RESUMEN
Pathogenic variants (PVs) in DNA repair-linked adult-onset cancer predisposition genes, including double heterozygosity, are increasingly identified in pediatric patients with cancer. Their role in childhood cancer, however, remains poorly understood. Integrating comprehensive tumor analysis is integral for understanding the contribution of such PVs in cancer development and personalized cancer care.
Asunto(s)
Edad de Inicio , Predisposición Genética a la Enfermedad , Neoplasias , Adulto , Niño , Humanos , Reparación del ADN , Neoplasias/genética , Neoplasias/patología , Neoplasias/etiologíaRESUMEN
PURPOSE: The development of a seroma after breast cancer surgery is a common postoperative complication seen after simple mastectomy and axillary surgery. We could recently demonstrate that breast cancer patients undergoing a simple mastectomy with subsequent seroma formation developed a T-helper cell increase within the aspirated fluid measured by flow cytometry. The same study revealed a Th2 and/or a Th17 immune response in peripheral blood and seroma fluid of the same patient. Based on these results and within the same study population, we now analyzed the Th2/Th17 cell associated cytokine content as well as the best known clinical important cytokine IL-6. METHODS: Multiplex cytokine measurements (IL-4, IL-5, IL-13, IL-10, IL-17, and IL-22) were done on 34 seroma fluids (Sf) after fine needle aspiration of patients who developed a seroma after a simple mastectomy. Serum of the same patient (Sp) and that of healthy volunteers (Sc) were used as controls. RESULTS: We found the Sf to be highly cytokine rich. Almost all analyzed cytokines were significantly higher in abundance in the Sf compared to Sp and Sc, especially IL-6, which promotes Th17 differentiation as well as suppresses Th1 differentiation in favor of Th2 development. CONCLUSION: Our Sf cytokine measurements reflect a local immune event. In contrast, former study results on T-helper cell populations in both Sf and Sp tend to demonstrate a systemic immune process.
Asunto(s)
Neoplasias de la Mama , Citocinas , Humanos , Femenino , Neoplasias de la Mama/cirugía , Interleucina-6 , Células Th17 , Células TH1 , Seroma/etiología , Mastectomía/efectos adversosRESUMEN
PURPOSE: Adjuvant imatinib treatment is recommended for patients with localized gastrointestinal stromal tumor (GIST) at high risk of recurrence. Almost half of high-risk patients are cured by surgery alone, indicating a need for improved selection of patients for adjuvant therapy. The aim of this study was to investigate if genomic tumor complexity could be used as a prognostic biomarker. METHODS: The discovery cohort consisted of patients who underwent resection of primary GIST at Oslo University Hospital between 1998 and 2020. Karyotypes were categorized as simple if they had ≤ 5 chromosomal changes and complex if there were > 5 chromosomal aberrations. Validation was performed in an independent patient cohort where chromosomal imbalances were mapped using comparative genomic hybridization. RESULTS: Chromosomal aberrations were detected in 206 tumors, of which 76 had a complex karyotype. The most frequently observed changes were losses at 14q, 22q, 1p, and 15q. The 5-year recurrence-free survival (RFS) in patients classified as very low, low, or intermediate risk was 99%. High-risk patients with a simple tumor karyotype had an estimated 5-year RFS of 94%, and patients with a complex karyotype had an estimated 5-year RFS of 51%. A complex karyotype was associated with poor RFS in patients with and without adjuvant imatinib treatment and in multivariable analysis adjusted for tumor site, size, mitotic count, and rupture. The prognostic impact of genomic complexity was confirmed in the validation cohort. In both cohorts, the 5-year disease-specific survival was > 90% for high-risk patients with genomically simple tumors. CONCLUSION: Genomic tumor complexity is an independent prognostic biomarker in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes. De-escalation of adjuvant imatinib treatment should be explored in patients with cytogenetically simple GISTs.
