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ABSTRACT: Transgenic (Tg) mice expressing Tax, a human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, develop mature T-cell leukemia/lymphoma. The leukemic cells in Tg mice expressing Tax show p53 dysfunction and nuclear factor-κB (NF-κB) activation, similar to that seen in adult T-cell leukemia/lymphoma (ATLL) cells from patients infected with HTLV-1. However, it is unclear when these effects occur in HTLV-1 carriers during the development of ATLL. Here, we examined p53 function and NF-κB activity before the onset of leukemia in Tax-expressing Tg (Tax-Tg) mice between 4 and 25 months of age. At 4-10 months of age, 71% of mice showed p53 inactivation, without evidence for NF-κB activation, even though tax expression was consistent from 4 to 25 months of age. The decline in p53 function resulted from decreased p53 accumulation after DNA damage. From 11 months of age onwards, 75% of mice showed p53 dysfunction and 37.5% showed constitutive NF-κB activation with the components of p50 and RelB. An NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced NF-κB activity (i.e., p50/RelB), but did not restore p53 function. In vivo experiments, treatment with DHMEQ until 24 months of age prevented the onset of T-cell leukemia in Tax-Tg mice. These results suggest that the Tax-induced decline in p53 function, which is independent of NF-κB activation in the early stage, might be the first stage in the onset of ATLL. NF-κB activity is involved in the later stages of ATLL onset.
Carcinogenesis 04/2013; · 5.70 Impact Factor
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ABSTRACT: Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.
Microbes and Infection 03/2013; · 3.10 Impact Factor
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ABSTRACT: Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection.
Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4-3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4-3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3' LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication.
The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4-3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle.
Retrovirology 05/2012; 9:38. · 6.47 Impact Factor
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ABSTRACT: Mammalian RNAi machinery facilitating transcriptional gene silencing (TGS) is the RNA-induced transcriptional gene silencing-like (RITS-like) complex, comprising of Argonaute (Ago) and small interfering RNA (siRNA) components. We have previously demonstrated promoter-targeted siRNA induce TGS in human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV), which profoundly suppresses retrovirus replication via heterochromatin formation and histone methylation. Here, we examine subcellular co-localization of Ago proteins with promoter-targeted siRNAs during TGS of SIV and HIV-1 infection. Analysis of retrovirus-infected cells revealed Ago1 co-localized with siRNA in the nucleus, while Ago2 co-localized with siRNA in the inner nuclear envelope. Mismatched and scrambled siRNAs were observed in the cytoplasm, indicating sequence specificity. This is the first report directly visualizing nuclear compartment distribution of Ago-associated siRNA and further reveals a novel nuclear trafficking mechanism for RITS-like components involving the actin cytoskeleton. These results establish a model for elucidating mammalian TGS and suggest a fundamental mechanism underlying nuclear delivery of RITS-like components.
Nucleic Acids Research 11/2011; 40(4):1579-95. · 8.03 Impact Factor
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Kazuo Suzuki, Takaomi Ishida,
Makoto Yamagishi,
Chantelle Ahlenstiel,
Sanjay Swaminathan,
Katharine Marks,
Daniel Murray,
Erin M McCartney,
Michael R Beard,
Marina Alexander,
Damian F J Purcell,
David A Cooper,
Toshiki Watanabe,
Anthony D Kelleher
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ABSTRACT: We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NF-kB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4, and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.
RNA biology 11/2011; 8(6):1035-46. · 5.56 Impact Factor
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ABSTRACT: To gain an insight into the microenvironmental regulation of nuclear factor (NF)-κB activity in the progression of leukemia, we established a bioluminescent imaging model of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) cells transduced with a NF-κB/luciferase (Luc) reporter and cocultured with murine stromal cells and cytokines. Stromal cells alone did not augment Luc activity, taken as an index of NF-κB, but Luc activity was synergistically upregulated by the combination of stromal cells and tumor necrosis factor (TNF)-α. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB DNA binding, rapidly induced the apoptosis of Ph+ALL cells, indicating that NF-κB is necessary for the growth and survival of these cells. However, the DHMEQ-induced suppression of NF-κB activity and the apoptosis of leukemia cells were attenuated by the presence of stromal cells and TNF-α. In NOD-SCID mice transplanted with NF-κB/Luc reporter-containing Ph+ALL cell lines and monitored periodically during the progression of the leukemia, murine TNF-α was significantly expressed in lesions in which the leukemia cells emitted a significant NF-κB signal. These results support the notion that TNF-α also triggers microenvironmental upregulation of NF-κB activity in vivo. Collectively, the results indicated that TNF-α-stimulated microenvironment may contribute to the survival and progression of Ph+ALL cells through the synergistic upregulation of NF-κB activity.
