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Porocarcinoma Écrino , Perfilação da Expressão Gênica , Neoplasias das Glândulas Sudoríparas , Humanos , Porocarcinoma Écrino/genética , Porocarcinoma Écrino/patologia , Neoplasias das Glândulas Sudoríparas/genética , Neoplasias das Glândulas Sudoríparas/patologia , Transcriptoma/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Feminino , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Masculino , Regulação Neoplásica da Expressão Gênica , Pessoa de Meia-IdadeRESUMO
Myeloid and lymphoid neoplasms share the characteristics of potential bone marrow infiltration as a primary or secondary effect, which readily leads to hematopoietic insufficiency. The mechanisms by which clonal malignant cells inhibit normal hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) have not been unraveled so far. Given the pivotal role of mesenchymal stromal cells (MSCs) in the regulation of hematopoiesis in the BM niche it is assumed that MSCs also play a relevant role in the pathogenesis of hematological neoplasms. We aimed to identify overlapping mechanisms in MSCs derived from myeloid and lymphoid neoplasms contributing to disease progression and suppression of HSPCs to develop interventions that target these mechanisms. MSCs derived from healthy donors (n = 44) and patients diagnosed with myeloproliferative neoplasia (n = 11), myelodysplastic syndromes (n = 16), or acute myeloid leukemia (n = 25) and B-Non-Hodgkin lymphoma (n = 9) with BM infiltration and acute lymphoblastic leukemia (n = 9) were analyzed for their functionality and by RNA sequencing. A reduced growth and differentiation capacity of MSCs was found in all entities. RNA sequencing distinguished both groups but clearly showed overlapping differentially expressed genes, including major players in the BMP/TGF and WNT-signaling pathway which are crucial for growth, osteogenesis, and hematopoiesis. Functional alterations in healthy MSCs were inducible by exposure to supernatants from malignant cells, implicating the involvement of these factors in disease progression. Overall, we were able to identify overlapping factors that pose potential future therapeutic targets.
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BACKGROUND AND OBJECTIVES: Malignant sweat gland tumors are rare, with the most common being eccrine porocarcinoma (EP). Approximately 18% of benign eccrine poroma (EPO) transit to EP. Previous research has provided first insights into the mutational landscape of EP. However, only few studies have performed gene expression analyses. This leaves a gap in the understanding of EP biology and potential drivers of malignant transformation from EPO to EP. METHODS: Transcriptome profiling of 23 samples of primary EP and normal skin (NS). Findings from the EP samples were then tested in 17 samples of EPO. RESULTS: Transcriptome profiling revealed diversity in gene expression and indicated biologically heterogeneous sub-entities as well as widespread gene downregulation in EP. Downregulated genes included CD74, NDGR1, SRRM2, CDC42, ANXA2, KFL9 and NOP53. Expression levels of CD74, NDGR1, SRRM2, ANXA2, and NOP53 showed a stepwise-reduction in expression from NS via EPO to EP, thus supporting the hypothesis that EPO represents a transitional state in EP development. CONCLUSIONS: We demonstrated that EP is molecularly complex and that evolutionary trajectories correspond to tumor initiation and progression. Our results provide further evidence implicating the p53 axis and the EGFR pathway. Larger samples are warranted to confirm our findings.
