Corticotropin-releasing factor stimulates accumulation of adenosine 3', 5'-monophosphate in rat pituitary corticotrophs.

The presence of synthetic ovine corticotropin-releasing factor leads to a rapid and marked stimulation of adenosine 3', 5'-monophosphate accumulation in an enriched population of rat pituitary corticotrophs in primary culture. The increase, observed as early as 60 seconds after the addition of corticotropin-releasing factor, suggests that changes in the intracellular concentration of the cyclic nucleotide coincide with or precede the secretion of adrenocorticotropic hormone in response to corticotropin-releasing factor.

Maintenance of androgen responsiveness by glucocorticoids in Shionogi mammary carcinoma cells in culture.

The possibility that glucocorticoids can delay or prevent loss of responsiveness to androgens in androgen-deprived cells was investigated. While a complete loss of responsiveness to dihydrotestosterone (DHT) was observed within 60 days of androgen deprivation, addition of the synthetic glucocorticoid dexamethasone (DEX) delayed both the loss of growth responsiveness to DHT and the increase in spontaneous growth rate by approximately equal to 60 days. When the growth response to DEX was studied, the changes found were similar to those described above for DHT, namely loss of response following steroid deprivation and preservation of response in cells preincubated with DHT or DEX. In addition, long-term incubation in the presence of DEX was accompanied by low-amplitude stimulation of cell growth at extremely low concentrations of DHT and DEX, suggesting that androgen- and glucocorticoid-hypersensitive cell clones developed during androgen deprivation. The present data show that long-term incubation of androgen-sensitive Shionogi cells leads not only to an increase in the spontaneous growth rate, but also to the appearance of androgen- and glucocorticoid-hypersensitive cells, which may well play an important role in the development of resistance to endocrine therapy in human hormone-sensitive cancers. Although it is not as efficient as DHT, the glucocorticoid DEX can delay the loss of androgen sensitivity in this cell line.

Low androgen levels induce the development of androgen-hypersensitive cell clones in Shionogi mouse mammary carcinoma cells in culture.

The influence of the concentration of androgen present on the development of heterogeneous growth responsiveness to androgens was studied in an androgen-sensitive clone (SEM-1) of the Shionogi mammary carcinoma cell line incubated for up to 6 months in the presence of 0, 0.01, 0.3, or 100 nM dihydrotestosterone (DHT). In the absence of added androgen, there was a rapid increase in spontaneous cell growth, the increase being delayed by 2, 4, and 6 months when the cells were incubated with 0.01, 0.3, and 100 nM DHT, respectively. Conversely, the mitogenic effect of DHT decreased rapidly when originally androgen-sensitive cells were incubated with less than maximal concentrations of DHT. The most significant finding was the marked heterogeneity in the androgen sensitivity of the clones obtained after incubation for 2 months in the presence of less than maximal concentrations of DHT. In cells exposed to a maximal concentration of DHT (100 nM), only 2 of 22 clones were more sensitive to DHT (lower Km value) than the original clone, while 8 subclones were hyposensitive to DHT. On the other hand, when the cells were incubated for 2 months with a low (0.3 nM) concentration of DHT, 29% (7 of 24) of the clones were hypersensitive to DHT. Clones derived from cells treated with 0 or 0.1 nM DHT lost most of their responsiveness to DHT during the same time interval, with a simultaneous increase in spontaneous growth rate. The present data show that incubation of a clone of androgen-sensitive Shionogi carcinoma cells in the presence of a low DHT concentration, comparable to the circulating levels of DHT in castrated men, induces the development of androgen-hypersensitive cell clones that are able to grow on minute amounts of androgens. Such androgen-hypersensitive cells are likely to be unresponsive or resistant to antihormonal therapy. The present data emphasize the major importance of the hormonal environment on the maintenance, loss, and increase of tumor cell responsiveness to androgens.

Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells.

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells.

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e., dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.

Direct effects of sex steroids on prolactin release at the anterior pituitary level: interactions with dopamine, thyrotropin-releasing hormone, and isobutylmethylxanthine.

