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Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Especificidade por SubstratoRESUMO
To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
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Biocatálise , Histonas/metabolismo , Oncogenes , Transcrição Gênica , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Linhagem Celular , Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Sequência Conservada , Evolução Molecular , Redes Reguladoras de Genes , Genoma , Histona Desacetilases/metabolismo , Humanos , Cinética , Metilação , Modelos Biológicos , RNA Polimerase II/metabolismoRESUMO
AIMS: The aims of this study were to evaluate the impact of tumour-associated necrosis (TAN) on metastasis-free survival for clear cell renal cell carcinoma (RCC), and to determine whether TAN provides survival information additional to World Health Organization (WHO)/International Society of Urological Pathology (ISUP) grading. METHODS AND RESULTS: The study consisted of 376 cases of clear cell RCC treated by nephrectomy, for which follow-up was available. WHO/ISUP grade was assigned, and sections were assessed for the presence of TAN. American Joint Committee on Cancer (AJCC) pT staging category and tumour size were also recorded. The development of metastatic disease was taken as the clinical endpoint, and survival analyses, utilising univariate and multivariate models, were performed. WHO/ISUP grades were: grade 1, 35 cases (9.3%); grade 2, 188 cases (50.0%); grade 3, 91 cases (24.2%); and grade 4, 62 cases (16.5%). Staging categories were pT1-pT2 [234 tumours (62.2%)] and pT3-pT4 [139 tumours (37.0%)]. TAN was seen in 128 cases (34.0%). Neither TAN nor metastases were seen in grade 1 tumours. Among grade 2-4 tumours, those with TAN had a significantly worse prognosis than those without TAN (P = 0.017, P = 0.04, and P = 0.006, respectively). Multivariate analysis (WHO/ISUP grade, pT staging category, and TAN) showed all three variables to be independently associated with outcome (P = 0.009, P = 0.005, and P = 0.001, respectively). For all tumour grades and pT staging categories, it was found that the presence of TAN was associated with a 2.91-fold greater risk of metastatic disease. CONCLUSION: Tumour-associated necrosis is an important prognostic factor for clear cell RCC, independently of WHO/ISUP grade. This supports the suggestion that TAN could be incorporated into tumour grading criteria.
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Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Rim/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/cirurgia , Feminino , Humanos , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Necrose/patologia , Gradação de Tumores , Nefrectomia , Prognóstico , Resultado do Tratamento , Organização Mundial da SaúdeRESUMO
AIMS: In 2012, the International Society of Urological Pathology (ISUP) introduced a novel grading system for clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma. This system is incorporated into the latest World Health Organization renal tumour classification, being designated WHO/ISUP grading. This study was undertaken to compare WHO/ISUP and Fuhrman grading and to validate WHO/ISUP grading as a prognostic parameter in a series of clear cell RCC. METHODS AND RESULTS: Analysis of 681 cases of ccRCC showed that 144 tumours could not be assigned a Fuhrman grade on the basis of ambiguous grading features. The application of WHO/ISUP grading resulted in a general down-grading of cases when compared with Fuhrman grading. In a sub-group of 374 cases, for which outcome data were available, 9.3% were WHO/ISUP grade 1, 50.3% were grade 2, 24.1% grade 3 and 16.3% grade 4, while the distribution of Fuhrman grades was 0.4% grade 1, 48.7% grade 2, 29.4% grade 3 and 21.5% grade 4. There were no recurrence/metastases amongst patients with WHO/ISUP grade 1 tumours and there was a significant difference in outcome for WHO/ISUP grades 2, 3 and 4. For Fuhrman grading the cancer-free survival was not significantly different for grade 2 and grade 3 tumours. On multivariate analysis WHO/ISUP grade and pT staging category were found to retain prognostic significance. CONCLUSIONS: The study demonstrates that FG cannot be applied in >20% of cases of ccRCC and the WHO/ISUP provides superior prognostic information.
