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2.
Acta Derm Venereol ; 102: adv00749, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35604238

RESUMEN

There is a need for biomarkers that predict the success of transplantation of venous leg ulcers (with autologous split-thickness skin grafts). The primary objective of this exploratory study was to investigate the association between split-thickness skin graft healing in venous leg ulcers and candidate wound fluid biomarkers representing inflammatory cell and endogenous proteinase activities, and bioactivity. A secondary objective was to compare biomarker levels of the 17 venous leg ulcers with sterile split-thickness skin graft donor-site wounds in another 10 patients with venous leg ulcers. Wound fluids were collected for 24 h using a validated method. The concentration of preoperative matrix metalloproteinase-9 in wound fluid was higher in venous leg ulcers showing good healing (n = 10) than in venous leg ulcers showing poor healing (n = 7) 12 weeks after transplantation with meshed split-thickness skin grafts. The diagnostic value of matrix metalloproteinase-9 was good according to receiver-operating characteristic curve analysis. Matrix metalloproteinase activity in wound fluids from split-thickness skin graft donor-site wounds increased as a function of time and healing, but was still lower than matrix metalloproteinase activity in venous leg ulcer wound fluids, which showed increased levels of most biomarkers except for matrix metalloproteinase-9 and matrix metalloproteinase-2. In conclusion, wound fluid matrix metalloproteinase-9 concentration is a potential predictive biomarker of split-thickness skin graft healing in venous leg ulcers.


Asunto(s)
Úlcera de la Pierna , Trasplante de Piel , Úlcera Varicosa , Biomarcadores/análisis , Humanos , Úlcera de la Pierna/cirugía , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Metaloproteinasas de la Matriz , Úlcera Varicosa/diagnóstico , Úlcera Varicosa/cirugía , Cicatrización de Heridas
3.
Biochem Biophys Rep ; 26: 101005, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34027132

RESUMEN

BACKGROUND: Immortalized mammalian cell lines are a valuable research tool, though they represent a highly simplified model. Due to accumulated mutations they may not reflect characteristics of the disease or even the tissue they derive from. OBJECTIVE: We aim to pinpoint factors distinguishing SeAx cells from two other cutaneous T-cell lymphoma (CTCL) cell lines, namely Hut78 and MyLa2000. Of note, these factors may influence cell sensitivity in an unspecific way and therefore should be taken under consideration. METHODS: We evaluated transcriptional levels of drug transporters across cell lines, cell membrane permeability, functionality of pathways related to DNA damage response and activation of G2/M block. RESULTS: Analysis of the transcriptional levels of genes coding drug efflux pumps indicated that they are not consistently down-regulated in SeAx. However, we noted that SeAx cell membrane is markedly more permeable than Hut78 and MyLa2000, which may contribute to increased chemosensitivity in an unspecific way.Moreover, though DNA damage response seemed to be at least partly functional in SeAx cells, they fail to activate G2/M block in response to psoralen + UVA treatment. Any DNA damage should be repaired before cells enter mitosis, in order to uphold genome integrity. Thus, a defective cell cycle block may contribute to cell sensitivity. CONCLUSIONS: We believe that factors such as increased membrane permeability or defective cell cycle block should be accounted for when comparing sensitivity of cell line panels to chemotherapeutics of interest. It is worth to exclude a simple, indiscriminative mechanisms of cell resistance or sensitivity before attempting comparisons. Cell lines that are indiscriminately sensitive to a broad range of chemicals may contribute to overestimating the cytotoxic potential of tested compounds if used in cytotoxicity studies.

