Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

Abstract

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.

Radioimmunoassay measurements of nuclear dihydrotestosterone in rat prostate. Relationship to androgen receptors and androgen-regulated responses

“…Although proof of the participation of androgen receptors in the direct control of cell proliferation, 5a-reductase activity and secretory acid phosphatase activity is contingent on a fuller understanding of the interaction of androgen receptors with chromatin acceptor sites (Davies & Griffiths, 1973;Mainwaring et al, 1976;Rennie, 1979), the results obtained in the present investigation favour the hypothesis that androgen receptors, rather than non-receptor-bound nuclear androgen, modulate cell proliferation and 5a-reductase activity. Since the association constant (Ka) for the binding of dihydrotestosterone to chromatin-bound receptor sites is of the order of 5 x 107M-1 (Rennie, 1979), and since the nuclear concentration of receptor sites is far lower than the concentration of dihydrotestosterone, the most obvious function of the excess nuclear dihydrotestosterone is to ensure that all the receptor sites within the nucleus are occupied with androgen.…”

“…Results from experiments performed in vitro by several groups (Davies & Griffiths, 1973;Mainwaring & Jones, 1975;Hu et al, 1975) have indicated that androgen-receptor complexes directly modulate the transcription of chromatin by RNA polymerase. Androgen receptors have also been implicated in the regulation of DNA synthesis and cell proliferation in the regenerating prostate (Coffey, 1974;Rennie et al, 1975;Van Doorn et al, 1976).…”

Nuclear components responsible for the retention of steroid–receptor complexes, especially from the standpoint of the specifcity of hormonal responses

“…Such a series of initial experiments is summarized in Fig. 1 (Mainwaring & Peterken, 1971;Davies & Griffiths, 1974). Degraded receptor, after treatment with N-ethylmaleimide (Mainwaring, 1969b), is totally inactive.…”

Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues.

“…To examine differences in physiological properties of receptor complexes which showed different characteristics of the nuclear binding, stimulation of RNA-polymerase in the rat ventral prostate by the DHT-prostate receptor complex was studied. However, Fig.3 this attempt was not successful, since, in our hand, no significant stimulation of RNApolymerase in the rat ventral prostate was obtained even with the DHT-prostate receptor complex which was not pretreated with heterogenous nuclei under the similar incubation conditions described by Davies and Griffiths (1973) (data not shown). Comparison of other properties of the receptor complex "absorbed" by heterogenous nuclei with those of the "unabsorbed" one in liver and prostate is under examination in this laboratory.…”