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BACKGROUND: Recently, we have identified a dysregulated protein signature in the esophageal epithelium of eosinophilic esophagitis (EoE) patients including proteins associated with inflammation and epithelial barrier function; however, the effect of proton pump inhibitor (PPI) treatment on this signature is unknown. Herein, we used a proteomic approach to investigate: (1) whether PPI treatment alters the esophageal epithelium protein profile observed in EoE patients and (2) whether the protein signature at baseline predicts PPI response. METHODS: We evaluated the protein signature of esophageal biopsies using a cohort of adult EoE (n = 25) patients and healthy controls (C) (n = 10). In EoE patients, esophageal biopsies were taken before (pre) and after (post) an 8-week PPI treatment, determining the histologic response. Eosinophil count PostPPI was used to classify the patients: ≥15 eosinophils/hpf as non-responders (non-responder) and < 15 eosinophils/hpf as responders (R). Protein signature was determined and differentially accumulated proteins were characterized to identify altered biological processes and signaling pathways. RESULTS: Comparative analysis of differentially accumulated proteins between groups revealed common signatures between three groups of patients with inflammation (responder-PrePPI, non-responder-PrePPI, and non-responder-PostPPI) and without inflammation (controls and responder-PostPPI). PPI therapy almost reversed the EoE specific esophageal protein signature, which is enriched in pathways associated with inflammation and epithelial barrier function, in responder-PostPPI. Furthermore, we identified a set of candidate proteins to differentiate responder-PrePPI and non-responder-PrePPI EoE patients before treatment. CONCLUSION: These findings provide evidence that PPI therapy reverses the alterations in esophageal inflammatory and epithelial proteins characterizing EoE, thereby providing new insights into the mechanism of PPI clinical response. Interestingly, our results also suggest that PPI response could be predicted at baseline in EoE.
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Introduction: Fanconi anemia (FA) is an inherited disorder characterized by bone marrow failure, congenital malformations, and predisposition to malignancies. Alterations in hematopoietic stem cells (HSC) have been reported, but little is known regarding the bone marrow (BM) stroma. Thus, the characterization of Mesenchymal Stromal Cells (MSC) would help to elucidate their involvement in the BM failure. Methods: We characterized MSCs of 28 FA patients (FA-MSC) before and after treatment (hematopoietic stem cell transplantation, HSCT; or gene therapy, GT). Phenotypic and functional properties were analyzed and compared with MSCs expanded from 26 healthy donors (HD-MSCs). FA-MSCs were genetically characterized through, mitomycin C-test and chimerism analysis. Furthermore, RNA-seq profiling was used to identify dysregulated metabolic pathways. Results: Overall, FA-MSC had the same phenotypic and functional characteristics as HD-MSC. Of note, MSC-GT had a lower clonogenic efficiency. These findings were not confirmed in the whole FA patients' cohort. Transcriptomic profiling identified dysregulation in HSC self-maintenance pathways in FA-MSC (HOX), and was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). Discussion: Our study provides a comprehensive characterization of FA-MSCs, including for the first time MSC-GT and constitutes the largest series published to date. Interestingly, transcript profiling revealed dysregulation of metabolic pathways related to HSC self-maintenance. Taken together, our results or findings provide new insights into the pathophysiology of the disease, although whether these niche defects are involved in the hematopoietic defects seen of FA deserves further investigation.
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Cytogenetic studies are essential in the diagnosis and follow up of patients with bone marrow failure syndromes (BMFSs), but obtaining good quality results is often challenging due to hypocellularity. Optical Genome Mapping (OGM), a novel technology capable of detecting most types chromosomal structural variants (SVs) at high resolution, is being increasingly used in many settings, including hematologic malignancies. Herein, we compared conventional cytogenetic techniques to OGM in 20 patients with diverse BMFSs. Twenty metaphases for the karyotype were only obtained in three subjects (15%), and no SVs were found in any of the samples. One patient with culture failure showed a gain in chromosome 1q by fluorescence in situ hybridization, which was confirmed by OGM. In contrast, OGM provided good quality results in all subjects, and SVs were detected in 14 of them (70%), mostly corresponding to cryptic submicroscopic alterations not observed by standard techniques. Therefore, OGM emerges as a powerful tool that provides complete and evaluable results in hypocellular BMFSs, reducing multiple tests into a single assay and overcoming some of the main limitations of conventional techniques. Furthermore, in addition to confirming the abnormalities detected by conventional techniques, OGM found new alterations beyond their detection limits.
