Studies on Androgen Receptor Mutations and Amplification in Human Prostate Cancer

Prostate cancer is a heterogenous neoplasm that is palliatively treated with androgen ablation therapy. Endocrine therapy aims to block the androgen receptor (AR), which has a central role in the transmission of extracellular signals and is therefore responsible for regulating proliferation and secretion. AR is expressed in all stages of prostate cancer and, therefore, the regulation of its expression and function are of great interest (1 ,2 ). AR point mutation was first described in the prostate cancer cell line LNCaP (3 ). This mutation leads to an enhanced activation of the AR by various steroidal and nonsteroidal AR antagonists. The presence of AR mutations was analyzed in the tissues of prostate cancer patients, and the protocols described in this chapter are designed to allow detection and functional analysis of these mutations (for review, see ref. 4 ). This chapter describes methods for analysis of AR structure in tissue samples; RNA isolation, synthesis of cDNA, polymerase chain reaction (PCR) amplification, and sequencing, as well as those needed for analysis of function of mutated receptors; and transient transfection, ligand-binding assay, and reporter gene assay.