RESUMO
Interleukin 33 (IL-33) signaling regulates most of the key processes of pregnancy, including decidualization, trophoblast proliferation and invasion, vascular remodeling, and placental growth. Accordingly, dysregulation of IL-33, its membrane-bound receptor (ST2L, transducer of IL-33 signaling), and its soluble decoy receptor (sST2, inhibitor of IL-33 signaling) has been linked to a wide range of adverse pregnancy outcomes that are common in women with obesity and polycystic ovary syndrome, that is, conditions associated with hyperandrogenism, insulin resistance, and compensatory hyperinsulinemia. To reveal if androgens and insulin might modulate uteroplacental IL-33 signaling, we investigated the effect of dihydrotestosterone (DHT) and/or insulin on the expression of ST2L and sST2 (along with the activity of their promoter regions), IL-33 and sIL1RAP (heterodimerization partner of sST2), during in vitro decidualization of endometrial stromal cells from 9 healthy women. DHT and insulin markedly upregulated sST2 secretion, in addition to the upregulation of its messenger RNA (mRNA) expression, while the proximal ST2 promoter, from which the sST2 transcript originates, was upregulated by insulin, and in a synergistic manner by DHT and insulin combination treatment. On the other hand, sIL1RAP was slightly downregulated by insulin and IL-33 mRNA expression was not affected by any of the hormones, while ST2L mRNA expression and transcription from its promoter region (distal ST2 promoter) could not be detected or showed a negligibly low level. We hypothesize that high levels of androgens and insulin might lead to subfertility and pregnancy complications, at least partially, through the sST2-dependent downregulation of uteroplacental IL-33 signaling.
Assuntos
Insulina , Interleucina-33 , Humanos , Feminino , Gravidez , Interleucina-33/genética , Interleucina-33/metabolismo , Interleucina-33/farmacologia , Insulina/farmacologia , Di-Hidrotestosterona/farmacologia , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Transdução de Sinais , Placenta/metabolismo , Androgênios/farmacologia , RNA Mensageiro , Células Estromais/metabolismoRESUMO
Autologous bone remains the gold standard bone substitute in clinical practice. Therefore, the microarchitecture of newly developed synthetic bone substitutes, which reflects the spatial distribution of materials in the scaffold, aims to recapitulate the natural bone microarchitecture. However, the natural bone microarchitecture is optimized to obtain a mechanically stable, lightweight structure adapted to the biomechanical loading situation. In the context of synthetic bone substitutes, the application of a Triply Periodic Minimum Surface (TPMS) algorithm can yield stable lightweight microarchitectures that, despite their demanding architectural complexity, can be produced by additive manufacturing. In this study, we applied the TPMS derivative Adaptive Density Minimal Surfaces (ADMS) algorithm to produce scaffolds from hydroxyapatite (HA) using a lithography-based layer-by-layer methodology and compared them with an established highly osteoconductive lattice microarchitecture. We characterized them for compression strength, osteoconductivity, and bone regeneration. The in vivo results, based on a rabbit calvaria defect model, showed that bony ingrowth into ADMS constructs as a measure of osteoconduction depended on minimal constriction as it limited the maximum apparent pore diameter in these scaffolds to 1.53 mm. Osteoconduction decreased significantly at a diameter of 1.76 mm. The most suitable ADMS microarchitecture was as osteoconductive as a highly osteoconductive orthogonal lattice microarchitecture in noncritical- and critical-size calvarial defects. However, the compression strength and microarchitectural integrity in vivo were significantly higher for scaffolds with their microarchitecture based on the ADMS algorithm when compared with high-connectivity lattice microarchitectures. Therefore, bone substitutes with high osteoconductivity can be designed with the advantages of the ADMS-based microarchitectures. As TPMS and ADMS microarchitectures are true lightweight structures optimized for high mechanical stability with a minimal amount of material, such microarchitectures appear most suitable for bone substitutes used in clinical settings to treat bone defects in weight-bearing and non-weight-bearing sites.
RESUMO
Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.
