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AR TA BRD: Appendix A3 October 2002 AR TA Assays Using CV-1 Cells Reference Hartig et al. (2002) Kelce et al. (1995) Characteristics of Cell Line Name of cell line Cell source Transfection of Cells with Plasmids CV-1 Monkey kidney CV-1 Monkey kidney Transient AR/Transient reporter Ad5 hAR human MMTV-Luc Luciferase activity none n.a. Transient AR/Transient reporter pCMVhAR human MMTV.luciferase Luciferase activity none n.a. 24 hours n.p. 4 hours 24 and 48 hours Ethanol 3 to 4 n.p. 48 hours n.p. n.p. At least 4 53 hours 5α-Dihydrotestosterone 0.1 nM n.a. n.a. 5α-Dihydrotestosterone 0.1 nM 5α-Dihydrotestosterone 0.1 nM Stable or transient tranfection AR expression vector AR source Reporter vector Endpoint measured Plasmid transfected for cell toxicity Endpoint measured for cell toxicity Preparation of Cells for Assay Pregrowth of cells before transient transfection Time from transient transfection to treatment of cells Transcriptional activation assay Test substance solvent No. of replicates No. of times assay repeated Test substance incubation time Agonism assay Reference ligand Final concentration of reference ligand Antagonism assay Reference ligand Final concentration of reference ligand Abbreviations: n.a. = not applicable; n.p. = not provided A3-3 AR TA BRD: Appendix A3 October 2002 AR TA Assays Using CV-1 Cells Reference Characteristics of Cell Line Name of cell line Cell source Transfection of Cells with Plasmids Stable or transient tranfection AR expression vector AR source Reporter vector Endpoint measured Plasmid transfected for cell toxicity Endpoint measured for cell toxicity Preparation of Cells for Assay Kemppainen and Wilson Kemppainen et al. (1992) (1996) CV-1 Monkey kidney CV-1 Monkey kidney Transient AR/Transient reporter pCMVhAR human MMTV-Luc Luciferase activity none n.a. Transient AR/Transient reporter pCMVhAR human pMTV29VTM CAT activity none n.a. n.p. 24 hours n.p. n.p. n.p. n.p. At least 3 30 hours n.p. n.p. At least 6 n.p. 5α-Dihydrotestosterone n.p. Methyltrienolone n.p. 5α-Dihydrotestosterone 0.1 nM Methyltrienolone n.p. Pregrowth of cells before transient transfection Time from transient transfection to treatment of cells Transcriptional activation assay Test substance solvent No. of replicates No. of times assay repeated Test substance incubation time Agonism assay Reference ligand Final concentration of reference ligand Antagonism assay Reference ligand Final concentration of reference ligand Abbreviations: n.a. = not applicable; n.p. = not provided A3-4 AR TA BRD: Appendix A3 October 2002 AR TA Assays Using CV-1 Cells Reference Kemppainen et al. (1999) Lambright et al. (2000) Characteristics of Cell Line Name of cell line Cell source Transfection of Cells with Plasmids CV-1 Monkey kidney CV-1 Monkey kidney Transient AR/Transient reporter pCMVhAR human MMTV-Luc Luciferase activity none n.a. Transient AR/Transient reporter pCMVhAR human MMTV-Luc Luciferase activity none n.a. 24 hours n.p. 4 hours 24 and 48 hours n.p. n.p. At least 3 4 hours n.p. n.p. n.p. 5-6 hours 5α-Dihydrotestosterone n.p. n.a. n.a. 5α-Dihydrotestosterone 0.1 nM 5α-Dihydrotestosterone 0.1 nM Stable or transient tranfection AR expression vector AR source Reporter vector Endpoint measured Plasmid transfected for cell toxicity Endpoint measured for cell toxicity Preparation of Cells for Assay Pregrowth of cells before transient transfection Time from transient transfection to treatment of cells Transcriptional activation assay Test substance solvent No. of replicates No. of times assay repeated Test substance incubation time Agonism assay Reference ligand Final concentration of reference ligand Antagonism assay Reference ligand Final concentration of reference ligand Abbreviations: n.a. = not applicable; n.p. = not provided A3-5 AR TA BRD: Appendix A3 October 2002 AR TA Assays Using CV-1 Cells Reference Van Dort et al. (2000) Characteristics of Cell Line Name of cell line Cell source Transfection of Cells with Plasmids CV-1 Monkey kidney Stable or transient tranfection AR expression vector AR source Reporter vector Endpoint measured Plasmid transfected for cell toxicity Endpoint measured for cell toxicity Preparation of Cells for Assay Pregrowth of cells before transient transfection Transient AR/Transient reporter mouse AR (not defined) mouse pSV2-CAT CAT activity none n.a. n.p. Time from transient transfection to treatment of cells Transcriptional activation assay Test substance solvent No. of replicates No. of times assay repeated Test substance incubation time Agonism assay Reference ligand Final concentration of reference ligand Antagonism assay Reference ligand Final concentration of reference ligand Abbreviations: n.a. = not applicable; n.p. = not provided A3-6 n.p. n.p. n.p. 3 n.p. 5α-Dihydrotestosterone n.p. n.a. n.a.
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