JOURNAL OF VIROLOGY, Sept. 2010, p. 8732–8742 0022-538X/10/$12.00 doi:10.1128/JVI.00487-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 17
Elevated Cyclic AMP Levels in T Lymphocytes Transformed by Human T-Cell Lymphotropic Virus Type 1䌤 Andrea K. Kress,* Grit Schneider, Klemens Pichler,† Martina Kalmer, Bernhard Fleckenstein, and Ralph Grassmann‡ Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universita ¨t Erlangen-Nu ¨rnberg, Erlangen, Germany Received 4 March 2010/Accepted 14 June 2010
Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4ⴙ T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence. hances viral mRNA synthesis by transactivating the HTLV-1 long terminal repeat promoter, Rex controls the synthesis of the structural proteins on a posttranscriptional level (29). Tax confers the transforming properties on HTLV-1, as it can immortalize T lymphocytes (24, 48) and induce leukemia in transgenic mice (26). Several Tax functions may contribute to its transforming capacity, including interference with cell cycle checkpoints, tumor suppressors, and DNA repair (11, 23). Tax can stimulate the expression of cellular proteins controlling proliferation and survival (23). In particular, Tax-mediated modulation of cellular gene expression may explain the resistance of HTLV-1-positive cells to various proapoptotic stimuli (13). Tax is capable of stimulating cellular transcription by interacting with various signaling pathways such as the canonical and the noncanonical nuclear factor kappa B (NF-B) pathways. Transactivation of another set of cellular promoters is mediated by Tax via direct contact with transcriptional activators CREB and SRF and with the coactivators p300/CBP (11, 23, 29, 34). The persistence of HTLV-1 in T-cell clones which are detectable over many years suggested that proteins mediating survival and proliferation of long-lived T-cell clones could be crucial for the lifelong persistence of the virus and thus be potential targets of its oncoprotein Tax. Costimulatory receptors such as GITR or 4-1BB are proteins typically sharing these properties. While being mostly absent from naive T cells, they are present on long-lived T lymphocyte clones such as memory (CD4⫹ CD45RO⫹) T cells (T-mem) and regulatory (CD4⫹ CD25high CD127low FOXP3⫹) T cells (T-reg). In these cell types, costimulatory receptors augment proliferation and survival. Phenotypic parallels between HTLV-transformed cells
Human T-cell lymphotropic virus type 1 (HTLV-1), a deltaretrovirus, is the causative agent of a severe and fatal lymphoproliferative disorder of CD4⫹ T cells, adult T-cell leukemia/lymphoma (ATLL). In addition, HTLV-1 causes a neurodegenerative, inflammatory disease, HTLV-1-associated myelopathy (HAM), also termed tropical spastic paraparesis (TSP) (22, 41, 44, 54). Both ATLL and HAM/TSP develop as a consequence of viral persistence in T cells over decades. HTLV-1 persists lifelong in the presence of an active immune system. The virus is propagated in its proviral form, stimulating cell division and thus inducing clonal amplification of infected cells. Virus-carrying clones can persist over many years in infected individuals irrespective of leukemia development (5). In addition to typical retroviral structural proteins and polymerase, HTLV-1 encodes the two regulatory proteins Rex and Tax as well as the accessory proteins p12, p30, p13, and HBZ. Structural and accessory proteins and polymerase are required for productive infection but not for lymphocyte immortalization (35, 39). Recent work found that HBZ promotes lymphocyte proliferation (3). The regulatory proteins Tax and Rex are both essential for viral replication. While Tax strongly en-
* Corresponding author. Mailing address: Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universita¨t Erlangen-Nu ¨rnberg, Schlossgarten 4, 91054 Erlangen, Germany. Phone: 49-91318523013. Fax: 49-9131-8522101. E-mail:
[email protected] -erlangen.de. † Present address: European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom. ‡ Deceased. 䌤 Published ahead of print on 23 June 2010. 8732
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and long-lived T-cell populations have been described earlier (4, 12, 25, 32, 37, 42). Besides expression of common surface differentiation markers, cellular microRNAs known to be overexpressed in natural T-reg have recently been found by our group in HTLV-transformed cells (43). Whether there is a functional resemblance between HTLV-transformed cells that do express FOXP3 and T-reg was still not settled (12), but very recent studies indicate that T-reg are distinct from leukemia cells in ATLL (51). Cyclic AMP (cAMP) plays a fundamental role in cellular responses to many external stimuli, including growth factors or chemokines (7). Receptors such as G protein-coupled receptors provide the extracellular signal for accumulation of the second messenger cAMP. Within a cell, cAMP levels are tightly regulated by the activities of two enzymes: (i) adenylate cyclases convert ATP to cAMP, and (ii) phosphodiesterases hydrolyze the phosphodiester bond in cAMP (8). Recent work found cAMP to be upregulated in murine natural T-reg and to contribute to the suppressive potential of these cells through intercellular transport of cAMP to responder cells via gap junctions, leading to interleukin-2 (IL-2) repression (10). Moreover, phosphodiesterase 3B (PDE3B) was described to be downregulated in murine T-reg (21, 55). This led us to ask whether cAMP concentrations are modulated in HTLV/Tax-carrying T-cell lines. We found elevated cAMP levels, which were dependent on Tax, and PDE3B downregulation as consistent features of HTLV-transformed cells. MATERIALS AND METHODS Cell culture. The in vitro-HTLV-1-immortalized IL-2-independent T-cell lines C8166, C91-Pl, and MT-2; the ATLL-derived cell lines HuT-102 (with no IL-2), ATL-3 and StEd (both with 40 U/ml IL-2), and Champ, Juana W, and PaBe (with 20 U/ml IL-2); and the HAM/TSP-derived cell lines Abgho and Nilu (both with 40 U/ml IL-2) and Eva and Xpos (both with 20 U/ml IL-2) were cultured as previously described. Media were supplemented with IL-2 (Roche Diagnostics, Mannheim, Germany) as indicated (42). The acute lymphoblastic leukemia (ALL) cell lines Jurkat, CCRF-CEM (CEM), HuT-78, and Molt-4 were cultured in RPMI 1640 M containing 45% Panserin 401 (PAN-Biotech, Aidenbach, Germany), 10% fetal calf serum (FCS), glutamine (0.35 g/liter), and gentamicin. Cell lines TRI (24 ) and Tesi (48) were cultured as described previously. TRI and Tesi are primary human T cells immortalized by a expression cassette for the Tax protein, which was transduced with a rhadinoviral vector; the latter cell line features tetracycline-repressible Tax expression. For complete Tax repression, Tesi cells were grown in medium containing 1 g/ml tetracycline for 10 days (Tesi/Tet). To exclude nonspecific tetracycline effects, the cell lines TRI, MT-2 (Tax⫹), and Jurkat were also cultured in the presence of 1 g/ml tetracycline for 10 days and served as controls in at least three biological replicates. Cord blood lymphocytes (CBL) transformed with the rhadinoviral oncoproteins StpC/Tip (herpesvirus saimiri; CBL/M124) (2) or Tio (herpesvirus ateles; CBL/M158) were kindly provided by J.C. Albrecht (Institute of Clinical and Molecular Virology, Erlangen, Germany) and cultured like Jurkat cells. Isolation and culture of CD4ⴙ lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors (Institut fu ¨r Transfusionsmedizin, Suhl, Germany) by Ficoll-Hypaque gradient centrifugation (Biocoll, Biochrom, Berlin, Germany). Thereafter, CD4⫹ lymphocytes were enriched using the CD4⫹ T-cell isolation kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity was at least 95% as determined by flow cytometry (FACSCalibur, Becton Dickinson [BD], Heidelberg, Germany) after staining cells with monoclonal antibodies to CD4 (clone SK3; BD) as described previously (43). CD4⫹ T cells were cultured in hTC culture medium (Amaxa, Cologne, Germany) supplemented with 10% FCS and 1 mM glutamine and stimulated with 2 g/ml phytohemagglutinin P (PHA-P) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). After 24 h, cells were washed and cultured in the presence of 20 U/ml IL-2. Quantitation of intracellular cAMP. To quantify intracellular cAMP concentrations, cells were washed and lysed in 0.1 M HCl (107 cells/ml), followed by a
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cAMP-specific enzyme-linked immunosorbent assay (ELISA) (Correlate-EIA direct cyclic AMP enzyme immunoassay kit; Assay Designs, Ann Arbor, MI). In all assays, Jurkat T cells served as controls. They were treated with the adenylate cyclase-stimulating agent forskolin (15 M, 30 min; Sigma-Aldrich) in dimethyl sulfoxide (DMSO) or with solvent alone. In addition, increasing amounts of forskolin ranging from 0.1 M to 15 M were tested. Cyclic AMP levels were assayed in at least three independent experiments in duplicate, and the mean ⫾ standard error (SE) was calculated. Transient transfection. HTLV-positive MT-2 and C8166 cells were transfected using Cell Line Nucleofector kit T VCA-1002 (Amaxa) as described earlier (27). Briefly, 3 ⫻ 106 cells/sample were used and transfected in a Nucleofector II device (Amaxa) with program O-17. For the small interfering RNA (siRNA) experiments, 4 g of double-stranded synthetic siRNA targeting the coding region of Tax (27) was transfected. A nonsense siRNA (GFP-22 siRNA; Qiagen, Hilden, Germany) was used as a negative control. After 24 h, cells were harvested to detect intracellular cAMP, Tax mRNA, and Tax protein. A Myc-tagged expression plasmid (TrueORF cDNA clones and PrecisionShuttle vector system) encoding PDE3B according to GenBank accession number NM_000922.2 was obtained from Origene (Rockville, MD). To overexpress PDE3B, 2 g of PDE3B_myc or of a mock control (pCMV6_ENTRY; Origene) was transfected. To analyze the impact of Tax on endogenous PDE3B expression, Jurkat T cells were transfected as described previously (53). Briefly, Jurkat T cells were transfected by electroporation (EasyJect plus; Equibio, Ashford, United Kingdom) at 290 V and 1500 F with increasing amounts of pcTax (47) (5, 15, 30 g) or 30 g pcDNA3 (Invitrogen). Cells were harvested at 48 h after transfection to isolate RNA for detection of PDE3B and Tax mRNAs and to perform immunoblotting. Immunoblots. Immunoblotting was performed as described previously (53) using anti--actin (ACTB) mouse monoclonal antibodies (1:2,500; Sigma, Taufkirchen, Germany), anti-HSP90␣/ mouse monoclonal antibodies (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse antibodies to Tax (1:50) which were derived from the hybridoma cell line 168B17-46-34 (provided by B. Langton through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) (33). To detect PDE3B_myc, supernatants of 9E10 mouse hybridoma cells were used (20). Secondary antibodies conjugated with horseradish peroxidase were obtained from GE Healthcare (Little Chalfont, United Kingdom). Peroxidase activity was detected by enhanced chemiluminescence using an LAS-1000 ImageReader (Fujifilm, Tokyo, Japan) charge-coupled device (CCD) camera and a Kodak Image Station 4000MM PRO camera (Kodak). Intensities of specific bands were quantitated using AIDA (Raytest Isotopenmessgera¨te GmbH, Straubenhardt, Germany). At least three independent experiments were performed. Transcriptome analysis. Total cellular RNA was extracted from an in vitroHTLV-1-transformed cell line (MT-2), from an ATLL-derived cell line (StEd), and from postmitotic CD4⫹ T lymphocytes (RNeasy; Qiagen). CD4⫹ T cells from a healthy donor were cultured as described in “Isolation and culture of CD4⫹ lymphocytes” above until they reached a postmitotic state (i.e., they ceased to divide). Tax expression was checked by real-time reverse transcriptionPCR (RT-PCR). Transcriptome analysis was carried out on the Affymetrix HGU133plus 2.0 platform (Affymetrix, Santa Clara, CA). A biological replicate was generated for each chip. Analysis procedures followed standard Affymetrix guidelines. Differential expression analysis using ArrayAssist software (Stratagene, La Jolla, CA) compared the mean log values of the replicated chips. mRNA detection. Total cellular RNA was isolated from cell lines (RNA isolation kit II; Macherey-Nagel, Du ¨ren, Germany) and reverse transcribed to cDNA (Superscript II; Invitrogen, Karlsruhe, Germany) using random hexamer primers (Invitrogen). Real-time