RESUMO
Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.
Assuntos
Leucemia Mieloide Aguda , Apoptose , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Histona Desacetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis.
Assuntos
Inflamação/imunologia , Interleucina-33/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos/imunologia , Mastócitos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Psoríase/imunologia , Pele/imunologia , Animais , Movimento Celular , Células Cultivadas , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
One of the main characteristics of cancer is the rapid proliferation of transformed cells. Cancer therapies aim to kill such cells. Cancer clones surviving therapy can be resistant to the treatment, but they can also lose the ability to proliferate. The ability of single cells to proliferate can be monitored in vitro and can provide insights into the sensitivity of tumor cells to chemotherapeutics. The following chapter describes how clonogenic hematopoietic cell growth can be determined with the colony formation assay.
Assuntos
Leucemia Mieloide Aguda , Humanos , Células Tumorais Cultivadas , Células Clonais/patologia , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Leucemia Mieloide Aguda/patologiaRESUMO
Genetic silencing of leukemia-associated proteins with small interfering RNAs (siRNAs) is a straightforward way to delineate their functions. It can be very challenging to deliver siRNAs to leukemia-derived cells with high transfection efficiency and without compromising their viability. This protocol describes an efficient approach to silence oncogenic feline McDonough sarcoma (FMS)-like tyrosine kinase-3 in leukemia cells using siRNAs that are delivered by electroporation. The protocol maintains high cell viability and is generally useful to decrease RNAs encoding proteins of interest. For complete details on the use and execution of this protocol, please refer to Beyer et al. (2022).
Assuntos
Leucemia , Animais , Gatos , Linhagem Celular , Leucemia/genética , Proteínas Tirosina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Tirosina Quinase 3 Semelhante a fms , Animais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
BACKGROUND: Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently. METHODS: We incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites. RESULTS: Chemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions. CONCLUSION: Drugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells. The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.
RESUMO
Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.
Assuntos
Linfoma de Células B , Tubulina (Proteína) , Acetilação , Animais , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Camundongos , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVES: To describe the epidemiology of rotavirus and norovirus infection among children with acute gastroenteritis in Sana'a, Yemen. METHODS: A cross-sectional study from November 2007 to March 2009 of children aged 1 month to 5 years attending the emergency and outpatient departments of two hospitals in Sana'a with acute gastroenteritis. Rotavirus was detected by ELISA and genotyped by RT-PCR. Norovirus was detected by End Point RT-PCR and nucleotide sequencing. RESULTS: A total of 290 children (48% in the emergency department and 52% in the outpatient department of the children respectively) were enrolled. Rotavirus infections were detected in 78 (27%) and norovirus infections in 30 (10%). Rotavirus genotypes included G1P[8] (55%), G9P[8] (21%) and G2P[4] (12%) with G12 comprising 3% of strain types. The main norovirus genotype was GII.4 (27%) with >10 other genotypes detected. CONCLUSIONS: Rotavirus and norovirus infections are common causes of gastroenteritis in Yemen. Rotavirus vaccines could play a significant role in the control of acute childhood diarrhoea in this setting.
Assuntos
Infecções por Caliciviridae/epidemiologia , Gastroenterite/epidemiologia , Norovirus/isolamento & purificação , Infecções por Rotavirus/epidemiologia , Doença Aguda , Infecções por Caliciviridae/virologia , Pré-Escolar , Estudos Transversais , Serviço Hospitalar de Emergência , Feminino , Gastroenterite/virologia , Genótipo , Humanos , Lactente , Masculino , Norovirus/classificação , Norovirus/genética , Ambulatório Hospitalar , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Iêmen/epidemiologiaRESUMO
Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.
Assuntos
Neoplasias Colorretais , Proteína Supressora de Tumor p53 , Acetilação , Animais , Apoptose , Benzamidas , Neoplasias Colorretais/tratamento farmacológico , Humanos , Irinotecano/farmacologia , Camundongos , Piridinas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutants of the FLT3 receptor tyrosine kinase (RTK) with duplications in the juxtamembrane domain (FLT3-ITD) act as drivers of acute myeloid leukemia (AML). Potent tyrosine kinase inhibitors (TKi) of FLT3-ITD entered clinical trials and showed a promising, but transient success due to the occurrence of secondary drug-resistant AML clones. A further caveat of drugs targeting FLT3-ITD is the co-targeting of other RTKs which are required for normal hematopoiesis. This is observed quite frequently. Therefore, novel drugs are necessary to treat AML effectively and safely. Recently bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. In order to optimize these agents we synthesized novel derivatives of these methanones with various substituents. Methanone 16 and its carbamate derivative 17b inhibit FLT3-ITD at least as potently as the TKi AC220 (quizartinib). Models indicate corresponding interactions of 16 and quizartinib with FLT3. The activity of 16 is accompanied by a high selectivity for FLT3-ITD.
Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/síntese química , Indóis/química , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors ß-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of ß-catenin. Indomethacin destabilizes ß-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of ß-catenin by WT1. In conclusion, reduced levels of ß-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.
