4-nonylphenol triggers apoptosis and affects 17-ß-estradiol receptors in calvarial osteoblasts.

The present research examines the effects of 4-nonylphenol (4-NP) on mouse primary calvarial osteoblasts (COBs). Incubation of the cells with 4-NP at 10(-5)M and 10(-6)M striking decreased osteoblasts viability and phosphatidylserine (PS) exposure, measured by Annexin V, was greatly enhanced. In addition, an up-regulation of Bax/Bcl2 ratio with a drop in ΔΨm and an increase of cleaved caspase 9 and 3 was found, suggesting that the alkylphenol induced osteoblast death via the mitochondrial-dependent apoptotic pathway. Interestingly, treatment with 4-NP was also able to increase cleaved caspase 8 in parallel with the truncated active Bid (t-Bid) suggesting that 4-NP-mediated apoptosis depends on cross talk between the extrinsic and intrinsic pathways. It is of relevance, that the apoptotic effects of 4-NP overcame 17-ß-Estradiol (17-ß E(2)) induced-survival on osteoblasts. Also, the alkylphenol interfered with 17-ß E(2) regulated estrogen receptors expression.

Proliferative responses to altered 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 expression in human breast cancer cells are dependent on endogenous expression of 17HSD type 1 and the oestradiol receptors.

The primary source of oestrogen in premenopausal women is the ovary but, after menopause, oestrogen biosynthesis in peripheral tissue is the exclusive site of formation. An enzyme group that affects the availability of active oestrogens is the 17beta-hydroxysteroid dehydrogenase (17HSD) family. In breast cancer, 17HSD type 1 and type 2 have been mostly investigated and seem to be the principal 17HSD enzymes involved thus far. The question whether 17HSD type 1 or type 2 is of greatest importance in breast tumour development is still not clear. The aim of this study was to investigate how the loss of 17HSD type 2 expression, using siRNA in the non-tumour breast epithelial cells HMEC (human mammal epithelial cells) and MCF10A, and gain of 17HSD type 2 expression, using transient transfection in the breast cancer derived cell lines MCF7 and T47D, affect oestradiol conversion and proliferation rate measured as S-phase fraction. We further investigated how this was related to the endogenous expression of 17HSD type 1 and oestradiol receptors in the examined cell lines. The oestradiol level in the medium changed significantly in the MCF7 transfected cells and the siRNA-treated HMEC cells, but not in T47D or MCF10A. The S-phase fraction decreased in the 17HSD type 2-transfected MCF7 cells and the siRNA-treated HMEC cells. The results seemed to be dependent on the endogenous expression of 17HSD type 1 and the oestradiol receptors. In conclusion, we found that high or low levels of 17HSD type 2 affected the oestradiol concentration significantly. However, the response was dependent on the endogenous expression of 17HSD type 1. Expression of 17HSD type 1 seems to be dominant to 17HSD type 2. Therefore, it may be important to investigate a ratio between 17HSD type 1 and 17HSD type 2.

Membrane 5alpha-pregnane-3,20-dione (5alphaP) receptors in MCF-7 and MCF-10A breast cancer cells are up-regulated by estradiol and 5alphaP and down-regulated by the progesterone metabolites, 3alpha-dihydroprogesterone and 20alpha-dihydroprogesterone, with associated changes in cell proliferation and detachment.

