RESUMEN

Objective: To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on extracellular matrix metalloproteinase (MMP in JAK2V617F positive myeloproliferative neoplasms (MPN) cells. Methods: ①Forty cases of newly diagnosed JAK2V617F positive MPN patients and 15 healthy volunteers as control in Baoding No.1 Hospital between January 2012 and December 2015 were enrolled in this study. JAK2V617F/JAK2 ratio was detected by real-time-PCR; the expression levels of phosphorylation protein tyrosine kinase 2 (p-JAK2) , MMP-2 and MMP-9 in pathological tissues of bone marrow were detected by immunohistochemistry. The bone marrow cells of JAK2V617F positive MPN patients were treated with ruxolitinib, then the migration ability and MMP-2, MMP-9 gene and protein expression levels were detected. ②The human erythroleukemia cell line HEL cells were treated with different concentrations of ruxolitinib (0, 50, 100, 250, 500, 1 000 nmol/L) . The cell viability was detected by CCK-8 test; cell migration ability was tested by transwell chambers. The mRNA expression levels of JAK2, MMP-2 and MMP-9 were detected by real-time-PCR. The protein expression levels of p-JAK2, MMP-2 and MMP-9 were detected by Western blot. Results: ①The expression levels of p-JAK2, MMP-2 and MMP-9 in the newly diagnosed group were significantly higher than control group respectively [ (78.56±24.55) % vs (41.59±17.29) %, P<0.05; (48.25±18.74) % vs (22.79±13.89) %, P<0.05; (53.29±19.28) % vs (15.56±14.96) %, P<0.05]. Spearman correlation analysis showed the positive correlation of MMP-2 and MMP-9 protein expression levels with JAK2V617F mutation (r=0.526, P=0.001; r=0.543, P=0.001) . ②The proliferation of HEL cells was inhibited by different concentrations of ruxolitinib in time and dose dependent manner. ③Cell migration test showed the number of cells leaked to the low chamber in MPN patients bone marrow cells and HEL cells treated with 5 nmol/L of ruxolitinib group were significantly lower than that without ruxolitinib treatment after 24 h [ (154.7±27.5) vs (320.3±67.3) , t=13.47, P<0.05; (70.7±10.5) vs (135.3±16.7) , t=13.89, P<0.05]. The mRNA and protein expression levels of JAK2, MMP-2 and MMP-9 decreased with the increased concentration of ruxolitinib. Conclusion: Ruxolitinib inhibits MPN cell migration and expression of MMP-2 and MMP-9 via JAK2 signal pathway.

Asunto(s)

Proliferación Celular , Trastornos Mieloproliferativos , Western Blotting , Médula Ósea , Movimiento Celular , Supervivencia Celular , Sinergismo Farmacológico , Humanos , Janus Quinasa 2 , Leucemia Eritroblástica Aguda , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Mutación , Nitrilos , Fosforilación , Pirazoles , Pirimidinas , Transducción de Señal

RESUMEN

Brain cancer stem cells are a subset of tumor cells present in several types of brain tumor that evade treatment regimens and are responsible for tumor recurrence. Recent reports on several tumors have suggested that Hoechst 33342 dye exclusion is a powerful technique for isolating cancer stem cell-like side population (SP) cells. In the present study, we attempted to isolate the SP cells from medulloblastoma, a malignant brain tumor in children. The tumor samples obtained were subjected to fluorescence-activated cell sorting analysis for SP cell isolation. Further, the SP cells were characterized by a sphere-formation assay and analyzed for expression of stem cell genes by reverse transcription-polymerase chain reaction (RT-PCR). Using flow cytometry, we isolated 2.9% of cancer stem-like SP cells from malignant medulloblastoma, which was reduced to 0.4% in the presence of verapamil, an inhibitor of ABC transporter. These SP cells undergo rapid proliferation and have a high tendency to form tumor spheres when compared with non-SP cells. Further, RT-PCR analysis revealed that the isolated SP cells have increased expression of neural stem cell markers such as nestin, Notch1, and the ABC transporter protein ABCG2. Therefore, our findings suggest that SP cells of medulloblastoma share the characteristics of cancer stem cells. The increased expression of stem cell markers and ABCG2 protein may function collectively and be responsible for drug and apoptosis resistance, as well as tumor recurrence and invasion.

Asunto(s)

Meduloblastoma/patología , Células Madre Neoplásicas/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Proliferación Celular , Preescolar , Expresión Génica , Humanos , Meduloblastoma/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Células de Población Lateral/metabolismo , Esferoides Celulares/metabolismo

RESUMEN

Patients with Crohn's disease have an increased risk of developing intestinal tumours. However, the carcinogenic mechanisms remain poorly understood. To address this question, this report describes an unusual case of Crohn's disease complicated by synchronous small intestinal and colonic adenocarcinomas. Genetic events in both the tumours and their adjacent mucosae were evaluated and the tumorigenesis of these cancers is discussed.