Asunto(s)
Antineoplásicos , Tumores del Estroma Gastrointestinal , Humanos , Mesilato de Imatinib/uso terapéutico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Hibridación Genómica Comparativa , Quimioterapia Adyuvante , Biomarcadores , Genómica , Aberraciones Cromosómicas/inducido químicamenteRESUMEN
The tripartite partition defect (PAR) polarity complex, which includes the proteins PAR3, atypical protein kinase C (aPKC), and PAR6, is a major regulator of cellular polarity. It is highly conserved and expressed in various tissues. Its largest component, PAR3, controls protein-protein interactions of the PAR complex with a variety of interaction partners, and PAR3 self-association is critical for the formation of filament-like structures. However, little is known about the structure of the PAR complex. Here, we purified non-filamentous PAR3 and the aPKC-PAR6 complex and characterized them by single-particle electron microscopy (EM). We expressed and purified an oligomerization-deficient form of PAR3, PAR3V13D,D70K, and the active aPKC-PAR6 dimer. For PAR3, engineering at two positions is sufficient to form stable single particles with a maximum dimension of 20 nm. aPKC-PAR6 forms a complex with a maximum dimension of 13.5 nm that contains single copies of aPKC. Thus, the data present a basis for further high-resolution studies of PAR proteins and PAR complex formation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Quinasa C , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Polaridad Celular , Humanos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismoRESUMEN
The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF-SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.
Asunto(s)
Proteínas Co-Represoras/genética , Inhibidores de Histona Desacetilasas/farmacología , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/metabolismo , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Complejo Correpresor Histona Desacetilasa y Sin3/aislamiento & purificación , Animales , Baculoviridae/metabolismo , Benzamidas/farmacología , Proteínas Co-Represoras/aislamiento & purificación , Proteínas Co-Represoras/metabolismo , Depsipéptidos/farmacología , Biblioteca de Genes , Histona Desacetilasas/metabolismo , Humanos , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/enzimología , Pirimidinas/farmacología , Proteínas Recombinantes , Elementos Reguladores de la Transcripción/genética , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Células Sf9 , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismoRESUMEN
The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.
Asunto(s)
Cromatina/aislamiento & purificación , ADN Nucleotidiltransferasas/química , Cromatina/química , Cromatina/metabolismo , ADN Nucleotidiltransferasas/metabolismo , HumanosRESUMEN
The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.
Asunto(s)
ADN Helicasas/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Factores de Transcripción/químicaRESUMEN
The PTEN hamartoma tumor syndrome (PHTS) is caused by heterozygous germline variants in PTEN. Here, we report two unrelated patients with juvenile polyposis, macrocephaly, intellectual disability, and hyperpigmented skin macules. Both patients were clinically suspected for the Bannayan-Riley-Ruvalcaba syndrome (BRRS), a PHTS subentity. By array-CGH analysis, we identified an interstitial 10q23.1q23.3 deletion in a buccal mucosa sample of Patient 1 that encompassed PTEN, BMPR1A, and KLLN, among others. In contrast, neither sequencing nor array-CGH analysis identified a pathogenic variant in PTEN or BMPR1A in a blood sample of Patient 2. However, in a surgical specimen of the thyroid gland high-level mosaicism for a 10q23.2q23.3 deletion was observed. Additionally, the pathogenic PTEN variant c.956_959delCTTT p.(Thr319LysfsTer24) was detected in his thyroid tissue. The frame shift variant was neither detected in the patient's blood nor in his buccal mucosa sample. Low-level mosaicism for the microdeletion was identified in a buccal swap sample, and reanalysis of the blood sample suggested marginal-level mosaicism for deletion. The 10q23.2q23.3 deletion mosaicism was also identified in a subsequently resected colonic polyp. Thus, in both cases, the diagnosis of a 10q23 deletion syndrome, which clinically presented as BRRS, was established. Overall, the study expands the BRRS spectrum and highlights the relevance of considering mosaicism in PHTS. We conclude that in all patients with a clear clinical suspicion of PHTS, in which genetic analyses of DNA from blood and buccal swap samples fail to identify causative genetic variants, genetic analyses of additional tissues are recommended.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Deleción Cromosómica , Cromosomas Humanos Par 10 , Síndrome de Hamartoma Múltiple/genética , Mosaicismo , Mutación , Fosfohidrolasa PTEN/genética , Adolescente , Femenino , Humanos , MasculinoRESUMEN
The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.
