ABSTRACT
Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.
Subject(s)
Extracellular Matrix/metabolism , Fibronectins/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Animals , Cell Culture Techniques , Down-Regulation/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Expression Regulation , Humans , Oncogene Protein pp60(v-src)/metabolism , Proteasome Inhibitors , Proto-Oncogene Proteins c-raf/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RatsABSTRACT
Antioxidants are important species which possess the ability to protect the body from damage caused by free radical-induced oxidative stress. A variety of free radical-scavenging antioxidants exist within the body many of which are derived from dietary sources. There is currently much interest in the antioxidant role of flavonoids and other polyphenols found in tea, wine, fruit and vegetables. Enhanced chemiluminescence is a simple technique which can be used as a rapid and sensitive assay for measuring the antioxidant activity of beverages such as green and black tea. This article examines the impact of water temperature, stewing time, leaf concentration and the addition of milk upon the antioxidant activity of black tea solutions. The antioxidant activity of a range of commercially available black and green teas has also been measured.