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Porocarcinoma Écrino , Perfilação da Expressão Gênica , Neoplasias das Glândulas Sudoríparas , Humanos , Porocarcinoma Écrino/genética , Porocarcinoma Écrino/patologia , Neoplasias das Glândulas Sudoríparas/genética , Neoplasias das Glândulas Sudoríparas/patologia , Neoplasias das Glândulas Sudoríparas/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Feminino , Masculino , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Idoso , Pessoa de Meia-IdadeRESUMO
Germ cell tumors (GCT) are the most common solid tumors in young men of age 15 - 40. In previous studies, we profiled the interaction of GCT cells with cells of the tumor microenvironment (TM), which showed that especially the 3D interaction of fibroblasts (FB) or macrophages with GCT cells influenced the growth behavior and cisplatin response as well as the transcriptome and secretome of the tumor cells, suggesting that the crosstalk of these cells with GCT cells is crucial for tumor progression and therapy outcome. In this study, we shed light on the mechanisms of activation of cancer-associated fibroblasts (CAF) in the GCT setting and their effects on GCT cells lines and the monocyte cell line THP-1. Ex vivo cultures of GCT-derived CAF were established and characterized molecularly and epigenetically by performing DNA methylation arrays, RNA sequencing, and mass spectrometry-based secretome analysis. We demonstrated that the activation state of CAF is influenced by their former prevailing tumor environment in which they have resided. Hereby, we postulate that seminoma (SE) and embryonal carcinoma (EC) activate CAF, while teratoma (TER) play only a minor role in CAF formation. In turn, CAF influence proliferation and the expression of cisplatin sensitivity-related factors in GCT cells lines as well as polarization of in vitro-induced macrophages by the identified effector molecules IGFBP1, LGALS3BP, LYVE1, and PTX3. Our data suggests that the vital interaction of CAF with GCT cells and with macrophages has a huge influence on shaping the extracellular matrix as well as on recruitment of immune cells to the TM. In conclusion, therapeutically interfering with CAF and / or macrophages in addition to the standard therapy might slow-down progression of GCT and re-shaping of the TM to a tumor-promoting environment. Significance: The interaction of CAF with GCT and macrophages considerably influences the microenvironment. Thus, therapeutically interfering with CAF might slow-down progression of GCT and re-shaping of the microenvironment to a tumor-promoting environment.
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Fibroblastos Associados a Câncer , Epigênese Genética , Macrófagos , Neoplasias Embrionárias de Células Germinativas , Neoplasias Testiculares , Microambiente Tumoral , Masculino , Humanos , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Regulação Neoplásica da Expressão Gênica , Metilação de DNA , Linhagem Celular Tumoral , Comunicação Celular , Cisplatino/farmacologia , Células THP-1 , Proliferação de CélulasRESUMO
Urothelial carcinoma (UC) urgently requires new therapeutic options. Histone deacetylases (HDAC) are frequently dysregulated in UC and constitute interesting targets for the development of alternative therapy options. Thus, we investigated the effect of the second generation HDAC inhibitor (HDACi) quisinostat in five UC cell lines (UCC) and two normal control cell lines in comparison to romidepsin, a well characterized HDACi which was previously shown to induce cell death and cell cycle arrest. In UCC, quisinostat led to cell cycle alterations, cell death induction and DNA damage, but was well tolerated by normal cells. Combinations of quisinostat with cisplatin or the PARP inhibitor talazoparib led to decrease in cell viability and significant synergistic effect in five UCCs and platinum-resistant sublines allowing dose reduction. Further analyses in UM-UC-3 and J82 at low dose ratio revealed that the mechanisms included cell cycle disturbance, apoptosis induction and DNA damage. These combinations appeared to be well tolerated in normal cells. In conclusion, our results suggest new promising combination regimes for treatment of UC, also in the cisplatin-resistant setting.
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Apoptose , Inibidores de Histona Desacetilases , Inibidores de Poli(ADP-Ribose) Polimerases , Neoplasias da Bexiga Urinária , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/patologiaRESUMO
BACKGROUND: Being implicated during tumor migration, invasion, clonogenicity, and proliferation, the nicotinamide adenine dinucleotide (NAD)/-phosphate (NADP)-dependent dehydrogenase/reductase member 2 (DHRS2) has been considered to be induced upon inhibition of histone deacetylases (HDACi). In this study, we evaluated the current knowledge on the underlying mechanisms of the (epi)genetic regulation of DHRS2, as well as its function during tumor progression. METHODS: DHRS2 expression was evaluated on mRNA- and protein-level upon treatment with HDACi by means of qRT-PCR and western blot analyses, respectively. Re-analysis of RNA-sequencing data gained insight into expression of specific DHRS2 isoforms, while re-analysis of ATAC-sequencing data shed light on the chromatin accessibility at the DHRS2 locus. Further examination of the energy and lipid metabolism of HDACi-treated urologic tumor cells was performed using liquid chromatography-mass spectrometry. RESULTS: Enhanced DHRS2 expression levels upon HDACi treatment were directly linked to an enhanced chromatin accessibility at the DHRS2 locus. Particularly the DHRS2 ENST00000250383.11 protein-coding isoform was increased upon HDACi treatment. Application of the HDACi quisinostat only mildly influenced the energy metabolism of urologic tumor cells, though, the analysis of the lipid metabolism showed diminished sphingosine levels, as well as decreased S1P levels. Also the ratios of S1P/sphingosine and S1P/ceramides were reduced in all four quisinostat-treated urologic tumor cells. CONCLUSIONS: With the emphasis on urologic malignancies (testicular germ cell tumors, urothelial, prostate, and renal cell carcinoma), this study concluded that elevated DHRS2 levels are indicative of a successful HDACi treatment and, thereby offering a novel putative predictive biomarker.
