RESUMEN

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Asunto(s)

Antígeno AC133/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , MicroARNs/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/mortalidad , Estudios de Casos y Controles , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/mortalidad , Carcinoma de Células Escamosas de Esófago , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad

RESUMEN

We report data showing that embryos of the zebrafish, Danio rerio, at 1.5 h post fertilization (hpf) subjected to a low-dose alpha-particle irradiation can release a stress signal into the water, which can be communicated to unirradiated bystander zebrafish embryos sharing the same water medium to induce a hormetic effect in the bystander embryos. Hormetic responses are characterized as biphasic dose-response relationships exhibiting a low-dose stimulation and a high-dose inhibition. The effects on the whole embryos were studied through quantification of apoptotic signals at 24 hpf through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. The results show that, for low alpha-particle dose, the number of apoptotic signals decreases in the irradiated embryos and also in the unirradiated bystander embryos having partnered with the irradiated embryos. These suggested that alpha-particle-irradiated zebrafish embryos could release a stress signal into the water, which could be communicated to unirradiated bystander zebrafish embryos sharing the same water medium to induce a hormetic effect in the bystander embryos.

Asunto(s)

Partículas alfa/efectos adversos , Comunicación Animal , Hormesis/fisiología , Estrés Fisiológico/fisiología , Pez Cebra/fisiología , Animales , Apoptosis/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Embrión no Mamífero/fisiología

RESUMEN

Epithelial-mesenchymal transition (EMT), characterized by cadherin switching, contributes to cancer metastasis. Our recent study showed that Id-1 (inhibitor of differentiation-1) promotes metastasis in esophageal cancer cells, but whether the invasive and metastatic dynamics can be induced early in the carcinogenesis process is still unclear. Immortalization is regarded as the initial stage in the malignant transformation of normal cells. In this study, we investigated the role and mechanisms of Id-1 in inducing EMT and cell invasiveness in immortalized esophageal epithelial cells. We found that immortalized epithelial cells expressed higher endogenous levels of Id-1 compared with normal cells. Ectopic Id-1 expression inhibited the differentiation of immortalized esophageal epithelial cells and promoted cadherin switching, which was accompanied by increased adhesiveness to extracellular matrix, cell motility, migratory potential and matrix metalloproteinase-dependent invasiveness. GTPase activity assays showed that over-expression or short-hairpin RNA knockdown of Id-1 led to corresponding changes in Rac1 activity, whereas RhoA activity was significantly decreased with Id-1 depletion. Inhibitors targeting Rac1, RhoA, and Rho kinase suppressed the invasiveness of Id-1-expressing NE2-hTERT cells. Knockdown of N-cadherin in Id-1-over-expressing cells inhibited cell invasiveness and down-regulated RhoA activity. These data suggest that the Id-1-induced invasive potential may be regulated through the N-cadherin-RhoA axis and Rac1 activation.

Asunto(s)

Cadherinas/metabolismo , Movimiento Celular , Transición Epitelial-Mesenquimal , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células HeLa , Humanos , Transducción de Señal

RESUMEN

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G(2) checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G(2) to M phase transition, impaired G(2) checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G(2) checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G(2) checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G(2) checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G(2) checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G(2) checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells.

Asunto(s)

Centrómero/metabolismo , Inestabilidad Cromosómica/genética , Ciclina B1/metabolismo , Fase G2/genética , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Estudios de Casos y Controles , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , División Celular/efectos de la radiación , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Centrómero/efectos de los fármacos , Ciclina B1/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Rayos gamma , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Índice Mitótico , Morfolinas/farmacología , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pironas/farmacología , Telomerasa/genética , Translocación Genética/genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética