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Hibridación Fluorescente in Situ , Humanos , Masculino , Femenino , Persona de Mediana Edad , Adulto , Anciano , Hibridación Fluorescente in Situ/métodos , Mapeo Cromosómico/métodos , Trastornos de Fallo de la Médula Ósea/genética , Aberraciones Cromosómicas , Adolescente , Análisis Citogenético/métodos , Enfermedades de la Médula Ósea/genética , Cariotipificación/métodos , Adulto JovenRESUMEN
Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.
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Neuroblastoma , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Fluoresceína-5-Isotiocianato , Citocinas/metabolismoRESUMEN
The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.
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OBJECTIVE: To characterize cell line CAE606 derived from a squamous cell carcinoma (SCC) of the external auditory canal (EAC) and to show its usefulness as a model for testing candidate therapeutic agents. STUDY DESIGN: Preclinical translational research. SETTING: Biomedical research institute. METHODS: The cell line was initiated from a moderately differentiated T2N0M0 EAC SCC. We studied its histologic and genetic features as well as growth and invasion parameters. Sensitivity to cell CDK4/6 cell cycle inhibitor palbociclib was analyzed. RESULTS: CAE606 cells expressed heavy molecular weight cytokeratin, p63, and vimentin. The population doubling time was 25.8 hours, and the cells showed fast collective cell migration in a wound-healing assay. Short tandem repeat analysis confirmed it to be derived from the primary tumor of the patient. Next-generation sequencing revealed alterations in cell cycle regulation genes, including inactivating mutations in CDKN2A and TP53 and high-level amplification of CCND1 and EGFR. CAE606 showed a strong decrease of phospo-Rb expression upon exposure to the CDK4/6 inhibitor palbociclib, causing significant growth inhibition with an IC50 of 0.46 µM. CONCLUSION: This is the first report of a stable EAC SCC cell line. Its genetic features make it a useful tool for preclinical testing of new therapeutic agents for EAC SCC, particularly those targeting cell cycle regulation in combination with radio- and chemotherapy or other specific signaling pathway inhibitors.
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Carcinoma de Células Escamosas , Conducto Auditivo Externo , Humanos , Conducto Auditivo Externo/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la CiclinaRESUMEN
BACKGROUND: Psoriasis can present different phenotypes and could affect diverse body areas. In contrast to the high effectiveness of biological drugs in the treatment of trunk and extremities plaque psoriasis, in palmoplantar phenotypes and in plaque scalp psoriasis, these same drugs usually have reduced efficacy. Anti-TNF drugs could induce the appearance of palmoplantar pustulosis (PPP) in patients with other inflammatory diseases. The objective of this study is to identify if there are DNA Copy Number Variations (CNVs) associated with these different clinical phenotypes, which could justify the differences found in clinical practice. Moreover, we intend to elucidate if anti-TNF-induced PPP has a similar genetic background to idiopathic PPP. METHODS: Skin samples were collected from 39 patients with different patterns of psoriasis and six patients with anti-TNF-induced PPP. The CNVs were obtained from methylation array data (Illumina Infinium Human Methylation) using the conumee R package. RESULTS: No significant CNVs were found between the different phenotypes and the locations of psoriasis compared. Nevertheless, we found two significant bins harboring five different genes associated with anti-TNF-induced PPP in patients with a different background other than psoriasis. CONCLUSIONS: Our results may help to predict which patients could develop anti-TNF-induced PPP.
