RESUMEN
Recently, possibilities of improving operation speed and force sensitivity in atomic-scale atomic force microscopy (AFM) in liquid using a small cantilever with an electron beam deposited (EBD) tip have been intensively explored. However, the structure and properties of an EBD tip suitable for such an application have not been well-understood and hence its fabrication process has not been established. In this study, we perform atomic-scale AFM measurements with a small cantilever and clarify two major problems: contaminations from a cantilever and tip surface, and insufficient mechanical strength of an EBD tip having a high aspect ratio. To solve these problems, here we propose a fabrication process of an EBD tip, where we attach a 2 µm silica bead at the cantilever end and fabricate a 500-700 nm EBD tip on the bead. The bead height ensures sufficient cantilever-sample distance and enables to suppress long-range interaction between them even with a short EBD tip having high mechanical strength. After the tip fabrication, we coat the whole cantilever and tip surface with Si (30 nm) to prevent the generation of contamination. We perform atomic-scale AFM imaging and hydration force measurements at a mica-water interface using the fabricated tip and demonstrate its applicability to such an atomic-scale application. With a repeated use of the proposed process, we can reuse a small cantilever for atomic-scale measurements for several times. Therefore, the proposed method solves the two major problems and enables the practical use of a small cantilever in atomic-scale studies on various solid-liquid interfacial phenomena.
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BACKGROUND: The transcription factor, zinc finger protein 143 (ZNF143), positively regulates many cell-cycle-related genes. The ZNF143 would show high expression of multiple solid tumours related closely to cancer cell growth, similar to the widely accepted Ki67 (MIB-1) protein, but the underlying mechanisms for ZNF143 remain unclear. We investigated the association of ZNF143 expression with clinicopathological features and prognoses of patients with lung adenocarcinoma. METHODS: Expressions of ZNF143 and MIB-1 were immunohistochemically analysed in 183 paraffin-embedded tumour samples of patients with lung adenocarcinoma. The ZNF143 expression was considered to be strong when >30% of the cancer cells demonstrated positive staining. RESULTS: Strong ZNF143+ expression showed a significantly close relationship to pathologically moderate to poor differentiation and highly invasive characteristics. The ZNF143 positivity potentially induced cell growth of lung adenocarcinoma, correlated significantly with high MIB-1 labelling index (⩾10%). Univariate and multivariate analyses demonstrated that both strong ZNF143+ and the high MIB-1 index group have only and significantly worse survival rates. CONCLUSIONS: The combination of strong ZNF143 expression and high MIB-1 index potentially predicts high proliferating activity and poor prognosis in patients with lung adenocarcinoma, and may offer a therapeutic target against ZNF143.
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Adenocarcinoma/química , Antígeno Ki-67/análisis , Neoplasias Pulmonares/química , Proteínas de Neoplasias/análisis , Transactivadores/análisis , Adenocarcinoma/mortalidad , Adenocarcinoma/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Diferenciación Celular , División Celular , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Índice Mitótico , Datos de Secuencia Molecular , Clasificación del Tumor , Invasividad Neoplásica , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Transactivadores/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Brigatinib is a next-generation tyrosine kinase inhibitor (TKI) targeting ALK and ROS1. The Barossa study is a multicenter, phase II basket study of brigatinib in patients with ROS1-rearranged solid tumors. ROS1 TKI-naive patients with ROS1-rearranged non-small-cell lung cancer (NSCLC) were enrolled in cohort 1, and ROS1-rearranged NSCLC patients treated previously with crizotinib were enrolled in cohort 2. Patients with ROS1-rearranged solid tumors other than NSCLC were enrolled in cohort 3. PATIENTS AND METHODS: Eligible patients received brigatinib at the dose of 180 mg once daily with a 7-day lead-in period at 90 mg. The primary endpoint was the objective response rate (RECIST 1.1) assessed by independent central review in cohorts 1 and 2. RESULTS: Between July 2019 and June 2021, 51 patients were enrolled into the study. Of the 51, 47 patients had ROS1-rearranged NSCLC; 28 and 19 of these patients were enrolled in cohort 1 and cohort 2, respectively. The remaining four patients had other ROS1-rearranged solid tumors, including rectal, brain, and pancreas tumor in one patient each, and primary unknown tumor in one patient. The confirmed objective response rate was 71.4% [95% confidence interval (CI) 51.3% to 86.8%] in cohort 1 (TKI-naive NSCLC patients) and 31.6% (95% CI 12.6% to 56.6%) in cohort 2 (NSCLC patients treated previously with crizotinib). The median progression-free survival was 12.0 months (95% CI 5.5-22.9 months) in cohort 1 and 7.3 months (95% CI 1.3-17.5 months) in cohort 2. None of the patients in cohort 3 showed any treatment response. Pneumonitis was observed in 9.8% of all the patients. CONCLUSIONS: Brigatinib was effective in TKI-naive patients with ROS1-rearranged NSCLC. The safety profile of brigatinib was consistent with that reported from previous studies.