Previous studies have shown that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), exhibits mitogenic and metastatic activity in breast cell lines and that specific, high affinity receptors for 5alphaP are located in the plasma membrane fractions of tumorigenic (ER/PR-positive) MCF-7 cells. The aim of this study was to determine the effects of the mitogenic (estradiol; 5alphaP) and anti-mitogenic (3alpha-hydroxy-4-pregnen-20-one, 3alphaHP; 20alpha-hydroxy-4-pregnen-3-one, 20alphaHP) endogenous steroid hormones on 5alphaP receptor (5alphaP-R) numbers and on cell proliferation and adhesion of MCF-7 and MCF-10A cells. Exposure of MCF-7 cells for 24h to estradiol or 5alphaP resulted in significant (p < 0.05-0.001) dose-dependent increases in 5alphaP-R levels. Conversely, treatment with 3alphaHP or 20alphaHP resulted in significant (p < 0.05-0.01) dose-dependent decreases in 5alphaP-R levels. Treatment with one mitogenic and one anti-mitogenic hormone resulted in inhibition of the mitogen-induced increases, whereas treatment with two mitogenic or two anti-mitogenic hormones resulted in additive effects on 5alphaP-R numbers. Treatments with cycloheximide and actinomycin D indicate that changes in 5alphaP-R levels depend upon transcription and translation. The non-tumorigenic breast cell line, MCF-10A, was also shown to posses specific, high affinity plasma membrane receptors for 5alphaP that were up-regulated by estradiol and 5alphaP and down-regulated by 3alphaHP. Estradiol binding was demonstrated in MCF-10A cell membrane fractions and may explain the estradiol action in these cells that lack intracellular ER. In both MCF-7 and MCF-10A cells, the increases in 5alphaP-R due to estradiol or 5alphaP, and decreases due to 3alphaHP or 20alphaHP correlate with respective increases and decreases in cell proliferation as well as detachment. These results show distribution of 5alphaP-R in several cell types and they provide further evidence of the significance of progesterone metabolites and their novel membrane-associated receptors in breast cancer stimulation and control. The findings that 3alphaHP and 20alphaHP down-regulate 5alphaP-R and suppress mitogenic and metastatic activity suggest that these endogenous anti-mitogenic progesterone metabolites deserve considerations in designing new breast cancer therapeutic agents.

Synthesis and biological properties of new stilbene derivatives of resveratrol as new selective aryl hydrocarbon modulators.

We developed new stilbene derivatives of resveratrol (E)-1-(4'-hydroxyphenyl)-2-(3,5-dihydroxyphenyl)ethene) selective for AhR and devoid of affinity for ER. Among the 24 stilbenes synthesized, all display a higher affinity than resveratrol for AhR. (E)-1-(4'-Trifluoromethylphenyl)-2-(3,5-ditrifluoromethylphenyl)ethene (4e), (E)-1-(4'-methoxyphenyl)-2-(3,5-dichlorophenyl)ethene (4j), and (E)-1-(4'-chlorophenyl)-2-(3,5-dichlorophenyl)ethene (4b) are selective, high-affinity AhR antagonists with, respective, K(i)s of 2.1, 1.4, and 1.2 nM. (E)-1-(4'-Trifluoromethylphenyl)-2-(3,5-dichlorophenyl)ethene (4i) displays a K(i) of 0.2 nM and is a selective and high-affinity agonist on AhR.

Effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells. Analysis of the underlying receptor pathways.

This study investigated the effects of testosterone and 17-beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) and the potential roles of hormone receptors involved in these actions. Human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-alpha in the presence or absence of testosterone or 17-beta-estradiol, and the expression of E-selectin and VCAM-1 was investigated. As shown by Western blot analysis, co-administration with testosterone or 17-beta-estradiol increased the expression of E-selectin and VCAM-1 induced by TNF-alpha at 6 h and 3 h, respectively. Similarly, RT-PCR analysis revealed a significant increase in the amount of mRNA for E-selectin and VCAM-1 after co-administration with testosterone or 17-beta-estradiol in TNF-alpha-stimulated HUVEC. The presence of mRNA and proteins for androgen receptor and estrogen receptor alpha in HUVEC was verified by RT-PCR and Western blot. Flow cytometric analysis showed that preincubation with androgen receptor antagonist cyproterone and estrogen receptor antagonist tamoxifen completely abrogated the upregulating effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression, respectively. Expression of TNF receptors in TNF-alpha-stimulated HUVEC was not influenced by testosterone and 17-beta-estradiol. The data indicate that both testosterone and 17-beta-estradiol increase TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells via a receptor-mediated system, and expression of TNF receptors are not changed in these actions. The implications of these results for the facilitory effects of both sex hormones on immune reactions are discussed.

Neuroprotective effects of estrogens on hippocampal cells in adult female rats after status epilepticus.

