RESUMEN
Objective: To determine whether clinically significant prostate cancer (PCa) and prostatitis in the peripheral zone can be distinguished using prostate imaging reporting and data system version 1 (PI-RADS V1) and version 2(PI-RADS V2). Methods: Between September 2010 and August 2016, mpMRI data of 77 patients with PCa and 29 prostatitis obtained at 3.0 T were collected in Zhangjiagang Hospital Affiliated to Soochow University. Every lesion was scored according to PI-RADS (V1 and V2), as well as a sum score and a PI-RADS V2 score. The non-parametric Kruskal-Wallis test was used to assess differences between PCa and prostatitis regarding PS3, PS4 and PI-RADS V2 score. The diagnostic performance of PI-RADS V1 and V2 for detection of prostatitis in peripheral zone was compared by analyzing ROC curve. Results: The PI-RADS V1 score for PS3, PS4 and the PI-RADS V2-score were all significantly higher for PCa (PS3:12.1±2.1; PS4:16.2±2.9; V2:4.6±0.8) than for prostatitis (PS3:8.0±0.7; PS4:10.6±1.0; V2:3.0±0.5) (all P<0.01). Of these parameters, PS4 achieved the highest predictive value for the presence of prostatitis with an AUC of 0.937, sensitivity and specificity were 87.0%, 97.0% with a threshold of 12.5. Conclusion: Prostatitis can be differentiated from clinically significant PCa in peripheral zone on mpMRI using PI-RADS system, PS4 achieved better results compared to PS3 and V2.
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Neoplasias de la Próstata , Prostatitis , Sistemas de Datos , Humanos , Imagen por Resonancia Magnética , Masculino , Estudios RetrospectivosRESUMEN
Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Bioconversion of l-proline using recombinant strain with proline-4-hydroxylase (P4H) is a preferred biocatalytic process in the economical production of trans-Hyp. In this study, a recombinant E. coli overexpressing hydroxylase (P4H), γ-glutamyl kinase and glutamate-semialdehyde dehydrogenase (ProBA) genes were constructed by knocking out the key genes in the metabolism. These key genes contained putA encoding proline dehydrogenase (PutA) in the l-proline metabolism and other catalytic enzyme genes, sucAB encoding α-ketoglutarate dehydrogenase (SucAB), aceAK encoding isocitratelyase (AceA) and isocitrate dehydrogenase kinase/phosphatase (AceK) in the TCA cycle. This recombinant strain coupled the synthetic pathway of trans-Hyp with TCA cycle of the host strain. It inhibited the consumption of l-proline completely and promoted the accumulation of 2-oxoglutarate (2-OG) as a co-substrate, which realized the highest conversion of glucose to trans-Hyp. A fed-batch strategy was designed, capable of producing 31·0 g l-1 trans-Hyp from glucose. It provided a theoretical basis for commercial conversion of glucose to trans-Hyp. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans-Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans-Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short-cut and efficient trans-Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans-Hyp.
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Escherichia coli , Glucosa/metabolismo , Hidroxiprolina/biosíntesis , Ingeniería Metabólica/métodos , Prolina/metabolismo , Biocatálisis , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato-5-Semialdehído Deshidrogenasa/genética , Glutamato-5-Semialdehído Deshidrogenasa/metabolismo , Hidroxiprolina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Prolil Hidroxilasas/genética , Prolil Hidroxilasas/metabolismoRESUMEN
We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrP(c)). Blocking P-gp activity or depletion of PrP(c) inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrP(c) clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrP(c) complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrP(c) interaction in this process.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Resistencia a Antineoplásicos , Paclitaxel/farmacología , Proteínas PrPC/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Clorometilcetonas de Aminoácidos/metabolismo , Apoptosis/fisiología , Neoplasias de la Mama/patología , Caveolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Proteínas PrPC/genéticaRESUMEN
Objective: To explore the effects of hypoxia inducible factor-1α (HIF-1α) on P311 and its influence on the migration of murine epidermal stem cells (ESCs) under hypoxia in vitro. Methods: Two kinds of murine ESCs were isolated and obtained from 15 neonatal wild-type C57BL/6J mice and 5 congeneric source P311 gene knock-out mice, respectively. The first passage of cells were used in the following experiments after morphologic observation and detection of expression of cell surface markers CD71 and CD49f with flow cytometer. (1) After cell scratch assay, according to the random number table (the same dividing method below), ESCs of P311 gene knock-out mice were divided into normoxia group (cells were cultured with complete medium in normoxic carbon dioxide incubator, and the subsequent normoxic treatments were the same) and hypoxia group (cells were cultured in hypoxic carbon dioxide incubator containing 1% oxygen, and the subsequent hypoxic treatments were the same), with 12 inserts in