Cancer Science 07/2011; 102(11):2014-21. · 3.33 Impact Factor
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ABSTRACT: HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein–protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax–SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus. (Cancer Sci 2011; 102: 260–266)
Cancer Science 11/2010; 102(1):260 - 266. · 3.33 Impact Factor
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ABSTRACT: Basal-like breast cancers are triple-negative (estrogen receptor negative, progesterone receptor negative, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) negative) tumors with an aggressive clinical behavior that lacks effective molecular targets for therapy. We reported previously that the basal-like subtype cell lines display high constitutive nuclear factor (NF)-κB activation, whose inhibition in the basal-like subtypes suppressed their proliferation. Moreover, NF-κB-inducing kinase (NIK) is involved in the constitutive NF-κB activation. Here, we report that enhanced NIK expression, which is exclusively observed in the basal-like subtype rather than the luminal-like subtype or non-tumorigenic mammary epithelial cells, is caused by epigenetic alteration of the NIK gene. The stability of NIK mRNA and transcriptional activity driven by the NIK promoter are similar in the basal-like and luminal-like subtypes. However, histone H3 acetylation levels were up-regulated in the basal-like subtype. Furthermore, treatment of the luminal-like subtype with a histone deacetylase inhibitor, valproic acid, significantly increased NIK expression. Although DNA methylation of the NIK locus was not detected, NIK expression also increased when the luminal-like subtype was treated with 5-azacytidine, which inhibits histone H3-Lys-9 dimethylation in addition to DNA methylation. Taken together, these results suggest that the closed chromatin structure mediated by histone H3 methylation and deacetylation suppresses NIK expression in the luminal-like subtype, whereas disruption of these suppression mechanisms leads to enhanced NIK expression and the constitutive NF-κB activation in the basal-like subtype. Thus, NIK and genes induced by the NIK-mediated constitutive NF-κB activation could be therapeutic targets of basal-like breast cancer.
Cancer Science 11/2010; 101(11):2391-7. · 3.33 Impact Factor
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ABSTRACT: HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus.
Cancer Science 09/2010; 102(1):260-6. · 3.33 Impact Factor
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ABSTRACT: Previous reports indicate that nuclear factor (NF)-kappaB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-kappaB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-kappaB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-alpha and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-kappaB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-kappaB is a molecular target for controlling active HIV-1 replication.
Microbes and Infection 02/2010; 12(5):400-8. · 3.10 Impact Factor
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ABSTRACT: Primary effusion lymphoma (PEL) is a refractory malignancy caused by human herpes virus 8 (HHV-8) in immunocompromised individuals. The tumor cells of PEL are characterized by constitutive NF-kappaB activation. Dehydroxymethylepoxyquinomicin (DHMEQ) is a new NF-kappaB inhibitor and is effective on various tumor cells with constitutively activated NF-kappaB. Thus, in search for a new therapeutic modality of PEL, we examined the effect of DHMEQ on PEL cells. We confirmed constitutive activation of NF-kappaB with subcomponents of p50 and p65 in PEL cell lines. DHMEQ quickly and transiently abrogated NF-kappaB activation and reduced the cell viability in dose- and time-dependent manners, inducing apoptosis through activation of both mitochondrial and membrane pathways. Array analysis revealed that DHMEQ down-regulated expression levels of NF-kappaB target genes, such as interleukin-6 (IL6), Myc, chemokine (C-C motif) receptor 5 (CCR5) and NF-kappaB1, whereas it up-regulated expression levels of some genes involved in apoptosis, and cell cycle arrest. DHMEQ did not reactivate HHV-8 lytic genes, indicating that NF-kappaB inhibition by DHMEQ did not induce virus replication. DHEMQ rescued CB-17 SCID mice xenografted with PEL cells, reducing the gross appearance of effusion. Thus, DHMEQ transiently abrogated the NF-kappaB activation, irreversibly triggering the apoptosis cascade without HHV-8 reactivation. In addition, DHMEQ could rescue the PEL-xenograft mice. Therefore, we suggest DHMEQ as a promising candidate for molecular target therapy of the PEL.
