Catherine B. Lazier

Androgen-Induced Regrowth in the Castrated Rat Ventral Prostate: Role of 5α-Reductase1

Testosterone (T), the major circulating androgen, must be converted to dihydrotestosterone (DHT) by the enzyme 5α-reductase (5α-R) to be maximally active in the prostate. The present study was designed to determine the relative potency of T and DHT on regrowth of the involuted prostate and to elucidate the role of 5α-R in the growing prostate. To create dose-response curves for intraprostatic T or DHT, rats were castrated for 2 weeks to allow their prostates to fully regress and then given T implants of various sizes in the presence or absence of the 5α-R inhibitor, finasteride. Markers for androgen effects on regrowth of the prostate were prostate weight, duct mass (a measure of secretory activity) and DNA content (a measure of cell number). To assess the relative uptake of T and DHT by the prostate, a comparison was made of intraprostatic DHT levels resulting from T and DHT implants. In the prostate, 1.6–1.9 times more T than DHT was required to achieve a half-maximal response for each of the three mark...

Hepatic estrogen receptors and plasma estrogen-binding activity in the Atlantic Salmon

Livers of male and female immature Atlantic Salmon (Salmo salar) contain specific high-affinity [3H]estradiol binding sites in cytosol (Kd 2-4 nM, concentration about 0.6 pmol/g liver). Low levels of high-affinity binding are detectable in salt extracts of nuclei of untreated fish, but injections of estradiol result in transient depletion of the cytosol binder and in accumulation of high levels of binding sites in nuclear salt extracts (Kd 5-6 nM; concentration about 6 pmol/g liver). Both the cytosol and nuclear binding sites are temperature sensitive and are optimally assayed by incubation at 2 degrees. Both are specific for estradiol and diethylstilbestrol (DES) and no significant competition by dihydrotestosterone (DHT), progesterone, or hydrocortisone is seen. The triphenylethylene nonsteroidal antiestrogen, 4-hydroxytamoxifen, exhibits an affinity comparable to that of estradiol. The nuclear binding activity sediments with a coefficient of 3.6 S in salt-containing sucrose density gradients, and is stable on storage at -20 degrees for several months. The cytosol binder on the other hand is not stable on sucrose density gradients or on prolonged storage. Salmon plasma contains two [3H]estradiol binding components, one with a relatively high affinity for [3H]estradiol (kd 13 nM) and the other having a much lower affinity but present in high concentrations. The high-affinity plasma binder exhibits distinctive specificity with no affinity for DES or 4-hydroxytamoxifen but some affinity for DHT and progesterone. These properties serve to distinguish the plasma activity from the intrahepatic estrogen binders. The salmon liver estrogen receptor system has many features in common with typical estradiol receptors from other vertebrates. Immature salmon liver appears to be the richest source of hepatic estrogen receptor so far found for any vitellogenic species.

Stimulation of estrogen receptor accumulation by estradiol in primary cultures of salmon hepatocytes

Hepatocytes of Salmo salar in primary culture form confluent monolayers and can be maintained at 11 °C in serum‐free medium for 8 days with minimal cell loss. Cultured hepatocytes from immature male salmon contain estrogen receptor both in nuclear and cytosol fractions (2000 and 2400 sites/cell, respectively). A single addition of estradiol results in an increase in the nuclear receptor to a level of 23 000 sites/cell after 24 h. This nuclear receptor concentration is similar to that in liver of estrogen‐treated salmon in vivo, and is much higher than has been found for any other egg‐laying vertebrate. The cultured salmon hepatocytes thus represent a highly sensitive system for the study of estrogen receptor dynamics and vitellogenesis in vitro.

Insulin‐like growth factor binding protein 5 is associated with involution of the ventral prostate in castrated and finasteride‐treated rats

Insulin‐like growth factor binding protein (IGFBP)‐5 has been proposed as a signal for apoptosis in the ovary. To determine the relationship between IGFBP‐5 and apoptosis during regression of the androgen‐deprived prostate, rats were castrated or treated with the 5α‐reductase inhibitor finasteride for 4, 9, 14, 21, and 28 days.

Prostatic involution in men taking finasteride is associated with elevated levels of insulin-like growth factor-binding proteins (IGFBPs)-2, -4, and -5

Insulin‐like growth factor‐binding proteins (IGFBPs)‐2, ‐4, and ‐5 are associated with upregulation of apoptosis in the ovary. The purpose of this study was to assess the roles of IGF‐I and IGFBPs during involution of the prostate. Frozen and fixed tissue was collected by transurethral prostatectomy from Caucasian men, aged 52–82 years, scheduled for prostatectomy for benign prostatic hyperplasia, who took either placebo (n = 7) or the 5α‐reductase inhibitor finasteride for 6 days to 6 years (n = 15) prior to surgery.

The utility of tissue transglutaminase as a marker of apoptosis during treatment and progression of prostate cancer.

