Effect of tamoxifen and tamoxifen derivatives on the conversion of estrone-sulfate to estradiol in the R-27 cells, a tamoxifen-resistant line derived from MCF-7 human breast cancer cells

R-27 cells, a tamoxifen-resistant clone of MCF-7 mammary cancer cells, were used to study the effect of tamoxifen and its derivatives (4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen) on the conversion of estrone sulfate to estradiol. The present data indicate that (1) tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen inhibit the uptake of the radioactivity after incubation of these triphenylethylene derivatives with [3H]-estrone sulfate; (2) there is a significant decrease of the conversion of estrone sulfate to estradiol by these antiestrogens; (3) the concentrations of estradiol (cytosol + 0.6 M KCl nuclear extract) which are 293 +/- 50 pg/mg DNA in the control studies (estrone sulfate alone), diminish to 26 +/- 5 pg/mg DNA after addition of tamoxifen, to 9 +/- 2 with 4-hydroxytamoxifen, to 24 +/- 7 with N-desmethyltamoxifen and to 32 +/- 6 with cis-tamoxifen. It is concluded that estrone sulfate can play an important role in the biological responses to estrogens in this breast cancer cell line and tamoxifen and its derivatives block the conversion of estrone sulfate to estradiol. The decrease in concentration of estradiol could be explained by the presence of the estrogen receptor system but other ways of the action of antiestrogens remain to be explored.     

An immunohistochemical study of the catecholamine synthesizing enzymes and neuropeptides in the female guinea-pig uterus and vagina

Summary The uterus and vagina of the guinea pig have been examined, region by region, for acetylcholinesterase, tyrosine hydroxylase, dopamine -hydroxylase and aromatic amino acid decarboxylase activity, as well as for the neuropeptides, neuropeptide Y, vasoactive intestinal peptide, substance P, enkephalin and somatostatin. No acetylcholinesterase activity was localized in the uterus, though it was present in associated paracervical ganglion tissues. Of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine -hydroxylase activity was found virtually throughout the reproductive tract, whereas aromatic amino acid decarboxylase activity was restricted in its distribution. Neuropeptide distribution was quite varied. Neuropeptide Y was found throughout the endometrium/submucosa but only in the muscularis of the vagina and not in the myometrium. Substance P was localized in the vagina and uterine horn, though not the body of the uterus. Vasoactive intestinal peptide was present in all regions of the endometrium/submucosa, but not in the myometrium of the uterine horn. Enkephalin and somatostatin were not localized in any part of the reproductive tract examined, apart from paracervical ganglion tissues. The types and significance of the nerves supplying the reproductive tract are discussed.     

Action of an antiestrogen agent, tamoxifen on the uterus and vagina of the ovariectomized rat]

Tamoxifen has more or less strong estrogen influence according to the targets : a light one on the uterus (1 mg being much less strong than 0.25 microgram of estradiol), a dynamic one on the vagina (50 microgram of tamoxifen make the vagina open in as short a time as 0.25 microgram of estradiol do but the keratinisation is still not completed even with 1 mg of tamoxifen). We can still see this influence four days after the end of the treatment. This influence is weak on the uterus until the 11 th day and it is much stronger on the vagina until about the 8 th day. Tamoxifen has an antiestrogenic action when opposed to 20 microgram of estradiol : this action is limited as soon as you give a dosis of 50 microgram on the uterus and it is nearly total with a dose of 1 mg ; we can notice it on the vaginal only from 200 microgram on.     

Biological responses and histological studies of estriol and estriol sulfate in fetal and newborn uteri of guinea pig

The biological responses of estriol (E₃) and of estriol-3-sulfate (Ea₃-S) in the fetal and newborn uteri of guinea pig were studied. After a treatment of E₃ (1 mg/kg/day) or E₃-S (1.4 mg/kg/day) to pregnant guinea pigs (49–60 days of gestation) for 6 days, both estrogens provoke a significant uterotrophic effect in the fetal uterus which increases in weight 1.8–2.5 times in relation to the non-treated animals. The stimulation of progesterone receptor (PR) is also very intense, 7–12 times in relation to the control animals.In another series of experiments newborn guinea pigs (2-day old) were treated with 100μg/animal of E₃ or 140 μg/animal of E₃-S for a short (2 days) or a long (12 days) period. Concerning the uterotrophic effect, the weight of the uterus increases 1.8–2.5 times (in relation to the non-treated animals) after a 2-day treatment and the effect continues to increase up to 4–5 times in the 12-day treated animals. In opposition to the fetal uterus, the effect on PR provoked by E₃ or E₃-S is very limited (only an increase of 1 time in relation to the non-treated animals); the effect is even less intense after the 12-day treatment. Histological studies show an intense hypertrophic effect particularly in the epithelium of the endometrium in the fetal and newborn uteri for both E₃ and E₃-S. In the newborns the effect is also intense on the epithelium of the uterine gland.It is concluded that: (1) Estriol and estriol sulfate are very active and with similar intensity on the uterine weight before and after birth; (2) The stimulatory effect on PR decreases very significantly after birth and after a long treatment; (3) E₃S can act as a potent hormonal precursor.     

