Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone.
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.
Siaussat
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Laboratoire de Physiologie Cellulaire des Invertébrés, Université Pierre et Marie Curie, Paris, France. David.Siaussat@snv.jussieu.fr
Mottier
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Bozzolan
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Porcheron
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Debernard
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002080:Butterflies / Q000502:physiology*; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D004441:Ecdysterone / Q000494:pharmacology*; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D006918:Hydroxyurea / Q000494:pharmacology; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004268:DNA-Binding Proteins; C087173:E75 protein, insect; D019476:Insect Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D004441:Ecdysterone; D006918:Hydroxyurea