Transcriptomics

Dataset Information

31

Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide


ABSTRACT: Methoxyfenozide (RH-2485) represents the group of non-steroidal ecdysteroid agonists with a dibenzoylhydrazine structure, that are used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ~1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis is also predicted to gain insights into the mechanism of resistance and the crosstalk between ecdysteroid signaling and cell proliferation-linked processes. In this microarray experiment three biological conditions were under study: (i) untreated S2 cells (S2), (ii) a subpopulation of these untreated cells, which were grown under continuous pressure of methoxyfenozide and which were shown to become resistant to this compound (S2res) and (iii) a subpopulation of the resistant cells, which were again cultured without methoxyfenozide added to the medium (S2res-woMF). For each condition, two subpopulations (which were treated as biological replicates, although they are not exactly independent), were isolated and used for the RNA extractions. The overall experimental design was an n+2 A-optimal design. When each hybridisation is respresented by an arrow (Cy5 → Cy3), the design can be respresented as follows: First loop = S2.1 → S2res.1 → S2.2 → S2res.2 → S2.1; Second loop = S2.1 → S2res-woMF.1 → S2.2 → S2res-woMF.2 → S2.1 (numbers following the condition name correspond to the biological replicate numbers). The two loops are thus connected by S2.1 and S2.2. Two analyses were performed: S2res compared to S2 and S2res-woMF compared to S2.

ORGANISM(S): Drosophila melanogaster  

SUBMITTER: Luc Swevers  Guy Smagghe   Hadi Mosallanejad   Jozef Vanden Broeck   Thomas Soin   Dries Knapen   Liesbeth Badisco    

PROVIDER: E-GEOD-17748 | ArrayExpress| 2014-05-02

SECONDARY ACCESSION(S): GSE17748PRJNA118351

REPOSITORIES: GEO, ArrayExpress

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Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide.

Mosallanejad Hadi H   Badisco Liesbeth L   Swevers Luc L   Soin Thomas T   Knapen Dries D   Vanden Broeck Jozef J   Smagghe Guy G  

Journal of insect physiology 20100909 12


Methoxyfenozide (RH-2485) is a non-steroidal ecdysteroid agonist with a dibenzoylhydrazine structure, representing a group used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ∼ 1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response  ...[more]

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