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Protective Roles of Epithelial Cells in the Survival of Adult T-Cell Leukemia/Lymphoma Cells

      Adult T-cell leukemia/lymphoma (ATL) is a highly invasive and intractable T-cell malignancy caused by human T-cell leukemia virus-1 infection. We demonstrate herein that normal tissue–derived epithelial cells (NECs) exert protective effects on the survival of leukemic cells, which may partially account for high resistance to antileukemic therapies in patients with ATL. Viral gene–silenced, ATL-derived cell lines (ATL cells) dramatically escaped from histone deacetylase inhibitor–induced apoptosis by direct co-culture with NECs. Adhesions to NECs suppressed p21Cip1 expression and increased a proportion of resting G0/G1 phase cells in trichostatin A (TSA)–treated ATL cells. ATL cells adhering to NECs down-regulated CD25 expression and enhanced vimentin expression, suggesting that most ATL cells acquired a quiescent state by cell-cell interactions with NECs. ATL cells adhering to NECs displayed highly elevated expression of the cancer stem cell marker CD44. Blockade of CD44 signaling diminished the NEC-conferred resistance of ATL cells to TSA-induced apoptosis. Co-culture with NECs also suppressed the expression of NKG2D ligands on TSA-treated ATL cells, resulting in decreased natural killer cell–mediated cytotoxicity. Combined evidence suggests that interactions with normal epithelial cells augment the resistance of ATL cells to TSA-induced apoptosis and facilitate immune evasion by ATL cells.
      Adult T-cell leukemia/lymphoma (ATL) is an intractable and fatal T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1).
      • Poiesz B.J.
      • Ruscetti F.W.
      • Gazdar A.F.
      • Bunn P.A.
      • Minna J.D.
      • Gallo R.C.
      Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma.
      After more than two decades of long-term latency, approximately 4% of HTLV-1 carriers develop ATL.
      • Yoshida M.
      • Miyoshi I.
      • Hinuma Y.
      Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease.
      • Uchiyama T.
      • Yodoi J.
      • Sagawa K.
      • Takatsuki K.
      • Uchino H.
      Adult T-cell leukemia: clinical and hematologic features of 16 cases.
      A striking feature of ATL is aggressive invasion of leukemia cells into the skin and epithelial linings of the gastrointestinal tract and lung.
      • Bittencourt A.L.
      • de Oliveira Mde F.
      Cutaneous manifestations associated with HTLV-1 infection.
      • Ohshima K.
      Pathological features of diseases associated with human T-cell leukemia virus type I.
      Leukemia cells that have invaded the tissues are resistant to chemotherapy, presenting a major obstacle to the effective treatment of ATL.
      • Nasr R.
      • El Hajj H.
      • Kfoury Y.
      • de Thé H.
      • Hermine O.
      • Bazarbachi A.
      Controversies in targeted therapy of adult T cell leukemia/lymphoma: ON target or OFF target effects?.
      Therefore, understanding the mechanisms by which tissue-infiltrating leukemia cells acquire resistance to chemotherapy is key to developing new promising treatments for patients with ATL.
      HTLV-1 proteins are generally undetectable in HTLV-1–infected cells isolated from HTLV-1 carriers because of viral gene silencing. Such silencing is observed not only in asymptomatic carriers but also in patients with ATL, indicating that it allows ATL cells to evade the host immune response in vivo.
      • Taniguchi Y.
      • Nosaka K.
      • Yasunaga J.
      • Maeda M.
      • Mueller N.
      • Okayama A.
      • Matsuoka M.
      Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms.
      • Furukawa Y.
      • Osame M.
      • Kubota R.
      • Tara M.
      • Yoshida M.
      Human T-cell leukemia virus type-1 (HTLV-1) Tax is expressed at the same level in infected cells of HTLV-1-associated myelopathy or tropical spastic paraparesis patients as in asymptomatic carriers but at a lower level in adult T-cell leukemia cells.
      However, the mechanisms leading to viral gene silencing are poorly understood. Freshly isolated HTLV-1–infected cells begin to express viral genes after overnight culture in vitro, implying that an unknown mechanism exists to suppress viral genes in vivo.
      • Gessain A.
      • Louie A.
      • Gout O.
      • Gallo R.C.
      • Franchini G.
      Human T-cell leukemia-lymphoma virus type I (HTLV-I) expression in fresh peripheral blood mononuclear cells from patients with tropical spastic paraparesis/HTLV-I-associated myelopathy.
      Recently, primary ATL cells were shown to be well maintained in vitro by co-culture in direct contact with stromal cells.
      • Nagai K.
      • Jinnai I.
      • Hata T.
      • Usui T.
      • Sasaki D.
      • Tsukasaki K.
      • Sugahara K.
      • Hishikawa Y.
      • Yamada Y.
      • Tanaka Y.
      • Koji T.
      • Mano H.
      • Kamihira S.
      • Tomonaga M.
      Adhesion-dependent growth of primary adult T cell leukemia cells with down-regulation of HTLV-I p40Tax protein: a novel in vitro model of the growth of acute ATL cells.
      Furthermore, type I interferon–induced HTLV-1 gag expression was suppressed in an ATL cell line when it was co-cultured with stromal cells.
      • Kinpara S.
      • Hasegawa A.
      • Utsunomiya A.
      • Nishitsuji H.
      • Furukawa H.
      • Masuda T.
      • Kannagi M.
      Stromal cell-mediated suppression of human T-cell leukemia virus type 1 expression in vitro and in vivo by type I interferon.
      These observations suggest that viral gene silencing occurs in ATL cells by interactions with the host microenvironment.
      Epigenetic regulations, such as histone acetylation, are also assumed to be involved in viral gene silencing in ATL cells.
      • Michael B.
      • Nair A.M.
      • Datta A.
      • Hiraragi H.
      • Ratner L.
      • Lairmore M.D.
      Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity.
      Thus, the trans-activity of the HTLV-1 long terminal repeats (LTRs) is induced by histone deacetylase (HDAC) inhibitors in HTLV-1–infected MT4 cells.
      • Ego T.
      • Ariumi Y.
      • Shimotohno K.
      The interaction of HTLV-1 Tax with HDAC1 negatively regulates the viral gene expression.
      Also, HTLV-1 Tax expression was doubled when primary ATL cells were treated with HDAC inhibitors.
      • Mosley A.J.
      • Meekings K.N.
      • McCarthy C.
      • Shepherd D.
      • Cerundolo V.
      • Mazitschek R.
      • Tanaka Y.
      • Taylor G.P.
      • Bangham C.R.
      Histone deacetylase inhibitors increase virus gene expression but decrease CD8+ cell antiviral function in HTLV-1 infection.
      These observations indicate that in ATL cells cultured in vitro, viral gene expression is suppressed by epigenetic regulation and that HDAC inhibitors can reactivate silenced viral genes. Taken together, available evidence indicates that viral gene expression is suppressed in ATL cells by the dual action of the microenvironment of the host-tumor interface and epigenetic regulation in host cells.
      Stromal fibroblasts constituting the bone marrow microenvironment prevent chemotherapy-induced apoptosis in acute myeloid leukemia and chronic lymphocytic leukemia cells.
      • Panayiotidis P.
      • Jones D.
      • Ganeshaguru K.
      • Foroni L.
      • Hoffbrand A.V.
      Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro.
      • Lagneaux L.
      • Delforge A.
      • Bron D.
      • De Bruyn C.
      • Stryckmans P.
      Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells.
      Recently, stromal cell–derived factor-1α and its cognate receptor CXCR4 have emerged as critical mediators of leukemic/stromal cell interactions.
      • Burger J.A.
      • Tsukada N.
      • Burger M.
      • Zvaifler N.J.
      • Dell’Aquila M.
      • Kipps T.J.
      Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1.
      Blockade of the stromal cell–derived factor-1α–CXCR4 axis by the CXCR4 antagonist AMD3100 suppresses the migration of cultured ATL cells,
      • Kawaguchi A.
      • Orba Y.
      • Kimura T.
      • Iha H.
      • Ogata M.
      • Tsuji T.
      • Ainai A.
      • Sata T.
      • Okamoto T.
      • Hall W.W.
      • Sawa H.
      • Hasegawa H.
      Inhibition of the SDF-1alpha-CXCR4 axis by the CXCR4 antagonist AMD3100 suppresses the migration of cultured cells from ATL patients and murine lymphoblastoid cells from HTLV-I Tax transgenic mice.
      suggesting that this axis may also be involved in formation of the stromal niche environment in ATL.
      The present study was undertaken to determine whether epithelial cells, another major component constituting the microenvironment of the host-tumor interface, affect the survival and phenotype of ATL cells. We show that co-culture with normal tissue–derived epithelial cells (NECs) increases a proportion of ATL cells in G0/G1 phase and rescues ATL cells from HDAC inhibitor–induced apoptosis. Adhesions to NECs induced prominent surface expression of CD44 on ATL cells; they also induced internalization of CD44 and nuclear translocation of cyclin D1 in a fraction of HDAC inhibitor–treated ATL cells, thus enabling such ATL cells to resume cell-cycle progression and leading to the ultimate survival of ATL cells. ATL cells co-cultured with NECs down-regulated the expression of NKG2D ligands, suggesting that this interaction also facilitates immune evasion by tumor cells.

