Induction of tamoxifen-dependent rat mammary tumors.

We have observed previously that animals given tamoxifen (TAM) and the carcinogen dimethylbenzanthracene develop exclusively hormone-independent tumors. Since our data implied that the TAM-associated tumors were different from control tumors, we undertook studies to examine the role of TAM in the induction and growth of these tumors. Following cessation of TAM administration, almost one-third [29.0 +/- 1.7% (SEM)] of the tumors regressed and more tumors appeared. Resumption of TAM administration resulted in regrowth of some tumors and regression of the new tumors. These studies demonstrate that some of the TAM-associated tumors are actually dependent upon TAM for growth, while the appearance of new tumors suggests that TAM does not totally prevent tumor formation but may only delay it.

Role of tamoxifen in the induction of hormone-independent rat mammary tumors.

Using the dimethylbenzanthracene-induced rat mammary tumor model, we examined the appearance and growth of tumors in animals given tamoxifen (TAM) either coincident with dimethylbenzanthracene or after initial tumor formation. While fewer tumors arose after coadministration of antiestrogen and carcinogen and TAM treatment caused regression of most existing tumors, new tumors developed in the presence of TAM in both studies. Since none of these new tumors regressed following ovariectomy, all were classified as hormone independent. Furthermore, these independent tumors grew more rapidly than both control-dependent and independent tumors, resulting in a greater average volume. These data suggest that a more aggressive form of hormone-independent tumor appears during TAM treatment.

Effect of 4-hydroxyandrostenedione on murine Leydig tumor cell steroidogenesis.

The murine Leydig cell tumor (M5480A) possesses high levels of estrogen receptor and is known to produce estrogens. In these studies we examined the effects of the potent aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) on Leydig tumor cell steroidogenesis both in vitro and in vivo. The addition of 4-OHA to Leydig tumor cells in primary culture resulted in a dose- and a time-dependent decrease in media progesterone levels. The observed decrease was most likely due to impaired synthesis of progesterone, inasmuch as no alteration in progesterone metabolism was seen when progesterone levels were diminishing. However, 4-OHA inhibited progesterone conversion to testosterone following 1 h of incubation, but this effect disappeared coincident with 4-OHA metabolism. Analysis of pregnenolone production revealed a biphasic dose-dependent effect of 4-OHA. At low doses (0.01-0.1 microM), 4-OHA was found to decrease pregnenolone concentrations, while at higher doses (1-10 microM) pregnenolone levels were elevated. Therefore, the actions of 4-OHA on Leydig cell steroidogenesis in vitro appear to be multifocal. Other experiments were performed to evaluate the effects of 4-OHA on tumor-bearing male mice in vivo. In these studies, the predominant effects of 4-OHA were to act as an aromatase inhibitor and to inhibit progesterone production. Thus, while 4-OHA is a potent aromatase inhibitor, we have found that this compound may alter steroidogenesis in Leydig tumor cells at several sites prior to aromatization.

Inhibition of Leydig tumor cell steroidogenesis by 10-propargylestr-4-ene-3,17-dione, an irreversible aromatase inhibitor.

The murine Leydig cell tumor (M5480A) was assayed for the presence of aromatase activity and for the effects of 10-propargylestr-4-ene-3,17-dione (PED), an aromatase inhibitor, on steroidogenesis. Microsomal preparations from these tumors contained low levels of aromatase activity which was PED sensitive. In addition, these Leydig tumor cells were maintained in primary culture and incubated under basal and gonadotropin-stimulated conditions with various doses of PED. Medium levels of progesterone, a major product of these cells, were found to decrease in a dose- and time-dependent manner upon addition of PED. To determine whether the observed effect was due to reduced synthesis or to increased metabolism of progesterone, tritiated progesterone was added to these cell cultures. Analysis of culture medium by high-performance liquid chromatography suggested that PED dramatically reduced the conversion of labeled progesterone to testosterone. Furthermore, examination of medium pregnenolone levels revealed diminished amounts of this steroid as well. Taken together, these results suggest that PED or its metabolites inhibit Leydig tumor cell steroidogenesis at several sites. Thus, in addition to its role as an aromatase inhibitor, this agent also has effects prior to pregnenolone production, as well as other effects in the pathway between progesterone and testosterone.

Induction of an estrogen-dependent early steroidogenic lesion in murine Leydig tumor cells.