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BACKGROUND: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). METHODS: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. RESULTS: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low ß-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. CONCLUSION: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , N-Acetilgalactosaminiltransferasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/enzimología , Línea Celular Tumoral , Estudios de Cohortes , Femenino , Humanos , Neoplasias Renales/enzimología , Masculino , Persona de Mediana Edad , N-Acetilgalactosaminiltransferasas/genética , Clasificación del Tumor , Pronóstico , Estudios Retrospectivos , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked in the raw milk whey were degraded, and naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. The RNA molecules in milk were stable. We also detected miRNA and mRNA in infant formulas purchased from Japanese markets. It is still unknown whether milk-derived RNA molecules play biological roles in infants; however, if milk-derived RNA do show functions in infants, our data will help guide future studies.
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MicroARNs/análisis , Leche/química , ARN Mensajero/análisis , Animales , Bovinos , Calostro/química , Femenino , Humanos , Lactante , Fórmulas Infantiles/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) is responsible for the altered glycosylation in cancer. The purpose of our study was to investigate the clinical significance of two isoforms, GalNAc-T6 and -T3, and their correlation with the prognosis of pancreatic cancer. METHODS: Immunohistochemistry was used to analyse GalNAc-T6 and -T3 expressions in 70 clinicopathologically characterised pancreatic cancer cases. RESULTS: Positive expressions of GalNAc-T6 and -T3 were immunohistochemically identified in 51% (36 of 70) and in 77% (54 of 70) of patients, respectively. A close relationship was noted between GalNAc-T6 positive expression and pathological well/moderate differentiated type (P=0.001), small tumour size (P=0.044), absence of vascular invasion (P=0.009), and low stage of the American Joint Committee on Cancer systems (P=0.043). The expression of GalNAc-T3 significantly correlated with good differentiation (P=0.001), but not with other clinicopathologic features. Furthermore, univariate and multivariate analyses revealed that GalNAc-T6 expression was an independent prognosis indicator for the disease, whereas GalNAc-T3 expression had no impact on clinical outcome, even though 33 of 36 GalNAc-T6-positive cases also had a positive expression of GalNAc-T3 (P=0.001, r=0.356). CONCLUSION: Both GalNAc-T6 and -T3 expressions correlated significantly with tumour differentiation, whereas only GalNAc-T6 expression predicted prognosis in pancreatic cancer.
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N-Acetilgalactosaminiltransferasas/análisis , Neoplasias Pancreáticas/mortalidad , Adulto , Anciano , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Pronóstico , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
From May 2005 to October 2010. 9 patients with severe emphysematous bullae suffered from uncontrolled pneumothorax had been successfully treated by a new surgical method in our hospital. By using direct instillation of fibrin glue into the ruptured bulla following ligation of the ruptured bulla hole, 8 of 9 patients revealed no recurrence of new rupture and pneumothorax. Although the ligation of ruptured bulla hole tended to increase tension of surface of the bulla around the ligation and caused new rupture of the bulla, the fully instilled glue reduced intra air pressure of the ligated bulla and prevented new rupture. Additionally, the direct instillation of the glue immediately stopped the air leakage by itself. This direct instillation method of the glue encouraged us to challenge the surgery for the patients suffered from uncontrolled pneumothorax with severe emphysematous bullae.
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Vesícula/cirugía , Adhesivo de Tejido de Fibrina/administración & dosificación , Neumotórax/cirugía , Enfisema Pulmonar/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Ligadura , Masculino , Persona de Mediana Edad , Neumotórax/etiologíaRESUMEN
Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Transcripción Genética/fisiología , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Unión Proteica , Proteína Tumoral p73RESUMEN
The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.
