RESUMO
Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Tirosina Quinase da Agamaglobulinemia/imunologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Masculino , Camundongos , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
CD103+ dendritic cells are critical for cross-presentation of tumor antigens. Here we have shown that during immunotherapy, large numbers of cells expressing CD103 arose in murine tumors via direct differentiation of Ly6c+ monocytic precursors. These Ly6c+CD103+ cells could derive from bone-marrow monocytic progenitors (cMoPs) or from peripheral cells present within the myeloid-derived suppressor cell (MDSC) population. Differentiation was controlled by inflammation-induced activation of the transcription factor p53, which drove upregulation of Batf3 and acquisition of the Ly6c+CD103+ phenotype. Mice with a targeted deletion of p53 in myeloid cells selectively lost the Ly6c+CD103+ population and became unable to respond to multiple forms of immunotherapy and immunogenic chemotherapy. Conversely, increasing p53 expression using a p53-agonist drug caused a sustained increase in Ly6c+CD103+ cells in tumors during immunotherapy, which markedly enhanced the efficacy and duration of response. Thus, p53-driven differentiation of Ly6c+CD103+ monocytic cells represents a potent and previously unrecognized target for immunotherapy.
Assuntos
Células Apresentadoras de Antígenos/fisiologia , Monócitos/fisiologia , Células Mieloides/metabolismo , Neoplasias/imunologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/metabolismo , Antígenos Ly/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Citometria de Fluxo , Humanos , Imunoterapia/métodos , Cadeias alfa de Integrinas/metabolismo , Camundongos , Monócitos/imunologia , Células Mieloides/fisiologiaRESUMO
Sickle cell disease (SCD) is a chronic hemolytic and systemic hypoxia condition with constant oxidative stress and significant metabolic alterations. However, little is known about the correlation between metabolic alterations and the pathophysiological symptoms. Here, we report that Nrf2, a master regulator of cellular antioxidant responses, regulates the production of the metabolite l-2-hydroxyglutarate (L2HG) to mediate epigenetic histone hypermethylation for gene expression involved in metabolic, oxidative, and ferroptotic stress responses in SCD. Mechanistically, Nrf2 was found to regulate the expression of L2HG dehydrogenase (L2hgdh) to mediate L2HG production under hypoxia. Gene expression profile analysis indicated that reactive oxygen species (ROS) and ferroptosis responses were the most significantly affected signaling pathways after Nrf2 ablation in SCD. Nrf2 silencing and L2HG supplementation sensitize human sickle erythroid cells to ROS and ferroptosis stress. The absence of Nrf2 and accumulation of L2HG significantly affect histone methylation for chromatin structure modification and reduce the assembly of transcription complexes on downstream target genes to regulate ROS and ferroptosis responses. Furthermore, pharmacological activation of Nrf2 was found to have protective effects against ROS and ferroptosis stress in SCD mice. Our data suggest a novel mechanism by which Nrf2 regulates L2HG levels to mediate SCD severity through ROS and ferroptosis stress responses, suggesting that targeting Nrf2 is a viable therapeutic strategy for ameliorating SCD symptoms.
Assuntos
Anemia Falciforme , Cromatina , Epigênese Genética , Ferroptose , Glutaratos , Fator 2 Relacionado a NF-E2 , Ferroptose/genética , Glutaratos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Cromatina/metabolismo , Metilação , Oxirredutases do Álcool/metabolismo , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Perfilação da Expressão GênicaRESUMO
Epigenetic regulations, including DNA methylation, are critical to the development and progression of kidney fibrosis, but the underlying mechanisms remain elusive. Here, we show that fibrosis of the mouse kidney was associated with the induction of DNA methyltransferases and increases in global DNA methylation and was alleviated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza). Genome-wide analysis demonstrated the hypermethylation of 94 genes in mouse unilateral ureteral obstruction kidneys, which was markedly reduced by 5-Aza. Among these genes, Hoxa5 was hypermethylated at its gene promoter, and this hypermethylation was associated with reduced HOXA5 expression in fibrotic mouse kidneys after ureteral obstruction or unilateral ischemia-reperfusion injury. 5-Aza prevented Hoxa5 hypermethylation, restored HOXA5 expression, and suppressed kidney fibrosis. Downregulation of HOXA5 was verified in human kidney biopsies from patients with chronic kidney disease and correlated with the increased kidney fibrosis and DNA methylation. Kidney fibrosis was aggravated by conditional knockout of Hoxa5 and alleviated by conditional knockin of Hoxa5 in kidney proximal tubules of mice. Mechanistically, we found that HOXA5 repressed Jag1 transcription by directly binding to its gene promoter, resulting in the suppression of JAG1-NOTCH signaling during kidney fibrosis. Thus, our results indicate that loss of HOXA5 via DNA methylation contributes to fibrogenesis in kidney diseases by inducing JAG1 and consequent activation of the NOTCH signaling pathway.