RT-PCR was performed in an ABI Prism 7500 sequence analyzer (Applied Biosystems, Foster City, CA) using 200 ng of cDNA. Primers and 6-carboxyfluorescein (FAM)/tetramethyl rhodamine (TAMRA)labeled probes for detection of -actin (ACTB) and Tax transcripts have been described before (46, 53). For quantitation of PDE3B transcripts, a TaqMan gene expression assay (Hs01057215_m1; Applied Biosystems) was used. Expression levels were computed by interpolation from standard curves generated from plasmids carrying the respective target sequences and calculation of the mean from triplicate samples. Each sample was measured in at least three biological replicates. ACTB was used for normalization. ChIP. To analyze chromatin modifications at the PDE3B promoter and in the ⫹1 region of PDE3B in HTLV-positive (MT-2 and Champ) and -negative (Jurkat) cells, 3 ⫻ 106 cells/sample were fixed and lysed and DNA-protein complexes were precipitated using 10 g anti-acetyl-histone H3 K9/14 (AcH3) (Millipore, Temecula, CA), anti-trimethyl-histone H3 K27 (Me3K27) (Millipore), or normal rabbit IgG (Millipore) as an isotype control. Chromatin immunoprecipitation (ChIP) was performed according to the manufacturer’s instruc-
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tions (Millipore). After recovery, identical amounts of precipitated or input DNA were measured by quantitative SYBR green real-time PCR (Platinum SYBR green qPCR SuperMix-UDG; Invitrogen) using the ⌬⌬CT method as described elsewhere (1). The percentage of specific binding to the PDE3B promoter (PDE3BP) and to a highly conserved region within the first intron (55) (PDE3B-I1) compared to input DNA was calculated and normalized to binding to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) promoter (GAPDHP), which served as a euchromatic control. The means from three independent experiments ⫾ SEs were calculated. Primers for amplification were PDE3BP-fwd (5⬘-TGCAGTCCGGTCATGAGG-3⬘), PDE3BP-rev (5⬘-GCTAT CTGTGAAGTACTGGTGAAAC-3⬘); PDE3B-I1-fwd (5⬘-TTTTGGCTACAT AGAGAACA-3⬘); and PDE3B-I1-rev (5⬘-CAGTGAAACATCAGCAGTACA A-3⬘). Primers for amplification of human PDE3B-I1 are homologous to a previously described conserved region in mice (R20) (55); primers for GAPDHP have been described earlier (1). Statistics. For statistical analysis, SPSS version 16.0.2 (SPSS, Chicago, IL) was used. The Mann-Whitney test was applied to evaluate differences between the different cell lines, whereas the t test (paired) was used in the tetracycline experiments. A P value of ⬍0.05 was considered to be significant. Microarray data. The microarray data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (16) and are accessible through GEO Series accession number GSE17718 (http://www.ncbi.nlm.nih.gov/geo /query/acc.cgi?acc⫽GSE17718).
RESULTS Elevated concentrations of intracellular cAMP in HTLV-1transformed T-cell lines. HTLV-1-transformed cells share some phenotypic properties with memory T cells and T-reg, both representing long-lived T-cell populations. To analyze whether HTLV-1-transformed cells also exhibit increased cAMP levels, similar to the case for murine T-reg (10), HTLV1-transformed cell cultures of various origins (in vitro transformed, ATLL derived, or HAM/TSP derived) were assayed for intracellular cAMP concentration using a competitive ELISA (Fig. 1A). HTLV-1-negative controls included four CD4⫹ ALL cell lines (CEM, HuT-78, Molt-4, and Jurkat) and primary CD4⫹ T cells from three healthy donors. As a positive control, Jurkat T cells were treated with increasing amounts of the adenylate cyclase-stimulating agent forskolin (0.1 M to 15 M) for 30 min, which is known to rapidly induce high cAMP levels. Overall, HTLV-1-infected cell lines exhibited high cAMP levels compared to those in ALL cells or primary CD4⫹ T cells. This was also true when CD4⫹ T cells were postmitotic (data not shown). Cyclic AMP concentrations in three of the infected cell lines (C8166, MT-2, and Xpos) reached levels measured in Jurkat cells treated with very large amounts of forskolin (10 M or 15 M). Moreover, these concentrations were comparable to cAMP levels measured in murine T-reg (10) (i.e., about 20 pmol/106 cells). Even though cAMP concentrations in HTLV-transformed cells of various origins (in vitro transformed, ATLL derived, or HAM/TSP derived) did not differ significantly, the values for HAM/TSP-derived cell lines Eva and Nilu were slightly lower than those for the ATLL-derived cell lines. The requirements of the HTLV-1transformed cultures for exogenous IL-2 did not influence cAMP levels, as there was no significant difference in cAMP between cell lines growing independently of IL-2 (C8166, C91Pl, MT-2, and HuT-102) and those that required IL-2 for their growth (P ⫽ 0.109). To test whether cAMP levels differ according to Tax expression levels, we measured Tax protein (Fig. 1B) and transcripts (Fig. 1C). Tax protein could be detected only in cell lines C91-Pl, C8166, MT-2 (with a known Tax-Env fusion of approximately 68 kDa [36, 50]), and HuT-
102 (Fig. 1B) in large amounts, while in all other HTLV-1transformed cell lines, expression levels were either very low or below the detection limit of Western blotting. Among the four cell lines expressing large amounts of Tax protein, three of them (C8166, C91-Pl, and MT-2) also exhibited high cAMP concentrations. In contrast, Tax transcripts were detectable in all types of HTLV-1-transformed cell lines (Fig. 1C). According to their Tax mRNA level being above or below the median of Tax mRNA, cell lines were grouped into Taxhigh and Taxlow cells. Although we could not detect a direct correlation between Tax mRNA and cAMP within the cell lines (r2 ⫽ 0.004; P ⫽ 0.851), a Mann-Whitney test on the results shown in Fig. 1A showed that cAMP concentrations were higher in the group of Taxhigh cells than in Taxlow cells (P ⫽ 0.02) (Fig. 1D). Moreover, the Mann-Whitney test confirmed a significant difference in cAMP levels in the group of all HTLV-1-infected cell lines compared to both ALL cells (P ⫽ 0.03) and primary CD4⫹ cells (P ⫽ 0.01). These data demonstrate high levels of intracellular cAMP as a consistent feature of HTLV-1-transformed T cells and give first hints on a possible relationship between Tax and