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Inibidores de Histona Desacetilases , Humanos , Inibidores de Histona Desacetilases/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Neoplasias Urológicas/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.
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Apresentação Cruzada , Células Dendríticas , Vetores Genéticos , Receptores Purinérgicos P2X7 , Vaccinia virus , Animais , Humanos , Camundongos , Apresentação de Antígeno/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos Endogâmicos C57BL , Receptores Purinérgicos P2X7/imunologia , Receptores Purinérgicos P2X7/metabolismo , Vaccinia virus/imunologiaRESUMO
Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling ß-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine, was protective against obesity and insulin resistance, but clinical development was halted by the emergence of CNTF antibodies. The chimeric cytokine IC7 used the framework of interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.
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Fator Neurotrófico Ciliar , Receptor gp130 de Citocina , Interleucina-6 , Transdução de Sinais , Animais , Humanos , Camundongos , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/genética , Receptor gp130 de Citocina/metabolismo , Receptor gp130 de Citocina/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Modelos Moleculares , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-6/genética , Receptores de OSM-LIF/metabolismo , Receptores de OSM-LIF/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Camundongos Endogâmicos C57BLRESUMO
ABSTRACT: The hallmark of multiple myeloma (MM) is a clonal plasma cell infiltration in the bone marrow accompanied by myelosuppression and osteolysis. Premalignant stages such as monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic stages such as smoldering myeloma (SMM) can progress to MM. Mesenchymal stromal cells (MSCs) are an integral component of the bone marrow microenvironment and play an important role in osteoblast differentiation and hematopoietic support. Although stromal alterations have been reported in MM contributing to hematopoietic insufficiency and osteolysis, it is not clear whether alterations in MSC already occur in MGUS or SMM. In this study, we analyzed MSCs from MGUS, SMM, and MM regarding their properties and functionality and performed messenger RNA sequencing to find underlying molecular signatures in different disease stages. A high number of senescent cells and a reduced osteogenic differentiation capacity and hematopoietic support were already present in MGUS MSC. As shown by RNA sequencing, there was a broad spectrum of differentially expressed genes including genes of the BMP/TGF-signaling pathway, detected already in MGUS and that clearly increases in patients with SMM and MM. Our data may help to block these signaling pathways in the future to hinder progression to MM.
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Células-Tronco Mesenquimais , Gamopatia Monoclonal de Significância Indeterminada , Mieloma Múltiplo , Mieloma Múltiplo Latente , Humanos , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Masculino , Feminino , IdosoRESUMO
The incidence of age-related neurodegenerative diseases is rising globally. However, the temporal sequence of neurodegeneration throughout adult life is poorly understood. To identify the starting points and schedule of neurodegenerative events, serotonergic and dopaminergic neurons were monitored in the model organism C. elegans, which has a life span of 2-3 weeks. Neural morphology was examined from young to old nematodes that were exposed to silica nanoparticles. Young nematodes showed phenotypes such as dendritic beading of serotonergic and dopaminergic neurons that are normally not seen until late life. During aging, neurodegeneration spreads from specifically susceptible ADF and PDE neurons in young C. elegans to other more resilient neurons, such as dopaminergic CEP in middle-aged worms. Investigation of neurodegenerative hallmarks and animal behavior revealed a temporal correlation with the acceleration of neuromuscular defects, such as internal hatch in 2-day-old C. elegans. Transcriptomics and proteomics of young worms exposed to nano silica showed a change in gene expression concerning the gene ontology groups serotonergic and dopaminergic signaling as well as neuropeptide signaling. Consistent with this, reporter strains for nlp-3, nlp-14 and nlp-21 confirmed premature degeneration of the serotonergic neuron HSN and other neurons in young C. elegans. The results identify young nematodes as a vulnerable age group for nano silica-induced neural defects with a significantly reduced health span. Neurodegeneration of specific neurons impairs signaling by classical neurotransmitters as well as neuropeptides and compromises related neuromuscular behaviors in critical phases of life, such as the reproductive phase.
