The aim of the studies described in the next chapters was, first, to prove that the AR in LNCaP cells is abnormal with respect to ligand binding characteristics, and to find an explanation for this defect. Second, it was investigated whether this aberration could account for the growth stimulating effects of antiandrogens on this cell line. Third, the effects of both androgens and antiandrogens were investigated at the biochemical level, with much emphasis on receptor interactions with other (heat-shock) proteins. Chapter 2 describes investigations to study the binding affmities of several steroidal and non-steroidal ligands for the AR in LNCaP cells. These binding affinities were compared with the binding affmities for the AR from other sources, including cells expressing wild type AR (Chang eta!., 1988; Lubalm eta!., 1988; Trapman eta!., 1988; Faber et a!., 1989). From studies with nuclear preparations, devoid of cytoplasmic contantinations, it was concluded that the binding affinity of the AR in LNCaP cells was abnormal. The third chapter describes that the AR gene in LNCaP cells contains a mutation. The expression ofthe mutant receptor in LNCaP cells was confirmed by eDNA sequence analysis. In transfection studies, the binding specificity of the mutant receptor was compared with the binding specificity of the wild type receptor expressed in the same cell type. Also the ability of both the mutant and wild type receptor to activate transcription from an AR responsive construct in response to androgens, antiandrogens, progestins and estrogens was investigated. One antiandrogen, ICI 176 334 ("casodex", a trade mark of!CI Pharmaceuticals), was found which could not stimulate growth of LNCaP cells, but inhibited the effect mediated by androgens (Chapter 4). It was investigated whether there is a difference between antiandrogens such as hydroxyflutamide, which induce growth of LNCaP cells, other hand. The ability of these compounds to provoke a dissociation of the AR-heatshock protein-complex was studied. In addition, it was investigated whether the three heat-shock proteins hsp90, hsp70, and hsp56 could be detected in the heteromeric complexes. In Chapter 5 of this thesis, the effects of incubation of LNCaP cells with androgens on the AR-heat-shock protein-complex is described. Both changes in complex-size and composition, and changes in affinity of the receptor for the nucleus were analyzed. In addition, the development of an antibody against part of the DNA-binding domain of the AR is described. This antibody was used to examine whether its epitope was exposed on the surface of untransformed and transformed ARs. It was also tested whether this antibody could be used to specifically precipitate wholly or partially transformed receptors. Finally, in Chapter 6, the results from the former chapters are discussed in a broader context. The effects of the mutation in the AR of LNCaP cells on results obtained with estrogens, progestins and antiandrogens are discussed. The possible role of the different heat-shock proteins in receptor transformation is considered. Suggestions are made for future investigations

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J.A. Grootegoed (Anton)
Erasmus University Rotterdam
hdl.handle.net/1765/39464
Erasmus MC: University Medical Center Rotterdam

Veldscholte, J. (1993, April 21). Mechanisms of action of androgen receptor agonists and antagonists. Retrieved from http://hdl.handle.net/1765/39464