Assuntos
Metilação de DNA , Fibrose , Proteínas de Homeodomínio , Proteína Jagged-1 , Regiões Promotoras Genéticas , Receptores Notch , Transdução de Sinais , Obstrução Ureteral , Animais , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Masculino , Obstrução Ureteral/complicações , Obstrução Ureteral/patologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Rim/patologia , Rim/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Epigênese Genética , Nefropatias/patologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/etiologia , Fatores de TranscriçãoRESUMO
BACKGROUND AND AIMS: Chimeric antigen receptor engineered T cells (CARTs) for HCC and other solid tumors are not as effective as they are for blood cancers. CARTs may lose function inside tumors due to persistent antigen engagement. The aims of this study are to develop low-affinity monoclonal antibodies (mAbs) and low-avidity CARTs for HCC and to test the hypothesis that low-avidity CARTs can resist exhaustion and maintain functions in solid tumors, generating durable antitumor effects. METHODS AND RESULTS: New human glypican-3 (hGPC3) mAbs were developed from immunized mice. We obtained three hGPC3-specific mAbs that stained HCC tumors, but not the adjacent normal liver tissues. One of them, 8F8, bound an epitope close to that of GC33, the frequently used high-affinity mAb, but with approximately 17-fold lower affinity. We then compared the 8F8 CARTs to GC33 CARTs for their in vitro function and in vivo antitumor effects. In vitro, low-avidity 8F8 CARTs killed both hGPC3high and hGPC3low HCC tumor cells to the same extent as high-avidity GC33 CARTs. 8F8 CARTs expanded and persisted to a greater extent than GC33 CARTs, resulting in durable responses against HCC xenografts. Importantly, compared with GC33 CARTs, there were 5-fold more of 8F8-BBz CARTs in the tumor mass for a longer period of time. Remarkably, the tumor-infiltrating 8F8 CARTs were less exhausted and apoptotic, and more functional than GC33 CARTs. CONCLUSION: The low-avidity 8F8-BBz CART resists exhaustion and apoptosis inside tumor lesions, demonstrating a greater therapeutic potential than high-avidity CARTs.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Glipicanas , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Commensal gut microflora and dietary fiber protect against colonic inflammation and colon cancer through unknown targets. Butyrate, a bacterial product from fermentation of dietary fiber in the colon, has been implicated in this process. GPR109A (encoded by Niacr1) is a receptor for butyrate in the colon. GPR109A is also a receptor for niacin, which is also produced by gut microbiota and suppresses intestinal inflammation. Here we showed that Gpr109a signaling promoted anti-inflammatory properties in colonic macrophages and dendritic cells and enabled them to induce differentiation of Treg cells and IL-10-producing T cells. Moreover, Gpr109a was essential for butyrate-mediated induction of IL-18 in colonic epithelium. Consequently, Niacr1(-/-) mice were susceptible to development of colonic inflammation and colon cancer. Niacin, a pharmacological Gpr109a agonist, suppressed colitis and colon cancer in a Gpr109a-dependent manner. Thus, Gpr10a has an essential role in mediating the beneficial effects of gut microbiota and dietary fiber in colon.