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Extracellular vesicles are membrane-enclosed secreted vesicles involved in cell-to-cell communication processes, identified in virtually all body fluids. Among extracellular vesicles, exosomes have gained increasing attention in recent years as they have unique biological origins and deliver different cargos, such as nucleic acids, proteins, and lipids, which might mediate various health processes. In particular, milk-derived exosomes are proposed as bioactive compounds of breast milk, which have been reported to resist gastric digestion and reach systemic circulation, thus being bioavailable after oral intake. In the present manuscript, we critically discuss the available evidence on the health benefits attributed to milk exosomes, and we provide an outlook for the potential future uses of these compounds. The use of milk exosomes as bioactive ingredients represents a novel avenue to explore in the context of human nutrition, and they might exert important beneficial effects at multiple levels, including but not limited to intestinal health, bone and muscle metabolism, immunity, modulation of the microbiota, growth, and development.
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Exosomas , Vesículas Extracelulares , MicroARNs , Microbiota , Animales , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Humanos , MicroARNs/metabolismo , Leche/metabolismo , Leche Humana/metabolismoRESUMEN
Biological drugs targeting tumour necrosis factor are effective for psoriasis. However, 30-50% of patients do not respond to these drugs and may even develop paradoxical psoriasiform reactions. This study search-ed for DNA copy number variations that could predict anti-tumour necrotic factor drug response or the appearance of anti-tumour necrotic factor induced psoriasiform reactions. Peripheral blood samples were collected from 70 patients with anti-tumour necrotic factor drug-treated moderate-to-severe plaque psoriasis. Samples were analysed with an Illumina 450K methylation microarray. Copy number variations were obtained from raw methylation data using conumee and Chip Analysis Methylation Pipeline (ChAMP) R packages. One copy number variation was found, harbouring one gene (CPM) that was significantly associated with adalimumab response (Bonferroni-adjusted p-value < 0.05). Moreover, one copy number variation was identified harbouring 3 genes (ARNT2, LOC101929586 and MIR5572) related to the development of paradoxical psoriasiform reactions. In conclusion, this study has identified DNA copy number variations that could be good candidate markers to predict response to adalimumab and the development of anti-tumour necrotic factor paradoxical psoriasiform reactions.
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Preparaciones Farmacéuticas , Psoriasis , Adalimumab/efectos adversos , Variaciones en el Número de Copia de ADN , Humanos , Infliximab , Psoriasis/diagnóstico , Psoriasis/tratamiento farmacológico , Psoriasis/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The epithelial-to-mesenchymal transition (EMT) and its reversion (MET) are related to tumor cell dissemination and migration, tumor circulating cell generation, cancer stem cells, chemoresistance, and metastasis formation. To identify chromatin and epigenetic factors possibly involved in the process of EMT, we compare the levels of expression of epigenetic genes in a transformed human breast epithelial cell line (HMEC-RAS) versus a stable clone of the same cell line expressing the EMT master regulator ZEB1 (HMEC-RAS-ZEB1). One of the factors strongly induced in the HMEC-RAS-ZEB1 cells was Transducin beta-like 1 (TBL1), a component of the NCoR complex, which has both corepressor and coactivator activities. We show that TBL1 interacts with ZEB1 and that both factors cooperate to repress the promoter of the epithelial gene E-cadherin (CDH1) and to autoactivate the ZEB1 promoter. Consistent with its central role, TBL1 is required for mesenchymal phenotypes of transformed breast epithelial and breast cancer cell lines of the claudin-low subtype. Importantly, a high expression of the TBL1 gene correlates with poor prognosis and increased proportion of metastasis in breast cancer patients, indicating that the level of TBL1 expression can be used as a prognostic marker.
