RESUMO
Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in sixteen PLHIV. 14/16 CSF cell samples had quantifiable CA-HIV-1 RNA, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T-cells; p<0.0001). In individual PLHIV, higher levels of HIV-1 transcripts in CSF cells were associated with greater brain injury in the frontal white matter (Std ß=-0.73; p=0.007) and posterior cingulate (Std ß=-0.61; p=0.03). 18-colour flow cytometry revealed that the CSF cells were 91% memory T-cells, equally CD4+ and CD8+ T-cells, but fewer B cells (0.4 %), and monocytes (3.1%). CXCR3+CD49d+integrin ß7-, CCR5+CD4+ T-cells were highly enriched in CSF, compared with PBMC (p <0.001). However, CA-HIV-1 RNA could not be detected in 10/16 preparations of highly purified monocytes from PBMC, and was extremely low in the other six. Our data show that elevated HIV-1 transcripts in CSF cells were associated with brain injury, despite suppressive ART. The cellular source is most likely memory CD4+ T cells from blood, rather than trafficking monocytes. Future research should focus on inhibitors of this transcription to reduce local production of potentially neurotoxic and inflammatory viral products.
Assuntos
Lesões Encefálicas , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , HIV-1/genética , Linfócitos T CD4-Positivos , Leucócitos Mononucleares , Infecções por HIV/tratamento farmacológicoRESUMO
Latency remains a barrier to achieving a sterilizing cure to HIV infection. It is thus important to find new host factor(s) to better understand maintenance of HIV latency and be exploited to develop new and more efficient latency reversing agents (LRAs). Here we employed RNA interference screening with a latently HIV-1-infected cell-line to identify Stathmin 1 (STMN1) as a host factor required for maintaining HIV-1 latency. Depletion of STMN1 significantly enhanced HIV-1 expression in a STMN1 depletion-dependent manner and forced expression of exogenous STMN1 suppressed it. We further showed that STMN1 depletion increases HIV-1 proviral transcriptional elongation. Moreover, chromatin immunoprecipitation (ChIP)-qPCR assays revealed STMN1 accumulation on/near the HIV-1 5' LTR region compared to other regions on the HIV-1 provirus, suggesting the possible contribution of STMN1 to HIV-1 transcription. These results suggest that STMN1 is required for the maintenance of HIV-1 latency and implicates STMN1 as a novel therapeutic target to eradicate HIV-1.
Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Estatmina/metabolismo , Latência Viral , Infecções por HIV/genética , Interações Hospedeiro-Patógeno , Humanos , Interferência de RNA , Estatmina/genética , Células THP-1RESUMO
The molecular mechanisms for IL2 gene-specific dysregulation during chronic human immunodeficiency virus type 1 (HIV-1) infection are unknown. Here, we investigated the role of DNA methylation in suppressing interleukin 2 (IL-2) expression in memory CD4(+) T cells during chronic HIV-1 infection. We observed that CpG sites in the IL2 promoter of CD4(+) T cells were fully methylated in naive CD4(+) T cells and significantly demethylated in the memory populations. Interestingly, we found that the memory cells that had a terminally differentiated phenotype and expressed CD57 had increased IL2 promoter methylation relative to less differentiated memory cells in healthy individuals. Importantly, early effector memory subsets from HIV-1-infected subjects expressed high levels of CD57 and were highly methylated at the IL2 locus. Furthermore, the increased CD57 expression on memory CD4(+) T cells was inversely correlated with IL-2 production. These data suggest that DNA methylation at the IL2 locus in CD4(+) T cells is coupled to immunosenescence and plays a critical role in the broad dysfunction that occurs in polyclonal T cells during HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/genética , Interleucina-2/genética , Adulto , Antígenos CD57/imunologia , Células Cultivadas , Ilhas de CpG , Metilação de DNA , Repressão Epigenética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Humanos , Memória Imunológica , Interleucina-2/biossíntese , Interleucina-2/imunologia , Leucócitos Mononucleares/imunologia , Pessoa de Meia-Idade , Regiões Promotoras GenéticasRESUMO
BACKGROUND: HIV-1 infected patients frequently have osteolytic bone disease, which is caused by the dysregulation of the bone remodeling system that involves the interaction between osteoblasts and osteoclasts, but the relationship between osteolytic disease and HIV-1 infection remains unclear. In this study we tested whether HIV-1 infection of osteoclasts affects their differentiation. RESULTS: We prepared human osteoclasts from CD14+ monocytes and examined them for their susceptibility to HIV-1. Furthermore, we investigated the effect of HIV-1 infection on osteoclast differentiation. CD14-derived osteoclasts were shown to express CD4, CCR5, and CXCR4 each at the similar level to that shown with macrophages. R5-tropic HIV-1 and X4-tropic HIV-1 were found to infect CD14-derived osteoclasts and replicate in them. Furthermore, HIV-1 infection induced formation of larger osteoclastst, enhanced the expression of mRNAs for three osteoclast specific marker molecules (tartrate-resistant acid phosphatase, cathepsin K, and the calcitonin receptor), and up-regulated osteoclast bone resorption activity. CONCLUSIONS: Our results suggest that osteoclasts serve as a novel target for HIV-1 infection, which may enhance the osteoclast differentiation contributing to the development of osteolytic disease in HIV-1-infected patients.