Assuntos
Carcinogênese/imunologia , Colite/imunologia , Colo/imunologia , Neoplasias do Colo/prevenção & controle , Células Epiteliais/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Butiratos/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colite/complicações , Colite/tratamento farmacológico , Colo/microbiologia , Colo/patologia , Neoplasias do Colo/etiologia , Células Dendríticas/imunologia , Suscetibilidade a Doenças , Células Epiteliais/efeitos dos fármacos , Interleucina-10/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , Niacina/administração & dosagem , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/imunologia , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
At sites of inflammation, certain regulatory T cells (Treg cells) can undergo rapid reprogramming into helper-like cells without loss of the transcription factor Foxp3. We show that reprogramming is controlled by downregulation of the transcription factor Eos (Ikzf4), an obligate corepressor for Foxp3. Reprogramming was restricted to a specific subset of "Eos-labile" Treg cells that was present in the thymus and identifiable by characteristic surface markers and DNA methylation. Mice made deficient in this subset became impaired in their ability to provide help for presentation of new antigens to naive T cells. Downregulation of Eos required the proinflammatory cytokine interleukin-6 (IL-6), and mice lacking IL-6 had impaired development and function of the Eos-labile subset. Conversely, the immunoregulatory enzyme IDO blocked loss of Eos and prevented the Eos-labile Treg cells from reprogramming. Thus, the Foxp3(+) lineage contains a committed subset of Treg cells capable of rapid conversion into biologically important helper cells.
Assuntos
Proteínas de Transporte/metabolismo , Fator de Transcrição Ikaros/metabolismo , Interleucina-6/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interleucina-6/genética , Ativação Linfocitária/imunologia , Camundongos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , TimoRESUMO
Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.
Assuntos
Neoplasias da Mama/metabolismo , Carcinogênese/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quinases Relacionadas a NIMA/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinogênese/patologia , Feminino , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Mitose/genética , Quinases Relacionadas a NIMA/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , Células PC-3RESUMO
Cancer cells always require more nutrients, energy, and biosynthetic activity to sustain their rapid proliferation than normal cells. Previous studies have shown the impact of THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), on transcription regulation and cell-cycle arrest in numerous cancers, but its effects on cellular metabolism in cancer cells remain unknown. In this study we elucidated the anticancer mechanism of THZ1 in human non-small-cell lung cancer (NSCLC) cells. We showed that treatment with THZ1 (10-1000 nM) dose-dependently suppressed the proliferation of human NSCLC cell lines H1299, A549, H292, and H23, and markedly inhibited the migration of these NSCLC cells. Furthermore, treatment with THZ1 (50 nM) arrested cell cycle at G2/M phase and induced apoptosis in these NSCLC cell lines. More importantly, we revealed that treatment with THZ1 (50 nM) blocked the glycolysis pathway but had no effect on glutamine metabolism. We further demonstrated that THZ1 treatment altered the expression pattern of glutaminase 1 (GLS1) isoforms through promoting the ubiquitination and degradation of NUDT21. Combined treatment of THZ1 with a glutaminase inhibitor CB-839 (500 nM) exerted a more potent anti-proliferative effect in these NSCLC cell lines than treatment with THZ1 or CB-839 alone. Our results demonstrate that the inhibitory effect of THZ1 on the growth of human NSCLC cells is partially attributed to interfering with cancer metabolism. Thus, we provide a new potential therapeutic strategy for NSCLC treatment by combining THZ1 with the inhibitors of glutamine metabolism.
Assuntos
Antineoplásicos/farmacologia , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Apoptose/efeitos dos fármacos , Benzenoacetamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Sinergismo Farmacológico , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Tiadiazóis/farmacologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
LTR retrotransposons are repetitive DNA elements comprising â¼10% of the human genome. However, LTR sequences are disproportionately present in human long, non-coding RNAs (lncRNAs). Whether and how the LTR lncRNAs serve biological functions are largely unknown. Here we show that in primary human erythroblasts, lncRNAs transcribed from the LTR retrotransposons of ERV-9 human endogenous retrovirus activated transcription of key erythroid genes and modulated ex vivo erythropoiesis. To dissect the functional mechanism of ERV-9 lncRNAs, we performed genome-wide RNA and ChIRP analyses before and after global knockdown or locus-specific deletion of ERV-9 lncRNAs in human erythroblasts carrying â¼4000 copies of the ERV-9 LTRs and in transgenic mouse erythroblasts carrying a single copy of the primate-specific ERV-9 LTR in the 100 kb human ß-globin gene locus. We found that ERV-9 lncRNAs acted in cis to stabilize assembly of the ERV-9 LTR enhancer complex and facilitate long-range LTR enhancer function in activating transcription of downstream, cis-linked globin genes. Our findings suggested that LTR lncRNAs transcribed from many of the 4000 copies of ERV-9 LTR retrotransposons acted by a similar cis mechanism to modulate LTR enhancer function in activating transcription of downstream genes critical to cellular processes including erythropoiesis.