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Carcinoma de Células Renais/classificação , Neoplasias Renais/classificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Rim/patologia , Neoplasias Renais/diagnóstico , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Sociedades Médicas , Organização Mundial da Saúde , Adulto JovemRESUMO
Neuroblastoma is the most common extra-cranial malignancy in childhood and accounts for â¼15% of childhood cancer deaths. Amplification of MYCN in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N-Myc and regulate numerous cellular processes via epigenetic modulation, including differentiation. In this study, we used the TH-MYCN mouse model of neuroblastoma to investigate the antitumor activity of the pan-HDAC inhibitor, panobinostat. In particular we sought to explore the impact of long term, continuous panobinostat exposure on the epigenetically driven differentiation process. Continuous treatment of tumor bearing TH-MYCN transgenic mice with panobinostat for nine weeks led to a significant improvement in survival as compared with mice treated with panobinostat for a three-week period. Panobinostat induced rapid tumor regression with no regrowth observed following a nine-week treatment period. Initial tumor response was associated with apoptosis mediated via upregulation of BMF and BIM. The process of terminal differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N-Myc expression, occurred following prolonged exposure to the drug. RNA-sequencing analysis of tumors from treated animals confirmed significant upregulation of gene pathways associated with apoptosis and differentiation. Together our data demonstrate the potential of panobinostat as a novel therapeutic strategy for high-risk neuroblastoma patients.
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Diferenciação Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Ácidos Hidroxâmicos/administração & dosagem , Indóis/administração & dosagem , Neuroblastoma/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neuroblastoma/genética , Neuroblastoma/patologia , Panobinostat , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas S100/biossíntese , Análise de SobrevidaRESUMO
UNLABELLED: Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01365065.
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Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Ativação Viral/efeitos dos fármacos , Adulto , Linfócitos T CD4-Positivos/imunologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Transcrição Gênica/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , VorinostatRESUMO
Immunomodulators are effective in controlling hematologic malignancy by initiating or reactivating host antitumor immunity to otherwise poorly immunogenic and immune suppressive cancers. We aimed to boost antitumor immunity in B-cell lymphoma by developing a tumor cell vaccine incorporating α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells. In the Eµ-myc transgenic mouse model, single therapeutic vaccination of irradiated, α-GalCer-loaded autologous tumor cells was sufficient to significantly inhibit growth of established tumors and prolong survival. Vaccine-induced antilymphoma immunity required NKT cells, NK cells, and CD8 T cells, and early IL-12-dependent production of IFN-γ. CD4 T cells, gamma/delta T cells, and IL-18 were not critical. Vaccine treatment induced a large systemic spike of IFN-γ and transient peripheral expansion of both NKT cells and NK cells, the major sources of IFN-γ. Furthermore, this vaccine approach was assessed in several other hematopoietic tumor models and was also therapeutically effective against AML-ETO9a acute myeloid leukemia. Replacing α-GalCer with ß-mannosylceramide resulted in prolonged protection against Eµ-myc lymphoma. Overall, our results demonstrate a potent immune adjuvant effect of NKT cell ligands in therapeutic anticancer vaccination against oncogene-driven lymphomas, and this work supports clinical investigation of NKT cell-based immunotherapy in patients with hematologic malignancies.
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Vacinas Anticâncer/uso terapêutico , Galactosilceramidas/administração & dosagem , Genes myc/genética , Imunoterapia , Linfoma de Células B/imunologia , Linfoma de Células B/prevenção & controle , Células T Matadoras Naturais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Citotoxicidade Imunológica/imunologia , Feminino , Citometria de Fluxo , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T/fisiologia , Humanos , Interferon gama/metabolismo , Interleucina-12/fisiologia , Interleucina-18/fisiologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Linfoma de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/patologia , VacinaçãoRESUMO
Chemical transformations of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) during wastewater treatment and sludge treatment must be characterized to accurately assess the risks that these nanomaterials pose from land application of biosolids. Here, X-ray absorption spectroscopy (XAS) and supporting characterization methods are used to determine the chemical speciation of Ag and Zn in sludge from a pilot wastewater treatment plant (WWTP) that had received PVP coated 50 nm Ag NPs and 30 nm ZnO NPs, dissolved metal ions, or no added metal. The effects of composting and lime and heat treatment on metal speciation in the resulting biosolids were also examined. All added Ag was converted to Ag2S, regardless of the form of Ag added (NP vs ionic). Zn was transformed to three Zn-containing species, ZnS, Zn3(PO4)2, and Zn associated Fe oxy/hydroxides, also regardless of the form of Zn added. Zn speciation was the same in the unamended control sludge. Ag2S persisted in all sludge treatments. Zn3(PO4)2 persisted in sludge and biosolids, but the ratio of ZnS and Zn associated with Fe oxy/hydroxide depended on the redox state and water content of the biosolids. Limited differences in Zn and Ag speciation among NP-dosed, ion-dosed, and control biosolids indicate that these nanoparticles are transformed to similar chemical forms as bulk metals already entering the WWTP.
