RESUMEN
While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes.
Asunto(s)
Proteínas Fúngicas/genética , Genes Reporteros , Luciferasas/genética , Proteínas Recombinantes de Fusión , Proteínas de Saccharomyces cerevisiae , Transducción de Señal , Transactivadores , Factores de Transcripción/genética , Sitios de Unión , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Expresión Génica , Células HeLa , Humanos , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Abnormalities of chromosome 9p have been reported in human leukemias and lymphomas, and in cell lines lacking the enzyme methylthioadenosine phosphorylase. It has been shown pCN2, the 3' nontranslated region of the N-ras oncogene, crosshybridizes with unknown DNA segments on chromosome 6, 9p, and 22, in addition to the N-ras oncogene itself on chromosome 1p. To use pCN2 to study chromosome 9p abnormalities in malignancies, we undertook to localize the pCN2 crosshybridizing region in chromosome 9p. By analyzing the copy numbers of the pCN2 crosshybridizing bands associated with chromosome 9p among various chromosomally aberrant human cell lines, we mapped the pCN2 hybridizing region to 9cen-p12. Since there is no other available probe in this region, pCN2 should prove very useful in studying abnormalities of chromosome 9p in human malignancies.
Asunto(s)
Cromosomas Humanos Par 9 , Genes ras , Southern Blotting , Células Cultivadas , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Mapeo Cromosómico , ADN/genética , Sondas de ADN , Humanos , Leucemia , Linfoma , Hibridación de Ácido Nucleico , Polimorfismo Genético , Biosíntesis de Proteínas , Células Tumorales CultivadasRESUMEN
Feeding cholesterol to rabbits produces an atherosclerotic model sharing common metabolic features with the disease in humans, including the vascular lipid accumulation. In coronary vascular cells, this lipid accumulation has been associated with decreased prostacyclin (PGI2) biosynthesis and acid cholesterol esterase activity. Unlike the coronary vascular bed, renal microvasculature appears relatively resistant to atherosclerotic injury. This study examined whether renal microvessels from cholesterol-fed rabbits demonstrated similar metabolic changes in coronary vascular cells. Rabbits were fed either a 0% or 2% cholesterol diet for 1 mo. Similar to coronary vascular cells, in renal microvessels from cholesterol-fed rabbits PGI2 biosynthesis decreased and tissue concentrations of cholesterol and cholesteryl esters increased. However, unlike coronary vascular cells, renal microvascular cholesterol esterase activity increased. Light and electron microscopy revealed sporadic lipid deposits in renal microvessels from cholesterol-fed rabbits and no foam cells or occlusive lesions. In vitro addition of prostanoids to normal renal microvessels had no effect on cholesterol esterase activity. It is inviting to speculate that the increased acid cholesterol esterase activity in renal microvessels from cholesterol-fed rabbits protected them from developing extensive microvascular lesions. These biochemical events may explain the relative resistance of human renal microvessels to the development of occlusive atherosclerotic microvascular lesions.