      Materials and Methods

      Cells

      The HTLV-1–positive ATL cell lines ATL-CR and ATL-TH were obtained from the Reference Center for HTLV Infection (Rio de Janeiro, Brazil). These cell lines were established from Brazilian patients with ATL after they provided informed consent. Briefly, peripheral blood mononuclear cells (PBMCs) isolated from patients with ATL were cultured in the presence of recombinant IL-2. After long-term culture, they acquired IL-2 independence, resulting in establishment of the ATL-CR and ATL-TH cell lines. Jurkat and HUT78, a T-cell lymphoma line derived from a patient with Sezary syndrome,
      • Wayner E.A.
      • Gil S.G.
      • Murphy G.F.
      • Wilke M.S.
      • Carter W.G.
      Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes.
      were used as HTLV-1–negative T-cell lymphoma cells. ATL-CR, ATL-TH, Jurkat, and HUT78 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. HEK293 and HEK293T, human embryo kidney epithelial-like cell lines, and primary normal human dermal fibroblasts were maintained in Dulbecco’s minimal essential medium supplemented with 10% FBS and penicillin/streptomycin. 1C3IKEI, a primary normal human embryonic pancreas–derived epithelial-like cell line, was purchased from the RIKEN BioResource Center (Tsukuba, Japan) and maintained in Dulbecco’s minimal essential medium supplemented with 15% FBS. KHYG-1, an NKG2D+ natural killer (NK) cell line,
      • Suck G.
      • Branch D.R.
      • Smyth M.J.
      • Miller R.G.
      • Vergidis J.
      • Fahim S.
      • Keating A.
      KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.
      was purchased from the Health Science Research Resources Bank (Osaka, Japan) and was maintained in Dulbecco’s minimal essential medium supplemented with 100 U of recombinant human IL-2 (Shionogi, Osaka, Japan), 10% FBS, and penicillin/streptomycin. Fresh ATL cells were obtained from patients with chronic or acute ATL after informed consent was provided. Briefly, PBMCs isolated from heparinized peripheral blood by Ficoll-Paque PLUS (GE Healthcare Life Sciences, Little Chalfont, UK) were resuspended in RPMI 1640 medium supplemented with 10% FBS and penicillin/streptomycin and were subjected to experiments immediately.

      Antibodies, Plasmids, and Reagents

      Rat anti-human/mouse CD44 antibody (clone IM7; eBioscience Inc., San Diego, CA) was used for flow cytometry (FCM) and immunofluorescence staining. Mouse anti-human MICA/B antibody (clone 6D4; eBioscience) was used for FCM. Mouse anti-human CD25 (clone BC96; eBioscience), polyclonal rabbit anti-human fibronectin (Dako, Glostrup, Denmark), mouse anti-human vimentin (clone V9; Dako), polyclonal rabbit anti-human p21Cip1 (eBioscience), and mouse anti-human Ki-67 (clone MIB-1; Dako) antibodies were used for immunofluorescence staining. HTLV-1 LTR luciferase plasmids have been described previously.
      • Sheehy N.
      • Lillis L.
      • Watters K.
      • Lewis M.
      • Gautier V.
      • Hall W.W.
      Functional analysis of human T lymphotropic virus type 2 Tax proteins.
      HDAC inhibitors, such as trichostatin A (TSA), valproic acid sodium salt (VPA), and sodium butyrate (NaB), were purchased from Sigma-Aldrich (St. Louis, MO).