Assuntos
Interferon gama , Leucemia Mielogênica Crônica BCR-ABL Positiva , Antígenos CD34/metabolismo , Antígenos CD34/farmacologia , Apoptose , Linhagem Celular Tumoral , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco/metabolismo , Regulação para CimaRESUMO
Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiofenos/farmacologia , Antígenos CD34/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mesilato de Imatinib/administração & dosagem , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína bcl-X/metabolismoRESUMO
Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR-8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin-43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.
Assuntos
Androgênios/metabolismo , Decídua/metabolismo , Insulina/metabolismo , Transdução de Sinais , Trofoblastos/metabolismo , Androgênios/farmacologia , Biomarcadores , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Endométrio/citologia , Endométrio/metabolismo , Feminino , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Insulina/farmacologia , Gravidez , Células Estromais/metabolismoRESUMO
The clinical outcome for patients with chronic myeloid leukemia (CML) has improved significantly with the introduction of tyrosine kinase inhibitors (TKIs). However, their curative potential appears limited, probably as a consequence of TKI-resistant leukemic stem cells (LSCs) that persist as a result of aberrant pathways independent of the well-established oncoprotein Bcr-Abl. One such pathway involves signaling through leukotrienes (LTs), bioactive compounds that have been suggested to play a role in several other malignancies. Cysteinyl LT1 receptor (CysLT1R) has been reported to be overexpressed in a number of solid cancers, and blocking of this receptor with the antagonist montelukast (treatment approved for bronchial asthma) has resulted in the killing of cancer cells. We recently demonstrated that montelukast, alone or in combination with imatinib, can effectively reduce the growth of CML cells, while normal bone marrow cells were left unaffected. Herein, we further investigated the importance of CysLT1R for the survival of CML cells and the mechanisms by which montelukast induces cell death. Knockdown of the CysLT1R of K562 cells with siRNA reduced their growth by 25%. Montelukast had no effect on these cells, while it killed more than 50% of CysLT1R-expressing cells. Growth inhibition exerted by imatinib was unaffected by CysLT1R status. Montelukast-induced killing of K562/JURL-MK1 CML cells was paralleled by Bax overexpression, cytochrome c release, PARP-1 cleavage, and caspase-3 activation, an event further increased in a setting where montelukast was added to imatinib. Wnt/ß-catenin signaling was activated by CysLT1R and we observed that montelukast could induce proteins in this pathway, a finding of relevance for LSC survival. Thus, montelukast, employed at in vivo-like concentrations, induces the killing of CML cells through apoptotic pathways and may provide an additional, novel therapeutic possibility in CML.
Assuntos
Acetatos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Antagonistas de Leucotrienos/farmacologia , Quinolinas/farmacologia , Receptores de Leucotrienos/metabolismo , Linhagem Celular Tumoral , Ciclopropanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , SulfetosRESUMO
B-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that can repress more than 1200 potential target genes. It exerts oncogenic effects through the inhibition of differentiation, DNA damage sensing and apoptosis in several human hematopoietic malignancies, including multiple myeloma (MM). The multifunctional cytokine interferon γ (IFNγ) exerts pro-apoptotic and anti-proliferative effects on MM cells in vitro, at least partially through the inhibition of the effects of interleukin 6 (IL6), one of the most important growth factor of MM and a strong inducer of BCL6 expression. However, IFNγ was also reported to directly upregulate BCL6 in several cell types. These observations prompted us to analyze the effect of IFNγ on BCL6 expression in MM cells. We discovered that among several myeloma growth/survival factors tested (including IL6, oncostatin M, insulin-like growth factor 1, tumor necrosis factor α and IFNα) IFNγ was the strongest inducer of BCL6 mRNA and protein expression in MM cell lines. IFNγ induced upregulation of BCL6 was dependent on the classical STAT1 signaling pathway, and affected both major BCL6 variants. Interestingly, although IFNα induced stronger STAT1 phosphorylation than IFNγ, it only slightly upregulated BCL6 in MM lines. We proved that IFNα induced BCL6 upregulation was limited by the concomitant activation of STAT5 signaling. We assume that BCL6 upregulation may represent a potentially pro-tumorigenic effect of IFNγ signaling in MM cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Interferon gama/metabolismo , Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Fator de Transcrição STAT1/metabolismo , Regulação para Cima , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