Cancer Science 05/2009; 100(4):737-46. · 3.33 Impact Factor
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ABSTRACT: We previously reported prolonged HIV-1 transcriptional gene silencing by an RNA duplex targeting a sequence located within the NF-kappaB binding motif of the HIV-1 promoter in a susceptible HeLa cell line. Here we report extremely prolonged suppression of productive HIV-1 infection in a T-cell line (Molt-4) by a retrovirally delivered short-hairpin RNA (shRNA) targeting the same region (shkappaB). Following retroviral delivery of an shRNA we established shRNA-expressing CD4(+) T-cell lines. HIV-1 gene expression was profoundly suppressed for 1 year. Results of nuclear run-on assays and HIV-1 LTR-luciferase reporter assays revealed that shkappaB acted by inhibition of HIV-1 transcription. The effect was reversed by a histone deacetylase inhibitor, trichostatin-A (TSA), but not by a DNA methyltransferase inhibitor, 5-azacytidine (5-AzaC). Furthermore, chromatin immunoprecipitation assays (ChIP) demonstrated rapid, sustained induction of heterochromatin structures within the HIV-1 promoter region, with enrichment of histone 3 lysine 27 tri-methylation (H3K27me3) and H3K9 methylation. H3K27me3 enrichment was the most pronounced. This prolonged suppression could not be recapitulated by either retrovirally delivered anti-sense or sense strands alone or in combination. Our data strongly suggest that shkappaB induces high level, sustained transcriptional gene silencing of HIV-1 and offers the possibility of new therapeutic strategies.
Microbes and Infection 03/2009; 11(4):500-8. · 3.10 Impact Factor
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ABSTRACT: Management strategies of chronic phase chronic myelogenous leukemia (CML) have been revolutionized due to the discovery of a selective tyrosine kinase inhibitor, imatinib (Gleevec, STI571), which is substantially improving median survival. However, emergence of imatinib-resistance has put up a serious problem that requires novel treatment methods. Catechins, polyphenolic compounds in green tea, are gathering much attention due to their potential antitumor effects. So far (-)-epigallocatechin-3-gallate (EGCG), the most abundant component of catechin, has been shown to cause typical apoptosis in several tumor cell lines in most cases through activation of caspases. In this study, we showed that EGCG predominantly caused necrosis-like cell death via a caspase-independent mechanism in CML cells, K562 and C2F8, whereas imatinib induced the typical apoptotic cell death. Moreover, this caspase-independent cell death partially mediated the release of apoptosis-inducing factor, AIF, and serine protease, HtrA2/Omi, from the mitochondria to cytosol. In addition, EGCG enhanced the imatinib-induced cell death (P < 0.01) resulting in additive cell death in K562 cells and EGCG alone, effectively reduced the viability of imatinib-resistant K562 cells (P < 0.01). Catechin is a possible candidate for an antitumor agent that causes cell death in CML cells via a caspase-independent mechanism.
Cancer Science 02/2009; 100(2):349-56. · 3.33 Impact Factor
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ABSTRACT: In some mammalian systems small interfering RNAs (siRNA) targeting homologous sequences in promoter regions of genes induce transcriptional gene silencing (TGS). We have previously reported the induction of TGS by an siRNA (prom-A siRNA) targeting the tandem NF-kappaB-binding motifs within the human immunodeficiency virus, type 1 (HIV-1), promoter region. Here we report that induction of TGS by prom-A siRNA is accompanied by immediate and sustained local recruitment of Argonaute-1 (Ago1), histone deacetylase-1 (HDAC1), and induction of dimethylation of histone 3 at lysine 9 (H3K9me2), processes known to be associated with transcriptional silencing. Elevated levels of H3K9me2 and HDAC1 spread upstream of the target sequence, and elevated H3K9me2 levels also spread downstream into the coding region. Moreover, this siRNA induces an immediate change in DNA accessibility to restriction enzyme digestion in the region of the transcription initiation site of the HIV-1. This change in accessibility is because of the relocation of a nucleosome known to be associated with this region of the integrated pro-virus. Although there is a theoretical possibility that the observed viral suppression could be mediated by the PTGS mechanism with this siRNA acting at the 3 (R)-long term repeat of the virus, we demonstrate that this siRNA, and three other U3 targeted siRNAs, are inefficient inducers of PTGS. These data strongly suggest that siRNA targeting the promoter region acts predominantly at a site within the 5 (R)-long term repeat of HIV to induce transcriptional silencing and alterations to chromatin structure of the HIV promoter region that extend well beyond the immediate siRNA target site. These induced changes are consistent with those described in latent HIV-1 infection.