PURPOSE: Estrogens have neuroprotective effects in ischemia, stroke, and other conditions leading to neuronal cell death (e.g., Alzheimer's disease). The present study examined whether estrogens may have neuroprotective effects after seizures. METHODS: The kainic acid model was used to determine if estrogens protect hippocampal cells after status epilepticus in adult female rats. Rats were ovariectomized 1 week before hormone replacement. beta-Estradiol benzoate (EB; 2 microg in 0.1 mL of oil) was injected subcutaneously 48 and 24 hours before seizure testing. We administered kainic acid (16 mg/kg intraperitoneally) and behaviorally monitored the rats for 5 hours. After this time, all rats were injected with pentobarbital (50 mg/kg intraperitoneally) irrespective of seizure severity. Some rats received two additional doses of EB, one immediately and one 24 hours after the seizures. Another group of rats received only these two doses of EB after the seizures, and yet another group of rats received pretreatment with the intracellular EB receptor antagonist tamoxifen before each of four EB injections. Control rats received oil instead of EB. Rats were killed 48 hours after seizures. Neuronal damage was evaluated in silver-impregnated and Nissl-stained sections. RESULTS: Estrogen treatment before kainic acid administration significantly delayed the onset of kainic acid-induced clonic seizures, whereas it did not change the onset of status epilepticus compared with oil-treated controls. Furthermore, estrogen treatment significantly protected against kainic acid-induced seizure-related mortality. In control rats, examination of Nissl-stained and silver-impregnated slides revealed severe neuronal damage in the vulnerable pyramidal neurons of the hippocampal CA3 subfield and in the hilus of the dentate gyrus. Estrogen pretreatment, as well as the combination of pretreatment and posttreatment, significantly reduced the number of argyrophilic neurons in both the CA3 and the dentate gyrus. Posttreatment only had no protective effects. The data indicate that intracellular EB receptors mediate this type of neuroprotective effect, because the tamoxifen pretreatment abolished EB neuroprotection. CONCLUSIONS: Our results suggest that estrogens can be beneficial in protecting against status epilepticus-induced hippocampal damage. Hormonal conditions may have differential effects on underlying epileptic state in some patients. Therefore, more studies are necessary to determine the prospective therapeutic advantage of hormonal treatment in seizure-related damage.

[An analysis of 3H-tamoxifen binding with the cell plasma membranes of the rat uterus]. / Analiz sviazyvaniia 3H-tamoksifena s plazmaticheskimi membranami kletok matki krys.

The effect of phencarol, ranitidine, propranolol, atropine, and diethylstilbestrol (known as modulators of the tamoxifen-binding sites in plasma membranes) on the 3H-tamoxifen binding to the plasma membrane fraction of white rat uterine cells was studied by determining the amounts of estradiol and H1- and H2-receptors. The interaction of tamoxifen with various receptors of uterine cells may be significant for evaluation of the tumor activity of this compound.

Reproductive effects of hexachlorobenzene in female rats.

Hexachlorobenzene (HCB) is a polyhalogenated aromatic hydrocarbon widely distributed in the environment. In animal testing, HCB has been shown to be a reproductive toxin. Previous investigations of the effects of HCB on ovarian function have yielded equivocal results. Thus, the effects of chronic administration of HCB (1 g kg(-1) body wt.) on the ovary and pituitary hormone levels, hepatic and uterine oestradiol receptors, ovarian histopathological changes and oestrus cycle characteristics were investigated in spontaneously cycling rats. Our data demonstrate that HCB treatment, under the conditions of the present study, reduced circulating levels of oestradiol and prolactin without differences in serum concentrations of progesterone. Follicle-stimulating hormone serum levels were elevated. Hexachlorobenzene treatment resulted in irregularity of cycles, characterized mainly as prolonged periods of oestrus with a reduced number of ova recovered. In addition, HCB administration resulted in significantly decreased uterine nuclear oestrogen receptor levels. Histopathological examination revealed degenerative changes of the ovarian follicles and germinal epithelium and increased numbers of atresic follicles.

Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells.