      Co-Culture

      ATL cells were co-cultured with NECs using a direct co-culture system that allowed for cell-cell contact. NECs were labeled with 1 μmol/L of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) at a concentration of 1 × 106 cells/mL in PBS for 20 minutes at 37°C and then washed three times in PBS. CFSE-labeled NECs were used immediately for each experiment. ATL cells, 5 × 105, were directly co-cultured with CFSE-labeled NECs growing as monolayers in 24-well plates. Cell culture inserts (pore size, 0.4 μm; Invitrogen, Camarillo, CA) were used for an indirect co-culture system. ATL cells were co-cultured with CFSE-labeled NECs using the indicated concentrations of TSA. After the indicated co-culture time, whole cells in co-culture were harvested for subsequent experiments. In some experiments, nonadhering ATL cells in the supernatant were collected separately from the ATL cells adhering to the monolayer of labeled NECs, and then the adhering ATL cells with labeled NECs were collected after rinsing twice gently with RPMI 1640 medium to remove nonadhering ATL cells. For the proliferation assay, ATL-CR cells were labeled with CFSE and then co-cultured with unlabeled HEK293T cells for 72 hours.
      • Green K.J.
      • Rowbottom D.G.
      Exercise-induced changes to in vitro T-lymphocyte mitogen responses using CFSE.

      FCM Analysis

      Cell-cycle distribution of ATL cells was analyzed by DNA content using the propidium iodide (PI) staining method. Co-cultured cells were harvested after the indicated durations of TSA treatment. After fixation in 70% ethanol at −20°C overnight, cells were incubated in a PI/RNase A staining buffer (50 μg/mL of PI and 20 μg/mL of RNase A in PBS). Cells were then analyzed by FCM using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). CFSE and PI were detected in the FL-1 and FL-2 channels, respectively. Co-cultured ATL cells gated on CFSE-negative cells were acquired in 10,000 events. Expression of cell-surface molecules was analyzed using FCM. Analysis was performed using CellQuest Pro software version 6.0 (Becton Dickinson Immunocytometry Systems, San Jose, CA). Sigmoid-like dose-response curves were drawn using the logistic curve-fitting software ImageJ, version 1.46 (NIH, Bethesda, MD).
      • Schneider C.A.
      • Rasband W.S.
      • Eliceiri K.W.
      NIH Image to ImageJ: 25 years of image analysis.

      Immunofluorescence Staining

      Cells cultured on chamber slides were fixed with 4% paraformaldehyde for 15 minutes. For intracellular staining, cells were treated with PBS containing 0.1% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) for 4 minutes and then fixed with ice-cold 70% methanol for 4 minutes. Nonspecific binding was blocked with 0.05% Tween 20 in PBS containing 0.1% goat serum for 10 minutes. After incubation with primary antibodies for each targeted protein, Alexa Fluor–conjugated goat polyclonal antibody was used as a secondary antibody. Images were acquired using an Olympus DP70 camera with its own Olympus DP controller software version 1.2.1.108 (Olympus, Tokyo, Japan). Three-dimensional analyses were reconstructed by images acquired by confocal microscopy using a laser scanning confocal microscope (FV-300; Olympus, Tokyo, Japan).

      Functional Assays

      Reporter genes were introduced into ATL cells directly using FuGENE transfection reagents (Roche Diagnostics, Branchburg, NJ) according to the manufacturer’s instructions. Briefly, 5 × 105 ATL-CR cells were cultured in 60-mm dishes and cotransfected with 1 μg of HTLV-1 LTR firefly luciferase reporter plasmid together with 50 ng of the Renilla luciferase reporter pRL-TK. Eighteen hours after transfection, ATL-CR cells were washed five times in PBS and then were co-cultured with HEK293T cells in the presence of TSA in triplicate wells of 24-well plates (2 × 105 cells per well). Reporter activities were measured using the Dual-Luciferase reporter assay system (Promega, San Luis Obispo, CA). Briefly, cells were lysed in 1× passive lysis buffer, and firefly and Renilla luciferase activities were measured using a Turner 20/20 luminometer (Promega). Reporter activities were normalized using Renilla luciferase values.

      Cytotoxicity Assays

      Cytotoxicity assays used a combination of two dyes: CFSE was used to label effector cells (KHYG-1 cells) and HEK293T cells and PI was used to stain target cells (ATL-CR cells). CFSE-labeled effector cells were incubated with nonlabeled target cells cultured alone or co-cultured with CFSE-labeled HEK293T cells at effector to target cell ratios of 3:1, 6:1, or 9:1 in triplicate wells of 24-well plates for 18 hours. Apoptotic ATL-CR cells were evaluated by the percentage of sub G0/G1 phase cells in CFSE-negative cells as determined by FCM. Mean values were calculated from three independent experiments.

      Statistical Analysis

      Data were analyzed using either the Student’s t-test or repeated-measures analysis of variance. A P < 0.05 was considered statistically significant.