each group. ESCs of wild-type mice were divided into normoxia group, pure hypoxia group, dimethyl sulfoxide (DMSO) control group (2 µL DMSO solvent was added for 1 h of normoxia treatment before hypoxia treatment), HIF-1α inhibitor group (cells were treated with 11 µmol/L HIF-1 inhibitor of 2 µL under normoxia condition for 1 h before hypoxia treatment), HIF-1α stabilizer group (the cells were treated with 2 µmol/L FG-4592 of 2 µL under normoxia condition for 1 h before hypoxia treatment), with 12 inserts in each group. Three inserts of each time point in each group were adopted respectively to measure the residual width of scratch under inverted phase contrast microscope at post scratch hour (PSH) 0 (immediately), 12, 24, and 48. (2) After hypoxia treatment, the protein level of HIF-1α in ESCs of wild-type mice was detected by Western blotting at post hypoxia hour (PHH) 0, 12, 24, and 48. (3) ESCs of wild-type mice were divided into pure hypoxia group, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group as that of experiment (1) with the same treatment. The mRNA expression of P311 and expression of P311 in ESCs were determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunocytochemical staining, respectively, at PHH 0 (immediately), 12, 24, and 48 (with sample numbers of 12). (4) The second passage of human embryonic kidney 293 (HEK-293) cells were divided into empty vector hypoxia group (cells were cultured under hypoxia condition after being transfected with empty vector plasmid), P311 normoxia group (cells were cultured under normoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia group (cells were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia+ HIF-1α inhibitor group (cells which were incubated with HIF-1α inhibitor were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid). The luciferase activity was detected at post culture hour (PCH) 0 and 12, respectively, and then the P311 transcriptional regulatory binding site of HIF-1α and the promoter sequence of P311 were predicted and searched by bioinformatics methods. Data were processed with factorial design variance analysis, one-way analysis of variance, LSD test and Bonferroni correction. Results: (1) The results of ESCs. The cells showed cobblestone-like pattern and different clonal morphology due to the different cell proliferation potential. The proportion of CD71(-)CD49f(+) cells accounted for about 85%. The identification results indicated that the cells showed strong stem cell properties and high purity. Compared with those in cells of normoxia group of P311 gene knock-out mice, the residual widths of scratch of cells in pure hypoxia group were smaller at PSH 12 and 24 (with P values below 0.05), and those in hypoxia group, normoxia group of wild-type mice, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group were smaller at PSH 12 (with P values below 0.05). Compared with those in cells of normoxia group of wild-type mice, the residual widths of scratch of cells in hypoxia group, pure hypoxia group, and DMSO control group were smaller at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α stabilizer group was smaller at PSH 12 (P<0.05). Compared with those of cells in pure hypoxia group, the residual widths of scratch of cells in hypoxia group were wider at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α inhibitor group was wider at PSH 12 (P<0.05), and those of cells in HIF-1α stabilizer group were smaller at PSH 12 and 24 (with P values below 0.05). There was no obvious difference in the width of scratch in cells among the 7 groups (F=19.02, P>0.05). The protein levels of HIF-1α in ESCs of wild-type mice at PHH 0, 12, 24, and 48 were respectively 1.02±0.05, 2.56±0.09, 1.60±0.17, and 1.17±0.03. Compared with that at PHH 0, the protein level of HIF-1α at PHH 12 was significantly enhanced (P<0.01). It began to decline at PHH 24 but was still higher than that at PHH 0 (P<0.05), and the protein level of HIF-1α at PHH 48 was close to the normoxia level (P>0.05). Compared with those of cells in pure hypoxia group, the mRNA expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), and those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the mRNA expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 0, 12, and 24 (with P values below 0.05). Compared with those of cells in pure hypoxia group, the expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), while those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). (2) The results of HEK-293 cells. At PCH 0, there was no significant difference in the luciferase activity among cells of empty vector hypoxia group, P311 normoxia group, P311 hypoxia group, and P311 hypoxia+ HIF-1α inhibitor group (F=13.33, P>0.05). At PCH 12, the luciferase activity of cells in P311 hypoxia group was higher than that in empty vector hypoxia group (P<0.01). The luciferase activity of cells in hypoxia group was higher than that in P311 normoxia group (P<0.05), while that of cells in P311 hypoxia+ HIF-1α inhibitor group was lower than that in P311 hypoxia group (P<0.01). Conclusions: HIF-1α may increase the migration of murine ESCs through inducing the expression of P311 at the early stage of hypoxia.
