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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 20, 2003 as doi:10.1096/fj.03-0627fje. |
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Institute of Biology, National Centre for Scientific Research "Demokritos," Aghia Paraskevi Attikis, 153 10 Athens;
* Vioryl S.A., Kifissia, 145 64 Athens, Greece;
Laboratory of Agrozoology, University of Ghent, B-9000 Ghent, Belgium;
Graduate School of Agriculture, Kyoto University, Katashirakawa, Kyoto 606-8502, Japan
2Correspondence: Institute of Biology, National Centre for Scientific Research "Demokritos," P.O. Box 60228, Aghia Paraskevi Attikis, 153 10 Athens, Greece. E-mail: swevers{at}bio.demokritos.gr and iatrou{at}bio.demokritos.gr
SPECIFIC AIMS
We describe the generation and use of a high-throughput (HT) screening system based on silk moth cell lines permanently transformed with a high-sensitivity ecdysone (insect molting hormone)-responsive reporter construct that allows easy, fast, and reliable detection of ecdysteroid agonistic and antagonistic activities in complex plant extracts and libraries of synthetic compounds.
PRINCIPAL FINDINGS
1. Ecdysone-dependent inducibility of a reporter cassette in silk moth Bm5 cells
Addition of the active molting hormone 20-hydroxy-ecdysone (20E) to Bm5 cells, a silk moth-derived cell line, caused a massive transcriptional induction of the primary response gene bmhr3, indicating that all elements of the 20E transduction pathway necessary for the induction of 20E primary response genes are present (Fig. 1
A).
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When a 20E-inducible expression cassette, pBmbA/ERE.cat, was introduced in Bm5 cells by transfection, addition of 20E resulted in dose-dependent stimulation of cat reporter gene activity (Fig. 1B
). Maximal levels of inducibility were 
1800-fold; the EC50 of 20E for the ecdysone-responsive element (ERE)-dependent reporter was between 75 and 100 nM.
2. Generation of reporter cell lines displaying 20E-induced fluorescence
Bm5 cells were transfected with an ecdysone-responsive GFP reporter cassette, pBmbA/ERE.gfp, and plasmid pBmA.hmb, which confers resistance to the antibiotic hygromycin B. After antibiotic selection, five semiclonal lines were established from the antibiotic-resistant (polyclonal) cell population by flow cytometry (FACS) and limited dilution.
3. Characterization of 20E-induced fluorescence responses in reporter cell lines
Experiments using 20E as the inducing agent revealed that 5000 cells plated onto a 96-well plate are sufficient to generate easily quantifiable fluorescence in a plate reader equipped with a probe for GFP fluorescence. 20E activates the 20E-dependent GFP reporter construct at a concentration of 10–100 nM, the same as that observed by the CAT assay in transient transfection experiments.
Subsequent measurements of fluorescence responses elicited by dibenzoylhydrazine compounds RH-0345, RH-2485, RH-5849, and RH-5992 revealed the following order of efficacy (ED50) in the reporter cell line: RH-2485>RH-5992>RH-0345>RH-5849. This order matches precisely the known order of potency determined by a variety of in vitro and in vivo biological and toxicity assays for lepidopteran insects.
4. High-throughput screening for synthetic and natural 20E agonists and antagonists
The HT screening scheme used to identify compounds with ecdysteroid mimetic (agonist and antagonist) activity in libraries of synthetic compounds and plant collections is presented diagrammatically in Fig. 2
.
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HTS of dibenzoylhydrazine derivatives: activity-based selection and toxicity test validation
The reporter cell lines were used to screen a library of 174 dibenzoylhydrazine derivatives. Based on HT screening, compounds were classified according to the minimal concentration required to induce a measurable response. Of particular interest was the identification of 29 compounds that were effective at concentrations of 10 nM or lower. KU-106 and KU-121, which showed the highest activity (Fig. 3
A), were chosen for further evaluation. In the HT assay, EC50 values of KU-106 and KU-121 (2.4 nM and 2.5 nM, respectively) were only marginally higher than those obtained for commercial compounds RH-5992 and RH-2485 (0.5 nM and 0.9 nM, respectively), which were examined in parallel (Fig. 3A
).