4.
Cells ; 9(11)2020 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-33182724

RESUMEN

DNA-damaging cancer therapies induce interferon expression and stimulate the immune system, promoting therapy responses. The immune-activating STING (Stimulator of Interferon Genes) pathway is induced when DNA or double-stranded RNA (dsRNA) is detected in the cell cytoplasm, which can be caused by viral infection or by DNA damage following chemo- or radiotherapy. Here, we investigated the responses of cutaneous T-cell lymphoma (CTCL) cells to the clinically applied DNA crosslinking photochemotherapy (combination of 8-methoxypsoralen and UVA light; 8-MOP + UVA). We showed that this treatment evokes interferon expression and that the type III interferon IFNL1 is the major cytokine induced. IFNL1 upregulation is dependent on STING and on the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS). Furthermore, 8-MOP + UVA treatment induced the expression of genes in pathways involved in response to the tumor necrosis factor, innate immune system and acute inflammatory response. Notably, a subset of these genes was under control of the STING-IFNL1 pathway. In conclusion, our data connected DNA damage with immune system activation via the STING pathway and contributed to a better understanding of the effectiveness of photochemotherapy.


Asunto(s)
Daño del ADN/fisiología , Interferones/metabolismo , Fotoquimioterapia/métodos , Línea Celular Tumoral , Humanos , Transfección , Interferón lambda
5.
Oncotarget ; 7(30): 48391-48400, 2016 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-27351221

RESUMEN

Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53-/-). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL.


Asunto(s)
Endopeptidasas/metabolismo , Linfoma Cutáneo de Células T/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Anciano , Anciano de 80 o más Años , Apoptosis/fisiología , Línea Celular Tumoral , Endopeptidasas/genética , Femenino , Humanos , Linfoma Cutáneo de Células T/genética , Linfoma Cutáneo de Células T/patología , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa
6.
Leuk Lymphoma ; 55(3): 639-44, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23697875

RESUMEN

Peripheral T-cell lymphomas (PTLs) represent an area of high medical need. Previously, we demonstrated high expression of Notch, a known oncogene, in primary cutaneous anaplastic large cell lymphoma (ALCL). In this study, we performed immunohistochemical staining for Notch1 in lymph nodes from PTL not otherwise specified (PTL-NOS) and systemic ALCL (ALK+ and ALK-) and report a similar distribution among the three subgroups: Negative, moderate and strong expression was, respectively, 18%, 27% and 55% for PTL-NOS (33 cases), 20%, 0% and 80% for ALCL ALK+ (10 cases) and 45%, 22% and 33% for ALCL ALK- (nine cases) (p > 0.05). In the ALK+ ALCL cell line, Karpas-299, pharmacological inhibition of Notch with γ-secretase inhibitor (GSI) I was far more potent than with GSI IX, XX and XXI with regard to cell viability and apoptosis. In conclusion, PTL tumor cells have prominent Notch1 expression and treatment with Notch inhibitors has cytotoxic effects.


Asunto(s)
Linfoma de Células T Periférico/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oligopéptidos/farmacología , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/metabolismo , Receptores Notch/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Adulto Joven
7.
J Mol Endocrinol ; 48(1): 11-23, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22049245

RESUMEN

The molecular determinants of thyroid follicular nodules are incompletely understood and assessment of malignancy is a diagnostic challenge. Since microRNA (miRNA) analyses could provide new leads to malignant progression, we characterised the global miRNA expression in follicular adenoma (FA) and follicular carcinoma (FC). Comparison of carcinoma and adenoma with normal thyroid revealed 150 and 107 differentially expressed miRNAs respectively. Most miRNAs were down-regulated and especially miR-199b-5p and miR-144 which were essentially lost in the carcinomas. Integration of the changed miRNAs with differentially expressed mRNAs demonstrated an enrichment of seed sites among up-regulated transcripts encoding proteins implicated in thyroid tumourigenesis. This was substantiated by the demonstration that pre-miR-199b reduced proliferation when added to cultured follicular thyroid carcinoma cells. The down-regulated miRNAs in FC exhibited a substantial similarity with down-regulated miRNAs in anaplastic carcinoma (AC) and by gene set enrichment analysis, we observed a significant identity between target mRNAs in FC and transcripts up-regulated in AC. To examine the diagnostic potential of miRNA expression pattern in distinguishing malignant from benign nodules we employed a supervised learning algorithm and leave-one-out-cross-validation. By this procedure, FA and FC were identified with a negative predicted value of 83% (data generated by microarray platform) and of 92% (data generated by qRT-PCR platform). We conclude that follicular neoplasia is associated with major changes in miRNA expression that may promote malignant transformation by increasing the expression of transcripts encoding tumourigenic factors. Moreover, miRNA profiling may facilitate the diagnosis of carcinoma vs adenoma.