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Antígenos CD/genética , Neoplasias de la Mama/genética , Cadherinas/genética , Células Madre Neoplásicas/metabolismo , Transducina/genética , Antígenos CD/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Línea Celular , Línea Celular Tumoral , Epigénesis Genética , Transición Epitelial-Mesenquimal , Femenino , Células HEK293 , Humanos , Células Madre Neoplásicas/patología , Fenotipo , Regiones Promotoras Genéticas , Transducina/metabolismo , Transfección , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.
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Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Interacciones Huésped-Patógeno , Bombas de Protones/metabolismo , Rodopsina/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidad , Ácidos Indolacéticos/farmacología , Neurospora/genética , Neurospora/metabolismo , Oryza/microbiología , Bombas de Protones/química , Bombas de Protones/genética , Rodopsina/química , Rodopsina/genética , Homología de Secuencia , Acetato de Sodio/farmacologíaRESUMEN
Stem cells hold enormous promise for regenerative medicine as well as for engineering of model systems to study diseases and develop new drugs. The discovery of protocols that allow for generating induced pluripotent stem cells (IPSCs) from somatic cells has brought this promise steps closer to reality. However, as somatic cells might have accumulated various chromosomal abnormalities, including aneuploidies throughout their lives, the resulting IPSCs might no longer carry the perfect blueprint for the tissue to be generated, or worse, become at risk of adopting a malignant fate. In this review, we discuss the contribution of aneuploidy to healthy tissues and how aneuploidy can lead to disease. Furthermore, we review the differences between how somatic cells and stem cells respond to aneuploidy.
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The COL11A1 human gene codes for the α1 chain of procollagen 11A1 and mature collagen 11A1, an extracellular minor fibrillar collagen. Under regular conditions, this gene and its derived products are mainly expressed by chondrocytes and mesenchymal stem cells as well as osteoblasts. Normal epithelial cells and quiescent fibroblasts from diverse locations do not express them. Mesenchyme-derived tumors and related conditions, such as scleroderma and keloids, are positive for COL11A1/(pro)collagen 11A1 expression, as well as high-grade human gliomas/glioblastomas. This expression is almost absent in benign pathological processes such as breast hyperplasia, sclerosing adenosis, idiopathic pulmonary fibrosis, cirrhosis, pancreatitis, diverticulitis, and inflammatory bowel disease. By contrast, COL11A1/(pro)collagen 11A1 is highly expressed by activated stromal cells of the desmoplastic reaction of different human invasive carcinomas, and this expression is correlated with carcinoma aggressiveness and progression, and lymph node metastasis. COL11A1 upregulation has been shown to be associated to TGF-ß1, Wnt, and Hh signaling pathways, which are especially active in cancer-associated stromal cells. At the front of invasive carcinomas, neoplastic epithelial cells, putatively undergoing epithelial-to-mesenchymal transition, and carcinoma-derived cells with highly metastatic capabilities, can express COL11A1. Thus, in established metastases, the expression of COL11A1/(pro)collagen 11A1 could rely on both the metastatic epithelial cells and/or the accompanying activated stromal cells. COL11A1/(pro)collagen 11A1 expression is a remarkable biomarker of human carcinoma-associated stromal cells and carcinoma progression.
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Carcinoma/genética , Colágeno Tipo XI/biosíntesis , Invasividad Neoplásica/genética , Neoplasias/genética , Carcinogénesis , Carcinoma/patología , Colágeno Tipo XI/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/patología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO(-) mutant and carO(+) control strains showed a faster development of light-exposed carO(-) germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.