Changes in the number of progesterone and oestradiol receptors in the endometrium are thought to play a role in the induction of luteolysis. The effect of oestradiol and progesterone on the regulation of their receptors in cultured bovine uterine epithelial and stromal cells was examined to determine the mechanisms involved in this process. Cells were obtained from cows at days 1-3 of the oestrous cycle and were cultured for 4 or 8 days in medium alone (RPMI medium + 5% (v/v) charcoal-dextran stripped newborn calf serum) or with oestradiol, progesterone or oestradiol and progesterone. At the end of culture, receptor binding was measured by saturation analysis. Specific binding of both [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor (6,7-3H) pregn-4-ene-3,20-dione) and [3H]oestradiol to epithelial and stromal cells showed high affinities (Kd = 1.1 x 10(-9) and 6 x 10(-10) mol l-1, respectively, for progesterone receptors; Kd = 5.5 x 10(-9) and 7 x 10(-10) mol l-1, respectively, for oestradiol receptors). In the stromal cells, oestradiol (0.1-10 nmol l-1) increased the number of oestradiol receptors from 0.21 +/- 0.06 to 0.70 +/- 0.058 fmol microgram-1 DNA and the number of progesterone receptors from 1.4 +/- 0.83 to 6.6 +/- 0.70 fmol microgram-1 DNA in a dose-dependent manner after 4 days of culture (P < 0.01). After culture for 8 days, the stimulatory effect of oestradiol increased. Progesterone (50 nmol l-1) had no effect on the number of oestradiol or progesterone receptors (P > 0.05). However, progesterone inhibited the stimulatory effect of oestradiol. In epithelial cells, the lower concentrations of oestradiol (0.1 and 1 nmol l-1) stimulated the number of progesterone receptors (P = 0.05) after 4 days culture, whereas the highest concentration of oestradiol (10 nmol l-1), progesterone (50 nmol l-1) and progesterone (50 nmol l-1) plus oestradiol (1 nmol l-1) had no effect. After culture for 8 days, the stimulatory effect of oestradiol decreased. In contrast to progesterone receptors, the number of oestradiol receptors increased with oestradiol concentration (P < 0.01). These data show that the number of progesterone receptors was higher in the stromal cells than in epithelial cells, whereas the number of oestradiol receptors was higher in the epithelial cells than in stromal cells. Oestradiol upregulates its own receptor and increases the number of progesterone receptors in both cell types in vitro, whereas progesterone has little effect, but inhibits the effects of oestradiol on progesterone receptors.

In vitro responses to 17beta-estradiol throughout pubertal maturation in female human bone cells.

To test the hypothesis that bone sensitivity to estrogens differ with the pubertal status, we cultured human osteoblasts (hOBs) from 14 girls (3-18 years) and examined the effects of repeated weekly doses of 17beta-estradiol (E2, 10 pM-10 nM) on estradiol receptor (ER) and progesterone receptor (PR) expression, type I procollagen (PICP) and osteocalcin (BGP; bone Gla protein) production, and alkaline phosphatase (ALP) activity. The bone samples were divided into two equal groups according to the pubertal status and plasma E2 level of the donor. The two groups were significantly different for age (9 +/- 1 and 15 +/- 1 years), pubertal status (Tanner stages I-III and IV-V), and plasma E2 concentrations (17 +/- 3 and 49 +/- 4 pg/ml). ER and PR were expressed and not influenced by the sexual maturation in untreated cells. E2 increased ER in the two groups with nanomolar doses. Picomolar doses did not significantly increase ER expression but led to significant differences in the percentage of cells expressing ER in premenarchial (33%) and postmenarchial (7%) hOB cultures. In the two groups, E2 had no clear effect on PR expression, ALP activity, nor BGP production. But repeated weekly doses of E2 significantly influenced PICP production at picomolar doses. This effect depended upon the sexual maturation of the donor. E2 decreased PICP in premenarchial cultures and increased PICP in postmenarchial cultures. Thus, E2 modulates in vitro human bone cell metabolism and probably their phenotype and has different effects, depending on the pubertal status of the donor. Unlike what could have been expected, prepubertal and early pubertal hOBs appear to be specifically sensitive to picomolar doses of E2, suggesting that this hormone is a crucial regulator of bone metabolism even before puberty.

Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor.

Impaired insulin secretion is a hallmark in both type I and type II diabetic individuals. Whereas type I (insulin-dependent diabetes mellitus) implies ss-cell destruction, type II (non-insulin dependent diabetes mellitus), responsible for 75% of diabetic syndromes, involves diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Although a clear demonstration of a direct effect of 17beta-estradiol on the pancreatic ss-cell is lacking, an in vivo insulinotropic effect has been suggested. In this report we describe the effects of 17beta-estradiol in mouse pancreatic ss-cells. 17beta-Estradiol, at physiological concentrations, closes K(ATP) channels, which are also targets for antidiabetic sulfonylureas, in a rapid and reversible manner. Furthermore, in synergy with glucose, 17beta-estradiol depolarizes the plasma membrane, eliciting electrical activity and intracellular calcium signals, which in turn enhance insulin secretion. These effects occur through a receptor located at the plasma membrane, distinct from the classic cytosolic estrogen receptor. Specific competitive binding and localization of 17beta-estradiol receptors at the plasma membrane was demonstrated using confocal reflective microscopy and immunocytochemistry. Gaining deeper knowledge of the effect induced by 17beta-estradiol may be important in order to better understand the hormonal regulation of insulin secretion and for the treatment of NIDDM. receptor.