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When compounds KU-106 and KU-121 were tested for larvicidal activity on silk moth last instar larvae with commercial compounds RH-2485, RH-5992, RH-0345, and RH-5849 (Fig. 3B
), the toxicity of the new compounds (LD50 of 17.1 ng and 12.7 ng for KU-106 and KU-121, respectively) was slightly less than that of the strongest compounds, RH-2485 and RH-5992 (LD50 of 7.5 ng and 3.6 ng for RH-5992 and RH-2485, respectively), but significantly enhanced compared with RH-0345 and RH-5849 (LD50 of 42.0 ng and 148.0 ng, respectively). Compound KU-136, inactive in the HT assay at a concentration of 10 µM, showed very little toxicity on silk moth larvae (20% mortality at a dose of 100 µg). The larvicidal tests therefore confirm the potency of KU-106 and KU-121 on whole larvae and validate the reliability of the specific reporter cell system as a high-throughput screening tool for compounds with ecdysone mimetic activity on lepidopteran insects.
HT screening of plant extracts
Extracts of 55 plant species were prepared and added at different concentrations to the reporter cells in 96-well plates. The screen revealed the presence of ecdysone mimetic and potentially antagonistic substances in 13 species belonging to 3 different families. Active extracts from two species, Spinacia oleracea (spinach, agonist) and Citrus aurantium (bitter orange, potential antagonist), were chosen for further analysis.
Additional fractionation indicated the presence of two agonistic and one potentially antagonistic fraction in spinach, of which the most abundant compound (agonist) was identified as 20E by RP-HPLC and mass spectrometry.
As toxic compounds could have an apparent inhibitory effect on RH-5992-induced fluorescence resembling the one caused by true antagonists, C. aurantium extracts were tested by a biochemical assay able to distinguish between general toxic effects on reporter cells and specific inhibitory ones at the level of the 20E receptor. Cells were transfected with the constitutive expression construct pIE1/153A.cat and treated with the C. aurantium extract at a concentration 100-fold higher than the one required for inhibition of RH-5992-induced fluorescence. Reporter enzyme (CAT) assays did not reveal the occurrence of any adverse physiological effects on the cells, suggesting that the antagonist(s) present in C. aurantium extracts is specific for the 20E response.
CONCLUSIONS AND SIGNIFICANCE
The reporter cell lines we developed can be used for initial assessment of multiple samples of complex natural mixtures of natural or synthetic compounds for the presence of compounds with ecdysone mimetic or antagonistic activities.
The results from our cell-based screening system provide reliable indications for the possible applicability of compounds for controlling larval growth in vivo: that dibenzoylhydrazine derivatives KU-106 and KU-121, which induced the appearance of green fluorescence at very low concentrations in our HT assay (Fig. 3A
), displayed a correspondingly high toxicity in whole insect assays (Fig. 3B
), validates use of the reporter cell lines for the identification of new high-performance ecdysteroid mimetics.
Dibenzoylhydrazine derivatives RH-2485, RH-5992, RH-0345, and ANS-118 are already used in insect pest control programs. Given the desirability of identifying compounds with bioactivity for specific insect pests, it will be of interest to determine the efficacy of KU-106 and KU-121 on insect species of different orders.
Our HTS system can also be used to screen complex plant extracts. Increasing interest in the use of plant secondary metabolites for insect pest management (green insecticides) has intensified the search for biologically active natural products with potent insect antifeedant activity, low mammalian toxicity, low persistence in the environment, and minimization of the development of resistance by the insect pest.
Another possible application for the ecdysone-responsive reporter cell lines could be for quantification of active ecdysteroids in biological extracts. The pBmbA/ERE.gfp gene construct in the reporter cell lines is activated by as little as 5 ng of 20E. The sensitivity and specificity of the reporter construct is high enough for 20E concentration measurements as an alternative to radioimmunoassays and bioassays.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0627fje 
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