Increased gap junctional intercellular communication is directly related to the anti-tumor effect of all-trans-retinoic acid plus tamoxifen in a human mammary cancer cell line

Additive effects against tumor cells might be achieved by combining anti-neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all-trans-retinoic acid (RA) and tamoxifen (Tx) in MCF-7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 +/- 3% of MCF-7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell-to-cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96-120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre-existing E-cadherin levels was observed after drug exposure along with a downregulation of Bcl-2 and C-myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx-induced GJIC with 18-beta-glycyrrhetinic acid (beta-Gly) prevented in 34% the inhibition of MCF-7 proliferation and the E-cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl-2, c-Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF-7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy.     

Adrenergic innervation of the tunica media in the saphenous artery of the fetal and newborn guinea-pig

Summary Previous studies have demonstrated that adrenergic nerves are located in the medial-adventitial border of the muscular arteries. Observations made in this study have revealed that adrenergic nerves penetrate into the outer medial layer of the saphenous artery in fetal and newborn guinea-pigs, while in the adult these nerves are located in the medial-adventitial border. It is proposed that the adrenergic nerves located in the tunica media may have a trophic effect on the medial smooth muscle. It is further suggested that the final refinement of the dual control system of arterial walls, by nerves and circulating catecholamines, involves exclusion of adrenergic nerves from the tunica media.     

Progestagen-concentrating cells in the brain, uterus, vagina and mammary glands of the galago (Galago senegalensis)

Progestagen-concentrating cells were localized in the oestrogen-primed ovariectomized galago by radioautography after injection of [3H]promegestone (R5020). In the brain, radioactivity was concentrated in the nuclei of neurones in the preoptic region and in the mediobasal hypothalamus. Labelled cells were also observed in the anterior pituitary. In the uterus (uterine horns and cervix), the muscle and stromal cells showed greater labelling than did the glandular and luminal epithelia. Labelled cells were present in the different cell layers of the vagina. The majority of glandular epithelial cells of the mammary glands exhibited a high degree of labelling. Pretreatment with an excess of unlabelled promegestone but not with an excess of nonradioactive testosterone reduced the nuclear concentration of radioactivity in these target tissues. These results show that there are no major differences in the distribution of progestagen-concentrating cells in rodents and galago.     

Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

Antiestrogen action in mammary cancer and in fetal cells

The present data confirm the very complicity of the response of antiestrogen when this compound is studied in different experimental conditions. The new and potent antiestrogen ICI 164,384, which is considered as a full antagonist in most models studied, concerning the progesterone receptor in the isolated cells of the uterus and vagina of guinea-pig acts as a real agonist. However, this compound antagonizes cell proliferation, progesterone receptor, and decreases the concentration of estradiol in different hormone-dependent mammary cancer cell lines. Another interesting aspect is the response of the antiestrogen 4-hydroxytamoxifen which in isolated cells of very close tissues such as the uterus and vagina is an antagonist for the former and agonist for the latter concerning the progesterone receptor. In conclusion, the present data added new information in the complicity of the mechanism of action of antiestrogens, but using new models interesting possibilities are opened to understand their responses and their mechanism.     

Environmental magnetic fields inhibit the antiproliferative action of tamoxifen and melatonin in a human breast cancer cell line

We have previously reported that environmental-level magnetic fields (1.2 μT [12 milligauss], 60 Hz) block the growth inhibition of the hormone melatonin (10⁻⁹ M) on MCF-7 human breast cancer cells in vitro. We now report that the same 1.2 μT, 60 Hz magnetic fields significantly block the growth inhibitory action of pharmacological levels of tamoxifen (10⁻⁷ M). In biophysical studies we have taken advantage of Faraday's Law of Current Induction and tested whether the 1.2 μT magnetic field or the associated induced electric field is responsible for this field effect on melatonin and tamoxifen. We observe that the magnetic field component is associated with the field blocking effect on melatonin and tamoxifen function. To our knowledge the tamoxifen studies represent the first experimental evidence for an environmental-level magnetic field modification of drug interaction with human breast cancer cells. Together, these findings provide support to the theory that environmental-level magnetic fields can act to modify the action of a drug or hormone on regulation of cell proliferation. Melatonin and tamoxifen may act through different biological pathways to down-regulate cell growth, and further studies are required to identify a specific biological site of interaction for the 1.2 μT magnetic field. Bioelectromagnetics 18:555–562, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Hedgehog signaling plays roles in epithelial cell proliferation in neonatal mouse uterus and vagina