RESUMEN

Telomeres, the terminal chromosomal structure crucial for maintaining genomic integrity, shorten with deoxyribonucleic acid replications in most human somatic cells. Chromosomes carrying critically short telomeres tend to form end-to-end fusions, which are subject to breakage during cell division. However, it remains obscure how such telomere-mediated fusions are resolved during the process of immortalization, which is an early and indispensable step toward cancer. It has been hypothesized that the breakage could occur at either the microtubule or chromatid, causing numerical or structural chromosome instability, respectively. In this paper, we show that although the distributions of chromosomal segment losses or gains involved in structural aberrations were significantly correlated with the profiles of critically short telomeres in human epithelial cells undergoing immortalization, no such association was detected for whole-chromosome losses or gains in either metaphase or interphase cells. By distinguishing between homologues, we further showed that the specific homologues with critically short telomeres and frequent end-to-end fusions were not preferentially involved in respective whole-chromosome losses or gains. Our data therefore demonstrate that microtubule breakage is not a major mechanism for resolving chromosomal end-to-end fusions in human cells undergoing immortalization. An important implication of this finding is that microtubule-kinetochore attachment is stronger than the chromosome structure.

Asunto(s)

Inestabilidad Cromosómica/genética , Aberraciones Cromosómicas , Cromosomas Humanos/genética , Microtúbulos/genética , Telómero/genética , Línea Celular Transformada , Transformación Celular Viral/genética , Humanos , Microtúbulos/patología , Modelos Genéticos , Telómero/patología

RESUMEN

Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-kappaB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

Asunto(s)

Aberraciones Cromosómicas , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Microtúbulos/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas de la Matriz Viral/fisiología , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Células HeLa , Humanos , Microtúbulos/patología , FN-kappa B/fisiología , Proteínas Supresoras de Tumor/biosíntesis

RESUMEN

Testicular germ cell tumour (TGCT) is the most common malignancy in young males. Although most TGCTs are sensitive to cisplatin-based chemotherapy, significant numbers of TGCT patients still relapse and die each year because of the development of resistance to cisplatin. Previously, we first reported that a key regulator of the mitotic checkpoint, mitotic arrest deficient-2 (MAD2), was a mediator of cisplatin sensitivity in human cancer cells. In this study, we investigated whether MAD2 played a role in cellular sensitivity to cisplatin in TGCT cells and the underlying molecular mechanisms responsible. Using 10 TGCT cell lines, we found that increased MAD2 expression was correlated with cellular sensitivity to cisplatin, which was associated with activation of the MEK pathway. Treatment of cells expressing high levels of MAD2 with an MEK inhibitor, U0126, led to cellular protection against cisplatin-induced apoptosis. Inactivation of MAD2 by transfecting a dominant-negative construct in TGCT cells with high levels of MAD2 resulted in the suppression of MEK pathway and resistance to cisplatin-induced cell death. These results support previous suggestion on the involvement of mitotic checkpoint in DNA damage response in human cancer cells and demonstrate a possible molecular mechanism responsible for the MAD2-mediated sensitivity to cisplatin in TGCT cells. Our results also suggest that downregulation of MAD2 may be an indicator for identification of TGCT cancer cells that are potentially resistant to cisplatin-based therapy.

Asunto(s)

Proteínas de Unión al Calcio/farmacología , Proteínas de Ciclo Celular/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , MAP Quinasa Quinasa 1/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Represoras/farmacología , Neoplasias Testiculares/tratamiento farmacológico , Butadienos/farmacología , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Proteínas Mad2 , Masculino , Modelos Biológicos , Neoplasias de Células Germinales y Embrionarias , Nitrilos/farmacología , Transducción de Señal , Transfección

RESUMEN

Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis.

Asunto(s)

Proteínas de Ciclo Celular/genética , Cromosomas Humanos Par 20/genética , Células Epiteliales/enzimología , Amplificación de Genes , Regulación Enzimológica de la Expresión Génica , Ovario/enzimología , Proteínas Quinasas/genética , Proteínas de Xenopus/genética , Aurora Quinasa A , Aurora Quinasas , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Cartilla de ADN , Femenino , Humanos , Hibridación Fluorescente in Situ , Ovario/citología , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