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Nanopartículas Metálicas/análise , Prata/análise , Águas Residuárias/análise , Poluentes Químicos da Água/análise , Óxido de Zinco/análise , Compostos de Cálcio/química , Temperatura Alta , Nanopartículas Metálicas/química , Óxidos/química , Prata/química , Solo/química , Eliminação de Resíduos Líquidos , Águas Residuárias/química , Poluentes Químicos da Água/química , Óxido de Zinco/químicaRESUMO
Introduction: Physical activity is commonly used for both measuring and treating dysfunction. While preclinical work has been historically biased towards males, the use of both male and female animals is gaining popularity after multiple NIH initiatives. With increasing inclusion of both sexes, it has become imperative to determine sex differences in common behavioral assays. The purpose of this study was to determine baseline sex differences in 3 activity assays: voluntary wheel running, forced treadmill running, and open field testing. Methods: This was a secondary analysis of sex differences in healthy mice in 3 different assays: Separate mice were used for each assay. Specifically, 16 mice underwent 28 days of voluntary wheel running, 178 mice underwent forced treadmill running, and 88 mice underwent open field testing. Differences between sex across several activity parameters were examined for each assay. Results: In voluntary wheel running, sex differences with larger effect sizes were observed in distance run, running time, and bout duration, with smaller effect size differences in speed, and no difference in total bouts. In forced treadmill running, differences were shown in time to exhaustion, but no difference in max speed attained. In open field, there were sex differences in active time but not in distance and speed in data aggregated over 30 minutes; however, distance and speed in male mice showed a downward trajectory over the final 20 minutes of testing, whereas females maintained the same trajectory. Conclusion: These data suggest that male mice demonstrate comparable activity intensity as female mice but do not match female's duration of activity, especially for volitional tasks. Researchers utilizing these assays should account for sex differences as they could potentially mask true findings in an experiment. Plain English Summary: Physical activity is a common measure to examine function in human subjects with and without disease. Animal models often use measures of physical activity to assess function, yet most of these measures have been done in males only, making interpretation and translation to females and humans difficult. Several measures have been used to measure activity in animals, including those examining voluntary running behavior, maximum capacity, and general activity levels; sex differences between these measures are unclear. We discovered sex differences throughout each of three activity tests. In voluntary running behavior there were large differences between sexes with females running a greater distance and spending more time running. There were small differences in the maximum capacity with females running for a longer period at high intensity. General activity levels showed small differences with females being less active than males. Thus, the greatest differences were found for voluntary running and small differences were found for maximum capacity and general activity levels; differences observed were dependent on the task. Researchers utilizing these assays should account for sex differences as they could potentially mask true findings in an experiment.
