: 17-DMAG treated; e, f: Dexamethasone treated) and
then harvested. Cells were lysed and fractionated into nuclear (N) or cytoplasmic (C)
fractions by NE-PER reagent (Promega). Ten μg of nuclear fraction or 20 μg of
cytoplasmic fraction of each sample was applied for IB. Monoclonal anti-Tax: Tax(M)
(upper panel), polyclonal anti-I-κBα: I-κBα(R) (middle panel), and monoclonal
anti-tubulin: Tubulin(M) (lower panel), were applied for each IB.
Supplement 2. IC50 of 17-DMAG against ATL cells and PBLs. Two million cells from
ATL cell lines established from ATL patient’s PBL (ATL4, red line); established by
co-culture of chord blood and ATL patient’s PBL (C8166 and MT4, orange lines) and
PBLs from healthy donors (PBL1, PBL2 and PBL3, blue lines) were treated with
17-DMAG at the indicated concentrations for 2 days. After 48 hours’ incubation,
triplicated 104 cells from five different lineages were transferred to each well of a 96 well
plate. One-tenth volume of Cell Counting Kit 8 (CCK8)-solution (Dojindo) was added to
each fraction. Thirty minutes after incubation, the absorbance at 465 nm was measured
using an E-max precision microplate reader (Molecular Divices). IC50 of 17-DMAG
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concentration values of leukemic cell lines were indicated in the right table.
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Supplement 3. Schematic representation of Hsp90/Cdc37/Tax ternary complex. (A)
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and function, we speculate that Hsp90’s role is limited to a scaffolding of the Cdc37-Tax
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Schematic modeling of Hsp90/Cdc37/CDK6 (Roe et al., 2001). Roe and his coworkers
analyzed the architecture of Hsp90-Cdc37 core protein complex by X-ray crystallography
and visualized hypothetical Hsp90/Cdc37/CDK6 complex structure. They described that
the client protein kinase (PK) is folded by the kinase binding domain (KBD) of Cdc37 and
middle domain (M) of Hsp90 equally. (B) Our proposed model of Hsp90/Cdc37/Tax
ternary complex. We demonstrated the crucial role of client binding domain (CBD) of
Cdc37 for Tax-binding and stbilization. Since Hsp90 mutants did not affect Tax’s stability
complex.
Supplement 4. Experimental scheme of 17-DMAG oral treatment for HTLV-1
infected mice. huNOG mice (established by transplantation of CD133+ human
hematopoietic stem cells) on 24 weeks age were inoculated with 106 of JEX (HTLV-1
producing Jurkat) cells. Two weeks after inoculation, two groups of mice (YZ12, YZ13,
YZ14 and YZ17, Experiment 1; Z617, Z618, Z619 and Z620: Experiment 2 ) were treated
with 500 or 300 microgram of 17-DMAG (25 or 15 mg/kg respectively) for 4 weeks (20
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times total) and other three (YZ15, YZ18 and YZ20: Experiment 1; Z611, Z612 and Z616:
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after infection and analyzed by FACS and real-time PCR (details in Materials and
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Supplement 5. siRNA transduction against Cdc37 or Hsp90 in ATL cells did not
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lysed and samples were applied for IB. (B) siRNAs against Luciferase (lane1), Cdc37-1
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Experiment 2) had PBS. Peripheral blood cells were routinely collected every 2 weeks
Methods).
.
induce Tax-degradation. (A) siRNAs against Cdc37-1 and the control Luciferase
(Sigma) were introduced into 3x105 MT4 cells by electroporation (Microporator,
DigitalBio) according to manufacutures instruction with modification. Briefly, cells were
pulsed at the indicated intensity (lane 1 and 3, 1,500 V, 10 mili seconds, 10 times; lane 2,
1,700 V, 10 mili seconds, 6 times). After 60 hours of incubation, cells were harvested and
(lane2), Cdc37-2 (lane3), Hsp90a (lane4) and Hsp90b (lane5) were introduced into 3x105
MT4 cells by electroporation method with intensity of 1,500 V, 10 mili seconds, 10 times.
After 60 hours of incubation, cells were harvested and lysed samples were applied for IB.
Supplement 6. Anti-ATL cell effects by the combined dosage of 17-DMAG and
LSG15. ATL cell lines C8166, MT-4, ATL4 and ATL9 were treated with either sub-optimal
single dose of 17-DMAG (0.1 μM) or LSG15 without prednisolone (vincristine, 37 ng/ml;
cyclophosphamide, 15 μg/ml; doxorubicin, 1.5μg/ml). Caspase/CCK values were
obtained as in Figure 7.