Asunto(s)
Adenocarcinoma Folicular/genética , Transformación Celular Neoplásica/genética , Regulación hacia Abajo , MicroARNs/genética , Neoplasias de la Tiroides/genética , Adenocarcinoma Folicular/clasificación , Adenocarcinoma Folicular/metabolismo , Adulto , Anciano , Línea Celular Tumoral , Proliferación Celular , Análisis por Conglomerados , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , ARN Mensajero/metabolismo , Transducción de Señal , Neoplasias de la Tiroides/clasificación , Neoplasias de la Tiroides/metabolismo
8.
Diabetes ; 58(10): 2419-24, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19587354

RESUMEN

OBJECTIVE: In the pancreatic beta-cell, ATP-sensitive K(+) (K(ATP)) channels couple metabolism with excitability and consist of Kir6.2 and SUR1 subunits encoded by KCNJ11 and ABCC8, respectively. Sulfonylureas, which inhibit the K(ATP) channel, are used to treat type 2 diabetes. Rare activating mutations cause neonatal diabetes, whereas the common variants, E23K in KCNJ11 and S1369A in ABCC8, are in strong linkage disequilibrium, constituting a haplotype that predisposes to type 2 diabetes. To date it has not been possible to establish which of these represents the etiological variant, and functional studies are inconsistent. Furthermore, there have been no studies of the S1369A variant or the combined effect of the two on K(ATP) channel function. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to study the nucleotide sensitivity and sulfonylurea inhibition of recombinant human K(ATP) channels containing either the K23/A1369 or E23/S1369 variants. RESULTS: ATP sensitivity of the K(ATP) channel was decreased in the K23/A1369 variant (half-maximal inhibitory concentration [IC(50)] = 8.0 vs. 2.5 mumol/l for the E23/S1369 variant), although there was no difference in ADP sensitivity. The K23/A1369 variant also displayed increased inhibition by gliclazide, an A-site sulfonylurea drug (IC(50) = 52.7 vs. 188.7 nmol/l for the E23/S1369 variant), but not by glibenclamide (AB site) or repaglinide (B site). CONCLUSIONS: Our findings indicate that the common K23/A1369 variant K(ATP) channel displays decreased ATP inhibition that may contribute to the observed increased risk for type 2 diabetes. Moreover, the increased sensitivity of the K23/A1369 variant to the A-site sulfonylurea drug gliclazide may provide a pharmacogenomic therapeutic approach for patients with type 2 diabetes who are homozygous for both risk alleles.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Diabetes Mellitus Tipo 2/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética , Canales KATP/fisiología , Canales de Potasio de Rectificación Interna/genética , Receptores de Droga/genética , Adenosina Trifosfato/farmacología , Sustitución de Aminoácidos , Homocigoto , Canales KATP/efectos de los fármacos , Canales KATP/genética , Polimorfismo de Nucleótido Simple , Compuestos de Sulfonilurea/farmacología , Receptores de Sulfonilureas
9.
Mol Endocrinol ; 23(6): 893-900, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19299446

RESUMEN

Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.


Asunto(s)
Calcio/metabolismo , Glutarredoxinas/metabolismo , Insulina/metabolismo , NADP/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Exocitosis/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Glucosa/farmacología , Inmunohistoquímica , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/enzimología , Oxidación-Reducción/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Tiorredoxinas/metabolismo
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