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Factor de Transcripción Activador 4/genética , Resistencia a Antineoplásicos/genética , Transactivadores/genética , Transcripción Genética , Factor de Transcripción Activador 4/metabolismo , Antineoplásicos/farmacología , Northern Blotting , Western Blotting , Proteínas CLOCK , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Cisplatino/farmacología , Etopósido/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glutatión/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Oxidación-Reducción , Interferencia de ARN , Transactivadores/metabolismoRESUMEN
Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.
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Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/farmacología , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transcripción Genética , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja YRESUMEN
Canine hepatocellular carcinoma (HCC) is the most common primary hepatic tumour in dogs. MicroRNA (miRNA) dysregulation has been reported in human HCC and shown to have diagnostic and prognostic value; however, there are no data on miRNA expression in canine HCC. The aim of the present study was to investigate differentially expressed miRNAs in canine HCC. Analysis of miRNA expression in canine HCC tissues and cell lines by quantitative reverse transcription PCR showed that miR-1, miR-122, let-7a, and let-7g were downregulated, whereas miR-10b and miR-21 were upregulated in canine HCC. MET is one of the target genes of miR-1. MET was upregulated in canine HCC at the gene and protein levels, and a significant correlation between the concomitant downregulation of miR-1 and upregulation of MET was observed. Fast/intermediate-proliferating canine HCC cell lines had higher MET gene and protein expression levels than the slow-proliferating cell line. These findings suggest that miRNAs are differentially expressed in canine HCC, and that the miR-1/MET pathway may be associated with canine HCC cell proliferation.
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Carcinoma Hepatocelular/veterinaria , Enfermedades de los Perros/genética , Neoplasias Hepáticas/veterinaria , MicroARNs/genética , Análisis de Varianza , Animales , Western Blotting/veterinaria , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Perros , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Abnormal amplification of centrosomes, commonly found in human cancer, is the major cause of mitotic defects and chromosome instability in cancer cells. Like DNA, centrosomes duplicate once in each cell cycle, hence the defect in the mechanism that ensures centrosome duplication to occur once and only once in each cell cycle results in abnormal amplification of centrosomes and mitotic defects. Centrosomes are non-membranous organelles, and undergo dynamic changes in its constituents during the centrosome duplication cycle. Through a comparative mass spectrometric analysis of unduplicated and duplicated centrosomes, we identified mortalin, a member of heat shock protein family, as a protein that associates preferentially with duplicated centrosomes. Further analysis revealed that mortalin localized to centrosomes in late G1 before centrosome duplication, remained at centrosomes during S and G2, and dissociated from centrosomes during mitosis. Overexpression of mortalin overrides the p53-dependent suppression of centrosome duplication, and mortalin-driven centrosome duplication requires physical interaction between mortalin and p53. Moreover, mortalin promotes dissociation of p53 from centrosomes through physical interaction. The p53 mutant that lacks the ability to bind to mortalin remains at centrosomes, and suppresses centrosome duplication in a transactivation function-independent manner. Thus, our present findings not only identify mortalin as an upstream molecule of p53 but also provide evidence for the involvement of centrosomally localized p53 in the regulation of centrosome duplication.
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Centrosoma/ultraestructura , Genes p53 , Proteínas HSP70 de Choque Térmico/fisiología , Ciclo Celular , Células Clonales , Fase G1 , Fase G2 , Células HeLa , Humanos , Mitosis , Fase S , Transcripción GenéticaRESUMEN
Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.
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Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Interleucina-8/fisiología , Linfocinas/fisiología , Neovascularización Fisiológica , Factor de Necrosis Tumoral alfa/fisiología , Animales , Células Cultivadas , Córnea/irrigación sanguínea , Regulación del Desarrollo de la Expresión Génica , Humanos , Técnicas Inmunológicas , Morfogénesis/efectos de los fármacos , FN-kappa B/fisiología , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-jun/fisiología , Conejos , Factor de Transcripción Sp1/fisiología , Factor de Crecimiento Transformador alfa/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined.