RESUMO
Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used media supplement worldwide. FCS contains a mixture of largely undefined growth factors and cytokines, which support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesired immune responses, possible contaminations, and because of its way of production an ethical concern. Thus, alternative, defined, valid, and reliable media supplements should be characterized in a large number of experiments. Human platelet lysate (hPL) is increasingly appreciated as an alternative to FCS. Since it is unclear whether cells respond differentially to clinically relevant chemotherapeutics inducing replicative stress and DNA damage (Hydroxyurea, Irinotecan), induction of reactive oxygen species (ROS), the tyrosine kinase inhibitor (TKi) Imatinib, and novel epigenetic modifiers belonging to the group of histone deacetylase inhibitors (HDACi), we investigated these issues. Here we show that cancer cells derived from leukemia and colon cancer grow very similarly in culture media with FCS or outdated hPL. Notably, cells have practically identical proteomes under both culture conditions. Moreover, cells grown with FCS or hPL respond equally to all types of drugs and stress conditions that we have tested. In addition, the transfection of blood cells by electroporation can be achieved under both conditions. Furthermore, we reveal that class I HDACs, but not HDAC6, are required for the expression of the pan-leukemic marker WT1 under various culture conditions. Hence, hPL is a moderately priced substitute for FCS in various experimental settings.
Assuntos
Antineoplásicos , Plaquetas/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Animais , Células Cultivadas , Humanos , SoroRESUMO
Various diseases are related to epigenetic modifications. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, and thus are involved in the regulation of gene expression. First generation histone deacetylase inhibitors (HDACi) are unselective, hinder all different kinds of zinc dependent HDACs and additionally cause several side effects. Subsequently, selective HDACi are gaining more and more interest. Especially, selective histone deacetylase 6 inhibitors (HDAC6i) are supposed to be less toxic. Here we present a successful optimization study of tubastatin A, the synthesis and biological evaluation of new inhibitors based on hydroxamic acids linked to various tetrahydro-ß-carboline derivatives. The potency of our selective HDAC6 inhibitors, exhibiting IC50 values in a range of 1-10â¯nM towards HDAC6, was evaluated with the help of a recombinant human HDAC6 enzyme assay. Selectivity was proofed in cellular assays by the hyperacetylation of surrogate parameter α-tubulin in the absence of acetylated histone H3 analyzed by Western Blot. We show that all synthesized compounds, with varies modifications of the rigid cap group, were selective and potent HDAC6 inhibitors.
Assuntos
Carbolinas/farmacologia , Desenho de Fármacos , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carbolinas/síntese química , Carbolinas/química , Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desacetilase 6 de Histona/deficiência , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Benzamidas/uso terapêutico , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/toxicidade , Antirreumáticos/síntese química , Antirreumáticos/farmacologia , Antirreumáticos/toxicidade , Artrite Experimental/induzido quimicamente , Artrite Reumatoide/induzido quimicamente , Benzamidas/líquido cefalorraquidiano , Benzamidas/farmacologia , Benzamidas/toxicidade , Sítios de Ligação , Carbolinas/síntese química , Carbolinas/farmacologia , Carbolinas/uso terapêutico , Carbolinas/toxicidade , Linhagem Celular Tumoral , Colágeno Tipo II , Células HEK293 , Desacetilase 6 de Histona/química , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/toxicidade , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Ácidos Hidroxâmicos/toxicidade , Masculino , Camundongos Endogâmicos DBA , Simulação de Acoplamento Molecular , Peixe-ZebraRESUMO
Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in their target proteins. This biochemical modification can have profound effects on the functions of these proteins and a dysregulation of HDAC activity contributes to severe diseases, including neoplastic transformation. In the following chapter, we present a strategy that allows to distinguish between the inhibition of the class I HDACs HDAC1, 2, and 3 and of the class IIb HDAC HDAC6. This method is based on Western blot and relies on the detection of hyperacetylated substrates of class I or class IIb HDACs in lysates from cells that were treated with histone deacetylase inhibitors (HDACi).
Assuntos
Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Benzamidas/farmacologia , Western Blotting/métodos , Linhagem Celular Tumoral , Epigênese Genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/genética , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Panobinostat , Piridinas/farmacologia , Ácido Valproico/farmacologiaRESUMO
The transcription factors NF-κB and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytokines promote signaling cascades that lead to the co-regulation of p53 and NF-κB. Consequently, nuclear p53/NF-κB signaling complexes activate NF-κB-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-κB, we addressed whether clinically relevant HDACi affect the NF-κB/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-κB-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class IIb deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-κB. HDACi decrease the basal and stress-induced expression of p53 and block NF-κB-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53.
Assuntos
Histona Desacetilases/metabolismo , NF-kappa B/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Dano ao DNA , DNA de Neoplasias/metabolismo , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hidroxiureia/farmacologia , Modelos Biológicos , Mutação/genética , Neoplasias/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca²âº-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation".This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33.We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo.Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.
Assuntos
Transformação Celular Neoplásica/patologia , Quinase I-kappa B/metabolismo , Interleucina-3/farmacologia , Mastócitos/patologia , NF-kappa B/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/fisiologia , Humanos , Quinase I-kappa B/genética , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitochondria are essential organelles with multiple functions, especially in energy metabolism. Recently, an increasing number of data has highlighted the role of mitochondria for cellular differentiation processes. Metabolic differences between stem cells and mature derivatives require an adaptation of mitochondrial function during differentiation. In this study we investigated alterations of the mitochondrial phenotype of human mesenchymal stem cells undergoing adipogenic differentiation. Maturation of adipocytes is accompanied by mitochondrial biogenesis and an increase of oxidative metabolism. Adaptation of the mt phenotype during differentiation is reflected by changes in the distribution of the mitochondrial network as well as marked alterations of gene expression and organization of the oxidative phosphorylation system (OXPHOS). Distinct differences in the supramolecular organization forms of cytochrome c oxidase (COX) were detected using 2D blue native (BN)-PAGE analysis. Most remarkably we observed a significant increase in the abundance of OXPHOS supercomplexes in mitochondria, emphasizing the change of the mitochondrial phenotype during adipogenic differentiation.