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Signal transducer and activator of transcription 1 (Stat1) mediates anti-viral responses and cytokine-driven anti-proliferative, apoptotic and immunomodulatory activities. As de-regulated Stat1 function can affect tumour progression we sought to elucidate the effects of tumour cell-intrinsic Stat1 expression on immunosurveillance. Knockout of Stat1 enhanced the development of sarcomas induced by the chemical carcinogen 3-methylcholanthrene (MCA). Growth of transplanted MCA-induced Stat1â»/â» sarcomas was suppressed in wild-type mice compared to growth in Stat1â»/â» and immunocompromised recipients. Co-depletion of NK and CD8⺠T cells from wild-type mice facilitated Stat1-deficient tumour growth whereas depletion of CD4⺠T cells and CD8⺠T cells did not. In vitro and in vivo analysis of the tumours implicated a role for NK cell-mediated, perforin-dependent killing of Stat1-deficient tumours. Interestingly, restoration of Stat1 expression in Stat1â»/â» tumours resulted in diminished involvement of NK cells and increased contribution of CD8⺠T cells in anti-tumour responses. Therefore, Stat1 expression within tumour cells modulated anti-tumour immune responses by altering the dominant immune effector cell involvement from NK cells to CD8⺠T cells in the absence or presence of Stat1 respectively.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Fator de Transcrição STAT1/metabolismo , Sarcoma Experimental/imunologia , Animais , Processos de Crescimento Celular/genética , Células Cultivadas , Citotoxicidade Imunológica , Regulação Neoplásica da Expressão Gênica , Humanos , Depleção Linfocítica , Metilcolantreno/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monitorização Imunológica , Transplante de Neoplasias , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Sarcoma Experimental/induzido quimicamenteRESUMO
Over the past twenty years, various commercial technologies have been deployed to remove ammonia (NH4-N) from anaerobic digestion (AD) liquors. In recent years many anaerobic digesters have been upgraded to include a pre-treatment, such as the thermal hydrolysis process (THP), to produce more biogas, increasing NH4-N concentrations in the liquors are costly to treat. This study provides a comparative techno-economic assessment of sidestream technologies to remove NH4-N from conventional AD and THP/AD dewatering liquors: a deammonification continuous stirred tank reactor (PNA), a nitrification/denitrification sequencing batch reactor (SBR) and thermal ammonia stripping process with an ammonia scrubber (STRIP). The SBR and PNA were based on full-scale data, whereas the STRIP was designed using a computational approach to achieve NH4-N removals of 90-95%. The PNA presented the lowest whole-life cost (WLC) over 40 years, with £7.7 M, while the STRIP had a WLC of £43.9 M. This study identified that THP dewatering liquors, and thus a higher ammonia load, can lead to a 1.5-3.0 times increase in operational expenditure with the PNA and the SBR. Furthermore, this study highlighted that deammonification is a capable and cost-effective nitrogen removal technology. Processes like the STRIP respond to current pressures faced by the water industry on ammonia recovery together with targets to reduce nitrous oxide emissions. Nevertheless, ammonia striping-based processes must further be demonstrated in WWTPs and WLC reduced to grant their wide implementation and replace existing technologies.
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ABSTRACT: Rigorous experimental design with transparent reporting in biomedical science reduces risk of bias and allows for scientists to judge the quality of the research. Basic factors of rigor such as blinding, randomization, power analysis, and inclusion of both sexes impact the reproducibility by reducing experimental bias. We designed a systematic study to analyze basic factors of rigor, inclusion of sex, and whether data were analyzed or disaggregated by sex over the past 10 years in the journal PAIN . Studies that included humans reported randomization in 81%, blinding in 48%, and the use of a power analysis calculation in 27% over the past 10 years. Studies that included mice reported randomization in 35%, blinding in 70%, and the use of a power analysis in 9%. Studies that included rats reported randomization in 38%, blinding in 63%, and the use of power analysis in 12%. This study also found that human studies consistently included both sexes over the past decade, but less than 20% of data were disaggregated or analyzed for sex differences. Although mouse and rat studies predominately used males only, there has been a slight increase in inclusion of both sexes over the past few years. Justification for single-sex studies was below 50% in both human and rodent data. In both human and animal studies, transparency in reporting of experimental design and inclusion of both sexes should be considered standard practice and will result in improved quality and reproducibility of published research.
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Projetos de Pesquisa , Caracteres Sexuais , Feminino , Humanos , Masculino , Animais , Camundongos , Ratos , Reprodutibilidade dos Testes , ViésRESUMO
The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.
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Anticorpos Monoclonais/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias Mamárias Experimentais/terapia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Animais , Apoptose , Western Blotting , Terapia Combinada , Sinergismo Farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Indóis , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Panobinostat , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Histona Desacetilases , Neoplasias Mamárias Experimentais/tratamento farmacológico , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Animais , Anticorpos Monoclonais/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , VorinostatRESUMO
Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.
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Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.
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Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Anergia Clonal/imunologia , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Linfopoese/fisiologia , Animais , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteína 2 de Resposta de Crescimento Precoce/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.
