Bridization [18]. Data analysis was performed with CisGenome software [19]. TC-AR APO866 biological activity binding regions were identified by comparison to total input control as well as IgG control using the TileMap peak detection tool [20]. Genomic locations of binding peaks were visualized in the CisGenome browser.not observed indicating that TC-AR does not form a heterodimer with FL-AR in the LN/TC-AR cell line.TC-AR is transciptionally active in the absence of DHTIn order to examine the ability of TC-AR to facilitate transcription at an AR-regulated promoter, a luciferase assay using the full-length PSA promoter was completed. Immediately following co-transfection of pPSA6.0-luc and pH 48-ren reporter plasmids, expression of TC-AR in LN/TC-AR was induced with various concentrations of doxycycline. Transfected, but uninduced, LN/TC-AR cells treated with either 1.0 nM DHT or vehicle (EtOH) serve as positive and negative controls, respectively. Luciferase FGF-401 site production (dependent upon activity of the upstream PSA promoter) was found to be significantly increased in all doxycycline-treated samples relative to untreated control (Figure 2A). Furthermore, transcriptional activity measured for each of the TC-AR expressing samples was three to seven fold higher than that found in the uninduced DHT-treated control in which luciferase production is controlled solely by DHT-bound endogenous AR.Results Titration of doxycycline induction yields a physiologically relevant level of TC-AR expression in the newly established LN/TC-AR cell lineLN/TC-AR is a newly developed cell line derived from the parental LNCaP line in which a truncated form of the androgen receptor (TC-AR) is expressed following doxycycline induction (Figure 1B). Titration of doxycycline levels showed that TC-AR expression was maximal when cells were cultured in complete media supplemented with 10 ng/mL doxycycline (data not shown). A second, more focused titration showed that a physiologically relevant level of TC-AR 1676428 expression (as defined here by similarity to AR expression in the CWR22Rv1 cell line) was achieved when cells were cultured in complete media supplemented with 4.5 ng/mL doxycycline (Figure 1C). In subsequent studies involving this cell line, induction of TC-AR with 4.5 ng/mL doxycycline (Low Dox) is used to approximate physiological levels of expression while increased doxycycline concentrations (High Dox) are used to induce “overexpression” of TC-AR.TC-AR localizes to the nucleus and is able to bind androgen response elements (AREs) in chromatin in the absence of DHTIn order to observe localization of TC-AR, immunostaining of LN/TC-AR was completed. Contrary to endogenous AR which has been shown to remain in the cytoplasm in the absence of DHT, TC-AR localized predominantly to the nucleus following induction with Low Dox (Figure 2B). Chromatin immunoprecipitation (ChIP) assay was performed to assess binding of TC-AR to the AR-regulated KLK3 promoter (Figure 2C). Occupancy of the KLK3 promoter by TC-AR following doxycycline induction of LN/TC-AR cells was observed. Unlike wild-type AR, DHT was not required for the binding of TC-AR to the KLK3 promoter [17]. RNA polymerase II was also found at the KLK3 promoter thus demonstrating the transcriptional activation of an endogenous androgen regulated gene by TC-AR in the 1662274 absence of DHT.Induction of exogenous AR causes a concomitant decrease in endogenous AR protein and mRNA levelsImmediately apparent in the doxycycline titrations is the inverse r.Bridization [18]. Data analysis was performed with CisGenome software [19]. TC-AR binding regions were identified by comparison to total input control as well as IgG control using the TileMap peak detection tool [20]. Genomic locations of binding peaks were visualized in the CisGenome browser.not observed indicating that TC-AR does not form a heterodimer with FL-AR in the LN/TC-AR cell line.TC-AR is transciptionally active in the absence of DHTIn order to examine the ability of TC-AR to facilitate transcription at an AR-regulated promoter, a luciferase assay using the full-length PSA promoter was completed. Immediately following co-transfection of pPSA6.0-luc and pH 48-ren reporter plasmids, expression of TC-AR in LN/TC-AR was induced with various concentrations of doxycycline. Transfected, but uninduced, LN/TC-AR cells treated with either 1.0 nM DHT or vehicle (EtOH) serve as positive and negative controls, respectively. Luciferase production (dependent upon activity of the upstream PSA promoter) was found to be significantly increased in all doxycycline-treated samples relative to untreated control (Figure 2A). Furthermore, transcriptional activity measured for each of the TC-AR expressing samples was three to seven fold higher than that found in the uninduced DHT-treated control in which luciferase production is controlled solely by DHT-bound endogenous AR.Results Titration of doxycycline induction yields a physiologically relevant level of TC-AR expression in the newly established LN/TC-AR cell lineLN/TC-AR is a newly developed cell line derived from the parental LNCaP line in which a truncated form of the androgen receptor (TC-AR) is expressed following doxycycline induction (Figure 1B). Titration of doxycycline levels showed that TC-AR expression was maximal when cells were cultured in complete media supplemented with 10 ng/mL doxycycline (data not shown). A second, more focused titration showed that a physiologically relevant level of TC-AR 1676428 expression (as defined here by similarity to AR expression in the CWR22Rv1 cell line) was achieved when cells were cultured in complete media supplemented with 4.5 ng/mL doxycycline (Figure 1C). In subsequent studies involving this cell line, induction of TC-AR with 4.5 ng/mL doxycycline (Low Dox) is used to approximate physiological levels of expression while increased doxycycline concentrations (High Dox) are used to induce “overexpression” of TC-AR.TC-AR localizes to the nucleus and is able to bind androgen response elements (AREs) in chromatin in the absence of DHTIn order to observe localization of TC-AR, immunostaining of LN/TC-AR was completed. Contrary to endogenous AR which has been shown to remain in the cytoplasm in the absence of DHT, TC-AR localized predominantly to the nucleus following induction with Low Dox (Figure 2B). Chromatin immunoprecipitation (ChIP) assay was performed to assess binding of TC-AR to the AR-regulated KLK3 promoter (Figure 2C). Occupancy of the KLK3 promoter by TC-AR following doxycycline induction of LN/TC-AR cells was observed. Unlike wild-type AR, DHT was not required for the binding of TC-AR to the KLK3 promoter [17]. RNA polymerase II was also found at the KLK3 promoter thus demonstrating the transcriptional activation of an endogenous androgen regulated gene by TC-AR in the 1662274 absence of DHT.Induction of exogenous AR causes a concomitant decrease in endogenous AR protein and mRNA levelsImmediately apparent in the doxycycline titrations is the inverse r.