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Background: Patients with muscle-invasive bladder cancer face a poor prognosis due to rapid disease progression and chemoresistance. Thus, there is an urgent need for a new therapeutic treatment. The tumor microenvironment (TME) has crucial roles in tumor development, growth, progression, and therapy resistance. TME cells may also survive standard treatment of care and fire up disease recurrence. However, whether specific TME components have tumor-promoting or tumor-inhibitory properties depends on cell type and cancer entity. Thus, a deeper understanding of the interaction mechanisms between the TME and cancer cells is needed to develop new cancer treatment approaches that overcome therapy resistance. Little is known about the function and interaction between mesenchymal stromal cells (MSC) or fibroblasts (FB) as TME components and bladder cancer cells. Methods: We investigated the functional impact of conditioned media (CM) from primary cultures of different donors of MSC or FB on urothelial carcinoma cell lines (UCC) representing advanced disease stages, namely, BFTC-905, VMCUB-1, and UMUC-3. Underlying mechanisms were identified by RNA sequencing and protein analyses of cancer cells and of conditioned media by oncoarrays. Results: Both FB- and MSC-CM had tumor-promoting effects on UCC. In some experiments, the impact of MSC-CM was more pronounced. CM augmented the aggressive phenotype of UCC, particularly of those with epithelial phenotype. Proliferation and migratory and invasive capacity were significantly increased; cisplatin sensitivity was reduced. RNA sequencing identified underlying mechanisms and molecules contributing to the observed phenotype changes. NRF2 and NF-κB signaling was affected, contributing to improved cisplatin detoxification. Likewise, interferon type I signaling was downregulated and regulators of epithelial mesenchymal transition (EMT) were increased. Altered protein abundance of CXCR4, hyaluronan receptor CD44, or TGFß-signaling was induced by CM in cancer cells and may contribute to phenotypical changes. CM contained high levels of CCL2/MCP-1, MMPs, and interleukins which are well known for their impact on other cancer entities. Conclusions: The CM of two different TME components had overlapping tumor-promoting effects and increased chemoresistance. We identified underlying mechanisms and molecules contributing to the aggressiveness of bladder cancer cells. These need to be further investigated for targeting the TME to improve cancer therapy.
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Alzheimer's disease is the most common age-associated neurodegenerative disorder and the most frequent form of dementia in our society. Aging is a complex biological process concurrently shaped by genetic, dietary and environmental factors and natural compounds are emerging for their beneficial effects against age-related disorders. Besides their antioxidant activity often described in simple model organisms, the molecular mechanisms underlying the beneficial effects of different dietary compounds remain however largely unknown. In the present study, we exploit the nematode Caenorhabditis elegans as a widely established model for aging studies, to test the effects of different natural compounds in vivo and focused on mechanistic aspects of one of them, quercetin, using complementary systems and assays. We show that quercetin has evolutionarily conserved beneficial effects against Alzheimer's disease (AD) pathology: it prevents Amyloid beta (Aß)-induced detrimental effects in different C. elegans AD models and it reduces Aß-secretion in mammalian cells. Mechanistically, we found that the beneficial effects of quercetin are mediated by autophagy-dependent reduced expression of Abl tyrosine kinase. In turn, autophagy is required upon Abl suppression to mediate quercetin's protective effects against Aß toxicity. Our data support the power of C. elegans as an in vivo model to investigate therapeutic options for AD.
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Introduction: The purpose of this feasibility study was to select targeted therapies according to "ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)". Data interpretation was further supported by a browser-based Treatment Decision Support platform (MH Guide, Molecular Health, Heidelberg, Germany). Patients: We applied next generation sequencing based whole exome sequencing of tumor tissue and peripheral blood of patients with metastatic breast cancer (n = 44) to detect somatic as well as germline mutations. Results: In 32 metastatic breast cancer patients, data interpretation was feasible. We identified 25 genomic alterations with ESCAT Level of Evidence I or II in 18/32 metastatic breast cancer patients, which were available for evaluation: three copy number gains in HER2 , two g BRCA1 , two g BRCA2 , six PIK3CA, one ESR1 , three PTEN , one AKT1 and two HER2 mutations. In addition, five samples displayed Microsatellite instability high-H. Conclusions: Resulting treatment options were discussed in a tumor board and could be recommended in a small but relevant proportion of patients with metastatic breast cancer (7/18). Thus, this study is a valuable preliminary work for the establishment of a molecular tumor board within the German initiative "Center for Personalized Medicine" which aims to shorten time for analyses and optimize selection of targeted therapies.