Asunto(s)

Adenocarcinoma/patología , Neoplasias del Colon/patología , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/patología , Neoplasias del Íleon/patología , Neoplasias Primarias Múltiples/patología , Adenocarcinoma/genética , Neoplasias del Colon/genética , Enfermedad de Crohn/genética , Proteínas del Citoesqueleto/análisis , Femenino , Expresión Génica , Humanos , Neoplasias del Íleon/genética , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Repeticiones de Microsatélite , Persona de Mediana Edad , Neoplasias Primarias Múltiples/genética , Transactivadores/análisis , beta Catenina

RESUMEN

The aim of the present study was to characterize expression and mutation of p53 during the neoplastic progression from Barrett's esophagus to adenocarcinoma and to test the reliability of immunohistochemistry for p53 overexpression as an indicator of p53 mutation in this context. The association of both gene mutation and protein accumulation with clinicopathological findings and survival was also studied. A total of 77 samples from 30 esophagectomy specimens with Barrett's esophagus and adenocarcinoma of patients in longitudinal clinical follow-up were analyzed. Different lesions (intestinal metaplasia, dysplasia, and adenocarcinoma) as well as normal squamous-cell esophageal epithelia were sampled from formalin-fixed, paraffin-embedded tissues by microdissection. Mutations in p53 Exons 5 to 9 were detected by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCP) and confirmed by direct DNA sequencing. Nuclear accumulation of p53 protein was analyzed immunohistochemically from tissue sections adjacent to those used for microdissection. p53 gene mutations were found in 17 and p53 protein accumulation were found in 20 tumor samples. Of the 17 adenocarcinomas with a p53 mutation, 16 stained positive for p53 protein. p53 mutations were detected significantly more frequently in high-grade dysplastic than in low-grade dysplastic lesions (77% versus 29%, P < 0.01). In contrast, nuclear accumulation of p53 was detected in 85% of high-grade and 71% of low-grade dysplastic lesions. In eight cases with p53 mutation, the mutation identified in the tumors was also detected in premalignant lesions, mainly in high-grade dysplasia. In four cases of p53-mutated tumors, clones with different p53 mutations were detected in premalignant lesions. Neither p53 mutations nor p53 protein accumulations were found in metaplastic lesions. In summary, we found that p53 mutations occurred mainly during the transition from low-grade to high-grade dysplasia in the neoplastic progression of Barrett's esophagus but not in the nondysplastic Barrett's mucosa. Mutational analysis of p53 by PCR-SSCP and p53 accumulation by immunohistochemistry were mostly concordant in adenocarcinoma and high-grade dysplastic lesions but frequently discordant in low-grade dysplastic lesions. No correlation between p53 gene mutation or p53 accumulation and clinicopathological findings was observed in this study.

Asunto(s)

Adenocarcinoma/genética , Esófago de Barrett/genética , Neoplasias Esofágicas/genética , Genes p53 , Mutación , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/etiología , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Anciano , Anciano de 80 o más Años , Esófago de Barrett/complicaciones , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Progresión de la Enfermedad , Neoplasias Esofágicas/etiología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Proteína p53 Supresora de Tumor/biosíntesis

RESUMEN

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

Asunto(s)

Metilación de ADN , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas/genética , ADN/genética , ADN/metabolismo , Disección , Femenino , Humanos , Neoplasias/genética , Neoplasias/patología , Adhesión en Parafina , Placenta/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Sulfitos , Fijación del Tejido , Células Tumorales Cultivadas

RESUMEN

Our aim was to characterize expression and mutation of beta-catenin in the progression of Barrett esophagus to adenocarcinoma. Immunohistochemical analysis of beta-catenin was performed on paraffin-embedded tissue from 30 cases with adenocarcinomas and premalignant lesions. To determine whether there is a correlation between beta-catenin nuclear accumulation and exon 3 mutation of this gene, mutational analysis by polymerase chain reaction-single-strand conformation polymorphism was performed on DNA extracted from the same 30 adenocarcinomas. As a result, the prevalence of reduced expression of beta-catenin on the membrane, with or without nuclear staining, increased significantly from low-grade (LG) to high-grade (HG) dysplasia. Focal nuclear staining for beta-catenin was present in 19 cases of adenocarcinoma, and nuclear staining was associated significantly with progression from metaplasia to LG dysplasia. In addition, in glands with clear histologic transition from metaplasia to LG dysplasia, nuclear accumulation of beta-catenin was found only in the LG dysplastic areas. No mutation in exon 3 of the beta-catenin gene was detected in adenocarcinomas. These results demonstrate that disturbance of the APC/beta-catenin pathway, as indicated by nuclear accumulation of beta-catenin, is a common and early event during neoplastic progression in Barrett esophagus.

Asunto(s)

Adenocarcinoma/metabolismo , Esófago de Barrett/metabolismo , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Neoplasias Esofágicas/metabolismo , Lesiones Precancerosas/metabolismo , Transactivadores , Adenocarcinoma/genética , Adenocarcinoma/patología , Esófago de Barrett/patología , Núcleo Celular/patología , Proteínas del Citoesqueleto/genética , Cartilla de ADN/química , ADN de Neoplasias/análisis , Progresión de la Enfermedad , Epitelio/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Esófago/citología , Esófago/metabolismo , Humanos , Técnicas para Inmunoenzimas , Metaplasia/metabolismo , Metaplasia/patología , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , beta Catenina

Asunto(s)

ADN de Neoplasias/análisis , ADN de Neoplasias/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Archivos , Carcinoma/genética , Carcinoma/patología , Cromosomas Humanos Par 18 , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , Humanos , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Adhesión en Parafina , Bancos de Tejidos