Assuntos
Elementos Facilitadores Genéticos , Eritroblastos/metabolismo , RNA Longo não Codificante/genética , Retroelementos , Sequências Repetidas Terminais , Globinas beta/genética , Animais , Sequência de Bases , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Eritroblastos/citologia , Eritropoese , Loci Gênicos , Genoma , Humanos , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , RNA Longo não Codificante/metabolismo , Transcrição Gênica , Globinas beta/metabolismoRESUMO
We previously observed that disruption of FK506-binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)-induced cardiac hypertrophy in mice, whereas the adenovirus-mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)-induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6-/- ) mice and cardiac-specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini-pump. The results showed that FKBP12.6 deficiency aggravated AngII-induced cardiac hypertrophy, while cardiac-specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII-induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII-induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca2+ ([Ca2+ ]i), in which the protein significantly inhibited the key Ca2+ /calmodulin-dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF-2, AKT/Glycogen synthase kinase 3ß (GSK3ß)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII-induced cardiac hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Cardiomegalia/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Angiotensina II/metabolismo , Angiotensina II/toxicidade , Animais , Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Linhagem Celular , Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Motivation: Chromatin accessibility plays a key role in epigenetic regulation of gene activation and silencing. Open chromatin regions allow regulatory elements such as transcription factors and polymerases to bind for gene expression while closed chromatin regions prevent the activity of transcriptional machinery. Recently, Methyltransferase Accessibility Protocol for individual templates-Bisulfite Genome Sequencing (MAPit-BGS) and nucleosome occupancy and methylome sequencing (NOMe-seq) have been developed for simultaneously profiling chromatin accessibility and DNA methylation on single molecules. Therefore, there is a great demand in developing computational methods to identify chromatin accessibility from MAPit-BGS and NOMe-seq. Results: In this article, we present CAME (Chromatin Accessibility and Methylation), a seed-extension based approach that identifies chromatin accessibility from NOMe-seq. The efficiency and effectiveness of CAME were demonstrated through comparisons with other existing techniques on both simulated and real data, and the results show that our method not only can precisely identify chromatin accessibility but also outperforms other methods. Availability and Implementation: CAME is implemented in java and the program is freely available online at http://sourceforge.net/projects/came/. Contacts: jechoi@gru.edu or khryu@dblab.chungbuk.ac.kr. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Metilação de DNA/genética , Nucleossomos/metabolismo , Análise de Sequência de DNA/métodos , Software , Algoritmos , Sequência de Bases , Neoplasias do Colo/genética , Simulação por Computador , Ilhas de CpG/genética , Bases de Dados Genéticas , Epigênese Genética , Células HCT116 , Humanos , Conformação de Ácido Nucleico , Curva ROC , Padrões de ReferênciaRESUMO
BACKGROUND: Haemophagocytic lymphohistiocytosis (HLH) is considered to be a large challenge for clinicians due to the variable overlaps of symptoms with other severe diseases and a high rate of mortality. Prompt diagnosis and treatment are crucial to avoid a fatal outcome. However, very limited reports have focused on HLH during chemotherapy (Ch-HLH) due to a low incidence and insufficient knowledge. CASE PRESENTATION: A 22-year-old male was diagnosed with acute monocytic leukemia with FLT3-ITD and DNMT3A mutations and pulmonary infection. He received IA regimen (Idarubicin, 8 mg/m2/d for 3 days and cytarabine, 100 mg/m2/d for 7 days) chemotherapy, anti-infection drugs and blood components transfusions. During the stage of bone marrow suppression, he presented with a fever, cytopenia (WBC, 0.43 × 109/L; Hb, 73 g/L and PLT, 1 × 109/L), refractory coagulation dysfunction (APTT, 104.0 s; PT, 30.5 s and Fbg, 0.87 g/L), splenomegaly (3 cm below the costal margin), hyperferritinemia (SF > 3000 µg/L), increased soluble interleukin-II receptors (sIL-2R > 7500 u/mL) and haemophagocytosis in the bone marrow and was diagnosed with HLH. After he was treated with methylprednisolone at 500 mg/d for 3 days, 120 mg/d for 3 days and 80 mg/d for 3 days, followed by a gradually reduced dose combined with powerful anti-infection drugs, his symptoms subsided and his abnormal parameters recovered to normal levels. CONCLUSION: Patients with HLH in acute leukemia have a high rate of mortality. This case report provides helpful clinical experiences relative to the recognition and treatment of Ch-HLH for clinicians.