Prokineticin 1 (PROK1), a hypoxia-regulated angiogenic factor, has emerged as a crucial regulator of embryo implantation and placentation. Dysregulation of PROK1 has been linked to recurrent pregnancy loss, pre-eclampsia, foetal growth restriction and preterm birth. These pregnancy complications are common in women with obesity and polycystic ovary syndrome, i.e. conditions associated with insulin resistance and compensatory hyperinsulinaemia. We investigated the effect of insulin on PROK1 expression during in vitro decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Insulin induced a significant dose-dependent up-regulation of PROK1 on both mRNA and protein level in decidualizing endometrial stromal cells. This up-regulation was mediated by hypoxia-inducible factor 1-alpha (HIF1α) via the phosphatidylinositol 3-kinase (PI3K) pathway. Furthermore, we demonstrated that PROK1 did not affect the viability, but significantly inhibited the migration of endometrial stromal cells and the migratory and invasive capacity of trophoblast cell lines. This in vitro study provides new insights into the regulation of PROK1 by insulin in human decidualizing endometrial stromal cells, the action of PROK1 on migration of endometrial stromal cells, as well as migration and invasion of trophoblasts. We speculate that hyperinsulinaemia may be involved in the mechanisms by which PROK1 is linked to placenta-related pregnancy complications.
Assuntos
Decídua/citologia , Decídua/metabolismo , Hormônios Gastrointestinais/genética , Insulina/farmacologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Adolescente , Adulto , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Coriocarcinoma/patologia , Feminino , Hormônios Gastrointestinais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Trofoblastos/citologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo , Cicatrização/efeitos dos fármacos , Adulto JovemRESUMO
Insulin resistance and compensatory hyperinsulinemia are characteristic features of obesity and polycystic ovary syndrome, and both are associated with reduced fertility and implantation. There is little knowledge about the effect of insulin on the decidualization process and previous findings are contradictory. We investigated the effect of insulin on the regulation of forkhead box protein O1 (FOXO1), one of the most important transcription factors during decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Gene expression levels of six putative FOXO1 target genes (including insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL)) were measured with Real-Time PCR following FOXO1 inhibition or insulin treatment. PI3K inhibition was used to identify the possible mechanism behind regulation. Subcellular localization of FOXO1 was analyzed with immunofluorescence. All the genes (IGFBP1, CTGF, INSR, DCN, LEFTY2), except prolactin, were evaluated as FOXO1 target genes in decidualizing stromal cells. Insulin caused a significant dose-dependent inhibition of the verified FOXO1 target genes. It was also demonstrated that insulin regulated FOXO1 target genes by transcriptional inactivation and nuclear export of FOXO1 via PI3K pathway. However, insulin did not inhibit the morphological transformation of endometrial stromal cells via transcriptional inactivation of FOXO1. This study provides new insights on the action of insulin on the endometrium via regulation of FOXO1. It is suggested that hyperinsulinemia results in dysregulation of a high number of FOXO1 controlled genes that may contribute to endometrial dysfunction and reproductive failure. Our findings may illuminate possible reasons for unexplained infertility.
Assuntos
Decídua/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Proteína Forkhead Box O1/genética , Insulina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Adolescente , Adulto , Bucladesina/farmacologia , Decídua/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Endométrio/efeitos dos fármacos , Feminino , Proteína Forkhead Box O1/metabolismo , Humanos , Acetato de Medroxiprogesterona/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Adulto JovemRESUMO
Characterization of viral DNA methylation patterns is essential to understand its function in viral pathogenesis. Bisulfite modification, followed by polymerase chain reaction (PCR) and sequencing is the most effective method for the high resolution methylation mapping of viral genomes. Since the bisulfite modification and PCR steps are the most critical ones, an optimized protocol for these two steps is presented, with special attention to potential pitfalls.
Assuntos
Epigênese Genética , Epigenômica , Genoma Viral , Análise de Sequência de DNA , Metilação de DNA , Epigenômica/métodos , Genótipo , Herpesvirus Humano 4/genética , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , SulfitosRESUMO
Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.