INTRODUCTION: The multifocal visual-evoked potential (mfVEP) has been widely used in the study of diseases of the visual system. However, the sensitivity of the mfVEP in the objective detection of relative field defects has not been determined. This study investigates variations in mfVEP responses while simulating relative field defects by using different luminous transmission masks [neutral density (ND) filters] on the stimulus pattern. METHODS: Simulated relative field defects with four different luminous transmissions were obtained by using 0.2, 0.4, 0.6, and 0.8 ND filters, 5 degrees in size, at two different retinal eccentricities (10 and 16 degrees) on a standard mfVEP dartboard stimulus. Eleven normal subjects were recruited for mfVEP measurements. The response amplitudes and latencies of the N1 and P1 of the mfVEP, with and without small simulated relative field defects, were compared. RESULTS: The mfVEP amplitudes of N1 and P1 decreased substantially when 0.6 and 0.8 ND filters were introduced. The effects were similar at both the 10- and 16-degree eccentricities but there was no change in latency with simulated field defects at either location. CONCLUSIONS: The mfVEP can detect a simulated relative field defect 5 degrees in size starting with 0.6 log unit reduction in luminance at both 10-degree and 16-degree eccentricities. This illustrates that the sensitivity of the mfVEP measurement is nearly comparable with that of the Humphrey Visual Field Analyser.

Asunto(s)

Potenciales Evocados Visuales/fisiología , Trastornos de la Visión/fisiopatología , Campos Visuales/fisiología , Adulto , Femenino , Humanos , Masculino , Estimulación Luminosa/métodos , Retina/fisiopatología , Percepción Visual/fisiología

RESUMEN

In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriólisis , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Bacteriólisis/efectos de los fármacos , Secuencia de Bases , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Octoxinol/farmacología , Penicilinas/farmacología , Análisis de Secuencia de ADN , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

Loss of E-cadherin (E-cad) has been associated with progression and poor survival in nasopharyngeal carcinoma (NPC). In this study, we investigated the role of methylation on E-cad inactivation in NPC cell lines, as well as in NPC tissue samples. Using 6 NPC cell lines, we found that methylation of the E-cad 5' CpG island promoter region was correlated with the loss of both mRNA and E-cad protein expression in these cell lines. In addition, using 29 NPC and 10 non-malignant nasopharyngeal samples, we also observed 5' CpG methylation of the E-cad gene in 52% (15 out of 29) NPC samples, but in only 10% (1 out of 10) of the non-malignant nasopharyngeal tissues. Our findings indicate that 5' CpG island methylation of the E-cad gene may play an important part in the inactivation of E-cad in NPC. Our results also suggest that reducing the methylation of the E-cad gene may be a potential therapeutic strategy for NPC.

Asunto(s)

Cadherinas/metabolismo , Islas de CpG , Neoplasias Nasofaríngeas/metabolismo , Western Blotting , Cadherinas/genética , Metilación de ADN , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Inmunohistoquímica , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Tumorales Cultivadas

RESUMEN

Galactosemia is a genetic disease with deficiency of galactose-1-uridyltransferase, resulting in the accumulation of galactose or galactose-1-phosphate in the blood and tissues. Rats were fed with normal rat chow and with a high-galactose diet for 4 weeks to give control and galactosemic groups, and their ovarian function was studied. The two groups of rats were injected with pregnant mare's serum gonadotrophin (PMSG) and were killed at different time points after human chorionic gonadotrophin (hCG) injection. The number of oocytes ovulated in the controls was significantly higher than in the galactosemic group. Morphometric studies of the ovaries also showed a higher number of corpora lutea in the controls. Western blot analysis of granulosa cells showed that the overall expressions of Fas and FasL were lower in the control group and their expressions of inhibitor of apoptosis proteins (IAPs) were higher than in the galactosemic group, especially at 8 h post hCG injection. TDT-mediated dUTP-biotin nick end-labeling (TUNEL) and immunohistochemical staining of ovarian sections with Ki-67 and IAPs showed more apoptotic granulosa cells in the galactosemic group and the expressions of IAPs in granulosa cells also confirmed the result of the Western blot. These findings support our hypothesis that ovarian dysfunction in galactosemic rats is due to increased apoptosis in granulosa cells of maturing follicles.

