RESUMEN
Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.
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Apoptosis/efectos de los fármacos , Bleomicina/farmacología , Daño del ADN/efectos de los fármacos , Pulmón/efectos de los fármacos , Nanopartículas/uso terapéutico , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Humanos , Pulmón/metabolismo , Estrés Oxidativo/efectos de los fármacos , Péptidos/farmacologíaRESUMEN
NHEJ1-patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous-NHEJ1 mutation identified by whole exome-sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1-patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1-deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA-PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient´s cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.
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Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Telomerasa/genética , Línea Celular , Niño , Enzimas Reparadoras del ADN/sangre , Proteínas de Unión al ADN/sangre , Regulación hacia Abajo , Expresión Génica , Humanos , Masculino , Mutación/genética , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero , Acortamiento del Telómero/genéticaRESUMEN
DUSP6/MKP3 is a dual-specific phosphatase that regulates extracellular regulated kinase ERK1/2 and ERK5 activity, with an increasingly recognized role as tumor suppressor. In silico studies from Gene expression Omnibus (GEO) and Cancer Genome atlas (TCGA) databases reveal poor prognosis in those Non-small cell lung cancer (NSCLC) patients with low expression levels of DUSP6. In agreement with these data, here we show that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth. We have found upregulation in the expression of several genes involved in epithelial to mesenchymal transition (EMT) in NSCLC-DUSP6 depleted cells. Data obtained in RNA-seq studies carried out in DUSP6 depleted cells identified EGFR, TGF-ß and WNT signaling pathways and several genes such as VAV3, RUNXR2, LEF1, FGFR2 whose expression is upregulated in these cells and therefore affecting cellular functions such as integrin mediated cell adhesion, focal adhesion and motility. Furthermore, EGF signaling pathway is activated via ERK5 and not ERK1/2 and TGF-ß via SMAD2/3 in DUSP6 depleted cells. In summary DUSP6 is a tumor suppressor in NSCLC and re-establishment of its expression may be a potential strategy to revert poor outcome in NSCLC patients.
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Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Fosfatasa 6 de Especificidad Dual/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Citoesqueleto de Actina/metabolismo , Adenocarcinoma del Pulmón/enzimología , Adenocarcinoma del Pulmón/patología , Uniones Adherentes/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Forma de la Célula/genética , Progresión de la Enfermedad , Fosfatasa 6 de Especificidad Dual/metabolismo , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas , Ratones Desnudos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Cadherin-17 (CDH17), a marker of differentiation in intestinal cells, binds and activates α2ß1 integrin to promote cell adhesion and proliferation in colorectal cancer (CRC) metastasis. Furthermore, CDH17 associates with p120- and ß-catenin in a manner yet to be fully elucidated. In this report, we explored the molecular mediators involved in this association, their contribution to CRC dissemination and potential therapeutic implications. METHODS: Proteomic and confocal analyses were employed to identify and validate CDH17 interactors. Functional characterization involved the study of proliferation, migration, and invasion in cell lines representative of various phenotypes. Immunohistochemistry was conducted on CRC tissue microarrays (TMA). In vivo animal experiments were carried out for metastatic studies. RESULTS: We found that desmocollin-1 (DSC1), a desmosomal cadherin, interacts with CDH17 via its extracellular domain. DSC1 depletion led to increased or decreased invasion in CRC cells displaying epithelial or mesenchymal phenotype, respectively, in a process mediated by the association with p120-catenin. Down-regulation of DSC1 resulted in an increased expression of p120-catenin isoform 1 in epithelial cells or a shift in cellular location in mesenchymal cells. Opposite results were observed after forced expression of CDH17. DSC1 is highly expressed in budding cells at the leading edge of the tumor and associates with poor prognosis in the stem-like, mesenchymal CRC subtypes, while correlates with a more favorable prognosis in the less-aggressive subtypes. In vivo experiments demonstrated that DSC1 silencing reduced tumor growth, liver homing, and metastasis in CRC mesenchymal cells. Furthermore, a synthetic peptide derived from CDH17, containing the NLV motif, effectively inhibited invasion and liver homing in vivo, opening up new possibilities for the development of novel therapies focused on desmosomal cadherins. CONCLUSIONS: These findings shed light on the multifaceted roles of CDH17, DSC1, and p120-catenin in CRC metastasis, offering insights into potential therapeutic interventions for targeting desmosomal cadherins in poorly-differentiated carcinomas.