Journal of Biological Chemistry 08/2008; 283(34):23353-63. · 4.77 Impact Factor
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ABSTRACT: Epstein-Barr virus (EBV) causes EBV-associated lymphoproliferative diseases in patients with profound immune suppression. Most of these diseases are life-threatening and the prognosis of AIDS-associated lymphomas is extremely unfavorable. Polyclonal expansion of virus infected B-cell predisposes them to transformation. We investigated the possibility of nuclear factor kappa B (NF-kappaB) inhibition by dehydroxymethylepoxyquinomicin (DHMEQ) for the treatment and prevention of EBV-associated lymphoproliferative diseases. We examined the effect of DHMEQ on apoptosis induction in four EBV-transformed lymphoblastoid cell lines as well as peripheral blood mononuclear cells infected with EBV under immunosuppressed condition. DHMEQ inhibits NF-kappaB activation in EBV-transformed lymphoblastoid cell lines and induces apoptosis by activation of mitochondrial and membranous pathways. Using an in vivo NOD/SCIDgammac mouse model, we showed that DHMEQ has a potent inhibitory effect on the growth of lymphoblastoid cells. In addition, DHMEQ selectively purges EBV-infected cells expressing latent membrane protein (LMP) 1 from peripheral blood mononuclear cells and inhibits the outgrowth of lymphoblastoid cells. These results suggest that NF-kappaB is a molecular target for the treatment and prevention of EBV-associated lymphoproliferative diseases. As a potent NF-kappaB inhibitor, DHMEQ is a potential compound for applying this strategy in clinical medicine.
Microbes and Infection 07/2008; 10(7):748-56. · 3.10 Impact Factor
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ABSTRACT: Adult T-cell leukemia (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type I (HTLV-I), which induces nuclear factor-kappaB (NF-kappaB), a molecule central to the ensuing neoplasia. The NF-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has been shown to inhibit NF-kappaB activation in Tax-expressing HTLV-I-infected cells. In this study, we used NOD/SCID beta2-microglobulin(null) mice to show that intraperitoneal inoculation with Tax-deficient ATL cell lines caused rapid death, whereas DHMEQ-treated mice survived. Furthermore, DHMEQ treatment after subcutaneous inoculation inhibited the growth of transplanted ATL cells. These results demonstrate that DHMEQ has therapeutic efficacy on ATL cells, regardless of Tax expression.
Cancer Letters 12/2007; 257(2):206-15. · 4.24 Impact Factor
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Harutaka Katano,
Yuko Sato,
Satomi Hoshino,
Natsuo Tachikawa,
Shinichi Oka,
Yasuyuki Morishita, Takaomi Ishida,
Toshiki Watanabe,
William N Rom,
Shigeo Mori,
Tetsutaro Sata,
Michael D Weiden,
Yoshihiko Hoshino
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ABSTRACT: Signal transducer and activator of transcription 3 (STAT3) is a DNA-binding transcription factor activated by multiple cytokines and interferons. High expression of STAT3 has also been implicated in cancer and lymphoma. Here, we show a case of B cell lymphoma in which a defective human immunodeficiency virus 1 (HIV-1) integrated upstream of the first STAT3 coding exon. The lymphoma cells with anaplastic large cell morphology formed multiple nodular lesions in the lung of an acquired immunodeficiency syndrome (AIDS) patient with Kaposi's sarcoma. The provirus had a 5' long terminal repeat (LTR) deletion, but the 3' LTR had stronger promoter activity than the STAT3 promoter in reporter assays. Immunohistochemistry showed increased expression of STAT3 in the nuclei of lymphoma cells. Transfection of STAT3 resulted in transient cell proliferation in primary B cells in vitro. Although this is a very rare case of HIV-1-integrated lymphoma, these data suggest that up-regulation of STAT3 caused by HIV-1 integration resulted in the development of B cell lymphoma in this special case.