Asunto(s)
Baculoviridae/crecimiento & desarrollo , Proteínas HSP70 de Choque Térmico/genética , Proteína SMARCB1/genética , Células Sf9/virología , Animales , Baculoviridae/genética , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Expresión Génica , Humanos , Regiones Promotoras Genéticas , Ingeniería de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
The loss of gamma-aminobutyric acid (GABA)-ergic medium spiny neurons (MSNs) in the striatum is the hallmark of Huntington disease (HD), an incurable neurodegenerative disorder characterized by progressive motor, psychiatric, and cognitive symptoms. Transplantation of MSNs or their precursors represents a promising treatment strategy for HD. In initial clinical trials in which HD patients received fetal neurografts directly into the striatum without a pretransplant cell-differentiation step, some patients exhibited temporary benefits. Meanwhile, major challenges related to graft overgrowth, insufficient survival of grafted cells, and limited availability of donated fetal tissue remain. Thus, the development of approaches that allow modeling of MSN differentiation and HD development in cell culture platforms may improve our understanding of HD and translate, ultimately, into HD treatment options. Here, recent advances in the in vitro differentiation of MSNs derived from fetal neural stem cells/progenitor cells (NSCs/NPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and induced NSCs (iNSCs) as well as advances in direct transdifferentiation are reviewed. Progress in non-allele specific and allele specific gene editing of HTT is presented as well. Cell characterization approaches involving phenotyping as well as in vitro and in vivo functional assays are also discussed.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Huntington/fisiopatología , Células-Madre Neurales/citología , Neuronas/citología , Animales , Diferenciación Celular , Humanos , Células-Madre Neurales/fisiología , Neuronas/fisiologíaAsunto(s)
Complemento C1r , Complemento C1s , Activación de Complemento , Complemento C1 , Complemento C1qRESUMEN
The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.
Asunto(s)
Sustitución de Aminoácidos , Piruvato Deshidrogenasa (Lipoamida)/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Pliegue de Proteína , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/metabolismo , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.
Asunto(s)
Proteínas Anfibias/sangre , Antioxidantes/metabolismo , Disulfuros/sangre , Hemoglobina A/metabolismo , Hipoxia/sangre , Estrés Oxidativo , Oxígeno/sangre , Tortugas/sangre , Adaptación Fisiológica , Animales , Biomarcadores/sangre , Cisteína , Hipoxia/fisiopatología , PolimerizacionRESUMEN
Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER). The vectors code for N-terminal and C-terminal affinity tags including 3 × FLAG and Twin-Strep, which represent tags compatible with efficient translocation to the ER as well as with purification under mild conditions that preserve protein structure and function. As a model, we used our system to express and purify the engineered high-affinity immunoglobulin gamma Fc receptor I (CD64) in complex with its gamma subunit (γ-chain). We demonstrate that CD64 expressed in complex with the γ-chain is functional in immunoglobulin G (IgG) binding. The sedimentation of CD64 in complex with IgG suggests individual CD64/IgG complexes in addition to formation of high-molecular weight complexes. In summary, our vectors can be used as a tool for expression of membrane proteins, other secretory pathway proteins and their protein complexes.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos , Insectos/citología , Receptores de IgG/genética , Proteínas Recombinantes/genética , Animales , Baculoviridae/metabolismo , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Insectos/genética , Ingeniería de Proteínas/métodos , Señales de Clasificación de Proteína/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3 × FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.