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Proteínas Fúngicas/metabolismo , Fusarium/crecimiento & desarrollo , Fusarium/efectos de la radiación , Luz , Bombas de Protones/metabolismo , Rodopsina/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , Secuencia de Aminoácidos , Southern Blotting , Ácidos Carboxílicos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de la radiación , Recuento de Colonia Microbiana , Oscuridad , Fenómenos Electrofisiológicos , Proteínas Fúngicas/química , Fusarium/efectos de los fármacos , Gluconatos/farmacología , Concentración de Iones de Hidrógeno , Iones , Microscopía Fluorescente , Datos de Secuencia Molecular , Bombas de Protones/química , Proteínas Recombinantes de Fusión/metabolismo , Rodopsina/química , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/efectos de la radiaciónRESUMEN
BACKGROUND: Patients with head and neck squamous cell carcinoma (HNSCC) have an unfavorable prognosis, with a 5-year survival rate of approximately 40%. Genetic analyses have revealed that the majority of HNSCCs carry complex, aneuploid karyotypes, showing numerical and structural chromosomal imbalances. New compounds are being developed that target chromosomal instability in general, specifically affecting cells with aneuploid karyotypes. METHODS: Two such compounds, 5-aminoimidazole-4-carboxamide riboside (AICAR) and 17-allylamino-17-demethoxygeldanamycin (17-AAG), were tested using a panel of stable diploid and unstable aneuploid HNSCC cell lines, and short-term cultures of normal keratinocytes as control. RESULTS: A significant growth inhibitory effect by both compounds was observed in the aneuploid compared to diploid HNSCC cell lines and to the normal keratinocytes. This effect was independent from the TP53 mutation status. Combination treatment with AICAR and 17-AAG demonstrated the strongest inhibition. CONCLUSION: Aneuploidy-targeted therapy may be a viable addition to the treatment options for HNSCC.
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Aminoimidazol Carboxamida/análogos & derivados , Aneuploidia , Benzoquinonas/farmacología , Terapia Genética/métodos , Lactamas Macrocíclicas/farmacología , Ribonucleósidos/farmacología , Proteína p53 Supresora de Tumor/genética , Anciano , Anciano de 80 o más Años , Aminoimidazol Carboxamida/farmacología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Estudios de Casos y Controles , Análisis Mutacional de ADN , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Queratinocitos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Mutación Missense , Sensibilidad y Especificidad , Carcinoma de Células Escamosas de Cabeza y Cuello , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
BACKGROUND: The human COL11A1 gene has been shown to be up-regulated in stromal cells of colorectal tumours, but, so far, the immunodetection of procollagen 11A1, the primary protein product of COL11A1, has not been studied in detail in human colon adenocarcinomas. Some cancer-associated stromal cells seem to be derived from bone marrow mesenchymal cells; the expression of the COL11A1 gene and the parallel immunodetection of procollagen 11A1 have not been evaluated in these latter cells, either. METHODS: We used quantitative RT-PCR and/or immunocytochemistry to study the expression of DES/desmin, VIM/vimentin, ACTA2/αSMA (alpha smooth muscle actin) and COL11A1/procollagen 11A1 in HCT 116 human colorectal adenocarcinoma cells, in immortalised human bone marrow mesenchymal cells and in human colon adenocarcinoma-derived cultured stromal cells. The immunodetection of procollagen 11A1 was performed with the new recently described DMTX1/1E8.33 mouse monoclonal antibody. Human colon adenocarcinomas and non-malignant colon tissues were evaluated by immunohistochemistry as well. Statistical associations were sought between anti-procollagen 11A1 immunoscoring and patient clinicopathological features. RESULTS: Procollagen 11A1 was immunodetected in human bone marrow mesenchymal cells and in human colon adenocarcinoma-associated spindle-shaped stromal cells but not in colon epithelial or stromal cells of the normal colon. This immunodetection paralleled, in both kinds of cells, that of the other mesenchymal-related biomarkers studied: vimentin and alpha smooth muscle actin, but not desmin. Thus, procollagen 11A1(+) adenocarcinoma-associated stromal cells are similar to "activated myofibroblasts". In the series of human colon adenocarcinomas here studied, a high procollagen 11A1 expression was associated with nodal involvement (p = 0.05), the development of distant metastases (p = 0.017), and advanced Dukes stages (p = 0.047). CONCLUSION: The immunodetection of procollagen 11A1 in cancer-associated stromal cells could be a useful biomarker for human colon adenocarcinoma characterisation.