cAMP. Effect of the viral transactivator Tax on intracellular cAMP levels. To determine whether the viral transactivator Tax causes increased cAMP levels in HTLV-transformed cells, we analyzed the impact of Tax expression on cAMP concentrations. Earlier studies have shown that Tax could not induce cAMP in Jurkat-derived cell systems (45), which we could confirm. We then extended our studies to the Tax-inducible cell line JPX9 (38) and to transiently Tax-transfected PBMC and CD4⫹ lymphocytes (data not shown). In none of the different model systems was Tax sufficient to induce cAMP. This led us to ask whether Tax expression is necessary for maintaining high intracellular cAMP concentrations. For this purpose, we transfected HTLV-positive C8166 cells with Tax siRNA. Levels of Tax (Fig. 1B and C) and intracellular cAMP (Fig. 1A) are high in this cell line. After transfection of the doublestranded Tax siRNA in C8166 cells, cAMP levels were reduced to 72% of those in cells transfected with a nonsense siRNA (Fig. 2A). Under these conditions, Tax mRNA expression was reduced to 26% (Fig. 2B) but could not be knocked down completely. On the protein level, the knockdown could also be demonstrated (Fig. 2C). These results gave the first indications that Tax expression is necessary for the maintenance of cAMP levels. As Tax expression could not be knocked down completely in C8166 cells and an influence of the Tax siRNA on other, overlapping viral genes could not be excluded, we further analyzed the role of Tax in maintaining intracellular cAMP in the cell line Tesi, which (i) carries only the Tax open reading frame and (ii) allows conditional Tax expression. The Tesi cell line had been established by transforming human cord blood lymphocytes (CBL) with a tetracycline-repressible HTLV-1 Tax gene using a rhadinoviral vector (48). After transformation, Tesi cells were in continuous culture for approximately 12 months. This system was chosen since it allows us to analyze the impact of Tax on cAMP levels of a human CD4⫹ T cell which is derived not from leukemia but directly from normal human lymphocytes. When Tax expression was repressed (Tesi/Tet), cAMP levels decreased (P ⫽ 0.007) (Fig. 2D). In the presence of Tax, cAMP levels were about 5-fold higher
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FIG. 1. Upregulation of intracellular cAMP in Tax/HTLV-1-positive cells. (A) Intracellular cAMP levels in Tax/HTLV-1-positive cell lines and HTLV-1-negative cells were measured by ELISA. Tax/HTLV-1-positive cell lines included in vitro-transformed, ATLL-derived, and HAM/TSPderived cells, while HTLV-1-negative cells included ALL-derived cell lines and primary CD4⫹ lymphocytes from healthy donors (HD). Jurkat cells treated with increasing amounts of forskolin (0.1 M to 15 M) for 30 min served as positive controls. Bars represent the means from at least three independent experiments, each performed in duplicate, ⫾ SEs. The dotted line indicates cAMP levels in natural murine T-reg as measured by Bopp et al. (10). (B) Tax protein was detected by immunoblotting. Detection of Hsp90␣/ served as loading control. (C) Transcripts of Tax were quantified by real-time RT-PCR. Relative copy number was determined by normalizing the Tax transcripts to those of ACTB. The means from three independent experiments ⫾ SEs are shown. The dotted line indicates the median (relative copy number ⫽ 0.35) of Tax mRNA. (D) Intracellular cAMP levels of the HTLV-1-positive cell lines shown in panel A were compared with those of HTLV-1 negative ALL cells and CD4⫹ lymphocytes from healthy donors using a two-tailed Mann-Whitney test. Tax/HTLV-1-positive cell lines were further subdivided into Taxhigh and Taxlow cell lines according to their Tax mRNA levels being above or below the median of Tax mRNA as depicted by the dotted line in panel C. Horizontal bars indicate the median; n.s. indicates not significant. f, Taxhigh/HTLV-1-positive cell lines; 䡺, Taxlow/HTLV-1-positive cell lines; F, Tax/HTLV-1-negative ALL cells; Œ, CD4⫹ lymphocytes.
than they were without Tax (Fig. 2E). Tax transcripts were completely repressed in the presence of tetracycline (Fig. 2F). In contrast, CBL/M124 and CBL/M158, which are fast-proliferating rhadinovirus-transformed cell lines immortalized by
the viral oncogenes StpC/Tip and Tio, respectively, showed only low cAMP levels (Fig. 2G) in comparison to HTLV-1-/ Tax-transformed cell lines (Fig. 1A and 2D). To exclude tetracycline effects on cAMP levels independent of Tax, TRI
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FIG. 2. Necessity of Tax for the maintenance of intracellular cAMP. (A to C) C8166 cells (Tax positive) were transfected either with Tax siRNA (si Tax) or nonsense siRNAs (si nons) and harvested after 24 h. Values obtained for cells treated with si nons were set as 100%, and the means from three independent experiments ⫾ SEs are shown. (A) Intracellular cAMP levels were measured by ELISA. (B) Transcripts of Tax were quantified by real-time RT-PCR. The relative copy number was determined by normalizing the Tax transcripts to those of ACTB. (C) Tax and ACTB proteins were detected by immunoblotting and quantitatively evaluated. (D to F) Tesi cells (Tax positive) were cultivated in the presence of 1 g/ml tetracycline for 10 days to repress Tax expression (Tesi/Tet). The means from three independent experiments ⫾ SEs are shown. (D) cAMP was measured by ELISA and compared for Tesi and Tesi/Tet cells using a paired t test (P ⫽ 0.007). (E) In addition to Tesi cells, cAMP levels were measured in Tax-positive cell lines TRI and MT-2 and in Tax-negative control cell line Jurkat after tetracycline treatment (1 g/ml; 10 days) and compared to those in untreated cells. The change of cAMP levels was calculated (fold change ⫽ cAMP level in untreated cells/cAMP level in tetracycline-treated cells). (F) Transcripts of Tax were quantified by real-time RT-PCR. The relative copy number was determined by normalizing the Tax transcripts to those of ACTB. (G) Cord blood lymphocytes transformed with the rhadinoviral oncoproteins Tio (CBL/M158) and StpC/Tip (CBL/M124) (2) were analyzed for intracellular cAMP by ELISA. The means ⫾ SEs from three independent experiments are shown.