Environmental xenobiotics may disrupt normal endocrine function by interfering with the binding of physiological ligands to steroid receptors and binding proteins.

The disruption of the reproductive system of male and female animals in the wild has been attributed to environmental chemicals (xenobiotics). The effects seen mirror alterations one might anticipate if the steroid hormone-dependent processes that regulate these systems were impaired. To determine whether xenobiotics (present at a concentration of 100 microM) exert their action through steroid-mediated pathways, we examined their ability to inhibit the binding of [3H]physiological ligands (present at a concentration of 7 nM) to the androgen and estrogen receptors, rat androgen-binding protein (ABP), and human sex hormone-binding globulin (hSHBG). The gamma- and delta-isomers of hexachlorocyclohexane, congeners of dichlorodiphenyl-trichloroethane (DDT; p,p'-DDT; p,p'-DDE; o,p'-DDT), dieldrin, atrazine, and pentachlorophenol, caused a statistically significant inhibition of specific binding of [3H]5 alpha-DHT to the androgen receptor that ranged from 100% (p,p'-DDE) to 25% (dieldrin). Methoxychlor, o,p'-DDT1, pentachlorophenol, and nonylphenol significantly reduced [3H]17 beta-estradiol binding to the estrogen receptor by 10, 60, 20, and 75%, respectively. The binding of [3H]5 alpha-DHT to ABP was inhibited 70% by the delta-isomer of hexachlorocyclohexane, but the gamma-isomer did not reduce binding significantly. Methoxychlor, p,p'-DDT, atrazine, and nonylphenol reduced [3H]5 alpha-DHT binding to ABP by approximately 40%. Nonylphenol reduced the binding of [3H]5 alpha-DHT to hSHBG by 70%. Hexachlorocyclohexane reduced [3H]5 alpha-DHT binding to hSHBG by 20%, but the stereospecific effects observed with ABP did not occur. o,p'-DDT and pentachlorophenol resulted in a statistically significant 20% inhibition of [3H]5 alpha-DHT binding to hSHBG. Some xenobiotics resulted in dissociation of [3H]ligands from their binding proteins that was statistically identical to that caused by the unlabeled natural ligand, whereas others resulted in slower or more rapid dissociation rates.

Regulation of oxytocin, oestradiol and progesterone receptor concentrations in different uterine regions by oestradiol, progesterone and oxytocin in ovariectomized ewes.

