Development of high throughput screening assay. For detection of E2F mediated signaling pathways

      Аннотация: E2Fs are transcription factors which regulate the cell cycle by a different mechanism. The E2F family contains eight members E2F1 – E2F8 and the disruption in the E2Fs signaling pathways is one of the reasons of cancer development. Theoretical part summarizes basic information about E2Fs, retinoblastoma protein, cell cycle and its control. It is also focused on the ways that cell death – apoptosis can be mediated by E2Fs. The experimental part is focused on testing three clones of cell line 293H transfected with E2F-luciferase construct. Clones with the best properties were selected for development and validation of homogenous high-throughput screening assays able to detect expression of E2F-mediated gene expression. Using this set up assays were validated on biologically active compounds of LOPAC library. Results summarized in the conclusion evaluate the role of activation or repression of E2F in relation to cell survival.

Ключевые слова: cell cuture, Compound dispension, LOPAC library, Luciferase assay