Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor.

To assess the function of androgen receptor in androgen-independent prostate cancer cells, human PC-3 prostate carcinoma cells, which lack androgen receptor (AR) expression, were transfected with a full length human AR cDNA sequence inserted into an episomal expression vector system. Several clonal lines of transfected cells expressing varying levels of a 110 kDa AR, as determined by immunoblotting and ligand binding assay, were isolated. The expressed ectopic receptors displayed nuclear binding following androgen treatment and mediated androgen inducibility of a mouse mammary tumor virus (MMTV)-luciferase reporter gene construct in a dose-dependent manner. 5 alpha-dihydrotestosterone (DHT) activation of luciferase activity was blocked by the AR antagonist hydroxyflutamide, and was promoter-specific based on the inability of the hormone-insensitive RSV promoter to respond to DHT. Treatment of AR-expressing PC-3 cells with physiological levels of DHT for 3 days resulted in paradoxical inhibition of cell growth. The growth-inhibitory effect was observed in clonal lines expressing low, moderate and high levels of AR, indicating that it was not the result of AR overexpression. To determine whether AR-expressing PC-3 cells had become androgen dependent, albeit with slowed growth, the effect of 1.0 nM DHT on the growth of two clonal lines expressing low and moderate receptor levels (PC-3(AR)13 and PC-3(AR)2, respectively) was examined on over an 18 day period. DHT removed after 3, 6, or 9 days and replaced with steroid-free medium. Surprisingly, after 6 days of DHT treatment, the number of PC-3(AR)2 cells began to decrease such that all cells were dead by 15 days after initiation of DHT treatment. A similar effect was observed in PC-3(AR)13 cells, but required a longer initial period of DHT exposure. PC-3(AR)2 cells were rescued from cell death if DHT was withdrawn 3 days but not 6 or 9 days after initiation of DHT treatment. As determined by DNA cell cycle analysis, the proportion of cells in the G1 phase was enhanced by DHT treatment, accompanied by a decrease in cells in the S and G2M phase of the cell cycle. After 6 days of DHT treatment, the proportion of cells in G1 decreased which was accompanied by an increase in cells in a subG1 population consistent with apoptosis. DNA fragmentation in PC-3(AR)2 cells after 3 or 6 days of DHT treatment was demonstrated by agarose gel electrophoresis, further indicating the cell death was apoptotic. Removal of DHT from PC-3(AR)2 cultures after 3 days, but not after 6 or 9 days, was followed by a large shift in cells from G1 to S and G2M. These data suggest that DHT blocks the progression of AR transfected PC-3 cells through the cell cycle, resulting in growth inhibition and apoptosis.

In vitro labeling of gonadal steroid hormone receptors in brain tissue sections.

Autoradiographic methods have been developed for measurement of gonadal steroid receptors in situ in brain tissue sections. Based on principles established previously for estrogen receptors in the rat brain using a 125I-labeled ligand, procedures have been developed for in vitro labeling of estrogen, androgen, and progestin receptors with commercially available tritiated ligands. Addition of protamine sulfate to the incubation buffer precipitates the receptors in situ in the tissue sections, allowing them to be detected autoradiographically after incubation with labeled steroid and subsequent washing to remove unbound and nonspecifically bound ligand. Occupied and unoccupied estrogen receptors can be measured selectively using appropriately modified incubation conditions. In the case of androgen and progestin receptors, unoccupied receptors are readily detected by in vitro labeling of tissue sections, but occupied receptors do not appear to label efficiently. Preliminary data suggest that these methods should be equally applicable to a variety of laboratory animals, including the rat, mouse, guinea pig, and monkey.

Vasopressin mediates hypoglycemia-induced inhibition of luteinizing hormone secretion in the ovariectomized rhesus monkey.

The objective of the present study was to examine the role of vasopressin in the regulation of LH secretion in the rhesus monkey. The effect of vasopressin administration on basal LH secretion and vasopressin antagonism on stress-induced inhibition of LH secretion were examined. Intracerebroventricular (i.c.v.) infusion of vasopressin (20 micrograms/h) to chair restrained ovariectomized rhesus monkeys (n = 5) decreased the area under the LH curve by -51.61 +/- 13.73 ng/ml/h compared to -8.35 +/- 7.11 ng/ml/h following infusion of artificial CSF (aCSF; p = 0.021). This effect was independent of any change in mean arterial pressure. Subsequently, the role of vasopressin in hypoglycemia-induced suppression of LH was examined. Administration of insulin (1 U/kg BW) to chair-restrained ovariectomized rhesus monkeys decreased the area under the LH curve by -60.88 +/- 19.77 ng/ml/h. The decrease in LH was significantly different from that observed in aCSF-infused euglycemic controls which exhibited a slight decrease in LH (-8.35 +/- 7.11 ng/ml/h; p = 0.036). In contrast, the area under the LH curve was increased slightly (1.42 +/- 11.93 ng/ml/h) when insulin administration was combined with i.c.v. infusion of the vasopressin antagonist [deaminopenicillamine1, O-methyl-tyrosine2, arginine8]-vasopressin (120 micrograms/h; p = 0.013 vs. insulin only). The demonstration that vasopressin administration inhibits LH secretion whereas vasopressin antagonism prevents hypoglycemia-induced LH suppression suggests that vasopressin is a physiological inhibitor of LH secretion in the rhesus monkey.