Assuntos
Diferenciação Celular , HIV-1/fisiologia , Osteoclastos/fisiologia , Osteoclastos/virologia , Replicação Viral , Antígenos CD4/análise , Células Cultivadas , Humanos , Receptores de Lipopolissacarídeos/análise , Osteoclastos/química , Receptores CCR5/análise , Receptores CXCR4/análiseRESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Argonautas/análise , Núcleo Celular/metabolismo , Inativação Gênica , RNA Interferente Pequeno/análise , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Argonautas/metabolismo , Linhagem Celular , Núcleo Celular/química , Citocalasina D/farmacologia , Fatores de Iniciação em Eucariotos/análise , Fatores de Iniciação em Eucariotos/metabolismo , HIV-1/fisiologia , Humanos , Membrana Nuclear/química , Regiões Promotoras Genéticas , Interferência de RNA , Vírus da Imunodeficiência Símia/fisiologia , Transcrição Gênica , Replicação ViralRESUMO
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 onward, 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, 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.
Assuntos
Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteína Supressora de Tumor p53/genética , Adulto , Animais , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Produtos do Gene tax/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Camundongos , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/fisiologia , RNA Antissenso/genética , RNA Viral/genética , Replicação Viral , Núcleo Celular/virologia , Genes Reporter , Células HEK293 , Infecções por HIV/virologia , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/patogenicidade , Humanos , Leucócitos Mononucleares/virologia , Mutação , NF-kappa B/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/genética , Regiões Promotoras Genéticas , Provírus/genética , Interferência de RNA , RNA Mensageiro/genética , Transcrição Reversa , Fatores de Tempo , TransfecçãoRESUMO
NPM-ALK characterizes anaplastic large cell lymphoma (ALCL), as does the high expression of CD30, a feature shared with H-RS cells of classic Hodgkin's lymphoma. In H-RS cells, ligand-independent signaling by overexpressed CD30 drives constitutive NF-kappaB activation, which is absent in ALCL cells. Here we show that NPM-ALK impedes CD30 signaling and NF-kappaB activation, dependent on both ALK kinase activity and the N-terminal NPM domain. NPM-ALK transduction into H-RS cell lines abrogates recruitment and aggregation of TRAF proteins, inducing an ALCL-like morphology and phenotype. TRAF2 associates with NPM-ALK at a consensus binding motif located in the kinase domain. Thus, NPM-ALK abrogates CD30-driven NF-kappaB activation and can also induce an ALCL phenotype, distinguishing ALCL cells from H-RS cells of T cell origin.
Assuntos
Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , NF-kappa B/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Motivos de Aminoácidos , Células Cultivadas , Sequência Consenso , Citoplasma/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas I-kappa B/metabolismo , Antígeno Ki-1/genética , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , NF-kappa B/genética , Proteínas de Fusão Oncogênica/genética , Fenótipo , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Fator 2 Associado a Receptor de TNF , Tirosina/metabolismoRESUMO
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.