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Endotelio Vascular/fisiología , FN-kappa B/fisiología , Neovascularización Fisiológica , Estrés Oxidativo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/química , ADN sin Sentido/farmacología , Regulación del Desarrollo de la Expresión Génica , Humanos , Interleucina-8/fisiología , Microcirculación/citología , Datos de Secuencia Molecular , Morfogénesis , Oxidación-Reducción , ARN Mensajero/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Volatile anesthetics have been known to suppress parasympathetic reflex vasodilatation in the lower lip and palate. However, in the submandibular gland, little is known about the effects of these anesthetics on the parasympathetic vasodilatation elicited by reflex and direct (i.e., non-reflex) activation of the parasympathetic vasodilator mechanisms. Although both parasympathetic vasodilatations were inhibited by isoflurane in a concentration- and time-dependent manner, the effects of continuous administration of the alpha(1)-adrenoceptor agonist methoxamine were markedly different: The reflex vasodilatation was not affected by methoxamine, while the direct vasodilatation was significantly reduced. Picrotoxin (GABA(A) receptor antagonist) attenuated the inhibitory effect of isoflurane on direct vasodilatation and the systemic arterial blood pressure. These findings suggest that the isoflurane-induced inhibitory effects on direct vasodilatation are produced by a decrease of peripheral vascular tone by GABAergic mechanisms, whereas those on the reflex vasodilatation are produced exclusively by the inhibition of the reflex center.
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Anestésicos por Inhalación/farmacología , Isoflurano/farmacología , Fibras Parasimpáticas Posganglionares/efectos de los fármacos , Glándula Submandibular/irrigación sanguínea , Glándula Submandibular/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Animales , Nervio de la Cuerda del Tímpano/efectos de los fármacos , Nervio de la Cuerda del Tímpano/fisiología , Estimulación Eléctrica , Nervio Lingual/efectos de los fármacos , Nervio Lingual/fisiología , Masculino , Metoxamina/farmacología , Ratas , Flujo Sanguíneo Regional/efectos de los fármacos , Glándula Submandibular/inervación , Simpatomiméticos/farmacologíaRESUMEN
We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.
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Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN , Exonucleasas/metabolismo , Factores de Transcripción , Sitios de Unión , Unión Competitiva , Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/genética , Cisplatino/farmacología , ADN/metabolismo , Dimerización , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Mutación , Factores de Transcripción NFI , Proteínas Nucleares , Oligonucleótidos/metabolismo , Unión Proteica/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja YRESUMEN
The nonhistone chromosomal protein, high mobility group 1 (HMG1), which is ubiquitously expressed in higher eukaryotic cells, preferentially binds to cisplatin-modified DNA. The observation that HMG1 is overexpressed in cisplatin-resistant human cancer cells suggests that cisplatin resistance may be closely associated with HMG1. To decipher the mechanism of HMG1 overexpression in cisplatin-resistant cells, we isolated two overlapping genomic DNA clones containing the entire human HMG1 gene. These clones, which span approximately 15 kb of contiguous DNA, include 5 kb of the 5' flanking region as well as the entire coding sequence. We sequenced 1500 bp upstream of the first exon. The segment proximal to the transcription initiation site did not contain a TATA box but did possess an activating transcription factor site, an activator protein-2 site, one CCAAT box, and two CCAAT-binding transcription factor/nuclear factor-1 (CTF/NF-1) sites. HMG1 promoter activity was 3-10-fold higher in cisplatin-resistant KB-CP20 cells than in parental KB cells. An in vivo footprint experiment showed several differences of dimethyl sulfate modifications between KB and KB-CP20 cells in the area around the CTF/NF-1 sites. In addition, electrophoretic gel mobility shift assays showed that binding of a nuclear factor from cisplatin-resistant cells to the CTF/NF-1 site was significantly higher than the binding of the same factor from parental cells. Semiquantitative reverse transcription-PCR and Western blot analysis also showed that expression of CTF/NF-1 was 3-20-fold higher in the resistant cell line than in its parental counterpart. These findings suggest that, in cisplatin-resistant cells, the expression of HMG1 gene product is enhanced at the transcriptional level and that this probably occurs through the enhanced expression of the CCAAT binding factor, CTF/NF-1.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Activación Transcripcional , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Clonación Molecular , Secuencia de Consenso , Huella de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos/genética , Proteínas del Grupo de Alta Movilidad/biosíntesis , Humanos , Células KB , Datos de Secuencia Molecular , Factores de Transcripción NFI , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Irsogladine used clinically as an anti-gastric ulcer agent, at 10(-6)-10(-4)M, inhibited cell proliferation and tubular morphogenesis of vascular endothelial cells, but the proliferation of human epidermoid cancer or glioma cells was not inhibited by this drug, even at 10(-4)M. In vivo studies demonstrated that p.o. administration of irsogladine significantly inhibited tumor growth of human glioma cells in mice, and histological analysis showed a dramatic decrease of the neovascularization in the tumors. In mice transplanted with chambers containing human glioma cells or hepatic cancer cells, irsogladine also inhibited angiogenesis. These in vivo and in vitro assays demonstrate that irsogladine may be a unique and potent inhibitor of tumor angiogenesis.
