Figure 4.

Mutations of the putative ARE site or the Sp1 sites reduce the ability of AR to down-regulate cyclin D1 transcriptional activity. (A) Representation of the putative ARE site location within the cyclin D1 promoter. A list of known high affinity AR-specific ARE sequences is listed in the lower panel. (B) Site-directed mutagenesis was performed as described in ‘Materials and Methods’ section. MCF-7 cells were co-transfected, as reported in ‘Materials and Methods’ section, by using the mutated promoter constructs, schematically represented in (C). Upon transfection, cells were treated with 10⁻⁷ M DHT or left untreated in PRF–CT for 24 h. Firefly luciferase activity was detected and expressed as percentage of Relative Luciferase Activity. Activation of reporter gene in the presence of PRF–CT is arbitrarily set at 100. Results represent the means ± SD of three separate experiments each in triplicate. Data were statistically analysed by Student’s t-test, *P < 0.05 and °P < 0.01 versus control (C); ^□P < 0.01 versus DHT treated D1Δ-944.