Both the uterus and vagina develop from the Müllerian duct but are quite distinct in morphology and function. To investigate factors controlling epithelial differentiation and cell proliferation in neonatal uterus and vagina, we focused on Hedgehog (HH) signaling. In neonatal mice, Sonic hh (Shh) was localized in the vaginal epithelium and Indian hh (Ihh) was slightly expressed in the uterus and vagina, whereas all Glioma-associated oncogene homolog (Gli) genes were mainly expressed in the stroma. The expression of target genes of HH signaling was high in the neonatal vagina and in the uterus, it increased with growth. Thus, in neonatal mice, Shh in the vaginal epithelium and Ihh in the uterus and vagina activated HH signaling in the stroma. Tissue recombinants showed that vaginal Shh expression was inhibited by the vaginal stroma and uterine Ihh expression was stimulated by the uterine stroma. Addition of a HH signaling inhibitor decreased epithelial cell proliferation in organ-cultured uterus and vagina and increased stromal cell proliferation in organ-cultured uterus. However, it did not affect epithelial differentiation or the expression of growth factors in organ-cultured uterus and vagina. Thus, activated HH signaling stimulates epithelial cell proliferation in neonatal uterus and vagina but inhibits stromal cell proliferation in neonatal uterus.     

Interaction of retinoids and tamoxifen on the inhibition of human mammary carcinoma cell proliferation

The growth of chemically induced mammary tumors is inhibited by both hormone manipulation as well as by retinoids. Numerous mammary carcinoma cell lines are also inhibited by retinoids. Co-treatment of estrogen receptor (ER)-positive breast cancer cells resulted in an additive effect in terms of inhibition of cellular proliferation. The addition of varying concentrations of retinoic acid (RA) to varying concentrations of tamoxifen (TMX) resulted in an additive effect on the inhibition of proliferation of the ER-positive human carcinoma cell lines (MCF-7). Co-treatment of MCF-7 cells over time with RA and TMX resulted in enhanced inhibition of growth. A similar phenomenon was observed when other synthetic retinoids were combined with TMX. This enhanced inhibition by the combination of retinoids and TMX was also observed with other ER-positive cell lines (ZR-75, T47-D), while no effect was noted on the ER-negative cell lines (MDA-MB-231, Hs578T).     

Histologic changes in ovary, uterus, vagina, and mammary gland of mature beagle dogs treated with the SERM idoxifene

BACKGROUND: Idoxifene is a selective estrogen receptor modulator similar to tamoxifen but is no longer in pharmaceutical development due to adverse genitourinary effects in the clinic. Histologic observations of the reproductive system and mammary glands are presented from female dogs treated with idoxifene for up to 12 months. METHODS: Studies were conducted as part of regulatory requirements to support clinical development. Idoxifene was given orally by capsule, once daily, for 1, 6, or 12 months to female Beagle dogs (n = 3 or 4/group) aged 11-14 months (start of dosing) at dosages 0, 0.03, 0.3, 1.5, or 3 mg/kg/day. Evaluations included the following: clinical observations, hematology, hemostasis, chemistry, toxicokinetics, and histology. RESULTS: Dose- and time-dependent findings were present in dogs given > or = 0.03 mg/kg/day and included abnormal vaginal discharge, minor increases of platelet and neutrophil counts, and microscopic observations in the ovary (atrophy and mesothelial [ovarian surface epithelium] hyperplasia), endometrium (edema, inflammation, glandular atrophy, squamous metaplasia, increased collagen), myometrium (edema, increased collagen), vagina (squamous hyperplasia, keratinization), and mammary gland (atrophy). CONCLUSION: Dogs given idoxifene exhibited estrogenic effects in ovary, uterus, and vagina but antiestrogenic effects in endometrial and mammary glands consistent with several observations in clinical trials in post-menopausal women treated with triphenylethylenes.     

Rapid modulation by progesterone and tamoxifen of estradiol effects on nuclear histone acetylation in the uterus of the fetal guinea pig

The effect of estradiol, progesterone, tamoxifen, estradiol + progesterone or estradiol + tamoxifen on the [³H]acetylation of histones in the fetal uterus of guinea pig was studied. The fetuses were injected subcutaneously ‘in situ’ with the hormones or $\text{tamoxifen}\text{+ [}{}^3\text{H}\text{]}\text{acetate}$ alone. In 10 min, estradiol stimulated the acetylation of histone 10–12-times with respect to the control animals. Progesterone and tamoxifen blocked this effect. It is suggested that histone acetylation is an early step induced by estrogen action during intrauterine life and that progesterone and tamoxifen suppress this mechanism very effectively.     

牦牛乳腺上皮细胞系的建立与生物学特性

本研究旨在建立牦牛乳腺上皮细胞体外培养体系。采用胶原酶消化法成功地建立了牦牛乳腺上皮细胞系(YMEC),通过免疫细胞化学、超微结构观察和RT-PCR 法对YMEC 细胞进行了鉴定,并研究了其形态、活力、生长曲线以及核型等生物学特性。结果表明,YMEC 细胞染色体2n = 60,群体倍增时间为45 ～ 48 h,持续培养25 代后出现细胞分化;细胞呈典型的“铺路石样”形态,其表面有丰富的微绒毛,细胞质内含丰富的线粒体和粗面内质网。污染检测结果为阴性。在激素诱导培养时,检测到了β - 酪蛋白mRNA 的表达。表明本研究成功建立了保留泌乳功能的牦牛乳腺上皮细胞系,为研究牦牛乳腺上皮细胞的功能提供了理想的工具。