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Quemaduras/metabolismo , Células Epiteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia , Animales , Western Blotting , Hipoxia de la Célula , Movimiento Celular , Proliferación Celular , Células Epiteliales/citología , Vectores Genéticos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso , Plásmidos , Células Madre/citología , TransfecciónRESUMEN
OBJECTIVE: To evaluate the feasibility of high-pitch spiral CT in imaging vascular anomalies (VAs) with affluent blood flow in the head and neck of infants and children. METHODS: For patients with suspected VAs and affluent blood flow pre-detected by ultrasound, CT was performed with high-pitch mode, individualized low-dose scan protocol and three-dimensional (3D) reformation. A five-point scale was used for image quality evaluation. Diagnostic accuracy was calculated with clinical diagnosis with/without pathological results as the reference standard. Radiation exposure and single-phase scan time were recorded. Treatment strategies were formulated based on CT images and results and were monitored through follow-up results. RESULTS: 20 lesions were identified in 15 patients (median age of 11 months). The mean score of image quality was 4.13 ± 0.74. 7 patients (7/15, 46.67%) were diagnosed with haemangiomas, 6 patients (6/15, 40%) were diagnosed with venous malformations and 2 patients (2/15, 13.33%) were diagnosed with arteriovenous malformations. The average effective radiation doses of a single phase and of the total procedure were 0.27 ± 0.08 and 0.86 ± 0.21 mSv. The average scanning time of a single phase was 0.46 ± 0.09 s. After treatment, 13 patients (13/15, 86.67%) achieved excellent results, and 2 patients (2/15, 13.33%) showed good results in follow-up visits. CONCLUSION: High-pitch spiral CT with an individualized low-dose scan protocol and 3D reformation is an effective modality for imaging VAs with affluent blood flow in the head and neck of infants and children when vascular details are needed and ultrasound and MRI could not provide the complete information. ADVANCES IN KNOWLEDGE: This study proposes an alternative modality for imaging VAs with affluent blood flow.
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Cabeza/irrigación sanguínea , Cuello/irrigación sanguínea , Enfermedades Vasculares/diagnóstico por imagen , Malformaciones Vasculares/diagnóstico por imagen , Niño , Preescolar , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/terapia , Hemangioma/diagnóstico por imagen , Hemangioma/terapia , Humanos , Lactante , Masculino , Variaciones Dependientes del Observador , Dosis de Radiación , Tomografía Computarizada Espiral , Enfermedades Vasculares/terapia , Malformaciones Vasculares/terapiaRESUMEN
Insulin-like growth factor (IGF)-II plays an important role in fetal growth and development. IGFs are potent mitogens for a variety of cancer cells. A paracrine/autocrine role of IGF-II in the growth of breast and prostate cancer cells has been suggested. To test the role of IGF-II in cancer cell growth, hammerhead ribozymes targeted to human IGF-II RNA were constructed. Single (R)- and double (RR)-ribozymes were catalytically active in vitro whereas mutant ribozymes (M or MM) did not cleave IGF-II RNA. RR was more active than R. In human prostate cancer PC-3 cells, both R and RR similarly suppressed IGF-II messenger RNA (mRNA) levels (approximately 40%) compared with the level in parental or M-expressing PC-3 cells. Polymerase II and III promoter-driven R similarly suppressed IGF-II mRNA levels. Suppression of IGF-II mRNA levels by R was associated with suppression of IGF-II protein levels. R- (or RR-) expressing PC-3 cells did not grow under serum-starved conditions and showed prolonged doubling times in the presence of 10% FCS compared with those of parental or M-expressing cells. These results substantiated that IGF-II plays a critical role in prostate cancer cell growth.