      Results

      Direct Co-Culture with NECs Rescues ATL Cells from TSA-Induced Apoptosis

      HTLV-1 gene expression is hardly detectable in ATL cells because of gene silencing. With the aim of eliminating viral gene silencing, we treated the IL-2–independent ATL-derived cell line ATL-CR with TSA. This treatment increased the amount of HTLV-1 p19 gag protein in the supernatants and lysates prepared from cultured ATL-CR cells (data not shown) and enhanced the transcriptional activity of HTLV-1 LTRs in a dose-dependent manner (Figure 1A), indicating that TSA effectively eliminates viral gene silencing. However, when ATL-CR cells were co-cultured with HEK293T cells (used as NECs), treatment with TSA did not increase the transcriptional activity of HTLV-1 LTRs, indicating that co-culture with NECs blocked TSA-induced viral gene reactivation (Figure 1A).
      Figure thumbnail gr1
      Figure 1Direct co-culture with NECs rescued ATL cells from TSA-induced apoptosis. A: Transcriptional activity of HTLV-1 LTRs in TSA-treated ATL-CR cells. ATL-CR cells were cotransfected with 1 μg of HTLV-1 LTR firefly luciferase reporter plasmid together with 50 ng of pRL-TK 1 day before co-culture with HEK293T cells. After 18 hours of co-culture with the indicated concentrations of TSA, reporter activities were measured and normalized to Renilla luciferase values. The values indicate the means ± SD fold increases obtained by three independent experiments normalized to the untreated control sample. B: Cell-cycle analysis in ATL-CR and ATL-TH cells. ATL cells, 5 × 105 per well, were cultured alone or with HEK293T cells in the presence of the indicated concentrations of TSA for 48 hours. HEK293T cells were labeled with CFSE 1 day before co-culture. After the whole co-cultured cells were harvested, cell-cycle analysis was performed by FCM. C: Percentage of apoptotic ATL-CR cells co-cultured with normal human dermal fibroblasts (NHDFs), 1C3IKEI cells, HEK293 cells, and HEK293T cells. The data are presented as the means ± SD of three independent experiments. P < 0.05, ∗∗P < 0.01.
      Next, we performed cell-cycle analysis to determine whether HEK293T cells affect the fate of TSA-treated ATL-CR and ATL-TH cells (Figure 1B). When ATL-CR cells were cultured alone in the presence of TSA, they displayed a dose-dependent increase in the percentage of apoptotic cells and a decrease in the percentage of G0/G1 phase cells; similar changes were observed in TSA-treated ATL-TH cells but not in TSA-treated HEK293T cells. ATL-CR and ATL-TH cells co-cultured with HEK293T cells showed no increase in the percentage of apoptotic cells; instead, the percentage of G0/G1 phase cells was increased. The ability of HEK293T cells to prevent apoptosis was evident even at an ATL-CR to HEK293T cell ratio of 25:1 (data not shown). Similar apoptosis-protective effects were also observed when ATL-CR cells were co-cultured with primary epithelial cells 1C3IKEI or primary dermal fibroblasts (Figure 1C). These results indicate that co-culture with NECs generally prevents ATL cells from TSA-induced apoptosis.

      Cell-Cell Contact–Dependent Interactions with NECs Are Required to Rescue ATL Cells from HDAC Inhibitor–Induced Apoptosis

      To examine whether cell-cell contact with NECs is required to rescue ATL cells from TSA-induced apoptosis, we co-cultured ATL-CR cells in direct or indirect contact with HEK293T cells in the presence of TSA. When ATL-CR cells were separated by 0.4-μm membranes from HEK293T cells, the ratio of apoptotic ATL-CR cells was lower than when they were cultured alone but showed a marked increase compared with when ATL-CR cells were cultured in direct contact with HEK293T cells (Figure 2A), indicating that efficient suppression of apoptosis requires cell-cell contact. Approximately 84% of ATL-CR cells adhered to a monolayer of HEK293T cells by 24 hours after co-culture with HEK293T cells (Figure 2, B and C). There was no correlation between the dose of TSA and the adhesiveness of ATL-CR cells (Figure 2B), suggesting that TSA treatment did not augment the adhesive activity of ATL cells. To examine whether the extracellular matrix can substitute for NECs, ATL-CR cells were cultured in an extracellular matrix (BD Matrigel Matrix; Becton Dickinson)–coated dish in the presence of TSA. These experiments showed that extracellular matrix alone was completely ineffective in reducing TSA-induced apoptosis in ATL cells (data not shown).
      Figure thumbnail gr2
      Figure 2Cell-cell contact–dependent interactions with NECs are required to rescue ATL cells from TSA-induced apoptosis. A: Percentage of apoptotic ATL-CR cells. ATL-CR cells were directly co-cultured with HEK293T cells or indirectly co-cultured with HEK293T cells using cell culture inserts (pore size, 0.4 μm) in the presence of the indicated concentrations of TSA for 48 hours. B: Means ± SD percentage of ATL-CR cells adhering to HEK293T cells after co-culture for 24 hours. C: Most of the ATL-CR cells (red) showed adhesion-dependent growth on a monolayer of HEK293T cells after 24 hours of direct co-culture. Scale bars: 50 μm. D: Percentage of apoptotic ATL-CR cells. ATL-CR cells were co-cultured with HEK293T cells in the presence of the indicated concentrations of TSA, VPA, and NaB. After co-culture, nonadhering ATL-CR cells in the supernatant were collected separately from the ATL-CR cells adhering to the monolayer of CFSE-labeled HEK293T cells. The data are presented as the means ± SD of three independent experiments.
      We next examined whether ATL-CR cells co-cultured with HEK293T cells acquire apoptosis resistance to treatment with other HDAC inhibitors, such as VPA and NaB (Figure 2D). TSA-treated ATL-CR cells directly co-cultured with HEK293T cells acquired dramatic apoptosis resistance regardless of whether they adhered to HEK293T cells. In contrast, apoptosis resistance to VPA or NaB was acquired only in adhering ATL-CR cells, and the extent of acquired resistance was diminished. These results indicate that cell-cell contact–dependent interactions with NECs make ATL-CR cells more or less resistant to apoptosis induced by HDAC inhibitors.