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Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Colágeno Tipo XI/genética , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Expresión Génica , Factor de Crecimiento Transformador beta1/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Línea Celular Transformada , Línea Celular Tumoral , Neoplasias del Colon/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Células del Estroma , Carga TumoralRESUMEN
Survival of micro-organisms in natural habitats depends on their ability to adapt to variations in osmotic conditions. We previously described the gene cut-1 of Neurospora crassa, encoding a protein of the haloacid dehalogenase family with an unknown function in the osmotic stress response. Here we report on the functional analysis of cutA, the orthologous gene in the phytopathogenic fungus Fusarium fujikuroi. cutA mRNA levels increased transiently after exposure to 0.68 M NaCl and were reduced upon return to normal osmotic conditions; deletion of the gene resulted in a partial reduction in tolerance to osmotic stress. ΔcutA mutants contained much lower intracellular levels of glycerol than the wild-type, and did not exhibit the increase following hyper-osmotic shock expected from the high osmolarity glycerol (HOG) response. cutA is linked and divergently transcribed with the putative glycerol dehydrogenase gene gldB, which showed the same regulation by osmotic shock. The intergenic cutA/gldB regulatory region contains putative stress-response elements conserved in other fungi, and both genes shared other regulatory features, such as induction by heat shock and by illumination. Photoinduction was also observed in the HOG response gene hogA, and was lost in mutants of the white collar gene wcoA. Previous data on glycerol production in Aspergillus spp. and features of the predicted CutA protein lead us to propose that F. fujikuroi produces glycerol from dihydroxyacetone, and that CutA is the enzyme involved in the synthesis of this precursor by dephosphorylation of dihydroxyacetone-3P.
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Fusarium/efectos de los fármacos , Fusarium/fisiología , Glicerol/metabolismo , Hidrolasas/metabolismo , Presión Osmótica , Estrés Fisiológico , Fusarium/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Orden Génico , Hidrolasas/genética , Cloruro de Sodio/metabolismoRESUMEN
DASH (Drosophila, Arabidopsis, Synechocystis, human) cryptochromes (cry-DASHs) constitute a subgroup of the photolyase cryptochrome family with diverse light-sensing roles, found in most taxonomical groups. The genome of Fusarium fujikuroi, a phytopathogenic fungus with a rich secondary metabolism, contains a gene encoding a putative cry-DASH, named CryD. The expression of the cryD gene is induced by light in the wild type, but not in mutants of the "white collar" gene wcoA. Targeted ΔcryD mutants show light-dependent phenotypic alterations, including changes in morphology and pigmentation, which disappear upon reintroduction of a wild-type cryD allele. In addition to microconidia, the colonies of the ΔcryD mutants produced under illumination and nitrogen starvation large septated spores called macroconidia, absent in wild-type colonies. The ΔcryD mutants accumulated similar amounts of carotenoids to the control strain under constant illumination, but produced much larger amounts of bikaverin under nitrogen starvation, indicating a repressing role for CryD in this biosynthetic pathway. Additionally, a moderate photoinduction of gibberellin production was exhibited by the wild type but not by the ΔcryD mutants. The phenotypic alterations of the ΔcryD mutants were only noticeable in the light, as expected from the low expression of cryD in the dark, but did not correlate with mRNA levels for structural genes of the bikaverin or gibberellin biosynthetic pathways, suggesting the participation of CryD in posttranscriptional regulatory mechanisms. This is the first report on the participation of a cry-DASH protein in the regulation of fungal secondary metabolism.
