| Androgen-independent growth of prostate cancer(PC) cells is associated with androgen receptor (AR) abnormalities. These include amplification and overexpression of the AR gene, disregulation of AR coregulators, activation of AR in a ligand-independent manner and AR mutation. Because mutations of the AR gene can alter receptor function, which may provide a growth advantage to PC cells expressing the mutants, determining the function of these mutant ARs is important for understanding their role in the development of androgen-independent PC.Previous years in our laboratory, we have identified five missense mutations(G142V, D221H, S296R, E872Q and M886I) in AR gene from nearly fifty PC samples by PCR-SSCP analysis. Dr Chen Guangchun has studied the transcriptional activity of four of the five mutants(G142V, D221H, E872Q and M886I) in CV-1 cells and found that compared with wild type AR(wtAR), the G142V and D221H variants demonstrated abnormally increased activities in response to DHT, and E872Q variant was more responsive to CPA, estradiol and progesterone. The effect of S296R on the structure and function of AR has not been studied.To investigate the AR mutant function, the S296R mutant was firstly constructed into an expression vector containing the human AR cDNA using overlap extension in vitro. The wtAR and S296R constructs were introduced into COS-7 cells by the calcium phosphate coprecipitation technique and then the cells were assayed by Western blot. The result showed the AR proteins could be clearly seen as a doublet of 112 and 110 kDa when visualized by a polyclonal antibody to the ARs, which meant this mutant could be expressed successfully and correctly. On this basis, the Scatchard assay was performed in the transfected COS-7 cells bywhole-cell ligand binding assay of wtAR and AR mutant S296R. The result showed similar equilibrium dissociation constants (Kd) and maximal binding capacity (5max) for wtAR and S296R, which meant the S296R mutation did not alter the binding capacity of AR to androgen.As a member of the nuclear receptor superfamily, AR is a ligand-dependent transcription factor which regulates the expression of target genes through binding to an androgen response element(ARE). Then we used reporter gene pMMTV-LUC to investigate whether the transcriptional activity of the AR mutant S296R had changed in CV-1 cells. In pMMTV-LUC, the reporter LUC gene is driven by a promoter consisting of mouse mammary tumor virus long terminal repeat called LTR in which there are AREs in front of a TATA box. So the LUC expression is driven by AR. CV-1 cells were transient cotransfected by Lipofectamine?2000 with the reporter pMMTV-LUC , pRLSV40-LUC and AR expression vector. Maximal stimulation of LUC activity was achieved by 2×10-9mol/L of dihydrotestosterone(DHT) in the presence of either normal or mutant AR. Compared with wtAR, S296R showed no obvious change in transactivity in response to DHT at 2×10-9mol/L and antiandrogen CPA at 2×10-6mol/L but was more responsive to estradiol(2×10-6mol/L) and progesterone(2-10-9mol/L), which indicated alteration of the ligand specificity or affinity of S296R.Ligand binding specificity or affinity does not alone reflect the functional capability of the AR. Recently, the "ligand-receptor-cofactors" tripartite system has been proposed to explain the molecular interactions of steroid receptors. It has become clear that the transcriptional activity of AR, as well as other members of the nuclear receptor superfamily, is modulated by coregulators. Coregulators are broadly defined as proteins that interact with nuclear receptors to enhance transactivation (coactivators) or reduce transactivation (corepressors) of target genes but do not significantly alter the basal transcription rate. In fact, amplification and/or overexpression of steroid hormone receptor cofactors, such as TIF-2,SRC-1 and ARA70, have been reported recently in PC. Hence, concomitant changes in coactivators or corepressors in addition to AR are also likely to be important molecular e... |
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