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Quimioterapia de Indução/efeitos adversos , Leucemia Monocítica Aguda/tratamento farmacológico , Linfo-Histiocitose Hemofagocítica/induzido quimicamente , Adulto , Anti-Infecciosos/uso terapêutico , Medula Óssea/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Humanos , Leucemia Monocítica Aguda/genética , Linfo-Histiocitose Hemofagocítica/sangue , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Masculino , Metilprednisolona/uso terapêutico , Mutação , Sequências de Repetição em Tandem/genética , Resultado do Tratamento , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
BACKGROUND: Total P16 methylation (P16M), including P16 hydroxymethylation (P16H) and true-P16M, correlates with malignant transformation of oral epithelial dysplasia (OED). Both true-P16M and P16H are early events in carcinogenesis. The aim of this study is to prospectively determine if discrimination of true-P16M from P16H is necessary for prediction of cancer development from OEDs. METHODS: Patients (n = 265) with mild or moderate OED were recruited into the double blind two-center cohort. Total-P16M and P16H were analyzed using the 115-bp MethyLight, TET-assisted bisulfite (TAB) methylation-specific PCR (MSP), and TAB-sequencing. Total-P16M-positive and P16H-negative samples were defined as true-P16M-positive. Progression of OEDs was monitored for a minimum 24 months follow-up period. RESULTS: P16H was detected in 23 of 73 (31.5%) total-P16M-positive OEDs. Follow-up information was obtained from 247 patients with an ultimate compliance rate of 93.2%. OED-derived squamous cell carcinomas were observed in 13.0% (32/247) patients during follow-up (median, 41.0 months). The cancer progression rate for total-P16M-positive patients was significantly increased when compared to total-P16M-negative patients [23.3% vs 8.6%; adjusted odds ratio = 2.67 (95% CI: 1.19-5.99)]. However, the cancer progression rates were similar between P16H- and true-P16M-positive OEDs [26.1% (6/23) vs 22.0% (11/50); odds ratio = 0.80 (95% CI: 0.22-2.92)]. The cancer-free survival was also similar for these patients. CONCLUSION: P16H and true-P16M are similar biomarkers for determining malignant potential of OEDs. Discrimination of P16H from true-P16M, at least in OED, may be not necessary in clinical applications. TRIAL REGISTRATION: This study is registered prospectively in the U.S. National Institutes of Health Clinical Trials Protocol Registration System (trial number NCT02967120, available at https://ClinicalTrials.gov/ct2/show/NCT02967120 ).
Assuntos
Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/etiologia , Idoso , Alelos , Biomarcadores Tumorais , Ilhas de CpG , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Prognóstico , Estudos Prospectivos , Fatores de RiscoRESUMO
CD38 was first identified as a lymphocyte-specific antigen and then has been found to be widely expressed in a variety of cell types. The functions of CD38 are involved in numerous biological processes including immune responses. Here, we showed the downregulations of both TLR2 mRNA and protein in macrophages from CD38-/- mice and in CD38 knockdown RAW264.7 cells. Several NF-κB-binding motifs in the promoter region of the TLR2 gene were identified by the bioinformatics analysis and were confirmed by the luciferase activity assay with the different truncated TLR2 promoters. CD38 deficiency resulted in the reduction of NF-κB p65 and acetyl-NF-κB p65 (Ac-p65) levels as determined by Western blot. The expression of Sirt1 did not change, but an increased activity of Sirt1 was observed in CD38-deficient macrophages. Inhibition of the Sirt1/NF-κB signaling pathway resulted in downregulation of TLR2 expression in RAW264.7 cells. However, re-expression of CD38 in the knockdown clones reversed the effect on Sirt1/NF-κB/TLR2 signaling, which is NAD-dependent. Moreover, the inflammatory cytokines including G-CSF, IL-1alpha, IL-6, MCP-1, MIP-1alpha, and RANTES were increased in CD38 knockdown RAW264.7 cells. Taken together, our data demonstrated that CD38 deficiency enhances inflammatory response in macrophages, and the mechanism may be partly associated with increased Sirt1 activity, which promoted NF-κB deacetylation and then inhibited expression of the TLR2 gene. Obviously, our study may provide an insight into the molecular mechanisms in CD38-mediated inflammation.