Microbes and Infection 11/2007; 9(14-15):1581-1589. · 3.10 Impact Factor
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ABSTRACT: C/EBPbeta plays a pivotal role in activation of human immunodeficiency virus type 1 (HIV-1) in monocytes/macrophages. However, mechanisms for functional regulation of C/EBPbeta remain uncharacterized. Previous studies indicated that NF-kappaB activation by tumor necrosis factor (TNF) receptor family, which activates TNF receptor associated factor (TRAF), induces HIV-1 expression. We found that TRAF signals activate HIV-1 LTR with mutations of NF-kappaB sites in promonocytic cell line U937, suggesting existence of an alternative HIV-1 activating pathway. In this study, we have characterized the signal transduction pathway of TRAF other than that leading to NF-kappaB, using U937 cell line, and its subline, U1, which is chronically infected by HIV-1. We show that signals downstream of TRAF2 and TRAF5 activate p38 MAPK, which directly phosphorylates C/EBPbeta, and that activation of p38 MAPK potently activates C/EBPbeta-mediated induction of HIV-1 gene expression. We also show TRAF2 and TRAF5 are expressed in monocytes/macrophages of spleen samples from HIV-1 infected patients. Identification of TRAF-p38 MAPK-CEBPbeta pathway provides a new target for controlling reactivation of latent HIV-1 in monocytes/macrophages.
Microbes and Infection 06/2007; 9(6):721-8. · 3.10 Impact Factor
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ABSTRACT: We previously described selective hypermethylation of the 5'-long terminal repeat (LTR) of HTLV-1 provirus in vivo and in vitro. This prompted us to analyze CpG methylation of the two LTRs of the HIV provirus in chronically infected cell lines. The results demonstrate selective hypermethylation of the 5' LTR of the HIV provirus in ACH-2 cells. Moreover, induction of viral gene expression by TNF-alpha resulted in demethylation of the 5'-LTR. These results suggest that selective epigenetic modification of the 5'LTR of the HIV-1 provirus may be an important mechanism by which proviral activity is suppressed.
Retrovirology 02/2006; 3:69. · 6.47 Impact Factor
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ABSTRACT: Tax is the oncoprotein of HTLV-1 which deregulates signal transduction pathways, transcription of genes and cell cycle regulation of host cells. Transacting function of Tax is mainly mediated by its protein-protein interactions with host cellular factors. As to Tax-mediated regulation of gene expression of HTLV-1 and cellular genes, Tax was shown to regulate histone acetylation through its physical interaction with histone acetylases and deacetylases. However, functional interaction of Tax with histone methyltransferases (HMTase) has not been studied. Here we examined the ability of Tax to interact with a histone methyltransferase SUV39H1 that methylates histone H3 lysine 9 (H3K9) and represses transcription of genes, and studied the functional effects of the interaction on HTLV-1 gene expression.
Tax was shown to interact with SUV39H1 in vitro, and the interaction is largely dependent on the C-terminal half of SUV39H1 containing the SET domain. Tax does not affect the methyltransferase activity of SUV39H1 but tethers SUV39H1 to a Tax containing complex in the nuclei. In reporter gene assays, co-expression of SUV39H1 represses Tax transactivation of HTLV-1 LTR promoter activity, which was dependent on the methyltransferase activity of SUV39H1. Furthermore, SUV39H1 expression is induced along with Tax in JPX9 cells. Chromatin immunoprecipitation (ChIP) analysis shows localization of SUV39H1 on the LTR after Tax induction, but not in the absence of Tax induction, in JPX9 transformants retaining HTLV-1-Luc plasmid. Immunoblotting shows higher levels of SUV39H1 expression in HTLV-1 transformed and latently infected cell lines.
Our study revealed for the first time the interaction between Tax and SUV39H1 and apparent tethering of SUV39H1 by Tax to the HTLV-1 LTR. It is speculated that Tax-mediated tethering of SUV39H1 to the LTR and induction of the repressive histone modification on the chromatin through H3 K9 methylation may be the basis for the dose-dependent repression of Tax transactivation of LTR by SUV39H1. Tax-induced SUV39H1 expression, Tax-SUV39H1 interaction and tethering to the LTR may provide a support for an idea that the above sequence of events may form a negative feedback loop that self-limits HTLV-1 viral gene expression in infected cells.
Retrovirology 02/2006; 3:5. · 6.47 Impact Factor