cells, which closely resemble Tesi cells in terms of their origin, growth characteristics, and phenotype (24), as well as HTLVpositive MT-2 cells and uninfected Jurkat cells were cultured in the presence of tetracycline for 10 days. None of the cell lines tested except Tesi exhibited changes in cAMP levels (Fig. 2E). Taking the results together, we demonstrated in two different Tax transformation systems that repression of the viral oncoprotein Tax is associated with downregulation of cAMP in Tax/HTLV-1-transformed cells. Thus, Tax appears to contribute to maintenance of intracellular cAMP. As Tax was not sufficient to induce cAMP, we searched for cellular determinants of cAMP metabolism by comparing the transcriptomes of HTLV-1-transformed cells to those of uninfected CD4⫹ lymphocytes. Expression profiles of cAMP-degrading PDEs in HTLV-1transformed human T lymphocytes. Cyclic AMP is produced from ATP by adenylate cyclases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs). Transcriptome analyses allowed us to compare HTLV-infected and uninfected T cells with regard to genes involved in regulation of cAMP levels.
Total cellular RNAs from the in vitro-HTLV-1-transformed cell line MT-2, from the ATLL-derived cell line StEd, and from postmitotic, uninfected primary CD4⫹ lymphocytes were assayed. The last were chosen since primary uninfected and HTLV-infected lymphocytes begin to differ in their growth properties at this time. While uninfected cells stop proliferating, some HTLV-infected lymphocytes continue to grow and can be established in continuous cell culture. Table 1 lists expression data of those PDEs, which are (i) present in lymphocytes and (ii) use cAMP as a substrate. Detection of PDE3B, PDE4A-D, and PDE7A confirmed earlier observations (17). In addition, we found expression of PDE8A. PDE1B and -3A were not expressed. Although activity of whole PDE4 and PDE3 families has been observed previously (17), quantitation of PDE transcripts in HTLV-transformed cells and comparison to uninfected CD4⫹ cells have not been described before. Most of the PDEs showed no differential expression when HTLV-transformed and uninfected CD4⫹ lymphocytes were compared, with the exception of PDE4D and PDE8A, which slightly differed in in vitro-transformed
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TABLE 1. Expression of cAMP-converting phosphodiesterases detected in microarrays of HTLV-1-transformed cell lines StEd and MT-2 in comparison to postmitotic HTLV-1-negative CD4⫹ lymphocytes StEd vs CD4⫹
Avg signala Gene
PDE1B PDE3A PDE3B PDE4A PDE4B PDE4C PDE4D PDE4D PDE7A PDE7B PDE8A PDE8A
MT-2 vs CD4⫹
MT-2
CD4⫹
Fold changeb
Regulation
P value for fold change
Fold change
Regulation
P value for fold change
Probe set
StEd
(5) (4) 32 53 695 264 98 641 46 (17) 86 (8)
(2) (6) 10 424 426 263 (12) 97 18 (10) 341 (6)
(15) (3) 331 441 495 247 44 783 31 36 58 (5)
NAc NA 10 8 1 1 2 1 2 NA 1 NA
NA NA Down Down NA NA Up NA Up Down NA NA
NA NA 0.001 0.002 0.067 0.648 0.039 0.065 0.145 NA 0.072 NA
NA NA 36 1 1 1 NA 8 2 NA 6 NA
NA NA Down NA NA NA NA Down Down NA Up NA
NA NA 0.018 0.770 0.233 0.621 NA 0.001 0.143 NA 0.000 NA
206444_at 234563_at 222317_at 204735_at 203708_at 206792_x_at 236610_at 204491_at 1552343_s_at 220343_at 212522_at 215635_at
a
Average fluorescence intensities are rounded to integers; values in parentheses were deemed absent calls. Values are rounded to integers. c NA, not applicable. b
MT-2 cells. Remarkably, among the PDEs described to be present in lymphocytes, PDE3B was the only phosphodiesterase found to be strongly downregulated in HTLV-1-carrying cells. ATLL-derived and in vitro-HTLV-transformed cells had 10-fold- and 36-fold-lower PDE3B expression levels than CD4⫹ lymphocytes, respectively. Downregulation of PDE3B in HTLV-1-infected cells. Downregulation of PDE3B in HTLV-1-infected cell cultures was verified by real-time RT-PCR. PDE3B-specific transcripts in RNAs from a series of HTLV-1-infected cultures and uninfected control samples were quantified (Fig. 3A). These analyses revealed expression of PDE3B in all samples, although expression levels were very low in some. Subsequent statistical analysis found PDE3B transcripts to be significantly downregulated in HTLV-1-transformed cells (P ⫽ 0.008) compared to uninfected leukemic cell lines and primary CD4⫹ T lymphocytes (P ⫽ 0.015) from uninfected individuals (Fig. 3B). In this respect, HTLV-1-infected cells resemble memory T cells (A. K. Kress, unpublished observations) and regulatory T cells (21, 55), which also express low levels of PDE3B. When cAMP concentrations and PDE3B mRNAs were sampled simultaneously, which was tested for 8 of 11 HTLV-1-transformed cell lines, their levels were inversely correlated in HTLV-1-positive cell lines (r2 ⫽ ⫺0.739; P ⫽ 0.006) but not in uninfected ones (r2 ⫽ 0.855; P ⬎ 0.05). Downregulation of PDE3B transcripts was independent of Tax, as transient transfection of Jurkat T cells with increasing amounts of Tax did not change the expression level of endogenous PDE3B although both Tax mRNA and protein expression rose. (Fig. 3C). This observation was also confirmed in Tesi cells and in PBMCs transfected with Tax (data not shown). Decrease of cAMP concentrations after overexpression of PDE3B in HTLV-1-infected cells. As PDE3B was downregulated in HTLV-1-transformed cells while cAMP concentrations were elevated, we asked whether a change of PDE3B expression levels would affect cAMP concentrations in HTLV1-transformed cells. Due to very low expression of PDE3B (Fig. 3A and B), knockdown experiments were not appropriate. Therefore, PDE3B was overexpressed in two HTLV-1-