Asunto(s)

Apoptosis/fisiología , Galactosemias/complicaciones , Células de la Granulosa/metabolismo , Enfermedades del Ovario/etiología , Proteínas/metabolismo , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Supervivencia Celular/fisiología , Gonadotropina Coriónica/farmacología , Modelos Animales de Enfermedad , Proteína Ligando Fas , Femenino , Alimentos Formulados/efectos adversos , Galactosa/efectos adversos , Galactosa/metabolismo , Galactosemias/metabolismo , Galactosemias/patología , Gonadotropinas Equinas/farmacología , Células de la Granulosa/patología , Proteínas Inhibidoras de la Apoptosis , Glicoproteínas de Membrana/metabolismo , Enfermedades del Ovario/metabolismo , Enfermedades del Ovario/patología , Ratas , Ratas Sprague-Dawley , Receptor fas/metabolismo

RESUMEN

E-cadherin and beta-catenin are cell-cell adhesion molecules, which are thought to play an important role in trophoblastic differentiation and remodelling during gestation. Their expression may be altered in pathological conditions with trophoblastic invasion. In this study, we used immunohistochemical methods to study the pattern of expression of E-cadherin and beta-catenin in villous trophoblastic tissue in normal and pathological pregnancies. In villous trophoblastic tissue, E-cadherin had a membranous distribution, whereas beta-catenin had a mixed-membranous and granular cytoplasmic distribution. The levels of expression of E-cadherin and beta-catenin correlated with each other. From first to third trimesters, the expression of both E-cadherin and beta-catenin showed a decreasing trend. In preeclampsia, there was an up-regulation of E-cadherin and beta-catenin expression. In placenta accreta, the level of expression of both did not differ from that in normal third-trimester placenta. In gestational trophoblastic diseases, there was a general trend of down-regulation of both E-cadherin and beta-catenin. Altered expression of E-cadherin and beta-catenin may play a role in the development of normal and pathological placentas.

Asunto(s)

Cadherinas/biosíntesis , Proteínas del Citoesqueleto/biosíntesis , Placenta Accreta/metabolismo , Preeclampsia/metabolismo , Transactivadores/biosíntesis , Neoplasias Trofoblásticas/metabolismo , Trofoblastos/metabolismo , Neoplasias Uterinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Placenta Accreta/patología , Preeclampsia/patología , Embarazo , Neoplasias Trofoblásticas/patología , Trofoblastos/patología , Neoplasias Uterinas/patología , beta Catenina

RESUMEN

Reactive oxygen species scavengers present in male accessory sex gland secretions might afford antioxidant protection to sperm DNA. This study was conducted to determine whether accessory sex gland secretions protect the genome and function of spermatozoa against oxidative damage in the uterus. Male golden hamsters were divided into four experimental groups: (i) all accessory sex glands removed; (ii) ampullary glands removed; (iii) ventral prostate gland removed and (iv) sham-operated controls. Ejaculated spermatozoa recovered from uteri 15-30 min after mating with experimental males and caput and cauda epididymal spermatozoa obtained from intact males were incubated in 0-20 mmol NADPH l(-1) for 2 h. These spermatozoa and untreated uterine spermatozoa were processed for two types of comet assay (single cell gel electrophoresis): alkaline comet assay (pH > 13) which revealed single-strand DNA breakage and neutral comet assay (pH 9) which revealed double-strand DNA breakage. In comparison with the sham-operated controls, spermatozoa that had not been exposed to accessory sex gland secretions had a higher incidence and more extensive single-strand DNA damage with increasing concentrations of NADPH. Spermatozoa from hamsters without ampullary glands and from hamsters without the ventral prostate glands were similar to those of the control group. After incubation with NADPH, the capacity of spermatozoa from hamsters without accessory glands and from sham-operated controls to fuse with oocytes in vitro was reduced. However, only hamsters without accessory glands showed a negative correlation between single-strand DNA damage and sperm-oocyte fusion. Cauda epididymal spermatozoa were less susceptible to NADPH treatment compared with caput epididymal spermatozoa. The results of the present study showed that male accessory sex gland secretions can preserve the integrity of the sperm genome.