Assuntos
Apoptose , Cerebelo/citologia , Cerebelo/metabolismo , Neurônios/citologia , Receptor de Endotelina B/deficiência , Células-Tronco/citologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Proliferação de Células , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Marcação In Situ das Extremidades Cortadas , Mutação/genética , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptor de Endotelina B/metabolismo , Células-Tronco/metabolismoRESUMO
In this study, the effects, fate and transport of ENPs in wastewater treatment plants (WWTP) were investigated using three parallel pilot WWTPs operated under identical conditions. The WWTPs were spiked with (i) an ENP mixture consisting of silver oxide, titanium dioxide and zinc oxide, and (ii) bulk metal salts. The third plant served as control (unspiked). ENP effects were evaluated for (i) bulk contaminant removal, (ii) activated sludge (AS) process performance, (iii) microbial community structure and dynamics and (iv) microbial inhibition. ENPs showed a strong affinity for biosolids and induced a specific oxygen uptake rate two times higher than the control. The heterotrophic biomass retained its ability to nitrify and degrade organic matter. However, non-recovery of ammonia- and nitrite-oxidizing bacteria such as Nitrosomonas, Nitrobacter or Nitrospira in the ENP spiked reactors suggests selective inhibitory effects. The results further suggest that ENPs and metal salts have antimicrobial properties which can reduce synthesis of extracellular polymeric substances and therefore floc formation. Scanning electron microscopy evidenced selective damage to some microbes, whereas lipid fingerprinting and 454 pyrosequencing indicated a temporal shift in the microbial community structure and diversity. Acidovorax, Rhodoferax, Comamonas and Methanosarcina were identified as nano-tolerant species. Competitive growth advantage of the nano-tolerant species influenced the removal processes and unlike other xenobiotic compounds, ENPs can hasten the natural selection of microbial species in AS.
Assuntos
Comamonas/metabolismo , Methanosarcina/metabolismo , Nanopartículas , Esgotos/microbiologia , Purificação da Água/métodos , Amônia/metabolismo , Biomassa , Processos Heterotróficos , Nitrificação , Nitritos/metabolismo , Nitrobacter/isolamento & purificação , Nitrosomonas/isolamento & purificação , Óxidos/química , Oxigênio/metabolismo , Compostos de Prata/química , Titânio/química , Óxido de Zinco/químicaRESUMO
Positron emission tomography (PET) can be used to monitor the uptake of the labeled glucose analog fluorodeoxyglucose (¹8F-FDG), a process that is generally believed to reflect viable tumor cell mass. The use of ¹8F-FDG PET can be helpful in documenting over time the reduction in tumor mass volume in response to anticancer drug therapy in vivo. In this protocol, we describe how to monitor the response of murine B-cell lymphomas to an inducer of apoptosis, the anticancer drug vorinostat (a histone deacetylase inhibitor). B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with vorinostat. The tracer ¹8F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because the uptake of ¹8F-FDG is not a direct measure of apoptosis, an additional direct method proving that apoptotic cells are present should also be performed.
Assuntos
Antineoplásicos/uso terapêutico , Fluordesoxiglucose F18 , Ácidos Hidroxâmicos/uso terapêutico , Linfoma de Células B/diagnóstico por imagem , Linfoma de Células B/tratamento farmacológico , Tomografia por Emissão de Pósitrons , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Positron emission tomography (PET) is used to monitor the uptake of the labeled glucose analogue fluorodeoxyglucose (¹8F-FDG) by solid tumor cells, a process generally believed to reflect viable tumor cell mass. The use of ¹8F-FDG exploits the high demand for glucose in tumor cells, and serves to document over time the response of a solid tumor to an inducer of apoptosis. The apoptosis inducer crizotinib is a small-molecule inhibitor of c-Met, a receptor tyrosine kinase that is often dysregulated in human tumors. In this protocol, we describe how to monitor the response of a solid tumor to crizotinib. Human gastric tumor cells (GTL-16 cells) are injected into recipient mice and, on tumor formation, the mice are treated with crizotinib. The tracer ¹8F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because ¹8F-FDG uptake varies widely among different tumor models, preliminary experiments should be performed with each new model to determine its basal level of ¹8F-FDG uptake. Verifying that the basal level of uptake is sufficiently above background levels will assure accurate quantitation. Because ¹8F-FDG uptake is not a direct measure of apoptosis, it is advisable to carry out an additional direct method to show the presence of apoptotic cells.