The regulation of oxytocin, oestradiol and progesterone receptors in different uterine cell types was studied in ovariectomized ewes. Animals were pretreated with a progestogen sponge for 10 days followed by 2 days of high-dose oestradiol to simulate oestrus. They then received either low-dose oestradiol (Group E), low-dose oestradiol plus progesterone (Group P) or low-dose oestradiol, progesterone and oxytocin (via osmotic minipump; Group OT). Animals (three to six per time-point) were killed following ovariectomy (Group OVX), at oestrus (Group O) or following 8, 10, 12 or 14 days of E, P or OT treatment. In a final group, oxytocin was withdrawn on day 12 and ewes were killed on day 14 (Group OTW). Oxytocin receptor concentrations and localization in the endometrium and myometrium were measured by radioreceptor assay, in situ hybridization and autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin. Oestradiol and progesterone receptors were localized by immunocytochemistry. Oxytocin receptors were present in the luminal epithelium and superficial glands of ovariectomized ewes. In Group O, endometrial oxytocin receptor concentrations were high (1346 +/- 379 fmol [3H]oxytocin bound mg protein-1) and receptors were also located in the deep glands and caruncular stroma in a pattern resembling that found at natural oestrus. Continuing low-dose oestradiol was unable to sustain high endometrial oxytocin receptor concentrations with values decreasing significantly to 140 +/- 20 fmol mg protein-1 (P < 0.01), localized to the luminal epithelium and caruncular stroma but not the glands. Progesterone treatment initially abolished all oxytocin receptors with none present on days 8 or 10. They reappeared in the luminal epithelium only between days 12 and 14 to give an overall concentration of 306 +/- 50 fmol mg protein-1. Oxytocin treatment caused a small increase in oxytocin receptor concentration in the luminal epithelium on days 8 and 10 (20 +/- 4 in Group P and 107 +/- 35 fmol mg protein-1 in Group OT, P < 0.01) but the rise on day 14 was not affected (267 +/- 82 in Group OT and 411 +/- 120 fmol mg protein-1 in Group OTW). In contrast, oestradiol treatment was able to sustain myometrial oxytocin receptors (635 +/- 277 fmol mg protein-1 in Group O and 255 +/- 36 in Group E) and there was no increase over time in Groups P, OT and OTW with values of 61 +/- 18, 88 +/- 53 and 114 +/- 76 fmol mg protein-1 respectively (combined values for days 8-14). Oestradiol receptor concentrations were high in all uterine regions in Group O. This pattern and concentration was maintained in Group E. In all progesterone-treated ewes, oestradiol receptor concentrations were lower in all regions at all time-points. The only time-related change occurred in the luminal epithelium in which oestradiol receptors were undetectable on day 8 but developed by day 10 of progesterone treatment. Progesterone receptors were present at moderate concentrations in the deep glands, caruncular stroma, deep stroma and myometrium in Group O. Oestradiol increased progesterone receptors in the luminal epithelium, superficial glands, deep stroma and myometrium. Progesterone caused the loss of its own receptor from the luminal epithelium and superficial glands and decreased its receptor concentration in the deep stroma and myometrium at all time-points. There was a time-related loss of progesterone receptors from the deep glands of progesterone-treated ewes between days 8 and 14. These results show differences in the regulation of receptors between uterine regions. In particular loss of the negative inhibition by progesterone on the oxytocin receptor by day 14 occurred only in the luminal epithelium, but is unlikely to be a direct effect of progesterone as no progesterone receptors were present on luminal epithelial cells between days 8 and 14.

Cyclosporin A potentiates estradiol-induced expression of the cathepsin D gene in MCF7 breast cancer cells.

Although the physiological role of the immunophilins cyclophilin-40 and FKBP52 is unknown, their identification as components of the unactivated estrogen receptor has raised the possibility that they might influence receptor activity in response to the binding of immunosuppressants cyclosporin A and FK506, respectively. We have used Northern analysis to determine the influence of cyclosporin A on the expression of the estrogen-inducible cathepsin D gene in human MCF7 breast cancer cells. We report that 1-3 microM cyclosporin A can potentiate cathepsin D mRNA expression by up to 2-fold in cells treated with 10(-12) to 10(-10) M estradiol. A decreased potentiation effect was noted at higher hormone concentrations. Cyclosporin A alone was unable to induce cathepsin D expression and the increased gene activation observed with combined estradiol/cyclosporin A treatment was negated by the antiestrogen ICI 164,384. Our results suggest that the increased potency of estradiol in the presence of cyclosporin A is associated with an enhanced transcriptional activity of the estrogen receptor and support a role for receptor-associated cyclophilin-40 in the activation process.

[Effect of L-TYR on the uterine cytosol estradiol and progesterone receptors in rat].

The effect of L-TYR on rat uterine cytosol estradiol and progesterone receptor contents was studied. The rats were divided into 4 groups: proestrus, estrus, metestrus and diestrus. One horn of each uterus was injected with L-TYR (2 mmol/L, 0.1 ml), while the other with 0.1 ml 0.9% NaCl serving as control. The mole concentration of receptor was calculated with RRA and cytosol receptor content was expressed as fmol/mg protein. It was shown that L-TYR decreased significantly the uterine estradiol and progesterone receptors in proestrus, estrus and diestrus phases of the estrus cycle, but without effect on metestrus. This was most probably due to competitive combination of TYR with some functional groups of the estradiol receptor because of similar chemical conformation of TYR to estradiol. Threonine could also decrease, to some extent, the uterine cytosol progesterone receptor content at estrus and dioestrus phases. Serine had no effect on the contents of uterine cytosol either estradiol or progesterone receptor in normal and ovariectomized rats. The present observation indicates that L-TYR appears to affect the synthesis of the cytosol estradiol and progesterone receptor in the rat uterus independent, however, of endogenous ovarian sex hormones, since the effect is still present in the ovariectomized animals.

Pubertal benzpyrene exposition decreases durably the sexual activity of the adult male and female rats.