Asunto(s)

Daño del ADN , Genitales Masculinos/metabolismo , Estrés Oxidativo , Espermatozoides/fisiología , Animales , Factores Biológicos/fisiología , Cricetinae , Relación Dosis-Respuesta a Droga , Epidídimo , Femenino , Masculino , Mesocricetus , NADP/farmacología , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/efectos de los fármacos , Útero

Asunto(s)

Proteínas Bacterianas/química , Staphylococcus aureus/química , Transactivadores , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalización , Cristalografía por Rayos X , Dimerización , Regulación Bacteriana de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Virulencia

RESUMEN

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible P(BAD) promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp(uvr) fusion carried on a shuttle plasmid, we demonstrated that dose-dependent tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

Asunto(s)

Antibacterianos/farmacología , Proteínas Portadoras , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Staphylococcus aureus/genética , Tetraciclina/farmacología , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Genes Reporteros , Ratones , Operón , Factor sigma/biosíntesis , Infecciones Estafilocócicas , Staphylococcus aureus/citología , Staphylococcus aureus/patogenicidad , Xilosa/metabolismo

RESUMEN

Two genes of the sigB operon, rsbU and rsbV, were deleted in an rsbU(+) strain (FDA486) to evaluate the contribution of these two genes to sigma(B) activity in Staphylococcus aureus. The sigma(B) protein level and the transcription of two sigma(B)-dependent promoters (sigB and sarA P3 transcripts) were analyzed in the constructed mutants. A deletion of the first gene (rsbU) within the sigB operon led only to a partial reduction in sigma(beta) activity. A deletion of the second gene (rsbV) resulted in a more dramatic reduction in the sigma(B) protein level and its activity than did the deletion of rsbU, thus indicating that RsbV can be activated independent of RsbU. In the parental strain, the sigma(B)-dependent transcript initiated upstream of rsbV was 28-fold higher than the sigma(A)-dependent transcript originating from the rsbU promoter. The level of the sigma(B)-dependent transcript decreased up to 50% in the rsbU mutant and up to 90% in the rsbV mutant compared with the transcript in the wild type. The yellow pigment of S. aureus colonies, a sigma(B)-dependent phenotype, was partially reduced in the rsbU and rsbV mutants, whereas alpha-hemolysin was increased. Additionally, the sarA P3 promoter activity of the parental strain was induced to a higher level in response to pH 5.5 than was that of the rsbU or rsbV mutant, indicating that RsbU is the major activator of the sigma(B) response to acid stress. Using a tetracycline-inducible system to modulate the expression of RsbW, we progressively repressed pigment production, presumably by reducing the free sigma(B) level. Collectively, our data indicated that RsbU and RsbV in S. aureus contributed to different levels of sigma(B) protein expression and varying sigma(B) activities. Although RsbV can activate sigma(B) independent of RsbU, RsbU remains the major activator of sigma(B) during acid stress.

Asunto(s)

Proteínas Bacterianas/metabolismo , Monoéster Fosfórico Hidrolasas , Factor sigma/metabolismo , Staphylococcus aureus/crecimiento & desarrollo , Transactivadores , Proteínas Bacterianas/genética , Toxinas Bacterianas/biosíntesis , Proteínas Portadoras/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/biosíntesis , Mutación , Operón , Pigmentación , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Eliminación de Secuencia , Transcripción Genética

RESUMEN

E-cadherin, a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Downregulation of E-cadherin is common in esophageal carcinoma and is associated with an increase in invasive and metastatic potential. To study the mechanisms responsible for inactivation of this gene in esophageal squamous cell carcinoma (ESCC), we investigated the methylation status around the 5' promoter region of E-cadherin gene of six ESCC cell lines by methylation-specific polymerase chain reaction, and compared it with E-cadherin protein and mRNA expression. We also studied the methylation status of 20 ESCC clinical specimens. Methylation was noted in four of the six cell lines (one fully methylated and three partially methylated). The completely methylated cell line lacked E-cadherin protein expression and mRNA transcription. E-cadherin expression and transcription were reduced in a partially methylated cell line but preserved in the other partially methylated cell lines. Treatment of E-cadherin-negative carcinoma cells with the demethylating agent, 5-aza-2'-deoxycytidine, induced re-expression of the gene. A high frequency of methylation (16/20, 80%) was also noted in the 20 ESCC clinical samples. Our results indicate that 5' CpG island methylation is common in esophageal carcinoma and may play an important role in downregulation of E-cadherin.

