Ecdysone-induced accumulation of mosquito cells in the G1 phase of the cell cycle

We have established baseline conditions for investigating the interaction of the insect steroid hormone 20-hydroxyecdysone (20E) with the cell cycle in the C7-10 cell line from the mosquito, Aedes albopictus. As is the case with Drosophila melanogaster cells, treatment of C7-10 cells with 20E inhibits proliferation. In the presence of 10⁻⁶ M 20E, a gradual decline in cell number is typically apparent at 24 h. Media components such as phenol red and the potential presence of endogenous steroids in serum have no effect on the response to 20E. Pre-treating the cells with 10⁻⁸ M 20E, with or without an intervening hormone-free period, did not alter the response to 10⁻⁶ M 20E. However, replenishment of the medium appeared to synchronize the response to 10⁻⁶ M 20E, causing an abrupt and complete cessation of cell division by 48 h. Flow cytometry over a 20 h period showed a decrease in the proportion of cells in S within 4-6 h after exposure to 20E. By 6-10 h, a transient increase in G2 was followed by the accumulation of more than 70% of the cells in G1. These data suggest that after treatment with 20E, cells complete the ongoing cycle before arresting in G1. Consistent with the decrease in the proportion of cells in S and G2, western blots show that levels of cyclin A, which is required during the S phase of the cycle, decreased in 20E-treated cells.