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Factor II del Crecimiento Similar a la Insulina/genética , Neoplasias de la Próstata/genética , ARN Catalítico/química , ARN Catalítico/metabolismo , ARN/metabolismo , Secuencia de Bases , Catálisis , División Celular , Expresión Génica , Humanos , Cinética , Masculino , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Polimerasa III/genética , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Subpopulations of infiltrating mononuclear cells in 51 resected pulmonary carcinoma specimens were studied with immunohistochemical technique (ABC and PAP methods). The major constituent of the mononuclears was T-cells, especially the OKT8+ cells (T suppressor/cytotoxic). The local infiltration of the mononuclears in pulmonary carcinoma was related to the prognosis of the case. The more the OKT8+ and B1+ lymphocytes infiltrated, the longer the survival (rate obtained) whereas, the infiltration of some kinds of plasma cells tended to have a negative correlation with the prognosis of the case. The significance of the mononuclears in pulmonary carcinoma is discussed.
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Leucocitos Mononucleares/patología , Neoplasias Pulmonares/patología , Adenocarcinoma/patología , Adulto , Anciano , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Linfocitos T/patologíaRESUMEN
The dynamic changes of extracellular matrix (ECM) and effected interstitial cells in experimental sclerosis (fibrosis) of the liver, lung and kidney glomeruli of rats were studied by immunohistochemical methods with monospecific antibodies against fibronectin, laminin, collagen type I, III, IV, V, desmin, vimentin and alpha-smooth muscle actin. The results suggest that there exists an organ (tissue) sclerosis effective cell system involved in ECM synthesis. The activation usually initiates from the Ito cell (liver), primitive mesenchymal call (lung), and mesangial cell (glomeruli), sometimes followed by fibroblast and myofibroblast differentiation with the transformation of cytoskeleton expression. Desmin is the most sensitive marker to reflect the functional state of the organ sclerosis effective cell system from the resting to the activating state.
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Matriz Extracelular/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Cirrosis Hepática Experimental/metabolismo , Fibrosis Pulmonar/metabolismo , Actinas/metabolismo , Animales , Antibióticos Antineoplásicos , Bleomicina , Intoxicación por Tetracloruro de Carbono , Desmina/metabolismo , Glomeruloesclerosis Focal y Segmentaria/etiología , Cirrosis Hepática Experimental/inducido químicamente , Fibrosis Pulmonar/inducido químicamente , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Vimentina/metabolismoRESUMEN
The diagnosis of leaks in pneumothorax was studied with radionuclide inhalation lung scan in animals. The animal pneumothorax models were made either by puncture or by operative method on nine dogs. The pneumothorax was proved by chest X-ray. The dogs were intubated and ventilated. Aerosol of 99mTc was delivered into the lung through tracheal cannula. 5-10 mGi: of 99mTc-phytafe with 150 ml of normal saline was used ultrasonics. The droplet size was estimated at 0.5-10 microns in diameter. The lung scan were obtained with GCA90B-gamma camera in dynamic or static way for about 0.5 hr. The air leaks (total 13) in tension pneumothorax were demonstrated by lung scan and were in accord with the fissures in the lung samples.
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Neumotórax/diagnóstico por imagen , Administración por Inhalación , Aerosoles , Animales , Perros , Femenino , Pulmón/diagnóstico por imagen , Masculino , Compuestos de Organotecnecio/administración & dosificación , Ácido Fítico/administración & dosificación , CintigrafíaRESUMEN
BACKGROUND: The balance between immune surveillance and immune escape determines the outcome of patients with primary mycosis fungoides (MF). FOXP3+ regulatory T cells (Tregs) and DC-SIGN+ immature dendritic cells (imDCs) play a central role in regulating the immune state in the progression of MF. However, whether the mechanisms used by these factors depend on MF stage is still underdetermined and even controversial. PATIENTS AND METHODS: FOXP3+ Tregs and DC-SIGN+ imDCs were detected by immunohistochemical staining of formalin-fixed, paraffin-embedded specimens obtained from the lesion biopsies of 89 patients with MF, comprising 69 patients at the patch stage, 12 at the plaque stage, and 8 at the tumor stage. The number of FOXP3+ Tregs and DC-SIGN+ imDCs in each stage was counted and compared. RESULTS: The expression of FOXP3 and DC-SIGN varied with the MF stage. The number of cells expressing FOXP3 was higher at the patch and plaque stages than at the tumor stage (p < 0.05), but no significant difference was noted between the patch and plaque stages (p = 0.715). DC-SIGN expression increased continuously, concomitant with tumor progression, through the three stages (p < 0.05). CONCLUSIONS: The predominant factor influencing the immune state is different for each MF stage. Therefore, therapeutic strategies that modulate the antitumor immune responses should be developed depending on MF progression.