PURPOSE To determine the extent of cell proliferation and apoptosis during treatment and progression of prostate cancer and to determine whether staining for tissue transglutaminase is a better histological marker than TUNEL for neoadjuvant androgen ablation treatment of localized prostate cancer. MATERIALS AND METHODS Immunocytochemistry techniques were used on archival prostate tissue from four groups of men: 14 men with BPH, 18 men with untreated, localized prostate cancer, 21 men with localized prostate cancer who received neoadjuvant hormone therapy prior to prostatectomy and 18 men with metastatic androgen-independent prostate cancer. Cell proliferation was evaluated by staining for the Ki67 nuclear antigen, and apoptosis was evaluated by staining for DNA fragmentation (TUNEL technique) and tissue transglutaminase (tTG). Image analysis was used to quantitate the results. RESULTS TUNEL staining increased by 37% in localized prostate cancer compared with BPH, with a further increase of 43% seen after neoadjuvant therapy, although variation was such that neither was statistically significant. In androgen-independent cancer, TUNEL staining was decreased compared with neoadjuvant hormone treated cancer (p = 0.02). Staining for tTG was not increased in untreated prostate cancer compared with BPH; however, staining more than doubled after neoadjuvant therapy, compared with untreated prostate cancer (p = 0.04). Staining for tTG was markedly decreased in androgen-independent cancer (p = 0.07 compared with BPH and p = 0.0004 compared with neoadjuvant hormone treated cancer). Ki67 immunoreactivity did not significantly change in localized prostate cancer, either before or after neoadjuvant therapy, compared with BPH, but it more than doubled in androgen-independent prostate cancer (p = 0.07 compared with BPH and p = 0.05 compared with untreated prostate cancer). CONCLUSIONS This study shows that cell proliferation increases and apoptosis decreases as prostate cancer progresses to androgen independence, and, that of the markers used in this study, tissue transglutaminase most accurately reflects the anticipated effect of neoadjuvant hormone therapy on localized prostate cancer. An assessment of these parameters provides a valuable tool for appraising new prostate cancer therapies.

[3H]-Estradiol binding by chick liver nuclear extracts: mechanism of increase in binding following estradiol injection

Specific high affinity binding of [3H]-estradiol by 0.5 M KCl extracts of chick liver nuclei is substantially increased by estradiol injection of the immature chick. The effect is observed shortly after estradiol injection, while the estradiol-induced production of serum phosphoproteins (vitellogenic response) is not detectable until about 24 hr. Cycloheximide given 90 min before estradiol inhibits the increase in nuclear binding for 12-15 hr. At 24-48 hr the levels of nuclear binding are similar to those in the estradiol-treated animals not given cycloheximide, but serum phosphoprotein levels are depressed by about 80% at 48 hr. By 75 hr however the serum of the cycloheximide-treated estrogenized chicks contains about twice as much phosphoprotein as does serum of chicks given estradiol alone. It is suggested that the inhibition of protein synthesis for 12-15 hr delays the vitellogenic response until sufficient levels of nuclear [3H]-estradiol binding protein can be synthesized. A correlation between the levels of nuclear [3H]-estradiol binding at 24 hr and phosphoprotein at 48 hr is shown in a dose-response experiment. In vitro, nafoxidine-HCl (Upjohn 11,100 A) inhibits binding of [3H]-estradiol by the chick liver nuclear extracts. In vivo, a single injection of nafoxidine with estradiol inhibits phosphoprotein production. Injection of nafoxidine alone results in a small but significant increase in [3H]-estradiol binding by nuclear extracts, but it is not estrogenic. A possible interpretation is that nafoxidine transfers low levels of a putative cytosol receptor to the nucleus, but is unable to induce the amplification mechanism required to give the levels of nuclear estradiol-binding protein needed for the vitellogenic response.

Interactions of tamoxifen in the chicken

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a Ki of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a Ki of 0.1 nM. The Kd of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity (Kd 2-4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogen-receptor-independent interactions of tamoxifen.

The effect of molybdate on the intracellular distribution of estrogen receptor in mammary tumors.

SummaryThe addition of sodium molybdate (20 mM) to human breast tumor homogenates results in an increased yield of estrogen receptor sites in the cytosol, but reduces the number of sites detectable in salt extracts of purified nuclei. Only about 25% of the increase in cytosol receptor can be accounted for by the loss of nuclear sites. Addition of molybdate to isolated nuclear salt extracts has no inhibitory effect on the estrogen receptor assay, but addition of the oxyanion to the cytosol after separation of the initial nuclear pellet still gives a significant increase in estradiol binding capacity. Furthermore, addition of molybdate to the suspension from the first nuclear pellet still results in loss of binding sites in the nuclear salt extract. This rules out the possibility that the negative effect of molybdate on nuclear receptor recovery is by its inhibition of cytosol receptor activation and translocation during tissue preparation.The number of nuclei isolated from estrogen receptor-containing tumors does not differ significantly in the presence or absence of molybdate. Estrogen receptor-negative tumors however yield fewer nuclei, particularly when they are processed in the presence of molybdate.We conclude that when homogenization of tumors is carried out in the presence of molybdate, a significant fraction of estrogen receptor which would normally be present in purified nuclei is lost, perhaps by leakage through the nuclear membrane.

Expression of the albumin gene in the yolk sac and liver during chick embryogenesis

1. Albumin mRNA is first detectable in vascular yolk sac on the third day of egg incubation, increases to peak level on day 14 and declines to zero by day 19. 2. Vascular yolk sac RNA contains a 6-10-fold higher level of albumin transcripts compared to non-vascularized yolk sac, suggesting a role for vascularization in promoting albumin gene expression. 3. Embryonic liver albumin transcripts are first detectable at day 6.5, increase 6-fold by day 8, continue to rise at a lower rate until day 14 and remain constant thereafter. 4. Albumin protein synthesis in liver cubes also exhibits a large increase over days 7-10. In contrast, another liver-specific constitutive protein, apolipoprotein B, shows a different biosynthetic pattern. 5. The data suggest development of hepatic albumin gene-specific regulatory factors over days 7-10.