Transcriptional activity of UGT1A1 5′ upstream and CDH2 intronic ARBSs. (a) Construction of luciferase reporter plasmids containing UGT1A1 5′ upstream regions. A 519-bp fragment of the 5′ upstream of UGT1A1 (−17 753/−17 235 bp) amplified from LNCaP cells, and the 5′ fragment of UGT1A1 with mutation at the positions −2C and +2G from the 3-bp spacer in ARE sequences (ARBS no. 1) were cloned into pGL3 vector containing SV40 promoter (SV40 Pro), designated as UGT1A1 5′-Luc and UGT1A1 5′ Mut-Luc, respectively. (b) Construction of luciferase reporter plasmid containing CDH2 intron 1. A genomic fragment of CDH2 intron 1 (ARBS no. 10) derived from LNCaP cells (+20 197/+21 260 bp), containing 15 repeats of palindromic ARE sequences with the half-site GGTACA, was cloned into pGL3 vector (CDH2 Int 1-Luc). Note that the number of ARE sequences in LNCaP cells was larger than that in the genome database. (c) Androgen-stimulated luciferase activities of ARE sequences involved in UGT1A1 5′ upstream and CDH2 intron 1. LNCaP cells were stimulated with R1881 (10 nM) or vehicle 12 h after transfection with indicated plasmids together with a Renilla luciferase reporter gene, and incubated for another 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity for each data set. MMTV luciferase reporter gene containing ARE sequences was used as a positive control. Data represent the mean±s.d., n=3. (d) Androgen-stimulated expression of UGT1A protein in LNCaP cells. Cells were stimulated with R1881 (10 nM) or vehicle for indicated times and whole cell lysates were separated by 8% SDS–PAGE. The PVDF membrane blotted with proteins were probed with anti-UGT1A or anti-β-actin antibodies.