Assuntos
ADP-Ribosil Ciclase 1/deficiência , Inflamação/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Sirtuína 1/metabolismo , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Animais , Western Blotting , Biologia Computacional , Inflamação/genética , Camundongos , Células RAW 264.7 , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sirtuína 1/genética , Receptor 2 Toll-Like/metabolismoRESUMO
DNA methylation is an epigenetic mechanism that regulates gene transcription without changing primary nucleotide sequences. In mammals, DNA methylation involves the covalent addition of a methyl group to the 5-carbon position of cytosine by DNA methyltransferases (DNMTs). The change of DNA methylation and its pathological role in acute kidney injury (AKI) remain largely unknown. Here, we analyzed genome-wide DNA methylation during cisplatin-induced AKI by reduced representation bisulfite sequencing. This technique identified 215 differentially methylated regions between the kidneys of control and cisplatin-treated animals. While most of the differentially methylated regions were in the intergenic, intronic, and coding DNA sequences, some were located in the promoter or promoter-regulatory regions of 15 protein-coding genes. To determine the pathological role of DNA methylation, we initially examined the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine and showed it increased cisplatin-induced apoptosis in a rat kidney proximal tubular cell line. We further established a kidney proximal tubule-specific DNMT1 (PT-DNMT1) knockout mouse model, which showed more severe AKI during cisplatin treatment than wild-type mice. Finally, interferon regulatory factor 8 (Irf8), a pro-apoptotic factor, was identified as a hypomethylated gene in cisplatin-induced AKI, and this hypomethylation was associated with a marked induction of Irf8. In the rat kidney proximal tubular cells, the knockdown of Irf8 suppressed cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. Thus, DNA methylation plays a protective role in cisplatin-induced AKI by regulating specific genes, such as Irf8.
Assuntos
Injúria Renal Aguda/genética , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Fatores Reguladores de Interferon/genética , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/genética , Decitabina , Modelos Animais de Doenças , Epigênese Genética , Técnicas de Silenciamento de Genes , Genoma , Humanos , Fatores Reguladores de Interferon/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/tratamento farmacológico , Ratos , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND: Methylation changes are frequent in cancers, but understanding how hyper- and hypomethylated region changes coordinate, associate with genomic features, and affect gene expression is needed to better understand their biological significance. The functional significance of hypermethylation is well studied, but that of hypomethylation remains limited. Here, with paired expression and methylation samples gathered from a patient/control cohort, we attempt to better characterize the gene expression and methylation changes that take place in cancer from B cell chronic lymphocyte leukemia (B-CLL) samples. RESULTS: Across the dataset, we found that consistent differentially hypomethylated regions (C-DMRs) across samples were relatively few compared to the many poorly consistent hypo- and highly conserved hyper-DMRs. However, genes in the hypo-C-DMRs tended to be associated with functions antagonistic to those in the hyper-C-DMRs, like differentiation, cell-cycle regulation and proliferation, suggesting coordinated regulation of methylation changes. Hypo-C-DMRs in B-CLL were found enriched in key signaling pathways like B cell receptor and p53 pathways and genes/motifs essential for B lymphopoiesis. Hypo-C-DMRs tended to be proximal to genes with elevated expression in contrast to the transcription silencing-mechanism imposed by hypermethylation. Hypo-C-DMRs tended to be enriched in the regions of activating H4K4me1/2/3, H3K79me2, and H3K27ac histone modifications. In comparison, the polycomb repressive complex 2 (PRC2) signature, marked by EZH2, SUZ12, CTCF binding-sites, repressive H3K27me3 marks, and "repressed/poised promoter" states were associated with hyper-C-DMRs. Most hypo-C-DMRs were found in introns (36 %), 3' untranslated regions (29 %), and intergenic regions (24 %). Many of these genic regions also overlapped with enhancers. The methylation of CpGs from 3'UTR exons was found to have weak but positive correlation with gene expression. In contrast, methylation in the 5'UTR was negatively correlated with expression. To better characterize the overlap between methylation and expression changes, we identified correlation modules that associate with "apoptosis" and "leukocyte activation". CONCLUSIONS: Despite clinical heterogeneity in disease presentation, a number of methylation changes, both hypo and hyper, appear to be common in B-CLL. Hypomethylation appears to play an active, targeted, and complementary role in cancer progression, and it interplays with hypermethylation in a coordinated fashion in the cancer process.