When present alone for 4 or 8 days, 5 alpha-dihydrotestosterone (DHT) or the pure progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) inhibits spontaneous PRL release by 33--50% in rat anterior pituitary cells in primary culture. This inhibitory effect of DHT and R5020 can only be partially reversed by 17 beta-estradiol (E alpha). DHT and R5020 inhibit spontaneous PRL release in E2-primed cells at ED50 values of 0.5 and 3 nM, respectively. While E2 diminishes by 30--60% the maximal inhibitory effect of dopamine on PRL release and increases by 10-fold the ED50 value of dopamine action, DHT and R5020 can prevent by 30--60% the action of E2 and thus increase the potency of dopamine to inhibit PRL release. The inhibitory action of DHT and R5020 as well as the stimulatory action of E2 on spontaneous PRL release are similarly expressed on TRH- and 3-isobutyl-1-methylxanthine-induced PRL release, thus suggesting that at least part of the highly effective modulatory effects of sex steroids are exerted at a step after cAMP formation.

Vasopressin rapidly stimulates phosphatidic acid-phosphatidylinositol turnover in rat anterior pituitary cells.

In cultured rat anterior pituitary cells, the agonist [Asu1,6, Arg8]vasopressin (AVP-A) increased by 1.5-fold 32Pi incorporation into phosphatidic acid (PA), as early as 15 s after its addition. Increased phosphatidylinositol (PI) labeling became significant 4 min after AVP-A addition. Dose-response measurements with AVP-A showed ED50 values of 76 and 62 nM for PA and PI labeling, respectively. Peptide corticotropin-releasing factor (CRF) (0.1 microM) did not affect the stimulatory effect of AVP-A on PA and PI labeling. These data suggest that stimulation of PI metabolism in corticotrophs may be one of the early events involved in the stimulation of ACTH release induced by vasopressin.

Synthesis and in vitro evaluation of 4-substituted N-(1,1-dimethylethyl)-3-oxo-4-androstene-17 beta-carboxamides as 5 alpha-reductase inhibitors and antiandrogens.

4-Substituted N-(1,1-dimethylethyl)-3-oxo-4-androstene-17 beta-carboxamides with the hydroxy (OH) 3d, mercapto (SH) 3e, chloro (Cl) 3f, and bromo (Br) 3g substituents at the 4-position were prepared in a two-step sequence with overall yields of 21%, 27%, 41%, and 37%, respectively. Compounds 3d-g showed weak inhibitory activity on human type I 5 alpha-reductase (IC50 > or = 700 nM) while they had intermediate inhibitory activity on human type II 5 alpha-reductase at IC50S of 172, 437, 192, and 387 nM, respectively. In androgen-sensitive Shionogi cells, the inhibition of dihydrotestosterone (DHT) stimulatory action on the proliferation of the androgen-sensitive cancer cells by all four compounds was high at IC50S of 170-279 nM compared with 117 nM for hydroxyflutamide. The present data show compounds having both moderate inhibition of human type II 5 alpha-reductase activity and relatively potent antiandrogenic action, two beneficial characteristics in the therapy of androgenic-sensitive diseases.

PHI stimulates ACTH release from pituitary tumor cell.

The identification of PHI (the 27-amino acid peptide (P) having an N-terminal histidine (H) and a C-terminal isoleucine amide (I] in median eminence suggested that PHI influences the secretory function of the anterior pituitary. The effects of PHI on ACTH release from clonal mouse pituitary corticotrophs were investigated. The secretory response to PHI was correlated with a prior increase in cyclic AMP accumulation. Both cyclic AMP synthesis and ACTH secretion were increased by PHI in a concentration-dependent manner. PHI was a less effective agonist of cyclic nucleotide synthesis and ACTH secretion than VIP. The secretory response to PHI was blocked by the calcium channel antagonist, nifedipine, and by dexamethasone. Somatostatin and oxotremorine blocked both PHI-stimulated cyclic AMP formation and ACTH secretion. The observation that VIP in high concentrations can elicit ACTH secretion from normal rat anterior pituitary suggested that a VIP-like substance may modulate ACTH secretion. However, the finding that PHI does not elicit ACTH release from primary cultures of dispersed anterior pituitary, coupled to its relatively lower potency compared to VIP, indicate that corticotrophs are not an important target for PHI.

Glucocorticoids stimulate the growth of mouse mammary carcinoma Shionogi cells in culture.