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Células Dendríticas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Micosis Fungoide/inmunología , Neoplasias Cutáneas/inmunología , Linfocitos T Reguladores/inmunología , Biomarcadores de Tumor/análisis , Biopsia , Progresión de la Enfermedad , Factores de Transcripción Forkhead/análisis , Humanos , Inmunohistoquímica , Recuento de Linfocitos , Micosis Fungoide/patología , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Neoplasias Cutáneas/patología , Escape del TumorRESUMEN
The TET (ten-eleven translocation) family of α-ketoglutarate (α-KG)-dependent dioxygenases catalyzes the sequential oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine, leading to eventual DNA demethylation. The TET2 gene is a bona fide tumor suppressor frequently mutated in leukemia, and TET enzyme activity is inhibited in IDH1/2-mutated tumors by the oncometabolite 2-hydroxyglutarate, an antagonist of α-KG, linking 5mC oxidation to cancer development. We report here that the levels of 5hmC are dramatically reduced in human breast, liver, lung, pancreatic and prostate cancers when compared with the matched surrounding normal tissues. Associated with the 5hmC decrease is the substantial reduction of the expression of all three TET genes, revealing a possible mechanism for the reduced 5hmC in cancer cells. The decrease of 5hmC was also observed during tumor development in different genetically engineered mouse models. Together, our results identify 5hmC as a biomarker whose decrease is broadly and tightly associated with tumor development.
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Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Citosina/análogos & derivados , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , 5-Metilcitosina/metabolismo , Animales , Citosina/metabolismo , Regulación hacia Abajo , Humanos , Hidroxilación , Ratones , Oxigenasas de Función MixtaRESUMEN
The epithelial-mesenchymal transition (EMT) induced by chemotherapeutic agents promotes malignant tumor progression; however, the mechanism underlying the drug-induced EMT remains unclear. In this study, we reported that miR-448 is the most downregulated microRNA following chemotherapy. Suppression of miR-448 correlated with EMT induction in breast cancer in vitro and in vivo. With the use of chromatin immunoprecipitation-seq analysis, we demonstrated that miR-448 suppression induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, leading to elevated levels of amphiregulin and thereby, increasing epidermal growth factor receptor (EGFR)-mediated Twist1 expression, as well as nuclear factor κB (NF-κB) activation. On the other hand, we also found that the adriamycin-activated NF-κB directly binds the promoter of miR-448 suppressing its transcription, suggesting a positive feedback loop between NF-κB and miR-448. Furthermore, all patients who received cyclophosphamide (CP), epirubicin plus taxotere/CP, epirubicin plus 5-fluorouracil chemotherapy showed miR-448 suppression, an increased SATB1, Twist1 expression and acquisition of mesenchymal phenotypes. These findings reveal an underlying regulatory pathway, in which the autoregulation between NF-κB and miR-448 is important for restrain miR-448 suppression upon chemotherapy and may have a role in the regulation of chemotherapy-induced EMT. Disruption of the NF-κB-miR-448 feedback loop during clinical treatment may improve the chemotherapy response of human breast cancers in which EMT is a critical component.
