The goal of this three-year proposal is to clarify how the insect steroid hormone 20-hydroxyecdysone (20E) interacts with cell cycle machinery in mosquito (Aedes albopictus C7-10 cells. A first Aim is to identify which phases of the mitotic cell cycle: G1, S, G2 or M, are affected by 20E, and whether the effect is stimulatory or inhibitory. These analyses will draw upon elegant studies with estrogen-responsive mammalian cells, in which hormone treatment predominantly affects the G1 phase of the cycle, where proteins known as the D1 cyclins relay information from the external environment to the intrinsic cell cycle machinery. Preliminary studies have shown that C7-10 cells respond to 20E: 20E-inducible proteins have been identified, and the cells express transcripts encoding the ecdysone receptor proteins EcR and USP. Available data suggest that in the presence of 20E, C7-10 mosquito cells complete the cycle in progress before the 20E response is detectable. A second Aim is to complete ongoing analyses of the EcR and USP isoforms expressed by these C7-10 cells and to further investigate the time course of appearance of 20E inducible proteins in the context of cell- cycle specific patterns of protein expression. The goal is to identify a component(s) of the cell cycle machinery that is involved in the early response to 20E. A third goal is to characterize a 40 kDa DNA binding protein that interacts with Drosophila DNA containing a known 20E response element. These studies will be based on an understanding of C7-10 cell cycle parameters and synchronization procedures that have been established in the PI's laboratory, as well as standard molecular biology techniques. Results of this study will provide novel insights into the effects of 20E on cell growth and proliferation, which are directly relevant to efforts to control vector-borne disease by genetic manipulation of insect vectors.