The effects of low doses (37 pM to 3.7 nM) of 17 beta-estradiol on Leydig tumor cell steroidogenesis were studied in primary culture. This gonadotropin-responsive Leydig tumor line (M5480A) produces progesterone as the major steroid and lower levels of testosterone. It was found that these tumor cells possess a relatively high level of estradiol receptors, but only low levels of estradiol. We, therefore, maintained dispersed Leydig tumor cells in culture under basal or hCG-stimulated conditions for varying periods of time with or without graded doses of estradiol. The media from these cultures were analyzed for pregnenolone, progesterone, and testosterone by specific RIAs. Although testosterone levels were similar to control values, both pregnenolone and progesterone levels were significantly decreased by low doses of estradiol in a dose- and time-dependent manner. For example, basal progesterone levels were diminished 36% by 0.37 nM estradiol, and this effect could be reversed by the antiestrogen LY117018 [6-hydroxy-2-(p-hydroxyphenol)benzo-b-thien-3-yl-p-2-(1-pyrr olidinyl)- ethoxyphenyl ketone]. To evaluate whether the decreased medium progesterone level was due to increased metabolism, [3H] progesterone was added to estrogen-treated and control cells, and ether-extracted media were analyzed for steroid metabolites by HPLC. No significant difference in progesterone metabolism, including its conversion to testosterone, was detected between control and treated cells. Thus, the estradiol-mediated decrease in progesterone concentrations most likely reflects decreased synthesis rather than increased metabolism. These results provide the first indication of an estrogen-mediated effect at an early site in Leydig tumor cell steroidogenesis.

Divergence between ovarian aromatase activity, estrogen, and androgen levels in the cycling rat.

Serum estrogen levels vary in a cyclic fashion during the rat estrous cycle, reaching peak values on proestrus and then rapidly declining. Daily measurements of ovarian aromatase activity appear to follow a similar pattern, suggesting a potential regulatory role for the enzyme. To examine this hypothesis, mature rats were killed at various times, and ovarian microsomes were assayed for aromatase activity, while androstenedione, testosterone, estradiol, and estrone levels were measured in ovarian cytosol by RIA. The ovarian aromatase activity reached a peak about 9 h after the onset of the light period on the day of proestrus, and then began to decrease gradually to 75% lower levels by the morning of estrus. Similarly, the estradiol levels also peaked at about 9 h, but then dropped sharply; at 18 h the estradiol levels were reduced 90-95%, while aromatase activity was still close to its peak value. In contrast, the androstenedione and testosterone levels remained high for 3-4 h after the estradiol levels began to decrease. Estrone levels varied considerably less than those of estradiol, reaching lower peak values, but then decreasing only 50-75%. The dissociation between product levels and enzyme activity cannot be explained by a change in the Km of aromatase, which was about 20 nM for androstenedione throughout this period. Therefore, it appears that the decrease in estradiol levels from the peak of the surge may in part be a consequence of a shift in the ratio of the estrogen products synthesized by the ovary, rather than a decrease in the ovarian capacity for estrogen production, although other factors appear to be involved as well.

Fate of human chorionic gonadotropin bound to rat corpora lutea.

The current studies were designed to ascertain the fate of hCG bound to rat corpora luteal cell receptors. Graded doses of highly purified hCG (CR119), ranging from 0.1-10.1 micrograms, were injected. Groups of pseudopregnant rats received iodinated hCG, unlabeled hCG, or both. Supraphysiological levels of hCG were used to enhance the internalization of hCG and its receptors. When ovarian membrane pellets (48,000 X g) were subjected to continuous sucrose density ultracentrifugation, two different ovarian membrane fractions (F1 and F2) bound hCG. Although an increase in hCG binding to the F2 membrane fraction was observed between 1-6 h after a single 0.1-microgram [125I]iodo-hCG injection, no subsequent enhanced binding to that fraction was observed. However, the F1 fraction bound at least 3 fold more hCG than did the F2 fraction 1, 6, 12, and 24 h after the injection of 0.1 micrograms [125I]iodo-hCG. When groups of animals were injected with 0.1, 1.0, or 10.0 micrograms unlabeled highly purified hCG, peak serum, ovarian plasma membrane, and ovarian intracellular hCG concentrations were observed at different times after hormone injection and suggested the progressive transfer of hCG from serum to ovarian fractions in a time- and dose-dependent relationship. Although no intracellular hCG was detected until 60 min after the single 0.1-microgram injection of hCG, both serum and membrane-bound levels were measurable within 15 min of that injection. From these observations, we suggest that ovarian intracellular hCG does not reflect significant contamination with serum or interstitial fluid or from significant dissociation of membrane-bound hCG during tissue handling. Finally, when intracellular hCG was subjected to continuous sucrose density gradient ultracentrifugation, a single major 135I peak was observed, and this comigrated with [125I]iodo-hCG. Our interpretation of the foregoing observations is that the major intracellular form of hCG is not receptor bound.