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Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Anfirregulina , Antibióticos Antineoplásicos/uso terapéutico , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo , Doxorrubicina/uso terapéutico , Familia de Proteínas EGF , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Femenino , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
We have studied the mutagenicity and toxicity of physical and chemical agents in the Chinese hamster ovary (CHO) cell line K1-BH4 and its transformant, AS52. The AS52 cells lack the normal X-linked mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine-guanine phosphoribosyltransferase (gpt) gene. We found that X-rays and neutrons appear to be equitoxic to both cell types; however, these physical agents are approximately 10 times more mutagenic to the gpt gene of AS52 cells than to the hprt gene of K1-BH4 cells. We reasoned that if reactive oxygens were to mediate the mutagenic effects of both radiomimetic chemicals and radiation, then reactive oxygen-producing chemicals, such as streptonigrin and bleomycin, and oxidizing agents such as potassium superoxide and hydrogen peroxide, would exhibit similar levels of toxicity but different frequencies of mutants when assayed with the two cell lines. Our experiments fulfill such predictions. We postulate that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi-locus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. Preliminary studies, using Southern blot and the polymerase chain reaction to analyze the mutational spectrum of the mutants, support our hypothesis that reactive oxygens induce deletion mutations in mammalian cells.
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Hipoxantina Fosforribosiltransferasa/genética , Mutación , Oxígeno/metabolismo , Animales , Línea Celular , Deleción Cromosómica , Cricetinae , Cricetulus , Mutágenos , Reacción en Cadena de la PolimerasaRESUMEN
Tissue fibrosis is frequently associated with an increase of mast cells, and mast cells are regarded as playing a role in the induction of tissue fibrosis. We attempted to examine whether mast cells influenced the induction of fibrosis using Ws/Ws mast cell-deficient rats. The mast cell deficiency of Ws/Ws rats is due to a 12-base pair deletion of the c-kit gene. The activity of c-kit receptor tyrosine kinase is remarkably reduced in Ws/Ws rats. Liver fibrosis was induced by the repeated injections of pig serum, and lung fibrosis was induced by the instillation of bleomycin. Marked fibrosis in the liver and lung did occur in the Ws/Ws rats, and the magnitude of fibrosis was more severe in Ws/Ws rats than in control normal (+/+) rats. The mast cell increase was observed in the liver of +/+ and Ws/Ws rats and in the lung of +/+ rats. However, the number of mast cells in the liver of treated Ws/Ws rats with marked fibrosis was comparable to that observed in the liver of nontreated +/+ rats without fibrosis. Histamine content increased in the liver and lung of +/+ rats after the treatment, but it remained in low levels even after the treatment in Ws/Ws rats. Mast cells and histamine did not appear to play important roles in the induction of fibrosis. Thus, an increase in mast cell number and histamine content may be associated with but not a cause of fibrosis.
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Cirrosis Hepática Experimental/patología , Hígado/patología , Pulmón/patología , Mastocitos/patología , Fibrosis Pulmonar/patología , Animales , Recuento de Células , Hidroxiprolina/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Ratas , Ratas MutantesRESUMEN
We have developed a rapid screening method using the polymerase chain reaction (PCR) for detecting deletion mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. DNA was extracted from spontaneous and ultraviolet (UV) light- and X-ray-induced hprt-deficient mutants. Two primer sets were used to amplify 276 bp and 344 bp fragments containing the entire exon 3 and exon 9 coding sequence, respectively. The PCR was performed using Taq DNA polymerase for 40 cycles, and the PCR product was directly analyzed for the presence of the respective amplified DNA using electrophoresis on agarose gels stained with ethidium bromide. With this assay, we have analyzed 39 independently derived hprt-deficient mutants. Four of ten spontaneous mutants were found to have deletions in exon 9. UV light produced mutants with predominantly wild-type amplification patterns (10/14). X-ray induced mostly deletion patterns (11/15); six of these occurred only in exon 9, and five occurred in both exons 3 and 9. These observations are consistent with the classical notion that UV light induces predominantly missense mutations and X-ray produces a high proportion of deletion mutations. Deletion mutations occurred most frequently at the 3' end of the hprt gene, suggesting the possible existence of hot spots for deletions in this region. The PCR assay for deletion detection has the advantage that it can be completed in less than 4 hr without using radioisotopes. This assay should be useful for routine deletion screening.