The relative potency of a series of glucocorticoids to stimulate the growth of a cloned cell line (SEM-1) derived from the androgen-sensitive Shionogi mouse mammary carcinoma is proportional to their known affinity for the glucocorticoid receptor. The stimulatory action of glucocorticoids is not inhibited by the pure antiandrogen hydroxyflutamide while the antiglucocorticoids RU25593 and RU38486 cause 100% and 80% inhibitions of the activity of triamcinolone acetonide, respectively, thus indicating that the stimulatory effect of glucocorticoids on Shionogi cell growth is mediated by the glucocorticoid receptor. Such data indicate that not only androgens but also glucocorticoids should be taken into account when assessing the endocrine control of the growth of these mammary carcinoma cells.

LHRH rapidly stimulates phosphatidylinositol metabolism in enriched gonadotrophs.

The effects of luteinizing hormone-releasing hormone (LHRH) and human pancreatic growth hormone-releasing factor (hpGRF(1-40)-NH2) on phospholipid metabolism were studied in rat anterior pituitary cells in primary culture. In a 4-fold enriched population of gonadotrophs, 30 nM LHRH increased 32Pi incorporation into phosphatidic acid (PA) as early as 1 min after its addition. Phosphatidylinositol (PI) labeling was increased 1 min later. The stimulatory action of LHRH was observed in both phospholipids up to 100 min, the last time interval studied. The decapeptide did not affect 32Pi labeling of phosphatidylcholine (PC), lysoPC, phosphatidylethanolamine or phosphatidylserine. Dose-response studies performed after 25 min of incubation showed an ED50 value of LHRH action at approximately 1 nM for PI labeling. In contrast, the addition of 0.1 microM GRF to anterior pituitary cells enhanced 32Pi incorporation only into PC after a 60 min incubation period. The present data suggest that stimulation of acidic phospholipid metabolism, particularly an increase in PA-PI turnover, may represent an early event in the mechanism of action of LHRH but not GRF in the anterior pituitary gland.

Regulation of Prolactin Messenger Ribonucleic Acid Levels by Estradiol and Dihydrotestosterone as Evaluated by in situ Hybridization Performed on Implanted Pituitary Glands and Anterior Pituitary Cells in Culture in the Male Rat.

We have recently demonstrated that 17ß-estradiol (E(2) ) administration increases protactin (PRL) mRNA levels in the male rat anterior pituitary gland and that this stimulatory effect is partially inhibited by concomitant administration of dihydrotestosterone. In order to gain more information about the site(s) of action of E(2) and dihydrotestosterone on PRL gene expression, we have studied the effects of these two hormones in pituitaries implanted under the kidney capsule as well as in anterior pituitary cells in culture. In implanted pituitaries, PRL mRNA levels were increased by 90% as compared to values obtained in the stalk-connected pituitaries from the same animals. Administration of E(2) induced a further increase of PRL mRNA levels in implanted pituitaries, while dihydrotestosterone did not produce any change in animals which had been treated or not with E(2) . In anterior pituitary cells in culture, addition of E(2) to the culture medium resulted in a 60% increase of PRL mRNA levels over control values. Supplementation with dihydrotestosterone did not induce any variation in the concentration of PRL mRNA in cells which were treated or not with E(2) . These results indicate that E(2) exerts a direct action on PRL cells at the pituitary level and strongly support the key role of the hypothalamus in the inhibitory effect of androgens on estrogen-induced stimulation of PRL mRNA in the male rat pituitary.

Differential androgen sensitivity is associated with clonal heterogeneity in steroid metabolism, ornithine decarboxylase regulation and IL-1alpha action in mouse mammary tumor cells.

Upon androgen deprivation, Shionogi (SC-115) mouse mammary tumors undergo phenotypic changes enabling their escape from growth dependence on androgens. Even within androgen-responsive cell populations, marked clonal heterogeneity is observed in the trophic effects of androgens. The present study compares several parameters of androgen action between three SC-115 cell clonal subpopulations exhibiting high (clone 107), low (clone S1A2) and no trophic response (clone 415) to androgens. These parameters pertain to (1) kinetics of androgen binding, (2) metabolism of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol), (3) ornithine decarboxylase (ODC) activity and (4) interleukin-1alpha (IL-1alpha) action on cell proliferation. Only marginal differences in the affinity and abundance of androgen-specific binding sites were detected between the three clones. While clone S1A2 degraded DHT to 3alpha-diol at a much faster rate than the highly androgen-sensitive 107 cells and androgen-insensitive 415 cells, differences in the rates of intracrine conversion of 3alpha-diol and 3beta-diol to DHT did not correlate with the ability of these steroids to stimulate cell proliferation. Induction of ODC activity at the onset of exponential growth was strongly DHT-dependent in 107 cells, whereas this dependence was markedly attenuated in androgen-hyposensitive cells. Unexpectedly, DHT strongly repressed the marked ODC induction resulting from fresh medium addition in 415 cells which show no growth response to androgens. Low IL-1alpha concentrations were mitogenic in all three SC-115 clones. Whereas the mitogenic action of IL-1alpha was completely androgen-dependent in 107 cells, this dependence was relieved in S1A2 cells, which responded to DHT and IL-1alpha in an additive fashion. Thus, clonal heterogeneity in the pattern of steroid metabolism within Shionogi tumors cannot solely account for loss of androgen dependence, which may rather correlate with the constitutive activation of transduction pathways controlling the expression of growth-associated genes (e.g. ODC) by serum growth factors, including IL-1alpha.