      Co-Culture with NECs Induces a Quiescent State in TSA-Treated ATL Cells

      To examine the cellular changes that follow co-culture with NECs more closely, we analyzed cell-cycle distribution in ATL cells cultured alone, co-cultured with NECs, or co-cultured with NECs in the presence of TSA (Figure 3A). Co-culture with NECs decreased the proportion of S and G2/M phase cells and increased the proportion of G0/G1 phase cells. Co-culture in the presence of 0.8 μmol/L TSA further strengthened these changes. In contrast, TSA-treated ATL-CR cells cultured alone did not show such cell-cycle changes and underwent apoptosis (Figure 3B). Furthermore, when CFSE-labeled ATL-CR cells were cultured alone or with HEK293T cells for 72 hours, co-cultured ATL-CR cells showed less proliferation than those cultured alone, in the presence and absence of 0.8 μmol/L TSA (Figure 3C). This is consistent with the observation that co-culture with NECs induces G0/G1 accumulation in ATL-CR cells and allows them to escape from TSA-induced apoptosis (Figure 3A).
      Figure thumbnail gr3
      Figure 3Co-culture with NECs induced a quiescent state in TSA-treated ATL-CR cells. A: Cell-cycle analysis in ATL-CR cells. ATL-CR cells co-cultured with HEK293T cells were treated with 0.8 μmol/L TSA for 24 hours. B: Changes in cell-cycle distribution were followed for 24 hours in ATL cells cultured alone in the presence or absence of TSA. C: Proliferation analysis in ATL-CR cells. CFSE-labeled ATL-CR cells were cultured alone or with HEK293T cells in the presence or absence of TSA for 72 hours. CFSE intensity was analyzed after gating on live cells. D: Recovery of TSA-pretreated ATL-CR cells by direct co-culture with NECs. ATL-CR cells were pretreated with the indicated concentration of TSA for 18 hours and then were cultured alone or with HEK293T cells in fresh RPMI 1640 medium for 24 hours. ATL-CR alone (input) indicates TSA-pretreated ATL-CR cells cultured alone. E: p21Cip1, Ki-67, and CD25 expression in ATL-CR cells. ATL-CR cells were cultured alone or with HEK293T cells in the presence of 0.8 μmol/L TSA. F: Expression of vimentin and fibronectin in ATL-CR cells co-cultured with HEK293T cells. Apo, apoptotic cells. The data are presented as means ± SD. P < 0.05, ∗∗P < 0.01. Scale bars: 50 μm (E and F).
      Next, to examine whether NECs have a protective role on ATL-CR cells pretreated with TSA, ATL-CR cells were incubated with TSA for 18 hours and then were transferred to the fresh medium free of TSA. Pretreatment of ATL-CR cells with TSA increased the proportion of G2/M phase cells in a dose-dependent manner (Figure 3D). These TSA-pretreated cells (shown as input cells) were transferred to the fresh TSA-free medium and were cultured for an additional 24 hours in the presence or absence of HEK293T cells. In the absence of HEK293T cells, the proportion of cells in the G0/G1, S, and G2/M phases continued to decline, and ATL-CR cells continued to undergo apoptosis. In contrast, the ratio of apoptotic cells did not increase appreciably in the presence of HEK293T cells. These results suggest that co-culture with HEK293T cells induced ATL-CR cells to enter the G0/G1 resting phase.
      Ki-67 antigen is present during all active phases of the cell cycle (G1, S, G2, and M) but is absent from cells in resting G0 phase. When ATL-CR cells were stained with anti–Ki-67 antibody, most cells were not stained; however, when they were treated with TSA, 44.2% of the cells became positive with Ki-67 and entered the cell cycle (data not shown). TSA increases p21Cip1 levels in cancer cells, leading to cell-cycle arrest.
      • Sowa Y.
      • Orita T.
      • Minamikawa-Hiranabe S.
      • Mizuno T.
      • Nomura H.
      • Sakai T.
      Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor.
      When ATL-CR cells cultured alone were treated with TSA, they exhibited markedly induced expression of p21Cip1 and Ki-67 and retained high expression of CD25 (Figure 3E). In contrast, when ATL-CR cells were treated with TSA and co-cultured with NECs, expression of p21Cip1 and Ki-67 remained low, and expression of CD25 was strongly diminished (Figure 3E).
      Vimentin is a mesenchymal intermediate filament supporting the structural integrity of quiescent cells while participating in adhesion, survival, growth regulation, and cell signaling processes.
      • Mani S.A.
      • Guo W.
      • Liao M.J.
      • Eaton E.N.
      • Ayyanan A.
      • Zhou A.Y.
      • Brooks M.
      • Reinhard F.
      • Zhang C.C.
      • Shipitsin M.
      • Campbell L.L.
      • Polyak K.
      • Brisken C.
      • Yang J.
      • Weinberg R.A.
      The epithelial-mesenchymal transition generates cells with properties of stem cells.
      It is a growth-regulated protein whose expression is induced in quiescent cells ≤2 hours after mitogenic stimulation, even when protein synthesis is inhibited.
      • Ferrari S.
      • Battini R.
      • Kaczmarek L.
      • Rittling S.
      • Calabretta B.
      • de Riel J.K.
      • Philiponis V.
      • Wei J.F.
      • Baserga R.
      Coding sequence and growth regulation of the human vimentin gene.
      We, therefore, examined the expression of vimentin and fibronectin, another mesenchymal marker, in ATL cells adhering to NECs. Expression of vimentin, but not fibronectin, was markedly elevated in ATL-CR cells co-cultured with HEK293T cells (Figure 3F). These results are consistent with the idea that adhesion to NECs induced cellular quiescence and resistance to TSA-induced apoptosis in ATL cells through vimentin expression.

      ATL Cells Adhering to NECs Augment CD44 Expression

      Cancer stem cell (CSC)–like cells often exhibit markers of epithelial-mesenchymal transition, such as vimentin.
      • Mani S.A.
      • Guo W.
      • Liao M.J.
      • Eaton E.N.
      • Ayyanan A.
      • Zhou A.Y.
      • Brooks M.
      • Reinhard F.
      • Zhang C.C.
      • Shipitsin M.
      • Campbell L.L.
      • Polyak K.
      • Brisken C.
      • Yang J.
      • Weinberg R.A.
      The epithelial-mesenchymal transition generates cells with properties of stem cells.
      Recent evidence indicates that CD44 directly reprograms stem cell properties in colon cancer cells.
      • Su Y.J.
      • Lai H.M.
      • Chang Y.W.
      • Chen G.Y.
      • Lee J.L.
      Direct reprogramming of stem cell properties in colon cancer cells by CD44.
      We, therefore, examined whether the cellular quiescence achieved by co-culture with NECs was accompanied by elevated expression of CD44, a hyaluronan receptor known to be involved in cellular quiescence and now recognized as a marker of CSC-like cells (Figure 4A). When we compared cell surface expression of CD44 on ATL cells co-cultured with NECs and cultured alone in the presence or absence of TSA, CD44 expression was markedly increased in ATL-CR cells co-cultured with HEK293T cells compared with ATL cells cultured alone regardless of the presence or absence of TSA. Staining for CD44 was more pronounced in ATL-CR cells located at the upper and central regions of colonies, whereas vimentin tended to be evenly expressed on all ATL-CR cells (Figure 4, B and C). These observations suggest the involvement of CD44-mediated interactions in the induction of cellular quiescence in co-cultured ATL cells.
      Figure thumbnail gr4
      Figure 4ATL cells adhering to NECs exhibited elevated CD44 expression. A: After 24 hours of co-culture with HEK293T cells, expression of CD44 and vimentin was elevated in ATL-CR cells adhering to the monolayer of HEK293T cells. B: Expression of CD44 and vimentin in ATL-CR cells co-cultured with HEK293T cells for 24 hours in the absence of TSA. C: Expression of CD44 and vimentin in ATL-CR cells co-cultured with HEK293T cells for 24 hours in the presence of 0.8 μmol/L TSA. Scale bars: 50 μm (A–C).

