Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone

• Published in: Insect molecular biology 2004-04, Vol.13 (2), p.179-187
• Main Authors: Siaussat, D, Mottier, V, Bozzolan, F, Porcheron, P, Debernard, S
• Format: Article
• Language: English
• Subjects: 20-hydroxyecdysone; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Butterflies - physiology; cell cycle; Cell Cycle - drug effects; Cell Cycle - physiology; cell cycle arrest; cell division; cell growth; Cell Line; cell lines; cell synchronization; complementary DNA; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; E75 receptors; ecdysterone; Ecdysterone - pharmacology; Flow Cytometry; Gene Expression Regulation, Developmental; gene induction; hormonal regulation; HR3 receptors; hydroxyurea; Hydroxyurea - pharmacology; Insect Proteins - genetics; Insect Proteins - isolation & purification; messenger RNA; Molecular Sequence Data; Plodia cell cycle; Plodia HR3 and E75; Plodia interpunctella; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Steroid - genetics; Receptors, Steroid - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; synchronization; transcription factors
• ISSN: 0962-1075; 1365-2583
• DOI: 10.1111/j.0962-1075.2004.00475.x

We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20-hydroxyecdysone (20E) using the IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL-PID2 cells in their division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mM for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at 10(-6) M induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These findings suggest that PiE75 and PHR3 could be involved in a 20E-induced genetic cascade leading to G2/M arrest.