Asunto(s)

Azacitidina/análogos & derivados , Cadherinas/genética , Carcinoma/genética , Islas de CpG , Metilación de ADN , Regulación hacia Abajo , Neoplasias Esofágicas/genética , Azacitidina/farmacología , Cadherinas/biosíntesis , Carcinoma/metabolismo , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Inhibidores Enzimáticos/farmacología , Neoplasias Esofágicas/metabolismo , Humanos , Regiones Promotoras Genéticas , ARN Neoplásico/biosíntesis , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

In searching the Staphylococcus aureus genome, we found several homologs to SarA. One of these genes, sarT, codes for a basic protein with 118 residues and a predicted molecular size of 16,096 Da. Northern blot analysis revealed that the expression of sarT was repressed by sarA and agr. An insertion sarT mutant generated in S. aureus RN6390 and 8325-4 backgrounds revealed minimal effect on the expression of sarR and sarA. The RNAIII level was notably increased in the sarT mutant, particularly in postexponential-phase cells, while the augmentative effect on RNAII was less. SarT repressed the expression of alpha-hemolysin, as determined by Northern blotting, Western blotting, and a rabbit erythrocyte hemolytic assay. This repression was relieved upon complementation. Similar to agr and sarA mutants, which predictably displayed a reduction in hla expression, the agr sarT mutant exhibited a lower level of hla transcription than the sarT mutant. In contrast, hla transcription was enhanced in the sarA sarT mutant compared with the single sarA mutant. Collectively, these results indicated that the sarA locus, contrary to the regulatory action of agr, induced alpha-hemolysin production by repressing sarT, a repressor of hla transcription.

Asunto(s)

Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Represoras/genética , Staphylococcus aureus/genética , Transactivadores , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/biosíntesis , Secuencia de Bases , Western Blotting/métodos , ADN Bacteriano , Proteínas Hemolisinas/biosíntesis , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Conejos , Proteínas Represoras/metabolismo , Staphylococcus aureus/metabolismo , Factores de Transcripción

RESUMEN

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). The sar (Staphylococcus accessory regulator) locus is composed of three overlapping transcripts (sarA P1, P3, and P2, transcripts initiated from the P1, P3, and P2 promoters, respectively), all encoding the 124-aa SarA protein. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of the sarA promoters. We previously partially purified a 13.6-kDa protein, designated SarR, that binds to the sarA promoter region to down-modulate sarA transcription from the P1 promoter and subsequently SarA expression. SarR shares sequence similarity to SarA, and another SarA homolog, SarS. Here we report the 2.3 A-resolution x-ray crystal structure of the dimeric SarR-MBP (maltose binding protein) fusion protein. The structure reveals that the SarR protein not only has a classic helix-turn-helix module for DNA binding at the major grooves, but also has an additional loop region involved in DNA recognition at the minor grooves. This interaction mode could represent a new functional class of the "winged helix" family. The dimeric SarR structure could accommodate an unusually long stretch of approximately 27 nucleotides with two or four bending points along the course, which could lead to the bending of DNA by 90 degrees or more, similar to that seen in the catabolite activator protein (CAP)-DNA complex. The structure also demonstrates the molecular basis for the stable dimerization of the SarR monomers and possible motifs for interaction with other proteins.

Asunto(s)

Proteínas Bacterianas/química , Staphylococcus aureus/química , Transactivadores , Secuencia de Aminoácidos , Cristalización , ADN/metabolismo , Dimerización , Datos de Secuencia Molecular