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Neoplasias Colorrectales , Desmocolinas , Animales , Catenina delta , Proteómica , CadherinasRESUMEN
Interleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/ß-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.
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Subunidad alfa2 del Receptor de Interleucina-13 , Neoplasias , Animales , Ratones , Interleucina-13 , Subunidad alfa2 del Receptor de Interleucina-13/genética , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias/genética , FN-kappa B/metabolismo , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , ProteómicaRESUMEN
The necessity to accurately predict recurrence and clinical outcome in early stage colorectal cancer (CRC) is critical to identify those patients who may benefit from adjuvant chemotherapy. Here, we developed and validated a gene-based risk-score algorithm for patient stratification and personalised treatment in early stage disease based on alterations in the secretion of metastasis-related proteins. A quantitative label-free proteomic analysis of the secretome of highly and poorly metastatic CRC cell lines with different genetic backgrounds revealed 153 differentially secreted proteins (fold-change >5). These changes in the secretome were validated at the transcriptomic level. Starting from 119 up-regulated proteins, a six-gene/protein-based prognostic signature composed of IGFBP3, CD109, LTBP1, PSAP, BMP1, and NPC2 was identified after sequential discovery, training, and validation in four different cohorts. This signature was used to develop a risk-score algorithm, named SEC6, for patient stratification. SEC6 risk-score components showed higher expression in the poor prognosis CRC subtypes: consensus molecular subtype 4 (CMS4), CRIS-B, and stem-like. High expression of the signature was also associated with patients showing dMMR, CIMP+ status, and BRAF mutations. In addition, the SEC6 signature was associated with lower overall survival, progression-free interval, and disease-specific survival in stage II and III patients. SEC6-based risk stratification indicated that 5-FU treatment was beneficial for low-risk patients, whereas only aggressive treatments (FOLFOX and FOLFIRI) provided benefits to high-risk patients in stages II and III. In summary, this novel risk-score demonstrates the value of the secretome compartment as a reliable source for the retrieval of biomarkers with high prognostic and chemotherapy-predictive capacity, providing a potential new tool for tailoring decision-making in patient care.
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Neoplasias del Colon , Neoplasias Colorrectales , Biomarcadores de Tumor/análisis , Neoplasias del Colon/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Fluorouracilo/uso terapéutico , Perfilación de la Expresión Génica , Humanos , Pronóstico , Proteómica , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/uso terapéutico , Secretoma , TranscriptomaRESUMEN
Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.
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Disqueratosis Congénita , Telomerasa , Animales , Proteínas de Ciclo Celular/genética , Disqueratosis Congénita/genética , Disqueratosis Congénita/terapia , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Mutación , Proteínas Nucleares/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin-mediated metastatic progression. CDH6 preferentially bound to αIIbß3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin Arginine-glycine-aspartic acid (RGD) motif. Furthermore, CDH6 and CDH17 were found to interact with α2ß1 in αIIbß3low cells. Transient silencing of CDH6, ITGA2B, or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion, and lung colonization through the downregulation of SRC, FAK, AKT, and ERK signaling. In ovarian and renal cancer cells, integrin αIIbß3 activation appears to be a prerequisite for proper α2ß1 activation. Interaction of αIIbß3 with CDH6, and subsequent αIIbß3 activation, promoted activation of α2ß1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically approved αIIbß3 inhibitors could block pro-metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbß3 regulates α2ß1-mediated adhesion and invasion of ovarian and renal cancer metastatic cells and constitutes a therapeutic target of broad potential for treating metastatic progression.