      ATL Cells that Form Colonies on the Monolayer of NECs Show Intracellular Expression of CD44

      ATL cells that formed colonies on the monolayer of NECs were more strongly positive for CD44 than were those that did not form colonies and floated in the medium (Figure 5A). To examine whether CD44 is involved in apoptosis resistance, we pretreated ATL-CR cells cultured alone with 40 μg/mL of anti-CD44 antibody or control IgG for 1 hour and then cultured for an additional 24 hours in the presence or absence of HEK293T cells (Figure 5B). Treatment with anti-CD44 antibody significantly reduced apoptosis resistance in ATL-CR cells cultured alone and co-cultured with NECs. These results indicate that elevated CD44 expression is involved in conferring apoptosis resistance on TSA-treated ATL-CR cells. Three-dimensional analysis by confocal microscopy revealed a population of ATL cells strongly positive for intracellular CD44; such cells were located on top of colony-forming, vimentin-positive ATL cells (Figure 5C). The proportion of cells showing strong intracellular staining for CD44 was significantly higher in ATL cells co-cultured with NECs than in ATL cells cultured alone (means ± SD: 10.68% ± 1.65% for TSA-treated co-cultured ATL cells and 1.91% ± 0.47% for TSA-treated ATL cells cultured alone; P < 0.001) (Figure 5D).
      Figure thumbnail gr5
      Figure 5A fraction of ATL cells forming colonies on the monolayer of NECs showed intracellular expression of CD44. A: Cell surface expression of CD44 on adhering and nonadhering ATL-CR cells. ATL-CR cells were co-cultured with HEK293T cells for 24 hours. B: Blocking antibody for CD44 inhibited the apoptosis-protective effects conferred by co-culture with HEK293T cells. ATL-CR cells cultured alone were pretreated with anti-CD44 antibody or control IgG for 1 hour and then were cultured for an additional 24 hours with or without HEK293T cells in the presence of 0.8 μmol/L TSA. C: Three-dimensional analysis by confocal microscopy. Expression of CD44 was observed on ATL-CR cells that formed colonies on the monolayer of HEK293T cells. Expression was particularly pronounced on ATL-CR cells located at the upper and central regions of the colonies. D: Percentage of intracellular CD44+ cells. E: CD44 and cyclin D1 expression. The TSA concentration used in D and E was 0.8 μmol/L. F: Nuclear localization of cyclin D1 in CD44+ high cells. ATL-CR cells were cultured in the absence of TSA. G: CD44 and vimentin expression on ATL-CR cells co-cultured with 1C3IKEI and normal human dermal fibroblast (NHDF) cells. The data are presented as the means ± SD of three independent experiments. P < 0.01. Scale bars: 10 μm (C and E); 50 μm (G).
      Recently, it was reported that nuclear translocation of CD44 promotes cell proliferation through direct binding to the promoter region of the cyclin D1 gene.
      • Lee J.L.
      • Wang M.J.
      • Chen J.Y.
      Acetylation and activation of STAT3 mediated by nuclear translocation of CD44.
      We, therefore, examined whether cells positive for intracellular CD44 express cyclin D1. In TSA-untreated ATL cells, CD44 expression was restricted to the cell surface, with no evidence of nuclear translocation of cyclin D1 (Figure 5E). In TSA-treated ATL cells cultured alone, CD44 surface expression was low, with little detectable nuclear translocation of cyclin D1. In contrast, a fraction of ATL cells co-cultured with NECs was strongly positive for intracellular CD44 and cyclin D1 regardless of whether they were treated with TSA. In ATL cells co-cultured with NECs, nuclear translocation of cyclin D1 was induced after nuclear translocation of CD44 (Figure 5F). These results suggest that nuclear translocation of cyclin D1 enables a fraction of ATL cells to enter the cell cycle from the resting G0/G1 phase, thus accounting for the slower, yet demonstrable cell proliferation observed in Figure 3C.
      We confirmed that as in co-culture with HEK293T cells, co-culture with primary epithelial 1C3IKEI cells induced expression of CD44 on a fraction of colony-forming ATL cells (Figure 5G). However, when ATL-CR cells were co-cultured with normal human dermal fibroblasts, they neither formed colonies nor increased CD44 expression (Figure 5G). These results suggest that NECs and fibroblasts confer apoptosis resistance on ATL cells through distinct mechanisms.

      Freshly Isolated ATL Cells Form Colonies when Co-Cultured with NECs, Escape TSA-Induced Apoptosis, and Augment CD44 Expression

