Characterization of subclones of the epithelial cell line from Chironomus tentans resistant to the insecticide RH 5992, a non-steroidal moulting hormone agonist

Selection of hormone resistant subclones in the continuous presence of the insecticide and ecdysteroid mimick RH 5992 (tefubenozide) resulted preferentially in clones with defects in ecdysteroid receptor function. RH 5992 is already degraded to polar products in wild-type cells; no increase in metab...

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Bibliographic Details
Published in:Insect biochemistry and molecular biology 2000-07, Vol.30 (7), p.591-600
Main Authors: Grebe, Marco, Rauch, Peter, Spindler-Barth, Margarethe
Format: Article
Language:English
Subjects:
Animals
Binding Sites - genetics
Cell Line
Chironomidae
Chironomidae - cytology
Chironomidae - genetics
Chironomus tentans
Cloning, Molecular
Ecdysteroid
Ecdysteroids
Epithelial Cells - drug effects
Epithelial Cells - physiology
Hydrazines - pharmacology
Insecticide Resistance
Insecticides - pharmacology
Ligands
Receptor
Selection, Genetic
Steroids - pharmacology
Tefubenozide
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Summary:Selection of hormone resistant subclones in the continuous presence of the insecticide and ecdysteroid mimick RH 5992 (tefubenozide) resulted preferentially in clones with defects in ecdysteroid receptor function. RH 5992 is already degraded to polar products in wild-type cells; no increase in metabolism of tefubenozide is observed in resistant clones. According to Western blots, ecdysteroid receptor (EcR) and its heterodimerization partner ultraspiracle (USP) are present in all resistant clones. The concentrations are comparable to wild-type cells, but in three clones the extent of phosphorylation of USP is diminished. With regard to hormone binding several types of hormone resistance are distinguished: (1) The same two high-affinity hormone recognition sites are present as in wild-type cells (K D1=0.31±0.28 nM, K D2=6.5±2.4 nM) but the number of binding sites is reduced. (2) The binding site with the lower affinity (K D2) is missing. (3) The binding site with the higher affinity (K D1) is missing. (4) No specific binding is observed. Ponasterone A binding can be rescued by addition of EcR but not by USP. (5) Ligand specificity is altered. RH 5992 can not compete [ 3H]-ponasterone A as efficient as in wild-type cells.
ISSN:0965-1748
1879-0240
DOI:10.1016/S0965-1748(00)00032-1