A long-acting gonadotropin-releasing hormone agonist inhibits the growth of a human ovarian epithelial carcinoma (BG-1) heterotransplanted in the nude mouse.

Human ovarian epithelial carcinoma (BG-1) was heterotransplanted in female nude athymic mice and growth was evaluated in intact, surgically castrated, and gonadotropin-releasing hormone (GnRH) agonist (Lupron-SR)-treated mice. Tumor volume was represented as percent of tumor volume on day 0 and measured every other day from the administration of drug and/or the attainment of a minimum tumor volume of 0.5 cm3 on each side of the animal. Tumors in surgically castrated mice had significantly accelerated growth compared with control tumors grown in intact mice (1810 +/- 247.2 versus 1253.6 +/- 44.6%, respectively; P less than .05). Treatment with GnRH agonist significantly reduced tumor growth in intact mice at days 16, 18, and 20 compared with normal control tumors and placebo-treated tumors in intact mice (P less than .02). Gonadotropin levels in pooled serum of mice were reduced from normal levels with GnRH agonist treatment (control: mLH 16.9 +/- 2.2 ng/mL, mFSH 6.6 +/- 0.3 ng/mL; GnRH agonist-treated: mLH 9.3 +/- 1.8 ng/mL, mFSH 4.9 +/- 0.7 ng/mL; surgically castrated: mLH 32.2 +/- 2.7 ng/mL, mFSH 19.4 +/- 0.5 ng/mL). This human tumor model appears responsive to gonadotropins as evidenced by the ability of a GnRH agonist to inhibit growth of BG-1. These results suggest that GnRH agonist therapy may be a useful adjuvant in the treatment of human ovarian epithelial carcinoma.

Inhibition of aromatase activity and of endocrine-responsive tumor growth by 10-propargylestr-4-ene-3, 17-dione and its 17-propionate derivative.

Two androstenedione derivatives, 10-propargylestr-4-ene-3,17-dione and its 17-propionated form, were administered to normal cycling rats, and both compounds led to an inhibition of ovarian aromatase. Under in vitro conditions, only the former compound exhibited high potency as an inhibitor of rat ovarian and human placental microsomal aromatase. At 1 mg/kg/day both compounds were effective in promoting regression of 9,10-dimethyl-1,2-benzanthracene-induced mammary tumors in rats without terminating their estrous cycle. PED also inhibited growth of a human ovarian carcinoma in athymic mice. The results with the 17-propionated compound testify to the necessity of in vivo assays in screening antitumor agents. In summary, PED and its propionated derivative inhibited ovarian aromatase in vivo and inhibited the growth of hormone-responsive tumors.

Endocrine characterization of a human ovarian carcinoma (BG-1) established in nude mice.

The steroid receptor-positive human ovarian cancer (BG-1) was evaluated to determine its usefulness as a tumor model. This tumor grows in intact male and female nude mice without hormone supplements. Moreover, its growth was significantly accelerated in ovariectomized mice, and the increased growth rate could be reversed by estradiol administration. Evaluation of tumor growth following endocrine therapy revealed that, while antiandrogens did not affect the tumor growth, both an aromatase inhibitor and a luteinizing hormone-releasing hormone agonist significantly impaired growth of this human ovarian tumor. Estradiol was also shown to up-regulate both estrogen and progesterone receptors in tumors grown in ovariectomized mice. Therefore, the BG-1 human ovarian carcinoma grows without hormonal supplements and yet responds to specific forms of endocrine therapy. Moreover, the steroid receptors present in this tumor respond to exogenous steroids. In conclusion, this tumor may serve as an ideal model for the study of hormonal regulation of ovarian tumor growth.

Characterization of oviductal aromatase in the northern leopard frog, Rana pipiens.