Assuntos
Metilação de DNA , Estudo de Associação Genômica Ampla/métodos , Leucemia Linfocítica Crônica de Células B/genética , Regiões Promotoras Genéticas/genética , Biomarcadores Tumorais/genética , Ilhas de CpG/genética , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Ontologia Genética , Heterogeneidade Genética , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Histonas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Metilação , Modelos GenéticosRESUMO
DNA methylation is an epigenetic modification that plays an important role in the regulation of gene expression. The function of RUNDC3B has yet to be determined, although its dysregulated expression has been associated with malignant potential of both breast and lung carcinoma. To elucidate the potential of using DNA methylation in RUNDC3B as a biomarker in lymphoid malignancies, the methylation status of six regions spanning the CpG island in the promoter region of RUNDC3B was determined in cancer cell lines. Lymphoid malignancies were found to have more prominent methylation and did not express RUNDC3B compared with myeloid malignancies and solid tumours, supporting the potential use of DNA methylation in this region as a biomarker for lymphoid malignancies. RUNDC3B contains a RUN domain in its N-terminal region that mediates interaction with Rap2, an important component of the mitogen-activated protein kinase (MAPK) cascade, which regulates cellular proliferation and differentiation. The protein sequence of RUNDC3B also contains characteristic binding sites for MAPK intermediates. Therefore, it is possible that RUNDC3B serves as a mediator between Rap2 and the MAPK signalling cascade. Three genes with MAPK-inducible expression were downregulated in a methylated leukaemia cell line (HSPA5, Jun and Fos). Jun and Fos combine to form the activating protein 1 transcription factor, and loss of this factor is associated with the dysregulation of genes involved in differentiation and proliferation. We hypothesize that the loss of RUNDC3B secondary to aberrant hypermethylation of the early growth response 3 transcription factor binding site results in dysregulated MAPK signalling and carcinogenesis in lymphoid malignancies. © 2015 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Células A549 , Sítios de Ligação , Biomarcadores Tumorais/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Chaperona BiP do Retículo Endoplasmático , Epigênese Genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Leucemia/metabolismo , Sistema de Sinalização das MAP Quinases , Domínios Proteicos , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Differential methylation of regulatory elements is critical in epigenetic researches and can be statistically tested. We developed a new statistical test, the generalized integrated functional test (GIFT), that tests for regional differences in methylation based on the methylation percent at each CpG site within a genomic region. The GIFT uses estimated subject-specific profiles with smoothing methods, specifically wavelet smoothing, and calculates an ANOVA-like test to compare the average profile of groups. In this way, possibly correlated CpG sites within the regulatory region are compared all together. Simulations and analyses of data obtained from patients with chronic lymphocytic leukemia indicate that GIFT has good statistical properties and is able to identify promising genomic regions. Further, GIFT is likely to work with multiple different types of experiments since different smoothing methods can be used to estimate the profiles of data without noise. Matlab code for GIFT and sample data are available at http://www.augusta.edu/mcg/biostatepi/people/software/gift.html.
Assuntos
Ilhas de CpG , Metilação de DNA , Modelos Genéticos , Sequências Reguladoras de Ácido Nucleico/genética , Simulação por Computador , Interpretação Estatística de Dados , Genômica , Humanos , Leucemia Linfocítica Crônica de Células B/genéticaRESUMO
The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.