transformed cell lines. MT-2 and C8166 cells were transfected with a PDE3B_myc expression plasmid and an empty control vector (mock). After 24 h, PDE3B and Tax expression (mRNA/protein) as well as cAMP concentrations were measured simultaneously. A strong increase of PDE3B transcript could be detected by real-time RT-PCR (Fig. 4A) in cells transfected with PDE3B_myc compared to mock-transfected cells, while Tax mRNA (Fig. 4A) and protein (Fig. 4B) expression levels remained unchanged. In addition, PDE3B_myc protein could be detected in immunoblots (Fig. 4B). To analyze whether overexpression of PDE3B had an impact on cAMP regulation in HTLV-transformed cells, cAMP was measured by ELISA. In both MT-2 and C8166 cells, cAMP concentrations were reduced to approximately 60% of those in mocktransfected cells (Fig. 4C). This indicates that PDE3B_myc was active and capable of cleaving cAMP in HTLV-1-transformed cells, while viral transcripts (Tax) remained unaffected. These observations support the hypothesis that PDE3B downregulation facilitates upregulation of cAMP in HTLV-1-transformed cells, as higher expression levels of PDE3B resulted in decreased cAMP concentrations. Chromatin modifications at the PDE3B promoter in HTLVtransformed cells. Endogenous PDE3B was downregulated in HTLV-1-transformed cells, and this could not be induced by Tax expression. We therefore searched for chromatin modifications at the PDE3B promoter (PDE3BP) and at the ⫹1 region of PDE3B in HTLV-1-transformed cells. On the one hand, a region upstream of the transcriptional start site of PDED3B (bp ⫺1421 to ⫺1242) (Fig. 5A) was chosen, because it is located in the center of a 2-kbp portion of 5⬘ flanking sequences with promoter activity (40) and contains in silicopredicted nuclear factor of activated T cells (NFAT) and overlapping NFAT/forkhead domain (FHD) binding sites. On the other hand, in the ⫹1 region of PDE3B a highly conserved region within the first intron (PDE3B-I1) that is known to be associated with inhibitory histone modifications and FOXP3 binding in murine T-reg (55), thereby potentially mediating PDE3B repression, was analyzed. Acetylation of histone 3 (H3) at K9/14 (AcH3) is associated with chromatin accessi-
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FIG. 3. Downregulation of PDE3B in HTLV-1-transformed cells. (A) Transcripts of PDE3B were quantified by real-time RT-PCR. The relative copy number was determined by normalizing the PDE3B transcripts to those of ACTB. The means from three independent experiments ⫾ SEs are shown. (B) The relative copy numbers of PDE3B mRNA in Tax/HTLV-1-positive cell lines as shown in panel A were compared to those in Tax/HTLV-1-negative ALL cell lines and uninfected primary CD4⫹ lymphocytes using a two-tailed Mann-Whitney test. Horizontal bars indicate the median, f, Tax/HTLV-1-positive cell lines; F, Tax/HTLV-1-negative ALL cells; Œ, CD4⫹ lymphocytes. (C) Jurkat cells were transfected with increasing amounts (5, 15, or 30 g) of Tax expression plasmid or 30 g of pcDNA as control. After 48 h, Tax and PDE3B transcripts were quantified by real time RT-PCR and normalized to those of ACTB. Relative copy numbers ⫾ SEs from one representative experiment of three are shown. In parallel, Tax and ACTB proteins were detected by immunoblotting.
bility and transcriptional activity, whereas trimethylation of H3 at K27 (Me3K27) is generally correlated with the repression of gene expression (9). Chromatin immunoprecipitation (ChIP) was performed with two HTLV-1-transformed cell lines (Champ, MT-2) and an uninfected control (Jurkat) (Fig. 5B). The percentage of specific binding to AcH3 or Me3K27 compared to input DNA was calculated and normalized to binding to the euchromatic GAPDH promoter (GAPDHP). As a specificity control, an isotype control antibody was used, which showed no binding. In contrast to the case for the transcriptionally active GAPDHP, acetylation of histone H3 at PDE3BP and PDE3B-I1 was weak, reaching intermediate levels in HTLV-negative Jurkat cells (PDE3BP) but only very low levels in HTLV-positive cells. However, trimethylation of H3K27 at both PDE3BP and PDE3B-I1 was much more pronounced in HTLV-transformed cells (35- to 197-fold
and 30- to 60-fold, respectively) than in uninfected cells (7-fold and 1-fold, respectively). Therefore, inhibitory histone modifications are strongly associated not only with the PDE3B promoter in HTLV-transformed cells but also with a highly conserved region within the ⫹1 region of PDE3B (PDE3B-I1). This is in accordance with decreased PDE3B gene expression. In summary, downregulation of phosphodiesterase PDE3B is a consistent feature of HTLV-1-infected cells in culture, strongly suggesting that it could contribute to Tax-mediated cAMP maintenance. DISCUSSION The present study has shown that the second messenger cAMP is consistently upregulated in HTLV-1-transformed cells and that the presence of the viral oncoprotein Tax is
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FIG. 4. Decrease of intracellular cAMP after overexpression of PDE3B in HTLV-1-transformed cells. HTLV-1-transformed cell lines MT-2 and C8166 were transfected with 2 g of a PDE3B_myc expression plasmid or a control plasmid (mock). After 24 h, cells were analyzed. (A) Tax and PDE3B transcripts were quantified by real time RT-PCR and normalized to those of ACTB. Relative copy numbers after transfection of PDE3B_myc were compared to those for mock-transfected cells. The means ⫾ SEs from three independent experiments are depicted. (B) PDE3B, Tax, and ACTB proteins were detected by immunoblotting. (C) Intracellular cAMP levels were measured by ELISA, and the value for mock-transfected cells was set as 100%. The mean change (⫾SE) of cAMP concentrations after PDE3B_myc transfection from three independent experiments is shown.