      To assess the clinical relevance of cell-cell interaction between ATL cells and NECs, we isolated PBMCs from two patients with ATL. Patient 1, with chronic-type ATL, had a white blood cell count of 6100/μL, of which 37.3% were lymphocytes. Patient 2, with acute-type ATL, had a white blood cell count of 29,520/μL, of which 88% were lymphocytes. Patient-derived PBMCs were cultured alone or with HEK293T cells with or without 0.8 μmol/L TSA, and then cycle analysis and immunofluorescence staining for CD44 and vimentin were performed. ATL cells co-cultured with HEK293T cells showed no increase in the percentage of apoptotic cells when treated with TSA for 48 hours, indicating that direct co-culture with NECs rescued ATL cells from TSA-induced apoptosis (Figure 6A). When co-cultured with HEK293T cells, a sizable proportion of ATL cells from both patients formed colonies on the monolayer of HEK293T cells and markedly increased CD44 and vimentin expression in the presence (Figure 6B) and absence (Figure 6, C and D) of TSA. Therefore, co-culture with NECs induced similar phenotypic changes in both ATL cell lines and freshly isolated ATL cells.
      Figure thumbnail gr6
      Figure 6Fresh ATL cells adhering to NECs formed colonies, escaped from TSA-induced apoptosis, and augmented CD44 expression. A–D: PBMCs derived from patients with ATL with chronic type (patient 1) and acute type (patient 2) were cultured alone or with HEK293T cells with or without 0.8 μmol/L TSA, and then cell-cycle analysis and immunofluorescence staining for CD44 and vimentin were performed. A: Percentage of apoptotic cells (Apo) and G0/G1 phase cells. B: Expression of CD44 and vimentin in TSA-treated ATL cells. C and D: Expression of CD44 and vimentin in TSA-untreated ATL cells derived from patient 1 (C) and patient 2 (D). E: Cell-cycle analysis in HTLV-1–negative T-cell lymphoma cells. HUT78 and Jurkat cells co-cultured with HEK293T cells were treated with 0.8 μmol/L TSA for 48 hours. F: Expression of CD44 and vimentin was examined in HTLV-1–negative T-cell lymphoma cells co-cultured with HEK293T cells for 24 hours in the presence of 0.8 μmol/L TSA. Scale bars: 50 μm (BD and F).
      Next, we examined whether NEC-induced protection occurs in leukemic cells other than ATL cells. When the HTLV-1–negative T-cell lymphoma cells Jurkat and HUT78 were cultured alone in the presence of TSA, they both displayed an increase in the percentage of apoptotic cells, and this increase was abrogated by co-culture with HEK293T cells (Figure 6E). Furthermore, similar to ATL cell lines and freshly isolated ATL cells, HUT78, an epidermotropic HTLV-1–negative T-cell lymphoma cell line established from a patient with Sezary syndrome,
      • Wayner E.A.
      • Gil S.G.
      • Murphy G.F.
      • Wilke M.S.
      • Carter W.G.
      Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes.
      formed colonies on the monolayer of HEK293T cells and markedly increased CD44 and vimentin expression (Figure 6F). In contrast, Jurkat cells co-cultured with HEK293T cells neither formed colonies nor increased CD44 expression, suggesting that they presumably escaped from TSA-induced apoptosis by mechanisms distinct from those operating in ATL and HUT78 cells (Figure 6F).

      Co-Culture with NECs Reduces the Expression of NKG2D Ligands on ATL-CR Cells

      MICA and MICB (MHC class I chain-related proteins A and B) are stress-inducible NKG2D ligands that interact with the activating NKG2D receptor expressed on NK cells.
      • Gleimer M.
      • Parham P.
      Stress management: MHC class I and class I like molecules as reporters of cellular stress.
      • Raulet D.H.
      • Guerra N.
      Oncogenic stress sensed by the immune system: role of natural killer cell receptors.
      • Bauer S.
      • Groh V.
      • Wu J.
      • Steinle A.
      • Phillips J.H.
      • Lanier L.L.
      • Spies T.
      Activation of NK cells and T cells by NKG2D, a receptor for stress inducible MICA.
      This interaction activates NK cells, thereby facilitating the elimination of tumor cells. To examine whether co-culture with NECs affects immunologic properties of ATL cells, we cultured ATL-CR cells in the presence or absence of HEK293T cells in a medium containing 0.2 μmol/L TSA and examined MICA/MICB expression on ATL-CR cells. ATL-CR cells co-cultured with HEK293T cells expressed decreased levels of MICA/MICB (Figure 7A). Consistent with this, they became less susceptible to NK cell–mediated cytotoxicity (Figure 7B). These results suggest that interactions with NECs help ATL cells evade NKG2D-mediated immune attack.
      Figure thumbnail gr7
      Figure 7Direct co-culture with NECs reduced the expression of NKG2D ligands on ATL-CR cells. A: Induction of MICA/B expression on ATL-CR cells treated with TSA for 18 hours. ATL-CR cells were cultured alone or with HEK293T cells in the presence of 0.2 μmol/L TSA or vehicle. The cell surface expression of MICA/B on ATL-CR cells was analyzed by FCM using an anti-human MICA/B antibody. B: Susceptibility of TSA- or vehicle-treated ATL-CR cells to NK cell–mediated cytotoxicity. ATL-CR cells were treated with 0.2 μmol/L TSA or vehicle for 18 hours. CFSE-labeled KHYG-1 cells were used as effector cells and ATL-CR cells as target cells in cytotoxicity assays. Effector cells were incubated with nonlabeled target cells cultured alone or with CFSE-labeled HEK-293T cells at the indicated effector to target cell (E:T) ratios in triplicate wells of 24-well plates for 18 hours. Apoptotic ATL-CR cells were evaluated by FCM for the percentage of sub G0/G1 phase cells in the CFSE-negative cells. The data are presented as the means ± SD of three independent experiments.

