(taken from the application) The goal of this project is to investigate the mechanism of activation and the function of the glycoprotein hormone alpha-subunit (subsequently referred to as alpha-subunit) in ectopic sites. Studies have shown that high levels of alpha-subunit may be ectopically produced by a variety of ectopic sites, but the mechanism of alpha-subunit gene activation in those sites has not yet been elucidated.
Under Specific Aim 1, the proposed studies will investigate the human alpha-subunit promoter cis-elements and the transcription factors important for activation of the promoter in two cell lines, ChaGo, derived from a bronchogenic epithelium, and HeLa, derived from a uterine cervix. First, we will alter the DNA sequence of the promoter, introduce luciferase-expression constructs containing the altered promoter into ChaGo and HeLa cells by electroporation, and measure the activity of the altered promoter in comparison to the wild type promoter sequence. Once we determine the regions important for alpha-subunit promoter activation, we will analyze the interactions between those regions and nuclear proteins from HeLa and ChaGo cells, in order to identify known and possibly as yet unknown transcription factors that may be playing a role in ectopic beta-subunit gene activation.
Under Specific Aim 2, we will investigate the notion that the alpha-subunit plays a role in the regulation of cell growth. The levels of alpha-subunit in ChaGo and HeLa cells will be modulated by stable transfection of alpha-subunit constructs that will result in the production of antisense RNA (decreasing endogenous production), and sense RNA (inducing over expression), both under the control of a muristerone-sensitive ecdysone system. Parameters of cell growth and cell death will be measured after induction with muristerone. For example, at various early time points, RNA samples will be assayed for the levels of various cell cycle regulators, including the cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors using microarray chips. Transcripts identified as responsive to manipulating the levels of alpha-subunit will be further quantitated by Northern blot analysis, and their corresponding proteins analyzed by Western blotting. Investigation of a specific function for the free alpha-subunit will advance the field of endocrinology.