Asunto(s)
Baculoviridae/genética , Biotecnología/métodos , ADN Nucleotidiltransferasas/genética , ADN/genética , Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Animales , Baculoviridae/metabolismo , Sitios de Unión , ADN/metabolismo , ADN Nucleotidiltransferasas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Edición Génica/métodos , Células HEK293 , Humanos , Mutación , Oligopéptidos/genética , Oligopéptidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , SpodopteraRESUMEN
PURPOSE: Medulloblastomas are aggressive brain malignancies. While considerable progress has been made in the treatment of medulloblastoma patients with respect to overall survival, these patients are still at risk of developing neurologic and cognitive deficits as a result of anti-cancer therapies. It is hypothesized that targeted molecular therapies represent a better treatment option for medulloblastoma patients. Therefore, the aim of the present study was to test a panel of epigenetic drugs for their effect on medulloblastoma cells under mild hypoxic conditions that reflect the physiological concentrations of oxygen in the brain. METHODS: Protein levels of histone deacetylase 1 (HDAC1) and DNA methyltransferase 1 (DNMT1) in medulloblastoma-derived cells (Daoy and D283 Med), as well as in developing and differentiated brain cells, were determined and compared. Class I and II histone deacetylase inhibitors (HDACi) and a DNMT inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), were applied to Daoy and D283 Med cells, and their effects were studied using viability, apoptosis and cancer sphere assays. RESULTS: We found that in HDAC1 and DNMT1 overexpressing medulloblastoma-derived cells, cell death was induced under various epigenetic drug conditions tested. At low HDACi concentrations, however, a pro-proliferative effect was observed. Parthenolide, a drug that affects cancer stem cells, was found to be efficient in inducing cell death in both cell lines tested. In contrast, we found that Daoy cells were more resistant to 5-aza-dC than D283 Med cells. When suberoylanilide hydroxamic acid (SAHA) and parthenolide were individually applied to both cell lines in combination with 5-aza-dC, a synergistic effect on cell survival was observed. CONCLUSIONS: Our current results suggest that the application of HDACi in combination with drugs that target DNMT may represent a promising option for the treatment of medulloblastoma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Cerebelosas/patología , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/antagonistas & inhibidores , Meduloblastoma/patología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Línea Celular Tumoral , Decitabina , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Sesquiterpenos/farmacologíaRESUMEN
In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.
Asunto(s)
Benzamidas , Elementos de Facilitación Genéticos , Hidrazinas , Complejos Multiproteicos , Proteínas de Neoplasias , Proteínas Represoras , Sulfonamidas , Acetilación , Animales , Benzamidas/química , Benzamidas/farmacología , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Hidrazinas/química , Hidrazinas/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Dominios Proteicos , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.
Asunto(s)
Complemento C1r/química , Complemento C1s/química , Vía Clásica del Complemento/fisiología , Complemento C1r/genética , Complemento C1r/metabolismo , Complemento C1s/genética , Complemento C1s/metabolismo , Activación Enzimática , Genes Sintéticos , Células HEK293 , Humanos , Inmunidad Innata , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Huntington disease (HD) is a devastating neurological disorder caused by an extended CAG repeat in exon 1 of the gene that encodes the huntingtin (HTT) protein. HD pathology involves a loss of striatal medium spiny neurons (MSNs) and progressive neurodegeneration affects the striatum and other brain regions. Because HTT is involved in multiple cellular processes, the molecular mechanisms of HD pathogenesis should be investigated on multiple levels. On the cellular level, in vitro stem cell models, such as induced pluripotent stem cells (iPSCs) derived from HD patients and HD embryonic stem cells (ESCs), have yielded progress. Approaches to differentiate functional MSNs from ESCs, iPSCs, and neural stem/progenitor cells (NSCs/NPCs) have been established, enabling MSN differentiation to be studied and disease phenotypes to be recapitulated. Isolation of target stem cells and precursor cells may also provide a resource for grafting. In animal models, transplantation of striatal precursors differentiated in vitro to the striatum has been reported to improve disease phenotype. Initial clinical trials examining intrastriatal transplantation of fetal neural tissue suggest a more favorable clinical course in a subset of HD patients, though shortcomings persist. Here, we review recent advances in the development of cellular HD models and approaches aimed at cell regeneration with human stem cells. We also describe how genome editing tools could be used to correct the HTT mutation in patient-specific stem cells. Finally, we discuss the potential and the remaining challenges of stem cell-based approaches in HD research and therapy development.