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Antiulcerosos/farmacología , Antineoplásicos/farmacología , Neovascularización Patológica/prevención & control , Triazinas/farmacología , Animales , Línea Celular , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológicoRESUMEN
The Y-box binding protein (YB-1) binds to inverted CCAAT box sequences that are present in the promoter region of many genes. We previously showed that YB-1 is overexpressed in human cancer cell lines that are resistant to cisplatin and that the depletion of YB-1 by transfection of a vector expressing YB-1 antisense RNA increases the sensitivity of human cancer cells to cisplatin. To determine whether YB-1 can bind to cisplatin-modified DNA, we fused YB-1 cDNA to glutathione S-transferase (GST) cDNA and purified the resulting GST fusion protein. When we tested the fusion protein with unmodified or cisplatin-modified oligonucleotides, we found that GST-YB-1 bound more strongly to cisplatin-modified oligonucleotides, as did GST fusion proteins of high mobility group 1 (HMG1), HMG2, and xeroderma pigmentosum group A protein. When we assayed the ability of proliferating cell nuclear antigen (PCNA) to interact with the GST fusion proteins, we observed binding to YB-1 but not to HMG1, HMG2, or xeroderma pigmentosum group A. Subsequent experiments demonstrated that YB-1 and PCNA interact directly via the COOH-terminal region of YB-1. Using immunochemical coprecipitation methods, we observed binding of YB-1 and PCNA in vivo. These results suggest that YB-1 can function as a recognition protein for cisplatin-damaged DNA and that it may be important in DNA repair or in directing the cellular response to DNA damage.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factores de Transcripción/metabolismo , Daño del ADN , Reparación del ADN , Humanos , Células KB , Factores de Transcripción NFI , Proteínas Nucleares , Proteína 1 de Unión a la Caja YRESUMEN
The UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 (Gal NAc-T3) gene, a member of the Gal NAc transferase gene family, is expressed in a tissue-specific manner. To elucidate the function of this gene, we have focused on the molecular mechanism underlying regulation of gene expression. We have cloned Gal NAc-T3 cDNA and used it to show that Gal NAc-T3 mRNA is expressed in tumor cell lines derived from secretory epithelial tissue adenocarcinomas but not in cell lines derived from bladder and epidermoid carcinomas. Using a polyclonal antibody to Gal NAc-T3, we observed protein expression in adenocarcinoma but not non-adenocarcinoma cell lines, and in breast carcinoma cells but not in normal breast tissue. We used Gal NAc-T3 cDNA to isolate three overlapping genomic clones containing the 5'-portion of the human Gal NAc-T3 gene, and we sequenced 1.6 kb around the first exon. A transient expression assay using the luciferase gene showed that promoter activity was much higher in MCF-7 cells than in KB cells. In vivo footprint experiments showed significant protection of a distal GC box, an NRF-1 site, and an AP-2 site in MCF-7 cells. A novel stem and loop structure extending from nucleotide -103 to nucleotide -165 and contiguous to these transcription factor binding sites seemed to be functional in regulating Gal NAc-T3 gene transcription, and a KMnO4 footprint experiment showed that this stem and loop structure could be formed in vivo. We also observed dimethyl sulfate hypersensitive sites in the untranslated region around nucleotide +50 in MCF-7 but not in KB cells. These findings indicate that Gal NAc-T3 gene expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure, and that this regulation is restricted to cell lines derived from epithelial gland adenocarcinomas but not cells derived from nonsecretory epithelial tissue carcinomas. In addition, our immunohistochemical results suggest that our anti-Gal NAc-T3 antibody may be useful for diagnostic purposes in the early stages of breast cancer.