necessary for the maintenance of high intracellular cAMP levels. Specific downregulation of the cAMP-degrading phosphodiesterase PDE3B in HTLV-1-transformed cells is associated with inhibitory histone modifications at both the promoter
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and the ⫹1 region and could therefore support maintenance of high cAMP levels. We found upregulation of the second messenger cAMP in a broad array of Tax/HTLV-1-transformed T-cell cultures, including cells derived from in vitro immortalization by a Tax expression cassette alone or by the entire virus and from ATLL or HAM/TSP patients (Fig. 1A and 2D). In general, cAMP is known to be a potent inhibitor of T-cell growth and proliferation (19). Although HTLV-transformed cells exhibit elevated cAMP levels, the proliferative capacities of most cell lines are similar to that of uninfected Jurkat cells, in which cAMP levels are low (Fig. 1A). Therefore, in HTLV-transformed cells, high cAMP levels do not seem to be associated with antiproliferative effects. As the HTLV long terminal repeat (LTR) is inducible by an increase of cAMP (45), the upregulation of cAMP may contribute to stimulating rather than antagonizing replication in cells that contain integrated HTLV proviruses. In addition, the antiproliferative function of cAMP in uninfected cells could be overcome by costimulation during T-cell activation in HTLV-infected cells. Interestingly, addition of cAMP to B cells was described to increase the expression of costimulatory molecules such as 4-1BBL and CD80 (14). These receptors are also expressed on HTLVtransformed cells (15, 42). Several selective advantages could arise from elevated cAMP levels in HTLV-carrying cells during latency and disease progression. (i) cAMP can maintain basal transcription from the viral LTR at low levels even without Tax expression (45). (ii) cAMP might be essential for maintenance of P-CREB and for phosphorylation of other protein kinase A (PKA)dependent target proteins, as CREB is maximally phosphorylated in HTLV-transformed cells (31). (iii) cAMP upregulation in HTLV-transformed cells could contribute to immune control of uninfected cells like in murine T-reg. These cells share phenotypic similarities with HTLV-transformed cells and are known to exhibit their suppressive potential by pumping cAMP via gap junctions to responder cells, which leads to IL-2 inhibition (10). This mechanism could also be used by HTLVtransformed cells to perform immune suppression, as earlier studies described a possible suppressive phenotype of ATLL cells in a cell-cell contact-dependent manner (12). In this respect, HTLV-transformed cells are known to form functional gap junctions with endothelial cells (18) and also with Jurkat T cells and PBMC (data not shown). The Tax-inducible expression of connexin 43 (6), a major component of lymphocytic gap junctions with enhanced selectivity for cAMP (28), strengthens this argument. On the other hand, cAMP may be secreted, as CD4⫹ lymphocytes can exert regulatory functions through the release of cAMP into the extracellular space (52). ATLL sera are known to suppress the production of IL-2 via unknown suppressive factors that are sensitive to acid treatment (49), which is also a feature of cAMP (30). Experiments are now in progress to test the possible functional consequences of the observed elevated cAMP levels. Although Tax was not sufficient to induce cAMP, the levels were increased in the presence of Tax, especially in the group of cell lines expressing large amounts of Tax (Fig. 1C). More importantly, repression of Tax in C8166 and Tesi cells led to a decrease of cAMP, which was much more pronounced when Tax mRNA was specifically and completely repressed as shown
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FIG. 5. Inhibitory chromatin modifications at the PDE3B promoter and the ⫹1 region of PDE3B in HTLV-transformed cells. (A) Schematic diagram of the PDE3B promoter and the PDE3B-coding region. Arrow pairs indicate primer binding sites, and ⫹1 indicates the transcriptional start site. (B) Histone modifications at the PDE3B promoter and the ⫹1 region of PDE3B were detected by ChIP using HTLV-positive (Champ and MT-2) and HTLV-negative (Jurkat) cell lines. Antibodies against acetylated H3 K9/14 (AcH3), trimethylated H3K27, or an isotype control were used. For SYBR green real-time PCR, primers corresponded to regions of the PDE3B and GAPDH promoters and to a highly conserved region in the first intron of PDE3B (PDE3B-I1). Identical amounts of precipitated or input DNA were used for each PCR. The percentage of specific binding compared to input DNA was calculated and normalized to binding to the GAPDH promoter. Results indicate the means from three independent experiments ⫾ SEs.
in Tesi cells (Fig. 2). Therefore, Tax/HTLV contributes to the phenotype, which appears to be dependent on a number of additional factors in transformation and tumor progression. As intracellular cAMP concentrations are also elevated in longlived T-cell populations (10), maintenance of cAMP by HTLV-1 Tax provides a causal linkage between Tax function and differentiation of infected lymphocytes into long-lived Tcell populations. Elevated cAMP levels do not seem to be a general feature of rhadinovirus-transformed T cells such as Tesi, as T cells transformed with other lymphotropic tumor viruses, such as herpesvirus saimiri (StpC/Tip) and herpesvirus ateles (Tio), harbor low cAMP concentrations as do uninfected CD4⫹ T cells (Fig. 1 and 2). Our data obtained in cell culture have suggested a role of elevated cAMP levels for virus persistence in vitro, and this may also be true for infected patients. Tax is necessary for cAMP maintenance in vitro. In primary ATLL cells, Tax expression is very low. Thus, detection of cAMP concentrations in patients by ELISA would require higher numbers of Tax-expressing cells than usually observed to guarantee sensitivity. Tax could not replace all necessary cellular signals which are required for induction of cAMP. Thus, we searched for the possible relevance of cellular components of cAMP metabolism by use of microarrays. We found PDE3B as the only phosphodiesterase to be strongly downregulated in the presence of virus infection, while most other PDEs that are known to be expressed in T lymphocytes remained unchanged. Downregulation of PDE3B was a consistent feature of HTLV-transformed cells. This is supported by observations describing low enzymatic activity of the whole PDE3 family, including PDE3A and PDE3B, in the context of HTLV-1-infection (17). How-
