The effects of the deregulated expression of the cloned transcription factor E2F-1 on Chinese hamster ovary cells

Citation

Lee, Kelvin H. (1995) The effects of the deregulated expression of the cloned transcription factor E2F-1 on Chinese hamster ovary cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/9dwx-6f88. https://resolver.caltech.edu/CaltechETD:etd-10172007-132158

Abstract

E2F-1 is one of a family of transcription factors known to be involved in cell cycle regulation at the G1/S phase transition. Several proteins intimately involved in DNA synthesis are regulated by the E2F-1 transcription factor family. It is believed that the presence of free E2F-1 in mammalian cells is required to activate transition of cells through the G1-S restriction point of the cell cycle. Stable clones from CHO K1 cells transfected with an expression vector for human E2F-1 were created. All of the clones expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Several clones were further studied by confocal microscopy and Southern analysis. Both of these studies also provide evidence for the expression of cloned E2F-1 in these CHO cells. CHO K1:E2F1 cells are able to proliferate in well-defined medium completely free from proteins or serum and flow cytometric analysis of CHO K1:E2F-1 cells indicates a prolonged S-phase compared to CHO cells grown by bFGF stimulation and by cyclin E overexpression. Two-dimensional gel electrophoresis (2DE) of CHO cellular proteins reveals increased expression of 236 spots indicating significant regulatory effects for cloned E2F-1 in CHO cells. These 2DE results also suggest new routes to identification of gene products regulated by a particular transcription factor. Expression of cloned E2F-1 is an important means for bypassing the serum requirement of mammalian cell culture. Metabolic engineering of cell cycle regulation bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing.

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