Previous work has shown that the oviduct of the northern leopard frog, Rana pipiens, can convert testosterone to estradiol. The present paper examines the characteristics of the aromatase responsible for this reaction and compares it with human placental aromatase. Microsomes were isolated from the oviducts, and aromatase activity was assayed by a tritiated water release method. The Km and the Vmax for androstenedione were 188.1 +/- 30.2 nM and 1.42 +/- 0.11 fmol of estrogen produced/min/mg protein, respectively. Using the same method, human placental aromatase had a Km of 123.0 nM and a Vmax of 113.57 fmol of estrogen produced/min/mg protein. When tested at four temperatures between 15 and 45 degrees C, the frog enzyme showed maximum activity at 37 degrees C. The enzyme had a broad pH optimum between 7.4 and 10.4. The aromatase inhibitor 4-hydroxyandrostenedione inhibited activity by 20% at 0.3 microM and 40% at 0.5 microM. The present study provides additional evidence that an aromatase is present in the frog oviduct. Characterization of this enzyme revealed similarities to human placental aromatase, but the specific activity was much lower in the frog oviduct.

Ovarian contents of immunoreactive beta-endorphin and alpha-N-acetylated opioid peptides in rats.

beta-Endorphin was measured by radioimmunoassay in homogenates of ovaries from immature Sprague-Dawley rats (21-29 days of age) and found to be present at levels of about 0.6-0.7 ng/ovary. After administration of PMSG there was approximately a 4-fold increase (2-3 ng/ovary) in total ovarian immunoreactive (ir) beta-endorphin 48 h after injection. Analysis of follicular fluid from similarly treated rats indicated about the same amount of ovarian ir-beta-endorphin (2-3 ng/ovary) as in ovarian homogenates, suggesting that most of the ir-beta-endorphin is localized in follicular fluid of PMSG-primed immature rats. Immature rats were made pseudopregnant by administration of hCG 48 h after PMSG, and at 24 h after injection of hCG there was a slight, but significant and reproducible, increase in the ovarian content of ir-beta-endorphin. The serum concentration of ir-beta-endorphin was in the range of 1-3 ng/ml and was unaffected by PMSG and PMSG/hCG; likewise, the pituitary content of ir-beta-endorphin did not change following administration of gonadotrophins to immature rats. In mature cyclic animals, levels of 2-4 ng ir-beta-endorphin/ovary were found, comparable to those in the ovaries of PMSG-primed immature rats, and there were only small changes during the oestrous cycle. In addition to ir-beta-endorphin, we also obtained evidence for the presence of alpha-N-acetylated opioid peptides (endorphins or enkephalins) in the ovaries of PMSG-primed immature and mature rats. The physiological role of the opioid peptides in reproductive tissue is unknown, but they are presumably acting in an autocrine or paracrine fashion.

The effects of in vivo administration of 10-propargylestr-4-ene-3,17-dione on rat ovarian aromatase and estrogen levels.

We have previously demonstrated that 10-propargylestr-4-ene-3,17-dione (PED) functioned as an irreversible inhibitor of rat ovarian aromatase in vitro. These studies were undertaken to examine the in vivo effects of PED on rat ovarian aromatase activity and estrogen production. In the current experiments, a single injection of PED (0.5 or 2.5 mg/kg) was found to maximally inhibit aromatase at 3 h regardless of dose. Significant inhibition of enzyme activity by PED was observed beyond 18 h, although some recovery was noted at the lower dose (0.5 mg/kg). Concomitantly, ovarian estrogen levels were also maximally reduced at 3 h, however ovarian estrogen levels returned toward control values prior to the recovery in enzyme activity. Even though significant inhibition of enzyme activity was observed at 12 h following a single injection of PED, the effect of double injections of the inhibitor at 12 h intervals was surprisingly not cumulative. Similarly, continued multiple injections of PED revealed significant inhibition of enzyme activity and estrogen production several hours after the injection, but variations in effectiveness were observed by 12 h which changed in accordance with a circannual cycle in aromatase. Apparently other factors are involved with maintaining aromatase levels and compensating for reduced enzyme activity. These mechanisms are evidenced by a continuation of the rat reproductive cycle with prolonged PED administration and a reduced influence of PED in regard to enzyme inhibition at certain times of the year. Despite these variations in the duration of action of PED, no comparable changes were observed in effectiveness as an anti-tumor agent. These results suggest that complex mechanisms exist which regulate the activity of aromatase in order to maintain estrogen production. Further research using compounds such as PED may assist in elucidating the factors that modulate ovarian estrogen production.

Divergent effects of phenothiazines on Leydig tumor cell steroidogenesis and adenylate cyclase activity.