ever, PDE3A is not expressed in HTLV-transformed cells (Table 1). As PDE3B is downregulated in murine T-reg (21, 55), its expression profile fits the long-lived phenotype of HTLV1-transformed cells (4, 12, 25, 32, 37, 42). The decrease of PDE3B was inversely correlated with cAMP levels in in vitro-transformed ATLL-derived and HAM/TSP-derived cells, which provides a possible explanation for the elevated cAMP levels in HTLV-transformed cells. This correlation is further corroborated by the decrease of cAMP in HTLV-1transformed cells after overexpression of PDE3B (Fig. 4). Transcriptional regulation of the PDE3B gene may be a key determinant of cAMP modulation in HTLV-1-transformed cells. Thus, we focused our analyses on the promoter of the PDE3B gene, which harbors potential binding sites for the transcription factors NFAT and FOXP3. In addition, we analyzed a region within the first intron of PDE3B (PDE3B-I1) which is homologous to a highly conserved region (PDE3BR20) in murine T reg cells. In these cells, transcriptional repression of PDE3B is mediated by FOXP3 binding to PDE3BR20 and associated with inhibitory histone modifications at this region (55). We obtained similar results when testing FOXP3 binding at PDE-I1 in the ATLL-derived cell line JuanaW (data not shown), but we focused our analyses on histone modifications. Inhibitory histone modifications, i.e., trimethylation of H3K27, were found at both the PDE3B promoter and PDE3B-I1 in HTLV-transformed cells (Fig. 5B). This could explain the transcriptional shutoff of PDE3B expression. In principle, PDE3B activity is rapidly regulated by phosphorylation, providing a fast supply of the active protein (8). Epigenetic silencing of PDE3B, however, provides stable long-term effects. In particular, H3K27 trimethylation is cata-
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lyzed by protein complexes which mediate mitotic inheritance of lineage-specific gene expression patterns (9). These results suggest that the long-lived phenotype of HTLV-transformed cells, namely, PDE3B downregulation, supports Tax-mediated cAMP maintenance. Apparently, HTLV-transformed cells assume biological features of a long-lived T-cell population, such as upregulation of the second messenger cAMP and downregulation of the cAMP-cleaving phosphodiesterase PDE3B. This may contribute to viral persistence, survival, and growth. ACKNOWLEDGMENTS This article is dedicated to the memory of Ralph Grassmann, who died on 1 July 2008. This work was supported by the EU (INCA, LSHC-CT-2005018704), by Deutsche Forschungsgemeinschaft (DFG GR 1224/3–1, DFG-GRK1071), and by Akademie der Wissenschaften und der Literatur Mainz. We thank Charles R. M. Bangham (Imperial College, London) for helpful discussions. A.K.K. performed research, analyzed, and interpreted data and wrote the manuscript; G.S. and M.K. performed research; K.P. and B.F. analyzed and interpreted data and participated in writing the manuscript; and R.G. analyzed and interpreted data and designed the research concept. The authors declare no competing financial interests. REFERENCES 1. Alberter, B., and A. Ensser. 2007. Histone modification pattern of the Tcellular herpesvirus saimiri genome in latency. J. Virol. 81:2524–2530. 2. Albrecht, J. C., B. Biesinger, I. Muller-Fleckenstein, D. Lengenfelder, M. Schmidt, B. Fleckenstein, and A. Ensser. 2004. Herpesvirus ateles Tio can replace herpesvirus saimiri StpC and Tip oncoproteins in growth transformation of monkey and human T cells. J. Virol. 78:9814–9819. 3. Arnold, J., B. Zimmerman, M. Li, M. D. Lairmore, and P. L. Green. 2008. Human T-cell leukemia virus type-1 antisense-encoded gene, Hbz, promotes T-lymphocyte proliferation. Blood 112:3788–3797. 4. Bal, H. P., J. Cheng, A. Murakami, A. S. Tallarico, W. Wang, D. Zhou, T. J. Vasicek, and W. A. Marasco. 2005. GITR overexpression on CD4⫹CD25⫹ HTLV-1 transformed cells: detection by massively parallel signature sequencing. Biochem. Biophys. Res. Commun. 332:569–584. 5. Bangham, C. R., and M. Osame. 2005. Cellular immune response to HTLV-1. Oncogene 24:6035–6046. 6. Bazarbachi, A., M. R. Abou, A. Gessain, R. Talhouk, H. El Khoury, R. Nasr, O. Gout, R. Sulahian, F. Homaidan, H. de The, O. Hermine, and M. E. El Sabban. 2004. Human T-cell lymphotropic virus type I-infected cells extravasate through the endothelial barrier by a local angiogenesis-like mechanism. Cancer Res. 64:2039–2046. 7. Beavo, J. A., and L. L. Brunton. 2002. Cyclic nucleotide research—still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3:710–718. 8. Bender, A. T., and J. A. Beavo. 2006. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol. Rev. 58:488–520. 9. Bernstein, B. E., A. Meissner, and E. S. Lander. 2007. The mammalian epigenome. Cell 128:669–681. 10. Bopp, T., C. Becker, M. Klein, S. Klein-Hessling, A. Palmetshofer, E. Serfling, V. Heib, M. Becker, J. Kubach, S. Schmitt, S. Stoll, H. Schild, M. S. Staege, M. Stassen, H. Jonuleit, and E. Schmitt. 2007. Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression. J. Exp. Med. 204:1303–1310. 11. Boxus, M., J. C. Twizere, S. Legros, J. F. Dewulf, R. Kettmann, and L. Willems. 2008. The HTLV-1 Tax interactome. Retrovirology 5:76. 12. Chen, S., N. Ishii, S. Ine, S. Ikeda, T. Fujimura, L. C. Ndhlovu, P. Soroosh, K. Tada, H. Harigae, J. Kameoka, N. Kasai, T. Sasaki, and K. Sugamura. 2006. Regulatory T cell-like activity of Foxp3⫹ adult T cell leukemia cells. Int. Immunol. 18:269–277. 13. Copeland, K. F., A. G. Haaksma, J. Goudsmit, P. H. Krammer, and J. L. Heeney. 1994. Inhibition of apoptosis in T cells expressing human T cell leukemia virus type I Tax. AIDS Res. Hum. Retroviruses 10:1259–1268. 14. DeBenedette, M. A., N. R. Chu, K. E. Pollok, J. Hurtado, W. F. Wade, B. S. Kwon, and T. H. Watts. 1995. Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp. Med. 181:985–992. 15. Dezzutti, C. S., D. L. Rudolph, and R. B. Lal. 1995. Infection with human T-lymphotropic virus types I and II results in alterations of cellular receptors, including the up-modulation of T-cell counterreceptors CD40, CD54, and CD80 (B7-1). Clin. Diagn. Lab. Immunol. 2:349–355.
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