01 July 2006
Androgen binding site in J111 cell line.Kazem Ahmadi, Alan B. McCruden
Med Sci Monit 2006; 12(7): BR239-244 :: ID: 452196
Background: The finding of sex steroid receptor protein in non classicalreproductive tissues suggested the possibility that sex steroids may have a relevance to the immune system.Material/Methods: The J111 cells were maintained in RPMI 1640 complete medium at 37 degrees C in 5% CO[sub]2[/sub] in air. Cells were resuspended at 1x10[sup]6[/sup] cells in 0.2 ml complete medium in 1.5 ml eppendorf tubes.A single saturating concentration 1x10[sup]-9[/sup] M of [[sup]3[/sup]H]5alpha-DHT was added to the cells suspension. Unlabelledsteroids (5alpha-DHT, 17-beta estradiol, or the synthetic glucocorticoid triamcinolone acetonid) wereadded over the range 1x10[sup]-8[/sup] to 1x10[sup]-9[/sup] M. Duplicate tubes were incubated at 37 degrees C for 1h. Forautoradiography, the supernatant was discarded and the pellet resuspended in 0.2 ml medium. For bindingassay, Labeled cells were separated from unbound steroid by immunomagnetic bead using anti-CD68 antibody.Results: In autoradiography, a population of approximately 96% of J111 cells that contain receptors forandrogen has been demonstrated. The results of immunomagnetic showed that binding identified in the J111cells was modest selective towards androgenic compounds. Schatchard analysis of data showed the KD valueof 2.5x10[sup]-9[/sup] M and the number of receptor in each cell was found to be 257+/-1. Little competition wasseen from 17 beta estradiol or the synthetic glucocorticoid triamcinolone acetonid. Conclusions: Thesedata indicate that androgen binding in J111 cells is of modest affinity and specific, due to the inabilityof 100-fold molar excess of estradiol to displace bound [[sup]3[/sup]H]-5alphaDHT.
Keywords: Androgens - metabolism, 3-Oxo-5-alpha-Steroid 4-Dehydrogenase - metabolism, Autoradiography, Binding Sites, Estradiol - metabolism, Triamcinolone Acetonide - metabolism
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