Single benzpyrene treatment of 5 week old male and female rats significantly decreased their sexual activity at 3 months of age. Among the hormone preparations used nandrolone was comparatively practically ineffective, while estradiol decreased the lordosis quotient of females. In males benzpyrene produced a total failure of ejaculation. These results draw attention to the wide time-scale of receptorine and behavioral effects of the aromatic hydrocarbon, benzpyrene. These effects are detectable following fetal, neonatal or pubertal treatments, especially in females. In males, the negative effect was manifested after neonatal and pubertal treatments only. The experiments suggest that in some cases hormonal imprinting does not develop solely in the perinatal period but it might develop later.

Growth of LNCaP human prostate cancer cells is stimulated by estradiol via its own receptor.

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.

[The effect of spermine on protein kinase activity and number of cytosolic estradiol receptors]. / Deistvie spermina na aktivnost' proteinkinaz i kolichestvo tsitozol'nykh retseptorov éstradiola.

Spermine decreased distinctly the activity of Ca2+ -phospholipid-dependent protein kinases, whereas not affected the protein kinase A activity. Content of cytosol estradiol receptors was drastically decreased in rat uterine in presence of the polyamine. The data obtained and literature data suggest that activation of estradiol receptors appears to occur after phosphorylation by means of Ca2+ - dependent protein kinases, which are regulated via phosphoinositide-involving mechanism, i.e. these receptors acquire the ability to bind hormones.

[Reductive effect of the sesquiterpenic lactone "Helenalin " and its metallic derivatives in the formation of the estradiol receptor complex in breast tumor tissue]. / Efecto reductor de la lactona sesquiterpénica "Helenalina" y sus derivados metálicos en la formación del complejo estradiol receptor en tejido tumoral mamario.

Chemotherapy for systemic therapy in breast cancer has been widely used, and has been supported by many varied compounds with different origins and different compositions. Nevertheless, all of them produce several side adverse effects which must be taken into account. For this reason we must study new possibilities in which the administered drug acts selectively on the tumour cell without injuring healthy tissue. For studying its effect, a gamma lactone called "Helenaline" and its metalic derivates He-Co, He-Hg and He-Cu were studied, which chemical composition allows them to react with -SH residues present in the tumour cell receptors, which when interspaced by a previous reaction, could modify its structural composition and finally its affinity by the hormone. The inhibition effect for formation of estradiol-receptor complex in breast tumour tissue using Helenaline 12 n M and 126 n M was studied, obtaining 14% and 56% inhibition effect respectively. When He-Co, He-Hg and He-Cu effect was studied, this effect was raised obtaining 11%, 10.5% and 60% with 12 nM and 44.5%, 74.4% and 86% with 126 nM respectively.

Efecto reductor de la lactona sesquiterpénica "Helenalina" y sus derivados metálicos en la formación del complejo estradiol receptor en tejido tumoral mamario / Reductive effect of sesquiterpenic lactone \"Helenaline\" and its metalic derivates in complex estradiol-receptor formation in breast tumoral tissue

Dentro de la terapia sistémica empleada para el tratamiento de cáncer mamario, ha sido extensamente utilizada la quimioterapia, la cual ha sido apoyada por muy diversos compuestos en cuanto a origen y composición química. Sin embargo, todos ellos, producen diversos efectos colaterales adeversos, dignos de tomarse en cuenta. Por este hecho, precisa estudiar nuevas posibilidades en donde el fármaco aplicado, actúa selectivamente sobre célula tumoral, sin lesionar tejido sano. Para su efecto, se estudió una gamma lactona llamada "Helenalina" y sus derivados metálicos He-Co, He-Hg y He-Cu, cuya composición química les permite reaccionar con residuos -SH presentes en el receptor de la célula tumoral, los cuales al intercalarse por una reacción previa, podría modificar su composición estructural y finalemente su afinidad por la hormona. Se investigó el efecto de inhibición para la formación del complejo estradiol-receptor en el citosol de tejido tumoral mamario empleando Helenalina a 12 n M y 126 n M, obteniéndose un efecto de inhibición de 14 por ciento y 56 por ciento respectivamente. Cuando se estudió He-Co, He-Hg y He-Cu este efecto se vió aumentado, obteniéndose 11 por ciento, 10.5 por ciento y 60 por ciento con 12 n m y 44.5 por ciento, 74.5 por ciento y 86 por ciento con 126 n M respectivamente