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Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a comprehensive transcriptome-wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase signaling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3' untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.
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Estabilidade de RNA , Regulon , RNA Mensageiro/genética , Regulon/genética , Regiões 3' não Traduzidas/genéticaRESUMO
BACKGROUND: Macrophages play an important role in maintaining liver homeostasis and regeneration. However, it is not clear to what extent the different macrophage populations of the liver differ in terms of their activation state and which other liver cell populations may play a role in regulating the same. METHODS: Reverse transcription PCR, flow cytometry, transcriptome, proteome, secretome, single cell analysis, and immunohistochemical methods were used to study changes in gene expression as well as the activation state of macrophages in vitro and in vivo under homeostatic conditions and after partial hepatectomy. RESULTS: We show that F4/80+/CD11bhi/CD14hi macrophages of the liver are recruited in a C-C motif chemokine receptor (CCR2)-dependent manner and exhibit an activation state that differs substantially from that of the other liver macrophage populations, which can be distinguished on the basis of CD11b and CD14 expressions. Thereby, primary hepatocytes are capable of creating an environment in vitro that elicits the same specific activation state in bone marrow-derived macrophages as observed in F4/80+/CD11bhi/CD14hi liver macrophages in vivo. Subsequent analyses, including studies in mice with a myeloid cell-specific deletion of the TGF-ß type II receptor, suggest that the availability of activated TGF-ß and its downregulation by a hepatocyte-conditioned milieu are critical. Reduction of TGF-ßRII-mediated signal transduction in myeloid cells leads to upregulation of IL-6, IL-10, and SIGLEC1 expression, a hallmark of the activation state of F4/80+/CD11bhi/CD14hi macrophages, and enhances liver regeneration. CONCLUSIONS: The availability of activated TGF-ß determines the activation state of specific macrophage populations in the liver, and the observed rapid transient activation of TGF-ß may represent an important regulatory mechanism in the early phase of liver regeneration in this context.
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Regeneração Hepática , Fator de Crescimento Transformador beta , Animais , Camundongos , Expressão Gênica , Hepatócitos/metabolismo , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
All except one cytokine of the Interleukin (IL-)6 family share glycoprotein (gp) 130 as the common ß receptor chain. Whereas Interleukin (IL-)11 signal via the non-signaling IL-11 receptor (IL-11R) and gp130 homodimers, leukemia inhibitory factor (LIF) recruits gp130:LIF receptor (LIFR) heterodimers. Using IL-11 as a framework, we exchange the gp130-binding site III of IL-11 with the LIFR binding site III of LIF. The resulting synthetic cytokimera GIL-11 efficiently recruits the non-natural receptor signaling complex consisting of gp130, IL-11R and LIFR resulting in signal transduction and proliferation of factor-depending Ba/F3 cells. Besides LIF and IL-11, GIL-11 does not activate receptor complexes consisting of gp130:LIFR or gp130:IL-11R, respectively. Human GIL-11 shows cross-reactivity to mouse and rescued IL-6R-/- mice following partial hepatectomy, demonstrating gp130:IL-11R:LIFR signaling efficiently induced liver regeneration. With the development of the cytokimera GIL-11, we devise the functional assembly of the non-natural cytokine receptor complex of gp130:IL-11R:LIFR.