Asunto(s)
Deleción Cromosómica , ADN Polimerasa Dirigida por ADN , Amplificación de Genes , Hipoxantina Fosforribosiltransferasa/genética , Animales , Células Cultivadas , ADN/genética , ADN/aislamiento & purificación , Electroforesis en Gel de Agar , Exones , Rayos Ultravioleta , Rayos XRESUMEN
Studies were done on the influence of radiotherapy or chemotherapy on peripheral lymphocyte SCE (sister chromatid exchange) frequency in patients with gynecologic cancer. The average value of spontaneous SCE frequency in 10 patients who had received radiotherapy was 6.94 +/- 2.57 (mean +/- S.D.), which was significantly lower than the control value of 8.59 +/- 2.99 ( p less than 0.001). The rate of decline of these frequency values was negatively correlated with the irradiation dosage. In contrast, the average spontaneous SCE frequency in the patients who had received combination chemotherapy was 12.48 +/- 4.02, which was significantly higher than the control value ( p less than 0.001). The average value of MMC-induced SCE frequency in patients under chemotherapy was much the same as the control value of 26.16 +/- 5.77. That is patients under radiotherapy was 23.88 +/- 5.65, which was slightly lower than the control value. There was, however, no significant difference in the MMC-induced SCE frequency value among these 3 groups.
Asunto(s)
Intercambio Genético/efectos de la radiación , Neoplasias de los Genitales Femeninos/radioterapia , Linfocitos/citología , Mitomicinas/efectos adversos , Intercambio de Cromátides Hermanas/efectos de la radiación , Coriocarcinoma/tratamiento farmacológico , Femenino , Neoplasias de los Genitales Femeninos/tratamiento farmacológico , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Mitomicina , Neoplasias Ováricas/tratamiento farmacológico , Embarazo , Intercambio de Cromátides Hermanas/efectos de los fármacos , Neoplasias del Cuello Uterino/radioterapia , Neoplasias Uterinas/tratamiento farmacológicoRESUMEN
Insulin-like growth factors (IGFs) are potent mitogens for a variety of cancer cells in vitro. A paracrine/autocrine role of IGF-II in the growth of breast and prostate cancer cells has been suggested. Information on cell-type-specific IGF-II expression in vivo in the breast and prostate is, however, limited. Thus, cell types expressing IGF-II mRNA and protein in tumors were identified by in situ hybridization and immunohistochemistry. Of 36 prostate, 17 breast, and 10 bladder cancers, and 9 paraganglioma tissues examined, IGF-II was expressed in more than 50% of prostate, breast, and bladder tumors, and in 100% of paraganglioma tumors. Expression levels of IGF-II were highest in the paraganglioma and bladder followed by prostate and breast tumors. In all the tumors expressing IGF-II, both mRNA and protein were localized to malignant cells, expression in the stroma being minimal. Since previous studies had indicated that an incompletely processed form of 15-kDa IGF-II exhibited higher mitogenic potency than the completely processed 7.5-kDa IGF-II form, the quantity and size of IGF-II proteins expressed in these tumors were analyzed by Western immunoblotting. Greater expression of 15-kDa IGF-II relative to the 7.5-kDa IGF-II form was clearly demonstrated in all six prostate cancers and in half of the two breast and four bladder cancers examined. The results are consistent with the hypothesis that the 15-kDa form of IGF-II expressed in cancerous cells contributes to autocrine cancer cell growth in vivo.
Asunto(s)
Neoplasias de la Mama/patología , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Paraganglioma/patología , Neoplasias de la Próstata/patología , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor II del Crecimiento Similar a la Insulina/análisis , Masculino , Paraganglioma/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Phosphodiesterase type 3 isoforms, PDE3A and 3B, are expressed primarily in cardiovascular and adipose tissues, respectively. We previously reported a shorter transcript of 4.4-kb PDE3A which is predominantly transcribed in human placenta, whereas a full-length 7. 6-kb transcript corresponding to the cardiac PDE3A cDNA has not been characterized. Due to unfortunate circumstances created by changes in PDE3 nomenclature, PDE3B gene structure previously reported used PDE3A in its title. Here, we describe PDE3A gene structure, which comprises 16 exons spanning over 130 kb on chromosome 12p12. Two PDE3 isoforms share similar gene organization, but localize to different chromosomes. The most distal transcription initiation site of the PDE3A gene is approximately 1071 bases upstream of the ATG site, suggesting that exon 1 consists of 1071 and 960 bp of untranslated and translated sequences, respectively. The proximal 5'-flanking region, which does not contain TATA-like sequences, exhibited weak but significant promoter activity. Results suggest potential involvement of distal promoter/enhancer and translational regulation for expression of the 7.6-kb transcript.