The dose and temporal (1-24 h) effects of two phenothiazines, chlorpromazine and trifluoperazine, on steroidogenesis and adenylate cyclase activity of gonadotropin-responsive Leydig tumor cells (M5480A) in primary culture were examined. At low doses (e.g. 0.1-1 microM) these antipsychotic drugs were slightly inhibitory (trifluoperazine) or without effect (chlorpromazine), while at 25 microM each drug was weakly stimulatory to basal testosterone production. Trifluoperazine was, in general, inhibitory to HCG-stimulated testosterone production, but chlorpromazine exhibited paradoxical effects. At 5 and 10 microM this neuroleptic agent increased HCG-stimulated steroidogenesis, while at 25 microM testosterone production was inhibited. In a particulate fraction prepared from the tumor the activity of adenylate cyclase was stimulated 3.4-fold in the presence of 10 microM 5'-guanylimidodiphosphate and 5-fold in the presence of HCG plus the non-hydrolyzable GTP analogue. Between doses of 1-100 microM neither drug altered the basal activity of adenylate cyclase. Trifluoperazine at doses of 1-100 microM inhibited 5'-guanylimidodiphosphate-stimulated adenylate cyclase activity both with and without added gonadotropin. At doses of 1-10 microM chlorpromazine had no effect on adenylate cyclase activity, but it stimulated activity in the dose range of 20-100 microM. Interestingly, in the presence of 5'-guanylimidodiphosphate this drug did not alter the stimulated enzymic activity achieved with a maximal dose of HCG. Therefore, these phenothiazines exhibit quite divergent dose-dependent effects and their actions must occur at multiple loci. Also, it seems unlikely that the effects of these agents on steroidogenesis and adenylate cyclase activity can be reconciled solely in terms of calmodulin-mediated processes.

Inhibition of growth and appearance of estrogen-dependent rat mammary tumors by 10-propargylestr-4-ene-3,17-dione, an aromatase inhibitor.

The aromatase inhibitor 10-propargylestr-4-ene-3,17-dione (PED) has been evaluated in vivo as an anticancer agent. Prolonged administration of PED to rats bearing dimethylbenzanthracene-induced mammary tumors resulted in significant regression of hormone-responsive tumors within several days. Greater than 50% regression was generally observed after 14 days of treatment, irrespective of dose (1, 5, or 50 mg/kg body weight/day). In addition to tumor regression, a significantly increased incidence in tumor stasis was observed over the course of PED treatment. While all doses of PED examined were equipotent for both tumor regression and stasis, a dose-dependent inhibition of new tumor formation was observed in PED-treated rats. In control animals an average of 1.2 new tumors was observed during the experimental period; in contrast, averages of 0.5 tumors appeared in animals receiving 1 mg PED/kg body weight/day, 0.1 tumors at 5 mg/kg, and at 50 mg of PED/kg body weight/day, no new tumors occurred during the time PED was administered. The effects of PED on both regression of existing tumors and appearance of new tumors were reversed by co-administration of estradiol. Thus, PED impairs estrogen-dependent mammary tumor growth, resulting in cessation of new growth and regression of responsive tumors.

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase.

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.

Characterization of pregnant mare's serum gonadotropin-stimulated rat ovarian aromatase and its inhibition by 10-propargylestr-4-ene-3,17-dione.

Aromatase, the important regulatory enzyme that converts androgens to estrogens, is found in relatively high levels in the human placenta. However, since the ovary is the major source of the estrogens in females, we undertook studies to compare the rodent ovarian enzyme with that from human placenta. Pregnant mare's serum gonadotropin (PMSG) markedly increases aromatase activity in the ovaries of immature rats, and this model was used in order to reproducibly obtain high enzyme levels. An injection of PMSG resulted in a specific stimulation of aromatase activity 12 times the increase in ovarian weight in 48 h. Kinetic studies demonstrated that, although the PMSG-stimulated ovarian microsomes had one-tenth the specific activity of the human placenta, the Km values were similar (about 33 and 44 nM, respectively). The potent inhibitor of placenta aromatase, 10-propargylestr-4-ene-3,17-dione, was used to further characterize the enzyme. It inhibited the rat aromatase with an I50 of 36 nM and exhibited time-dependent inhibition with a half-life of inactivation of 16 min and a Ki of 15 nM. These values are similar to those we obtained with the human enzyme (10 nM, 12 min, and 5 nM, respectively). The enzyme parameters in the presence and absence of the inhibitor suggest that the enzymes from the two sources are kinetically quite similar.