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Hepatectomia , Interleucina-11 , Camundongos , Animais , Humanos , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Interleucina-11/genética , Receptores de Interleucina-11 , Antígenos CD/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais , Subunidade alfa de Receptor de Fator Inibidor de LeucemiaRESUMO
In contrast to class I/IIb/pan histone deacetylase inhibitors (HDACi), the role of class IIa HDACi as anti-cancer chemosensitizing agents is less well understood. Here, we studied the effects of HDAC4 in particular and the class IIa HDACi CHDI0039 on proliferation and chemosensitivity in Cal27 and cisplatin-resistant Cal27CisR head and neck squamous cell cancer (HNSCC). HDAC4 and HDAC5 overexpression clones were generated. HDAC4 overexpression (Cal27_HDAC4) increased proliferation significantly compared to vector control cells (Cal27_VC). Chicken chorioallantoic membrane (CAM) studies confirmed the in vitro results: Cal27_HDAC4 tumors were slightly larger than tumors from Cal27_VC, and treatment with CHDI0039 resulted in a significant decrease in tumor size and weight of Cal27_HDAC4 but not Cal27_VC. Unlike class I/pan-HDACi, treatment with CHDI0039 had only a marginal impact on cisplatin cytotoxicity irrespective of HDAC4 and HDAC5 expression. In contrast, the combination of CHDI0039 with bortezomib was synergistic (Chou-Talalay) in MTT and caspase 3/7 activation experiments. RNAseq indicated that treatment with CHDI0039 alters the expression of genes whose up- or downregulation is associated with increased survival in HNSCC patients according to Kaplan-Meier data. We conclude that the combination of class IIa HDACi with proteasome inhibitors constitutes an effective treatment option for HNSCC, particularly for platinum-resistant cancers.
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Antineoplásicos , Neoplasias de Cabeça e Pescoço , Humanos , Inibidores de Histona Desacetilases/farmacologia , Bortezomib/farmacologia , Cisplatino , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genéticaRESUMO
Hypertrophic scarring in burn wounds is caused by overactive fibroblasts and myofibroblasts. Blue light reveals wavelength- and dose-dependent antibacterial and antiproliferative effects and may serve as a therapeutic option against wound infection and fibrotic conditions. Therefore, we evaluated in this study the effects of single and multiple irradiations with blue light at 420 nm (BL420) on the intracellular ATP concentration, and on the viability and proliferation of the human skin fibroblast (HDFs). In addition, possible BL420-induced effects on the catalase expression and differentiation were assessed by immunocytochemical staining and western blot analyses. Furthermore, we used RNA-seq analyses to identify BL420-affected genes. We found that BL420 induced toxicity in HDFs (up to 83%; 180 J/cm2). A low dose of 20 J/cm2 reduced the ATP concentration by ~50%. Multiple irradiations (4 × 20 J/cm2) inhibited proliferation without visible toxicity and reduced catalase protein expression by ~37% without affecting differentiation. The expression of about 300 genes was significantly altered. Many downregulated genes have functions in cell division/mitosis. BL420 can strongly influence the fibroblast physiology and has potential in wound therapy. However, it is important to consider the possible toxic and antiproliferative effects, which could potentially lead to impaired wound healing and reduced scar breaking strength.
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N-hydroxypipecolic acid (NHP) accumulates in pathogen-inoculated and distant leaves of the Arabidopsis shoot and induces systemic acquired resistance (SAR) in dependence of the salicylic acid (SA) receptor NPR1. We report here that SAR triggered by exogenous NHP treatment requires the function of the transcription factors TGA2/5/6 in addition to NPR1, and is further positively affected by TGA1/4. Consistently, a tga2/5/6 triple knockout mutant is fully impaired in NHP-induced SAR gene expression, while a tga1/4 double mutant shows an attenuated, partial transcriptional response to NHP. Moreover, tga2/5/6 and tga1/4 exhibited fully and strongly impaired pathogen-triggered SAR, respectively, while SA-induced resistance was more moderately compromised in both lines. At the same time, tga2/5/6 was not and tga1/4 only partially impaired in the accumulation of NHP and SA at sites of bacterial attack. Strikingly, SAR gene expression in the systemic tissue induced by local bacterial inoculation or locally applied NHP fully required functional TGA2/5/6 and largely depended on TGA1/4 factors. The systemic accumulation of NHP and SA was attenuated but not abolished in the SAR-compromised and transcriptionally blocked tga mutants, suggesting their transport from inoculated to systemic tissue. Our results indicate the existence of a critical TGA- and NPR1-dependent transcriptional module that mediates the induction of SAR and systemic defence gene expression by NHP.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Ácidos Pipecólicos/farmacologia , Ácidos Pipecólicos/metabolismo , Ácido Salicílico/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.