      Discussion

      We demonstrated that in ATL cells cultured alone, TSA treatment induces p21Cip1 accumulation, leading to cell-cycle arrest and apoptosis, whereas co-culture with NECs induces G0/G1 accumulation in TSA-treated ATL cells, enabling them to stay in a quiescent state and to acquire apoptosis resistance (Figures 1, 2, and 3). This resistance was acquired even when ATL cells cultured alone were pretreated with TSA and then co-cultured with NECs in fresh TSA-free medium, indicating that the contact with NECs subsequent to exposure to TSA enabled ATL-CR cells that otherwise were destined to undergo apoptosis to recover and resume growth (Figure 3). HDAC inhibitors, such as TSA, VPA, and NaB, induce not only transcriptional activation of viral and host genes but also genomic instability by a variety of mechanisms.
      • Marks P.A.
      • Richon V.M.
      • Rifkind R.A.
      Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells.
      • Shakespear M.R.
      • Halili M.A.
      • Irvine K.M.
      • Fairlie D.P.
      • Sweet M.J.
      Histone deacetylases as regulators of inflammation and immunity.
      • Eot-Houllier G.
      • Fulcrand G.
      • Magnaghi-Jaulin L.
      • Jaulin C.
      Histone deacetylase inhibitors and genomic instability.
      Thus, the present data suggest that NECs have a key role in guarding ATL cells from genomic instability, including reactivation of viral genes.
      Co-culture with NECs induced cellular quiescence in ATL cells as assessed by cell-cycle analysis and staining with Ki-67 (Figure 3). ATL-CR cells that adhered to NECs displayed enhanced expression of CD44 and vimentin (Figures 3, 4, and 5). In this regard, treatment of CSCs with high concentrations of hyaluronan induces cellular quiescence, epithelial-mesenchymal transition, and a multidrug-resistant phenotype.
      • Solis M.A.
      • Chen Y.H.
      • Wong T.Y.
      • Bittencourt V.Z.
      • Lin Y.C.
      • Huang L.L.
      Hyaluronan regulates cell behavior: a potential niche matrix for stem cells.
      Expression of vimentin and CD44 might be induced by hyaluronan fragments in the extracellular matrix produced by NECs. Accumulating evidence indicates a close link between epithelial-mesenchymal transition and cancer stemness.
      • Mani S.A.
      • Guo W.
      • Liao M.J.
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      • Zhang C.C.
      • Shipitsin M.
      • Campbell L.L.
      • Polyak K.
      • Brisken C.
      • Yang J.
      • Weinberg R.A.
      The epithelial-mesenchymal transition generates cells with properties of stem cells.
      It is, therefore, possible that direct contact with NECs induces CSC-like phenotypes in ATL cells.
      Co-culture with NECs induced expression of CD44 and vimentin in not only ATL cell lines but also ATL cells freshly isolated from patients (Figure 6, A–C), strongly suggesting the potential clinical importance of our observation. Indeed, recent work has shown that CD44 is expressed on skin-infiltrating tumor cells in patients with ATL.
      • Chagan-Yasutan H.
      • Tsukasaki K.
      • Takahashi Y.
      • Oguma S.
      • Harigae H.
      • Ishii N.
      • Zhang J.
      • Fukumoto M.
      • Hattori T.
      Involvement of osteopontin and its signaling molecule CD44 in clinicopathological features of adult T cell leukemia.
      Taken together, the present study suggests that interactions with epithelial cells induce CSC-like phenotypes in ATL cells and make them highly resistant to chemotherapies. Co-culture with NECs exerted antiapoptotic effects on two HTLV-1–negative T-cell lymphoma cells, Jurkat and HUT78 (Figure 6E), and induced the expression of CD44 and vimentin in HUT78 cells (Figure 6F). These observations suggest that NECs may play a protective role in lymphomas other than ATL.
      Co-culture with NECs reduced expression of the stress-inducible NKG2D ligands MICA and MICB in TSA-treated ATL cells (Figure 7A), presumably because it reduced cellular stresses incurred by epigenetic changes. Reduced MICA and MICB expression made ATL cells less susceptible to cytotoxicity mediated by NKG2D+ NK cells (Figure 7B). These results suggest that NECs not only induce CSC-like phenotypes in tissue-infiltrating ATL cells but also facilitate immune evasion by tumor cells.
      HDAC inhibitors have emerged as a new class of promising chemotherapeutic agents against cancer.
      • Marks P.A.
      • Richon V.M.
      • Rifkind R.A.
      Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells.
      However, monotherapeutic clinical trials with HDAC inhibitors have met with only limited success in most types of cancers.
      • Olsen E.A.
      • Kim Y.H.
      • Kuzel T.M.
      • Pacheco T.R.
      • Foss F.M.
      • Parker S.
      • Frankel S.R.
      • Chen C.
      • Ricker J.L.
      • Arduino J.M.
      • Duvic M.
      Phase IIb multicenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutaneous T-cell lymphoma.
      • Piekarz R.L.
      • Frye R.
      • Prince H.M.
      • Kirschbaum M.H.
      • Zain J.
      • Allen S.L.
      • Jaffe E.S.
      • Ling A.
      • Turner M.
      • Peer C.J.
      • Figg W.D.
      • Steinberg S.M.
      • Smith S.
      • Joske D.
      • Lewis I.
      • Hutchins L.
      • Craig M.
      • Fojo A.T.
      • Wright J.J.
      • Bates S.E.
      Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma.
      The therapeutic efficacy of HDAC inhibitors in patients with ATL is still a controversial issue.
      • Nishioka C.
      • Ikezoe T.
      • Yang J.
      • Komatsu N.
      • Bandobashi K.
      • Taniguchi A.
      • Kuwayama Y.
      • Togitani K.
      • Koeffler H.P.
      • Taguchi H.
      Histone deacetylase inhibitors induce growth arrest and apoptosis of HTLV-1-infected T-cells via blockade of signaling by nuclear factor kappaB.
      • Hasegawa H.
      • Yamada Y.
      • Tsukasaki K.
      • Mori N.
      • Tsuruda K.
      • Sasaki D.
      • Usui T.
      • Osaka A.
      • Atogami S.
      • Ishikawa C.
      • Machijima Y.
      • Sawada S.
      • Hayashi T.
      • Miyazaki Y.
      • Kamihira S.
      LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway.
      Furthermore, the safety of this treatment has not been established because it could activate viral genes.
      • Mosley A.J.
      • Meekings K.N.
      • McCarthy C.
      • Shepherd D.
      • Cerundolo V.
      • Mazitschek R.
      • Tanaka Y.
      • Taylor G.P.
      • Bangham C.R.
      Histone deacetylase inhibitors increase virus gene expression but decrease CD8+ cell antiviral function in HTLV-1 infection.
      The present work suggests that the therapeutic effectiveness of HDAC inhibitors will be reduced against leukemia cells that have invaded epithelial tissues and, thereby, acquired resistance to the inhibitors.
      In conclusion, the results of the present study suggest that a propensity of leukemia cells to infiltrate epithelial tissues might produce at least two pathologic outcomes in patients with ATL: survival of leukemia cells through the acquisition of CSC-like phenotypes and evasion of the host immune response through reduced expression of NKG2D ligands.

      Acknowledgments

      We thank Carlo Bidoia, Virginie W. Gautier, and Noreen Sheehy for helpful discussions, Jonathan Dean for help with FCM analysis, and Masumi Tsuda and Shinya Tanaka for help with confocal microscopic analysis.

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