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Criptocromos/genética , Criptocromos/metabolismo , Fusarium/metabolismo , Animales , Arabidopsis/metabolismo , Secuencia de Bases , Drosophila/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Giberelinas/biosíntesis , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Análisis de Secuencia de ADN , Synechocystis/metabolismo , Xantonas/metabolismoRESUMEN
BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is a rare, inheritable, multisystem disorder characterized by numerous basal cell carcinomas (BCCs), maxillary keratocyst, and musculoskeletal malformations. Occasionally, it is associated with malignancies like rhabdomyoma, melanoma, and sinonasal undifferentiated carcinoma, to name a few. METHODS: A patient presented with NBCCS with a medullary thyroid carcinoma. Clinical, surgical details, and germline genetic analysis are herein described. RESULTS: A 32-year-old woman was referred to our department with suspicion of medullary thyroid carcinoma, which was confirmed by histopathological examination. The patient was diagnosed as also having NBCCS. Germline mutation analysis indicated wild-type genes PTCH1 and RET. DNA copy number analysis by high resolution microarray comparative genomic hybridization (CGH) revealed a small interstitial loss at chromosomal band 2q37.3. CONCLUSION: To our knowledge, this is the first described patient with NBCCS carrying a medullary thyroid carcinoma and a 2q37 deletion, which confirms that this syndrome can be associated with many different malignancies.
Asunto(s)
Síndrome del Nevo Basocelular/complicaciones , Deleción Cromosómica , Neoplasias de la Tiroides/complicaciones , Adulto , Carcinoma Neuroendocrino , Cromosomas Humanos Par 2 , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Femenino , Mutación de Línea Germinal , Humanos , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
Introducción y objetivos: La literatura sobre la participación de la inestabilidad de microsatélites en el carcinoma de células escamosas de cabeza y cuello muestra una gran variabilidad, probablemente debido a las diferencias en la metodología de las pruebas. Utilizando un sistema de detección consensuado, nos planteamos como objetivo llegar a una estimación fiable de la prevalencia de la inestabilidad de microsatélites en un subconjunto de carcinomas de células escamosas de cabeza y cuello. Métodos: Se analizó el estado de inestabilidad de microsatélites en 43 pacientes no tratados previamente y diagnosticados de un carcinoma primario de células escamosas de laringe mediante una prueba de PCR múltiple, incluyendo 5 marcadores repetidos de mononucleótidos. Resultados: En 36 casos se observó un fenotipo estable o microsatélites estables (83,7%), y en 7 casos (16,3%) se mostró un fenotipo positivo de inestabilidad de microsatélites. Uno de los casos mostró inestabilidad en 3 de los 5 marcadores, otro mostró inestabilidad en 2 marcadores y 5 casos en un marcador. Entre los casos de inestabilidad de microsatélites positiva y los casos estables no hubo diferencias con respecto a la edad, el estadio del tumor, la afectación de los ganglios linfáticos o las metástasis a distancia. Conclusiones: Nuestros datos muestran que una parte de los carcinomas de células escamosas de laringe presentan inestabilidad de microsatélites. El conocimiento sobre el estado de inestabilidad de microsatélites de los pacientes permitirá el ajuste de la terapia anti-cancerígena a nivel individual(AU)
Introduction and objectives: The literature on the involvement of microsatellite instability in head and neck squamous cell carcinoma shows great variability, probably due to differences in the testing methods. Using a consensus detection system, we aimed to reach a reliable estimate of microsatellite instability prevalence in a subset of head and neck squamous cell carcinoma cases. Methods: The microsatellite instabilityI status of 43 patients with previously untreated primary laryngeal squamous cell carcinomas was analyzed by a multiplex polymerase chain reaction assay including 5 mononucleotide repeat markers. Results: Thirty-six cases showed a stable phenotype or a microsatellite stable phenotype (83.7%) and 7 cases (16.3%) showed an microsatellite instability-positive phenotype. One case showed instability in 3 of 5 markers, 1 case in 2 markers and 5 cases in 1 marker. The microsatellite instability-positive and stable cases did not differ with respect to age, tumour stage, lymph node or distant metastases. Conclusions: Our data showed that a proportion of laryngeal squamous cell carcinomas are microsatellite instability positive. Knowledge of microsatellite instability patient status will allow adjusting anticancer therapy at an individual level(AU)