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Neoplasias Renales , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Cadherinas/metabolismo , Adhesión Celular , Femenino , Humanos , Integrina alfa2beta1/metabolismo , Neoplasias Renales/genética , Neoplasias Ováricas , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismoRESUMEN
The mechanistic basis of liver metastasis in colorectal cancer remains poorly understood. We previously reported that the sclerostin domain containing-1 (SOSTDC1) protein is overexpressed in the secretome of metastatic colorectal cancer cells and can inhibit liver homing. Here, we investigated the mechanisms of SOSTDC1 for promoting invasiveness and progression of colorectal cancer liver metastasis. SOSTDC1 inhibition of BMP4 maintains the expression of cancer stem cell traits, including SOX2 and NANOG. Immunoprecipitation and mass spectrometry analyses reveal the association of SOSTDC1 with ALCAM/CD166, which was confirmed by confocal microscopy and competition ELISA. Interaction with ALCAM is mediated by the N-terminal region of SOSTDC1, which contains a sequence similar to the ALCAM-binding motif used by CD6. Knocking down either SOSTDC1 or ALCAM expression, or using blocking antibodies, reduces the invasive activity by inhibiting Src and PI3K/AKT signaling pathways. In addition, ALCAM interacts with the α2ß1 and α1ß1 integrins, providing a possible link to Src activation. Finally, inoculation of SOSTDC1-silenced metastatic cells increases mouse survival by inhibiting liver metastasis. In conclusion, SOSTDC1 promotes invasion and liver metastasis in colorectal cancer, by overcoming BMP4-specific antimetastatic signals and inducing ALCAM-mediated Src and PI3K/AKT activation. These experiments underscore the potential of SOSTDC1 as a therapeutic target in metastatic colorectal cancer.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Proteínas Fetales/metabolismo , Neoplasias Hepáticas/secundario , Actinas/química , Actinas/metabolismo , Animales , Biomarcadores de Tumor , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Mitogen-activated protein kinases (MAPK) such as p38 and the c-Jun N-terminal kinases (JNKs) are activated during the cellular response to stress signals. Their activity is regulated by the MAPK-phosphatase 1 (DUSP1), a key component of the anti-inflammatory response. Stress kinases are well-described elements of the response to otic injury and the otoprotective potential of JNK inhibitors is being tested in clinical trials. By contrast, there are no studies exploring the role of DUSP1 in hearing and hearing loss. Here we show that Dusp1 expression is age-regulated in the mouse cochlea. Dusp1 gene knock-out caused premature progressive hearing loss, as confirmed by auditory evoked responses in Dusp1-/- mice. Hearing loss correlated with cell death in hair cells, degeneration of spiral neurons and increased macrophage infiltration. Dusp1-/- mouse cochleae showed imbalanced redox status and dysregulated expression of cytokines. These data suggest that DUSP1 is essential for cochlear homeostasis in the response to stress during ageing.
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Fosfatasa 1 de Especificidad Dual/deficiencia , Pérdida Auditiva/fisiopatología , Estimulación Acústica , Animales , Cóclea/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-ReducciónRESUMEN
Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.
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Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/metabolismo , Telómero/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Roturas del ADN de Doble Cadena , Daño del ADN , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Nanopartículas/química , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrés Oxidativo/fisiología , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Telomerasa/metabolismo , Telómero/genética , Telómero/patologíaRESUMEN
BACKGROUND: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. METHODS: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. RESULTS: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening. CONCLUSION: Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.
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Anemia Aplásica/genética , Reparación del ADN/genética , Disqueratosis Congénita/genética , Fibrosis Pulmonar/genética , Acortamiento del Telómero/genética , Telómero/genética , Adolescente , Adulto , Niño , Preescolar , Exones/genética , Femenino , Humanos , Lactante , Masculino , Linaje , ARN/genética , Telomerasa/genética , Adulto JovenRESUMEN
In vitro, tenofovir and abacavir induced a significant dose-dependent inhibition of telomerase activity at therapeutic concentrations in peripheral blood mononuclear cells of healthy subjects. Median inhibition of telomerase activity by tenofovir at 0.5 and 1 µM was 29% [Interquartile range (IQR) 29%-34%, P = 0.042] and 28% (IQR 28%-41%, P = 0.042), respectively. Abacavir inhibition was 12% (IQR 9%-13%, P = 0.043) at 3 µM and 14% (IQR 10%-29%, P = 0.043) at 10 µM. Tenofovir and abacavir did not change human telomerase reverse transcriptase (hTERT) levels or mRNA levels of other telomerase complex genes. Exposure to emtricitabine or darunavir did not affect telomerase activity, hTERT protein levels, or mRNA levels of telomerase/shelterin genes.