EM-652 (SCH 57068), a third generation SERM acting as pure antiestrogen in the mammary gland and endometrium.

Breast cancer is the most frequent cancer in women while it is the second cause of cancer death. Estrogens are well recognized to play the predominant role in breast cancer development and growth and much efforts have been devoted to the blockade of estrogen formation and action. The most widely used therapy of breast cancer which has shown benefits at all stages of the disease is the use of the antiestrogen Tamoxifen. This compound, however, possesses mixed agonist and antagonist activity and major efforts have been devoted to the development of compounds having pure antiestrogenic activity in the mammary gland and endometrium. Such a compound would avoid the problem of stimulation of the endometrium and the risk of endometrial carcinoma. We have thus synthesized an orally active non-steroidal antiestrogen, EM-652 (SCH 57068) and the prodrug EM-800 (SCH57050) which are the most potent of the known antiestrogens. EM-652 is the compound having the highest affinity for the estrogen receptor, including estradiol. It has higher affinity for the ER than ICI 182780, hydroxytamoxifen, raloxifene, droloxifene and hydroxytoremifene. EM-652 has the most potent inhibitory activity on both ER alpha and ER beta compared to any of the other antiestrogens tested. An important aspect of EM-652 is that it inhibits both the AF1 and AF2 functions of both ER alpha and ER beta while the inhibitory action of hydroxytamoxifen is limited to AF2, the ligand-dependent function of the estrogen receptors. AF1 activity is constitutive, ligand-independent and is responsible for mediation of the activity of growth factors and of the ras oncogene and MAP-kinase pathway. EM-652 inhibits Ras-induced transcriptional activity of ER alpha and ER beta and blocks SRC-1-stimulated activity of the two receptors. EM-652 was also found to block the recruitment of SRC-1 at AF1 of ER beta, this ligand-independent activation of AF1 being closely related to phosphorylation of the steroid receptors by protein kinase. Most importantly, the antiestrogen hydroxytamoxifen has no inhibitory effect on the SRC-1-induced ER beta activity while the pure antiestrogen EM-652 completely abolishes this effect, thus strengthening the need to use pure antiestrogens in breast cancer therapy in order to control all known aspects of ER-regulated gene expression. In fact, the absence of blockade of AF2 by hydroxytamoxifen could explain why the benefits of tamoxifen observed up to 5 years become negative at longer time intervals and why resistance develops to tamoxifen. EM-800, the prodrug of EM-652, has been shown to prevent the development of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat, a well-recognized model of human breast cancer. It is of interest that the addition of dehydroepiandrosterone, a precursor of androgens, to EM-800, led to complete inhibition of tumor development in this model. Not only the development, but also the growth of established DMBA-induced mammary carcinoma was inhibited by treatment with EM-800. An inhibitory effect was also observed when medroxyprogesterone was added to treatment with EM-800. Uterine size was reduced to castration levels in the groups of animals treated with EM-800. An almost complete disappearance of estrogen receptors was observed in the uterus, vaginum and tumors in nude mice treated with EM-800. EM-652 was the most potent antiestrogen to inhibit the growth of human breast cancer ZR-75-1, MCF-7 and T-47D cells in vitro when compared with ICI 182780, ICI 164384, hydroxytamoxifen, and droloxifene. Moreover, EM-652 and EM-800 have no stimulatory effect on the basal levels of cell proliferation in the absence of E2 while hydroxytamoxifen and droloxifene had a stimulatory effect on the basal growth of T-47D and ZR-75-1 cells. EM-652 was also the most potent inhibitor of the percentage of cycling cancer cells. (ABSTRACT TRUNCATED)

Mast cell increase in the duodenum and kidney of magnesium-deficient rats.

