Altered androgen regulation of differentiated ventral prostate function during aging of the AXC rat.

We have examined testosterone regulation of differentiated androgen responsive ventral prostate function in aging AXC rats as a possible means of identifying age-related changes in prostate gene function. Concerning cellular content of functional macromolecules, we identified three categories of age-related change in ventral prostate: (1) diminutions completely reversible by short-term chronic testosterone treatment, as exemplified by androgen receptor content; (2) diminutions partially reversible by testosterone treatment, as exemplified by prolactin receptor content and L-ornithine-decarboxylase (ODC) activity; and (3) diminutions not reversed by exogenous testosterone treatment, as exemplified by S-adenosyl-L-methionine decarboxylase (AMDC) activity. A fourth category of alteration revealed apparent age-related changes in polyamine homeostasis. The anabolic products of ODC and AMDC activities, putrescine, spermidine and spermine, showed an age-related diminished dependence between the activities of ODC and AMDC and ventral prostate polyamine content. The possibility that these changes reflect age-related alterations in ventral prostate gene activity is under investigation.

Creatine kinase and lactate dehydrogenase: stability of isoenzymes and their activity in stored human plasma and prostatic tissue extracts and effect of sample dilution.

We examined the effect of storing human plasma or extracts of prostate at -90 degrees C on the activity of creatine kinase and lactate dehydrogenase and isoenzyme distribution. Enzyme activities were unaltered during storage for as long as six weeks. If these preparations were thawed only once at 2 to 4 degrees C, they could be stored for as long as 165 days at -90 degrees C with no change in isoenzyme distribution. Inexplicably, apparent isoenzyme distribution of prostatic lactate dehydrogenase was sensitive to sample dilution, whereas the isoenzyme distribution of lactate dehydrogenase in plasma was not. Our observations emphasize the importance of validating details of analytical protocols that are to be used for quantification of new types of specimens.

Human prostate androgen receptor quantitation: effects of temperature on assay parameters.

When cytoplasmic extracts of human prostatic tissues were split to permit quantitation of total androgen receptor (RCT) content by saturation analysis at 15 degrees and 2 degrees, we observed that 30% (10 of 32) of the specimens yielded statistically increased values for RCT following incubation at 15 degrees as compared to 2 degrees. Considering only those specimens (13 of 32) showing statistically differentiated RCT yield, 77% (10 of 13) yielded greater RCT content following incubation at 15 degrees. The families of association constants (Ka) obtained for RCT determinations at 2 degrees and 15 degrees were not statistically differentiated. The increased yield of RCT content determined at 15 degrees was 95% (mean) and 20 to 350% (range). Nuclear androgen receptor content determined at 15 degrees was greater for 25% (2 of 8) of the patient specimens when compared to split determinations performed at 2 degrees. Incubation of nuclear extracts at 15 degrees resulted in a significant 3-fold reduction in receptor Ka for methyltrienolone (R1881). This did not appear to affect assay precision. These studies showed that incubation at 15 degrees is preferable to incubation at 2 degrees for quantitation of RCT and nuclear androgen receptor content by saturation analysis. Single saturating dose determinations of RCT consistently yielded underestimates. The extent of underestimate was variable from specimen to specimen and was both ligand concentration and assay temperature dependent. Our data suggest that results of single saturating dose determinations of RCT require cautious interpretation.

A x C rat prostate adenocarcinoma: initial characterization of testosterone regulation of hormone receptors of cultured cancer cells and derived tumors.

A x C rat prostate cancer cells were established in continuous culture. The polygonal epithelial cells had granular cytoplasm and well-defined cell margins, contained round to oval nuclei with prominent nucleoli, and were tumorigenic when inoculated into A x C male rats. The tumors produced by the injected prostate cancer cells grew as well-vascularized, solid, cribriform adenocarcinomas. The rat prostate cancer cells and derived tumors contained cytoplasmic and nuclear androgen receptors and prolactin receptors. Androgen regulation of prolactin receptor content and androgen receptor distribution in A x C rat prostate cancer cells were comparable to those of the normal ventral prostate gland. These studies suggest that the A x C rat prostate cancer cells and tumors may represent a unique model for studies of hormonal regulation of prostate cancer cell behavior.

AXC rat prostatic adenocarcinoma: characterization of cells in culture.

We have described the establishment of AXC rat prostatic cancer cells in continuous culture. When injected into isogeneic male rats, these cells produce prostatic adenocarcinomas. The response of androgen and prolactin receptors and ODC in LSC-AXC prostatic cancer cells and tumors to androgen ablation is indistinguishable from that of ventral prostate. In addition, LSC-AXC prostatic tumors retain levels of secretory acid phosphatase comparable to those of ventral prostate of aged AXC rats. These data demonstrate that LSC-AXC prostatic cancer cells and tumors retain a high degree of differentiation, androgen regulated function. The LSC-AXC prostatic cancer cells and tumors appear to represent a unique model system for combined in vivo and in vitro studies of androgen regulation of prostate cancer cell function.

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats.

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730-740 day old) rats is decreased 50% when compared to intact, young mature (150-170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 microgram testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate.

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.

Androgen receptor content of the normal and hyperplastic canine prostate.

A procedure was developed for measurement of androgen receptors in cytoplasmic extracts of prostates from intact dogs. The protocol utilized exchange saturation analysis at 15 degrees C employing the synthetic androgen R1881 (17beta-hydroxy-17alpha-methylestra-4,9,11-trien-3-one) as the ligand probe and quantitatively detected total cytoplasmic androgen receptor (R(c), androgen-free receptor, and R(c)A, androgen-occupied receptor) present at the initiation of the assay. This protocol was employed in conjunction with a tissue mince saturation analysis procedure (for quantitation of nuclear androgen receptor) to quantitate total androgen receptor content of normal and hyperplastic prostates obtained from young (2.5- or 4.6-yr old) and aged (12.5-yr old) purebred dogs of known birth date. The total cytoplasmic androgen receptor content (picomoles per prostate) of hyperplastic prostates was 4.6-fold greater than that of normal prostates. The total nuclear androgen receptor content of hyperplastic prostates (picomoles per prostate measured in crude nuclear preparations) was either 5.0- (4.6-yr-old dogs) or 7.8-fold (2.5-yr-old dogs) greater than that of normal prostates. However, androgen receptor content per cell was identical for hyperplastic and normal canine prostates, with the exception that nuclear androgen receptor was diminished in prostates from 2.5-yr-old dogs. The cell content per gram dry weight was identical for hyperplastic and normal canine prostates. We conclude that canine prostate hyperplasia is characterized by coordinate proliferation of androgen receptor-positive and androgen receptor-negative cells and is not a consequence of increased accumulation of 5alpha-dihydrotestosterone due to proliferation of androgen receptors per prostate cell.