Assuntos
Produtos do Gene tax/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Transporte Ativo do Núcleo Celular , Células Cultivadas , Produtos do Gene tax/análise , Histona-Lisina N-Metiltransferase/análise , Histona-Lisina N-Metiltransferase/química , Humanos , NF-kappa B/metabolismo , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , NF-kappa B/biossíntese , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Microambiente Tumoral/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Medula Óssea/metabolismo , Linhagem Celular Tumoral/metabolismo , Cicloexanonas/farmacologia , Progressão da Doença , Genes Reporter , Humanos , Mesilato de Imatinib , Medições Luminescentes , Camundongos , NF-kappa B/análise , NF-kappa B/genética , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pirimidinas/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Baço/metabolismo , Células Estromais/fisiologia , Transgenes , Transplante Heterólogo , Carga Tumoral , Microambiente Tumoral/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
Assuntos
Inativação Gênica , HIV-1/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Sequência de Bases , Sítios de Ligação/genética , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Linhagem Celular Tumoral , Exonucleases/genética , Exorribonucleases , Citometria de Fluxo , Regulação da Expressão Gênica , HIV-1/imunologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Leucemia , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/virologia , RNA Interferente Pequeno/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , eIF-2 Quinase/metabolismoRESUMO
OBJECTIVE: Some HIV+ patients, virally suppressed on ART, show occasional 'blips' of detectable HIV-1 plasma RNA. We used a new highly sensitive assay of cell-associated HIV-1 RNA to measure transcriptional activity in PBMCs and production of infectious virus from the viral reservoir, in patients with and without 'blips'. DESIGN/METHODS: RNA and DNA extracted from cells in 6âml of peripheral blood, from suppressed patients with one to two 'blip' episodes over the past 2âyears of ART (nâ=â55), or no 'blips' (nâ=â52), were assayed for HIV-1 RNA transcripts and proviral DNA targeting the highly conserved 'R' region of the LTR. Follow-up samples were also collected. Purified CD4+ T cells were cultured with anti-CD3/CD28/CD2 T-cell activator to amplify transcription and measure replication competent virus. RESULTS: HIV-1 RNA transcripts ranged from 1.3 to 5415âcopies/106 white blood cells. 'Blip' patients had significantly higher levels vs. without blips (median 192 vs. 49; Pâ=â0.0007), which correlated with: higher levels of inducible transcripts after activation in vitro, sustained higher HIV-1 transcription levels in follow-up samples along with increasing HIV-1 DNA in some, and production of replication-competent HIV-1. CONCLUSION: Viral 'blips' are significant reflecting higher transcriptional activity from the reservoir and contribute to the reservoir over time. This sensitive assay can be used in monitoring the size and activity of the HIV-1 reservoir and will be useful in HIV-1 cure strategies.
Assuntos
Infecções por HIV , HIV-1 , HIV-1/genética , Humanos , Provírus/genética , RNA , RNA Viral , Carga ViralRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Serina-Treonina Quinases/genética , Acetilação/efeitos dos fármacos , Azacitidina/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Valproico/farmacologia , Quinase Induzida por NF-kappaBRESUMO
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.
Assuntos
Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Catequina/análogos & derivados , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Chá/química , Western Blotting , Catequina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Técnicas Imunoenzimáticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necrose , Frações SubcelularesRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Cicloexanonas/farmacologia , Herpesvirus Humano 8/metabolismo , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/prevenção & controle , NF-kappa B/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Células Jurkat , Células K562 , Linfoma de Efusão Primária/virologia , Fatores de TempoRESUMO
Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5' LTR to the end of the 3' LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.
Assuntos
DNA Viral/genética , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Provírus/genética , Linhagem Celular , Quimera , Células Clonais , Infecções por HIV/virologia , Humanos , Integração Viral/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/virologia , Benzamidas/farmacologia , Cicloexanonas/farmacologia , Herpesvirus Humano 4/fisiologia , Fatores Imunológicos/farmacologia , NF-kappa B/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Transtornos Linfoproliferativos/tratamento farmacológico , Camundongos , Camundongos SCIDRESUMO
HIV-1 latent reservoirs harbouring silenced but replication-competent proviruses are a major obstacle against viral eradication in infected patients. The "shock and kill" strategy aims to reactivate latent provirus with latency reversing agents (LRAs) in the presence of antiretroviral drugs, necessitating the development of effective and efficient LRAs. We screened a chemical library for potential LRAs and identified two dual Polo-like kinase (PLK)/bromodomain inhibitors, BI-2536 and BI-6727 (volasertib), which are currently undergoing clinical trials against various cancers. BI-2536 and BI-6727 significantly reactivated silenced HIV-1 provirus at both the mRNA and protein level in two latently infected model cell lines (ACH2 and U1). BI-2536 dramatically reactivated transcription of latent HIV-1 provirus in peripheral blood mononuclear cells derived from infected patients. Long terminal repeat activation by the inhibitors was associated with bromodomain rather than PLK inhibition. We also found that BI-2536 synergistically activates the latent provirus in combination with SAHA, a histone deacetylase inhibitor, or the non-tumour-promoting phorbol ester prostratin. Our findings strongly suggest that BI-2536 and BI-6727 are potent LRAs for the "shock and kill" HIV-1 eradication strategy.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Pteridinas/farmacologia , RNA Viral/genética , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Combinação de Medicamentos , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Inibidores de Histona Desacetilases/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/virologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/virologia , Ésteres de Forbol/farmacologia , Cultura Primária de Células , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/agonistas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/agonistas , RNA Viral/metabolismo , Células THP-1 , Transcrição Gênica/efeitos dos fármacos , Ativação Viral/genética , Latência Viral/genética , Vorinostat/farmacologiaRESUMO
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.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/fisiologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , HIV-1/genética , Humanos , Macrófagos/virologia , Monócitos/virologia , Fosforilação , Transdução de Sinais/genética , Baço/imunologia , Ativação Viral , Latência ViralRESUMO
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.