Rats were maintained on a magnesium-deficient diet for 1 to 5 weeks to study the mast cell (MC) populations in their duodenum and kidney. A marked increase of intestinal subepithelial mast cells was observed in these animals as compared with normal controls. The cells in both groups showed an identical reaction for mucopolysaccharides but the 5-hydroxytryptamine content tended to be higher in the cells of magnesium-deficient animals. Proliferation of MC was also observed in the renal cortex of the magnesium-deficient rats. This finding is significant because MC are known to be virtually absent from normal kidneys. Magnesium deprivation resulted in numerous MC not only in the intertubular spaces but also within the glomeruli. Possible correlations between these and other pertinent observations are discussed with regard to certain renal diseases. The discussion is extended to the possible mechanism through which magnesium could influence secretory processes in MC.

A wide range of sensitivities to androgens develops in cloned Shionogi mouse mammary tumor cells.

Clones obtained in soft agar from a Shionogi mouse mammary carcinoma show marked heterogeneity of growth characteristics and sensitivities to androgens. These data pertain to spontaneous growth in the absence of androgens, maximal response to dihydrotestosterone (DHT) and Km values of the stimulatory action of DHT ranging from 0.008 to 10 ng/ml (1,250-fold range). Following 13 months in culture in the presence of 10 nM DHT, recloning of one original cell clone led to an even greater variation of androgen-free growth and of the maximal responses to DHT, while the Km values of DHT action ranged from 0.05 to 10 nM (200-fold range). The present demonstration of a marked heterogeneity of Km values of DHT action in subpopulations of tumors grown in a controlled environment has major implications for the efficient antihormonal treatment of androgen-sensitive diseases such as prostate cancer. Such data indicate that cell clones having a high degree of sensitivity to DHT (androgen-hypersensitive) can continue to grow in the presence of castration levels of androgens, thus suggesting that an antiandrogen is required in order to achieve a more complete androgen blockage and to induce a regression of these androgen-hypersensitive tumors.

Variation for 2n pollen production in clones of Solanum phureja Juz. and Buk.

Frequency of unreduced pollen grains was estimated for five genotypes of Solanum phureja (2n=24) growing in three environments; (E1) cool (7.2-13.3°C) and (E2) warm (12.2-17.2°C) growth chambers and (E3) field conditions. Highly variable frequencies were found, with genotype, environment, and genotype x environment interaction as significant components of variance. The frequency of unreduced gametes for two additional genotypes was studied over time in two growth chamber environments (cool and warm). One genotype, characterized by mostly fused spindles at the second meiotic division, expressed a high frequency of big pollen (BP) in both environments, whereas the second, characterized by fused, parallel and tripolar second division spindless was found to increase in BP frequency over time in the cool chamber, but remained consistently low in the warm chamber. The identification of specific environmental components with general effect on the expression of un-reduced gametes is not possible because of the large genotype x environment interaction component of variance. A genetic hypothesis based on incomplete penetrance and variable expressivity of a dominant gene is presented as an alternative to the currently accepted theory of control of parallel spindles by a single recessive gene.

Solid-phase synthesis and some biological properties of ovine corticotropin-releasing factor.

Ovine corticotropin-releasing factor (CRF) was synthesized by solid-phase method and isolated using two purification steps: gel filtration and high performance liquid chromatography. The synthetic peptide is a potent stimulator of ACTH release, as well as cyclic AMP accumulation and release in rat adenohypophyseal cells in culture and shows highly specific binding to bovine anterior pituitary plasma membranes.

Monoclonal antibodies for use with 125iodine-labeled radioligands in progesterone radioimmunoassay.

The interactions of heterologous and homologous 125I-iodinated radioligand with polyclonal and monoclonal antibodies directed against 11-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin were compared. Our data show that, with a polyclonal antibody, the use of the same bridge in the tracer and the antigen results in a low sensitivity while a heterologous tracer can decrease markedly the titer of antibody but increases the sensitivity of the radioimmunoassay. Due to the high specificity of monoclonal antibodies, the interaction with heterologous and homologous iodinated tracers is extremely variable from one antibody to another but the present results demonstrate that radioimmunoassays of very high sensitivity can be obtained with homologous and heterogeneous tracers. However, an appropriate tracer has to be selected to meet the requirement of such an assay.