RESUMEN

We investigated whether the repulsive SLIT/ROBO pathway is expressed in the endometrium and is negatively regulated during implantation. We also examined whether deficient expression in the Fallopian tube (FT) may predispose to ectopic pregnancy (EP). Endometrium (n = 21) and FT (n = 17) were collected across the menstrual cycle from fertile women with regular cycles. Decidualized endometrium (n = 6) was obtained from women undergoing termination, and FT (n = 6) was obtained from women with EP. SLIT/ROBO expression was quantified by reverse transcription-PCR and protein localized by immunohistochemistry. The regulation of SLIT/ROBO expression in vitro, by sex steroids and hCG, was assessed in endometrial (hTERT-EEpC) epithelial cells, and the effects of Chlamydia trachomatis infection and smoking were studied in oviductal (OE-E6/E7) epithelial cells. Endometrial SLIT3 was highest in the mid-secretory phase (P = 0.0003) and SLIT1,2 and ROBO1 showed a similar trend. ROBO2 was highest in proliferative phase (P = 0.027) and ROBO3,4 showed a similar trend. SLIT2,3 and ROBO1, 4 were lower in decidua compared with mid-secretory endometrium (P < 0.05). SLITs and ROBOs, excepting ROBO2, were expressed in FT but there were no differences across the cycle or in EP. SLIT/ROBO proteins were localized to endometrial and FT epithelium. Treatment of hTERT-EEpC with a combination of estradiol and medroxyprogesterone acetate inhibited ROBO1 expression (P < 0.01) but hCG had no effect. Acute treatment of OE-E6/E7 with smoking metabolite, cotinine, and C. trachomatis had no effect. These findings imply a regulated role for the endometrial SLIT/ROBO interaction during normal development and pregnancy but that it may not be important in the aetiology of EP.

Asunto(s)

Endometrio/metabolismo , Trompas Uterinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal/genética , Células Cultivadas , Infecciones por Chlamydia/metabolismo , Decidua/metabolismo , Implantación del Embrión/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Embarazo , Embarazo Ectópico/genética , Embarazo Ectópico/metabolismo , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/análisis , Receptores Inmunológicos/genética , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Fumar/efectos adversos , Proteínas Roundabout

RESUMEN

RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras-association domain. RASSF2 resides at 20p13, a region frequently lost in human cancers. In this report we investigated methylation status of the RASSF2 promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2 was frequently methylated in breast tumour cell lines (65%, 13/20) and in primary breast tumours (38%, 15/40). RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5'-aza-2'deoxycytidine. RASSF2 was also frequently methylated in NSCLC tumours (44%, (22/50). The small number of corresponding normal breast and lung tissue DNA samples analysed were unmethylated. We also did not detect RASSF2 methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2 in these ovarian tumours. We identified a highly conserved putative bipartite nuclear localization signal (NLS) and demonstrated that endogenous RASSF2 localized to the nucleus. Mutation of the putative NLS abolished the nuclear localization. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished. Hence we demonstrate that RASSF2 has a functional NLS that is important for its tumour suppressor gene function. Our data from this and a previous report indicate that RASSF2 is frequently methylated in colorectal, breast and NSCLC tumours. We have identified RASSF2 as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.

Asunto(s)

Neoplasias de la Mama/genética , Epigénesis Genética/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor/fisiología , Neoplasias Pulmonares/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular , Línea Celular Tumoral , Metilación de ADN , Perros , Femenino , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Neoplasias Ováricas/genética , Proteínas/metabolismo , Ratas , Proteínas Supresoras de Tumor

RESUMEN

In Drosophila, the Slit gene product, a secreted glycoprotein, acts as a midline repellent to guide axonal development during embryogenesis. Three human Slit gene orthologues have been characterised and recently we reported frequent promoter region hypermethylation and transcriptional silencing of SLIT2 in lung, breast, colorectal and glioma cell lines and primary tumours. Furthermore, re-expression of SLIT2 inhibited the growth of cancer cell lines so that SLIT2 appears to function as a novel tumour suppressor gene (TSG). We analysed the expression of SLIT3 (5q35-34) and SLIT1 (1q23.3-q24) genes in 20 normal human tissues. Similar to SLIT2 expression profile, SLIT3 is expressed strongly in many tissues, while SLIT1 expression is neuronal specific. We analysed the 5' CpG island of SLIT3 and SLIT1 genes in tumour cell lines and primary tumours for hypermethylation. SLIT3 was found to be methylated in 12 out of 29 (41%) of breast, one out of 15 (6.7%) lung, two out of six (33%) colorectal and in two out of (29%) glioma tumour cell lines. In tumour cell lines, silenced SLIT3 associated with hypermethylation and was re-expressed after treatment with 5-aza-2'-deoxycytidine. In primary tumours, SLIT3 was methylated in 16% of primary breast tumours, 35% of gliomas and 38% of colorectal tumours. Direct sequencing of bisulphite-modified DNA from methylated tumour cell lines and primary tumours demonstrated that majority of the CpG sites analysed were heavily methylated. Thus, both SLIT2 and SLIT3 are frequently methylated in gliomas and colorectal cancers, but the frequency of SLIT3 methylation in lung and breast cancer is significantly less than that for SLIT2. We also demonstrated SLIT1 promoter region hypermethylation in glioma tumour lines (five out of six; 83%), the methylation frequency in glioma tumours was much lower (two out of 20; 10%). Hence, evidence is accumulating for the involvement of members of the guidance cues molecules and their receptors in tumour development.

Asunto(s)

Epigénesis Genética , Proteínas de la Membrana/genética , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Bases , Línea Celular Tumoral , Islas de CpG , Metilación de ADN , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

An analysis is made of the likely contribution of smoke particles from biomass burning to the global radiation balance. These particles act to reflect solar radiation directly; they also can act as cloud condensation nuclei, increasing the reflectivity of clouds. Together these effects, although uncertain, may add up globally to a cooling effect as large as 2 watts per square meter, comparable to the estimated contribution of sulfate aerosols. Anthropogenic increases of smoke emission thus may have helped weaken the net greenhouse warming from anthropogenic trace gases.