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Inhibidores de la Proteasa del VIH/efectos adversos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Inhibidores de la Transcriptasa Inversa/efectos adversos , Telomerasa/antagonistas & inhibidores , Voluntarios Sanos , HumanosRESUMEN
The elucidation of mechanisms involved in resistance to therapies is essential to improve the survival of patients with malignant gliomas. A major feature possessed by glioma cells that may aid their ability to survive therapy and reconstitute tumors is the capacity for self-renewal. We show here that glioma stem cells (GSCs) express low levels of MKP1, a dual-specificity phosphatase, which acts as a negative inhibitor of JNK, ERK1/2, and p38 MAPK, while induction of high levels of MKP1 expression are associated with differentiation of GSC. Notably, we find that high levels of MKP1 correlate with a subset of glioblastoma patients with better prognosis and overall increased survival. Gain of expression studies demonstrated that elevated MKP1 impairs self-renewal and induces differentiation of GSCs while reducing tumorigenesis in vivo. Moreover, we identified that MKP1 is epigenetically regulated and that it mediates the anti-tumor activity of histone deacetylase inhibitors (HDACIs) alone or in combination with temozolomide. In summary, this study identifies MKP1 as a key modulator of the interplay between GSC self-renewal and differentiation and provides evidence that the activation of MKP1, through epigenetic regulation, might be a novel therapeutic strategy to overcome therapy resistance in glioblastoma.
RESUMEN
Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.
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Proteínas de Ciclo Celular/genética , Senescencia Celular , Daño del ADN , Mutación/genética , Proteínas Nucleares/genética , Estrés Oxidativo , Péptidos/metabolismo , Telomerasa/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Disqueratosis Congénita/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Señales de Localización Nuclear , Fragmentos de Péptidos , Regiones Promotoras Genéticas/genéticaRESUMEN
The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.
Asunto(s)
Materiales Biocompatibles/química , Proteínas de Ciclo Celular/química , Sistemas de Liberación de Medicamentos , Reactivadores Enzimáticos/química , Nanopartículas/química , Proteínas Nucleares/química , Fragmentos de Péptidos/química , Animales , Materiales Biocompatibles/efectos adversos , Transporte Biológico , Proteínas de Ciclo Celular/administración & dosificación , Proteínas de Ciclo Celular/efectos adversos , Proteínas de Ciclo Celular/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/efectos adversos , Péptidos de Penetración Celular/química , Células Cultivadas , Fenómenos Químicos , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/efectos adversos , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/uso terapéutico , Composición de Medicamentos , Sistemas de Liberación de Medicamentos/efectos adversos , Liberación de Fármacos , Estabilidad de Medicamentos , Disqueratosis Congénita/tratamiento farmacológico , Reactivadores Enzimáticos/administración & dosificación , Reactivadores Enzimáticos/efectos adversos , Reactivadores Enzimáticos/uso terapéutico , Humanos , Ácido Láctico/efectos adversos , Ácido Láctico/química , Ratones , Nanopartículas/efectos adversos , Proteínas Nucleares/administración & dosificación , Proteínas Nucleares/efectos adversos , Proteínas Nucleares/genética , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/genética , Poliaminas/efectos adversos , Poliaminas/química , Polielectrolitos , Polietilenglicoles/efectos adversos , Polietilenglicoles/química , Poliglactina 910/efectos adversos , Poliglactina 910/química , Ácido Poliglicólico/efectos adversos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Estabilidad Proteica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéuticoRESUMEN
The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.