Hypoglycemia-induced inhibition of luteinizing hormone secretion in the rhesus monkey is not mediated by endogenous opioid peptides.

A role for endogenous opioid peptides in stress-induced inhibition of LH secretion has been suggested based on the observation in rats, humans, and nonhuman primates that LH inhibition in response to a variety of different stresses could be blocked by the administration of opiate antagonists. In the present study, we have examined in rhesus monkeys whether suppression of LH secretion by insulin-induced hypoglycemia is prevented by administration of the opiate antagonist naloxone. The administration of 1.0 U insulin/kg to chair-restrained ovariectomized monkeys (n = 6) decreased blood glucose levels from 4.98 +/- 0.17 to 2.08 +/- 0.05 mmol/L and increased cortisol levels from 1279 +/- 205 to 2191 +/- 475 nmol/L. LH levels declined to 62% of the levels observed in the pretreatment control period (P < 0.05). Infusion of naloxone (2-mg bolus plus 2 mg/h or 10-mg bolus plus 10 mg/h) did not reverse the effects of insulin-induced hypoglycemia on LH concentrations. The administration of 1.0 U insulin/kg to nonrestrained monkeys produced a similar hypoglycemic state. Blood glucose levels declined from 4.08 +/- 0.11 to 2.45 +/- 0.05 mmol/L, while cortisol concentrations increased from 577 +/- 53 to 1324 +/- 294 mmol/L. However, LH concentrations did not decline in response to hypoglycemia. These data indicate that hypoglycemia-induced inhibition of LH secretion in chair-restrained ovariectomized monkeys is not mediated by endogenous opiates, since naloxone failed to reverse this effect. The observation that hypoglycemia inhibited LH levels only during a period of restraint suggests either an additive or synergistic effect of these two stresses on LH secretion.

Progesterone inhibits the estrogen-induced gonadotropin surge in the rhesus monkey independent of endogenous opiates.

Administration of an estrogen challenge during the luteal phase, a time when progesterone concentrations are elevated, fails to elicit a gonadotropin-positive feedback response. The purpose of the present study was to determine if endogenous opiates are involved in the mechanism by which progesterone blocks the estrogen-induced gonadotropin surge in monkeys. To this end, rhesus monkeys in the luteal phase were pretreated with either saline or various regimens of nalmefene, a long-acting opiate antagonist, before being given an estrogen challenge. Three groups of animals were given nalmefene (10 mg, iv) every 12 h beginning 24, 48, or 96 h before an estrogen challenge and continued until 48 h after the start of the estrogen challenge. A fourth group received a continuous sc infusion of nalmefene (20 mg/day) via osmotic minipumps beginning 48 h in advance of the estrogen challenge. In a second experiment, monkeys in the follicular phase received progesterone implants at the time of an estrogen challenge and iv injections of nalmefene every 12 h for 48 h. Gonadotropin and steroid levels were monitored in both experiments by collecting blood samples by saphenous venipuncture at intervals of 6-12 h. The majority of luteal phase animals that were pretreated with saline were unresponsive to the estrogen challenge. Only 2 of 16 (12.5%) had an increase in LH concentrations that could be classified as a surge. Animals pretreated with iv nalmefene every 12 h beginning 48 h before the estrogen challenge exhibited a higher incidence of positive feedback responses (8 of 12 or 66.7%). A concomitant FSH surge was observed in 3 of these instances. However, when progesterone concentrations, which declined before the estrogen challenge in the nalmefene-treated group, were supplemented with exogenous progesterone, nalmefene failed to evoke any LH surges. Six of 8 animals that received nalmefene by sc infusion exhibited LH responses. However, the amplitude and duration of these LH responses were diminished, and no FSH responses were observed. Monkeys pretreated with nalmefene for either shorter (24 h) or longer (96 h) periods before the challenge were less responsive (0 responses out of 6 trials and 1 response out of 4 trials, respectively). Nalmefene was equally ineffective in preventing progesterone inhibition of the estradiol-induced LH surge in follicular phase animals (0 of 15 animals had LH surge). These results indicate that nalmefene antagonism of endogenous opiates does not enable estrogen to exert positive feedback effects on LH release when progesterone levels are high, such as during the luteal phase or after progesterone administration.(ABSTRACT TRUNCATED AT 400 WORDS)