Assuntos
Retroalimentação Fisiológica , Imunidade , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linfócitos T/imunologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Imunidade/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Modelos Biológicos , Piperazinas/farmacologia , Receptores Notch/metabolismo , Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tiazóis/farmacologia , Proteína Supressora de Tumor p14ARF/metabolismoRESUMO
Latent Epstein-Bar virus genomes undergo epigenetic modifications which are dependent on the respective tissue type and cellular phenotype. These define distinct viral epigenotypes corresponding with latent viral gene expression profiles. Viral Latent Membrane Proteins 1 and 2A can induce cellular DNA methyltransferases, thereby influencing the methylation status of the viral and cellular genomes. Therefore, not only the viral genomes carry epigenetic modifications, but also the cellular genomes adopt major epigenetic alterations upon EBV infection. The distinct cellular epigenotypes of EBV-infected cells differ from the epigenotypes of their normal counterparts. In Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC) significant changes in the host cell methylome with a strong tendency towards CpG island hypermethylation are observed. Hypermethylated genes unique for EBVaGC suggest the existence of an EBV-specific "epigenetic signature". Contrary to the primary malignancies carrying latent EBV genomes, lymphoblastoid cells (LCs) established by EBV infection of peripheral B cells in vitro are characterized by a massive genome-wide demethylation and a significant decrease and redistribution of heterochromatic histone marks. Establishing complete epigenomes of the diverse EBV-associated malignancies shall clarify their similarities and differences and further clarify the contribution of EBV to the pathogenesis, especially for the epithelial malignancies, NPC and EBVaGC.
Assuntos
Transformação Celular Viral , Epigênese Genética , Infecções por Vírus Epstein-Barr/metabolismo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Animais , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.
Assuntos
Linfócitos B/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Prolinfocítica/patologia , Linfócitos B/imunologia , Linfócitos B/virologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Evolução Clonal/imunologia , Células Clonais/imunologia , Células Clonais/patologia , Células Clonais/virologia , Progressão da Doença , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Expressão Gênica , Herpesvirus Humano 4/fisiologia , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/virologia , Leucemia Prolinfocítica/imunologia , Leucemia Prolinfocítica/virologia , Contagem de Linfócitos , Fatores de Tempo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
UNLABELLED: Human B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cells in vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ(-/-) mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferated in vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that the in vitro growth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8(+) cell-depleted mice, and it was absent from CD4(+) cell-depleted mice. These results indicate that CD4(+) T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4(+) T cells contributed to this process through their CD40L expression. IMPORTANCE: At primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4(+) T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Linfócitos T/imunologia , Latência Viral , Animais , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regiões Promotoras Genéticas , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Viral/genética , Metilação de DNA/genética , Herpesvirus Humano 4/genética , Linfócitos B/virologia , Carcinogênese , Ilhas de CpG/genética , DNA Viral/genética , Genoma Humano , Humanos , Regiões Promotoras GenéticasRESUMO
We analyzed epigenetic marks at the CD23 regulatory regions in well characterized Epstein-Barr virus (EBV)-carrying cell lines covering the major latency types. Bisulfite sequencing showed that DNA methylation is not a major regulator of EBV-induced CD23 transcription, although a wide hypomethylated DNA sequence in the regulatory regions is always present in the cell lines with high CD23 expression. Acetylated histone H3 levels at the CD23b promoter showed strong correlation with CD23b expression, while a weaker correlation could be observed at the CD23a core promoter. DMS in vivo footprinting at the intronic EBV-responsive enhancer and the intermediate-affinity CBF1 site at the CD23a core promoter did not reveal any significant sign of in vivo protein-DNA interactions, despite the presence of strong, characteristic footprints in the same DMS-treated DNA samples at the two CBF1 sites of the LMP2A-promoter. Our in vivo results suggest a minor role for DNA methylation, while a more important role for histone acetylation in the regulation of EBV-induced CD23 expression. Furthermore, our in vivo footprinting results support the complex model of CD23 induction by EBV, rather than a simple model with direct transactivation of CD23 by EBNA-2.
Assuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Receptores de IgE/genética , Acetilação , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologia , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Although the microRNA miR-146a is an important regulator of immunological processes and contributes to the pathogenesis of certain B cell lymphoma types, in B cells the epigenetic regulation of miR-146a expresion has not been studied yet. To elucidate the mechanisms controlling miR-146a expression in B lymphoid cells we analysed epigenetic marks, including CpG methylation and histone modifications, at the miR-146a promoter in well characterized Epstein-Barr virus (EBV) positive and EBV negative B cell lines. In addition, EBV positive epithelial cell lines were also studied as controls. In cells with a silent miR-146a promoter the 5' regulatory sequences comprising a CpG island were devoid of activating histone modifications, independently of the methylation pattern of the regulatory region. The regulatory sequences flanking the inactive miR-146 promoter were hypermethylated at CpG dinucleotides in the EBV positive Burkitt's lymphoma (BL) cell lines of memory B cell phenotype (Rael and Akata), partially methylated in the mammary carcinoma cell lines C2G6 and C4A3, and completely unmethylated in the nasopharyngeal carcinoma cell line C666-1. In contrast, in EBV positive cell lines of activated B cell phenotype, and EBV negative BL cell lines the invariably unmethylated 5' regulatory sequences of active miR-146a promoters were enriched in the euchromatic histone modification marks acetylated histone H3, acetylated histone H4, and histone H3 dimethylated at lysine 4. The euchromatic histone modification marks extended over the immediate vicinity of the transcriptional initiation site to the 3' intron, too. We concluded that similarly to the promoters of protein coding genes, both DNA methylation and histone modifications contribute to the host cell dependent expression of miR-146a.
Assuntos
Região 5'-Flanqueadora , Linfócitos B/virologia , Metilação de DNA , Histonas/metabolismo , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Acetilação , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores/metabolismo , Northern Blotting , Linfoma de Burkitt/patologia , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ilhas de CpG , Epigênese Genética , Eucromatina/genética , Eucromatina/metabolismo , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Histonas/genética , Humanos , Íntrons , Lisina/metabolismo , MicroRNAs/genética , Sítio de Iniciação de Transcrição , Latência ViralRESUMO
Lack of functional SAP protein, due to gene deletion or mutation, is the cause of X-linked lymphoproliferative disease (XLP), characterized by functionally impaired T and NK cells and a high risk of lymphoma development. We have demonstrated earlier that SAP has a pro-apoptotic function in T and B cells. Deficiency of this function might contribute to the pathogenesis of XLP. We have also shown that SAP is a target of p53 in B cell lines. In the present study, we show that activated primary T cells express p53, which induces SAP expression. p53 is functional as a transcription factor in activated T cells and induces the expression of p21, PUMA and MDM2. PARP cleavage in the late phase of activation indicates that T cells expressing high levels of SAP undergo apoptosis. Modifying p53 levels using Nutlin-3, which specifically dissociates the MDM2-p53 interaction, was sufficient to upregulate SAP expression, indicating that SAP is a target of p53 in T cells. We also demonstrated p53's role as a transcription factor for SAP in activated T cells by ChIP assays. Our result suggests that p53 contributes to T cell homeostasis through the induction of the pro-apoptotic SAP. A high level of SAP is necessary for the activation-induced cell death that is pivotal in termination of the T cell response.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos T/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Homeostase/genética , Homeostase/fisiologia , Humanos , Imidazóis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Piperazinas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Proteína Supressora de Tumor p53/genéticaRESUMO
We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.
Assuntos
Linfócitos B/metabolismo , Herpesvirus Humano 4/genética , Interferon Tipo I/metabolismo , Proteínas da Matriz Viral/genética , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Linhagem Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/imunologia , Humanos , Interferon Tipo I/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , Proteínas da Matriz Viral/metabolismo , Latência ViralRESUMO
Following infection with Epstein-Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4(+) T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4(+) T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4(+) T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4(+) T cells can control the EBV-induced B-cell proliferation.