Characterization of MNAR expression

We have previously demonstrated that modulator of nongenomic action of estrogen receptor (MNAR) integrates action of estrogen receptor alpha (ERalpha), and potentially some other nuclear receptors (NRs), in regulation of Src/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathway. MNAR is a scaffolding protein that contains 10 LXXLL type motifs that can interact with NRs and 3 PXXP type motifs that can bind to SH3 domains present in kinases and other signaling molecules. Formation of ER-MNAR-cSrc complex leads to activation of Src and downstream Ras/Raf/MAPK pathway. The goal for this study was to compare MNAR expression in various cell lines, to optimize methods that can be used to manipulate its expression and to evaluate MNAR cellular distribution. We found that MNAR is differentially expressed. The highest levels of its expression were found in fast proliferating cells, such as breast adenocarcinoma (MCF-7)-, T cell lymphoma (Jurkat)-, prostate carcinoma (LNCaP)- and osteosarcoma (SaOS2)-derived cell lines. MNAR was undetectable in African green monkey kidney cells (COS-7) and Chinese hamster ovary cells (CHO-K1). We established and optimized a protocol to knockdown MNAR using siRNA and to overexpress it in MCF-7 cells. Exogenously expressed MNAR was found in both cytoplasmic and nuclear fractions, the majority of MNAR, however, was found in the cytoplasmic fraction. Presence of MNAR in the cell nucleus indicates that it may play a role in regulation of gene expression.     

Impact of physical aquatic parameters on the annual rhythmicity of sex steroid and cortisol and their interrelationship in two distantly related fish population

A chronological relationship between the annual profiles of stress hormone cortisol and male (testosterone and 11-keto testosterone) and female (17β-estradiol) sex steroids, the key regulator of annual reproductive cycle has been sought in two different group of fish (Mystus gulio and Parambassis ranga) under natural photothermal conditions. The serum samples were collected at two different times in each month (from January to December) and the same was repeated for two consecutive years throughout an annual cycle. The fluctuations of major physical factors (temperature, salinity, pH, dissolved oxygen and carbon dioxide) and presence of three important heavy metals were also estimated accordingly. Therefore, the present study aims to explore the rhythmic responses of sex steroids and cortisol to assess the impact of different environmental stressors on selected fish species. We tried to develop a realistic conceptual idea to analyze and predict the effect of changing environmental parameters on the possible shift in the rhythmicity of aforesaid hormones in two different groups of fish and their adaptive responses to thrive in such environment. Our results indicated that the fluctuation of circannual rhythms of testosterone, 17-β estradiol and 11-KT varied according to species, was related with the physical factors of the aquatic system and temperature was the most important factor among them. This information might help to frame the reproductive strategies for different fish species, as well.     

Targeted brain delivery of 17β-estradiol via nasally administered water soluble prodrugs

The utility of the nasal route for the systemic delivery of 17β-estradiol was studied using watersoluble prodrugs of 17β-estradiol. This delivery method was examined to determine if it will result in preferential delivery to the brain. Several alkyl prodrugs of 17β-estradiol were prepared and their physicochemical properties were determined. In vitro hydrolysis rate constants in buffer, rat plasma, and rat brain homogenate were determined by high-performance liquid chromatography. In vivo nasal experiments were carried out on rats. Levels of 17β-estradiol in plasma and cerebral spinal fluid (CSF) were determined with radioimunoassay using a gamma counter. The study revealed that the aqueous solubilities of the prodrugs were several orders of magnitude greater than 17β-estradiol with relatively fast in vitro conversion in rat plasma. Absorption was fast following nasal delivery of the prodrugs with high bioavailability. CSF 17β-estradiol concentration was higher following nasal delivery of the prodrugs compared to an equivalent intravenous dose. It was determined that water-soluble prodrugs of 17β-estradiol can be administered nasally. These prodrugs are capable of producing high levels of estradiol in the CSF and as a result may have a significant value in the treatment of Alzheimer's disease. Published: March 25, 2002.     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

Paradoxical effects of 17β-estradiol on glucose transport in primary cell cultures of a rat mammary tumor

Primary cell cultures of rat mammary carcinoma R3230AC exhibited a rapid, reversible and dose-related inhibition of carrier-mediated 3-0-methyl-D-glucose transport when estrone, 17β-estradiol, estriol, diethylstilbestrol or phloretin was present during the transport assay. With 17β-estradiol, maximal transport inhibition (66%) was observed at 40μM, a concentration also effective in preventing cell growth when present in the media. Cultures preincubated in growth media containing 5mM glucose plus 40μM 17β-estradiol for one day displayed enhanced rates of carrier mediated 3-0-methyl-D-glucose transport. This increase was prevented by 50mM glucose and can be explained as an adaptive response to a condition simulating glucose starvation.     

Fluoroenzymatic cycling assay (FECA) for the determination of catechol estrogen monomethyl ethers in human urine

In the present study a method is described for the quantitative determination of the methylated metabolites of catechol estrogens in human urine. Following initial enzymatic hydrolysis the urine samples are extracted with ethyl acetate. The monomethyl ethers of catechol estrogens are then selectively fractionated with straight phase chromatography on Lipidex-5000 gel. Finally, samples are quantitated using enzymatic cycling with 17-estradiol dehydrogenase combined with fluorometry. The method is sensitive, reproducible and reasonably rapid for routine analysis and avoids the hazards of radioisotopes. Preliminary values of normal males and non-pregnant females are presented.Special Issue dedicated to Dr. O. H. Lowry.     

Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxy resins, has previously been reported to induce micronuclei containing whole chromosomes in Chinese hamster V79 cells. In the present study, the aneuploidogenic potential of BPA was investigated in cultured human AG01522C fibroblasts. In contrast to the known aneugens diethylstilbestrol (DES) and 17beta-estradiol, which caused mitotic arrest and the induction of kinetochore-positive micronuclei, BPA did not induce micronuclei and inhibited the proliferation of AG01522C cells in G2 phase and probably also in G1 phase. Fluorescence microscopy of the BPA-treated cells after immunofluorescent staining of microtubules revealed structural abnormalities of the cytoplasmic microtubule complex (CMTC): densely stained rings and loops of tubulin were observed, which increased in number with increasing BPA concentration and were more stable against low temperature than normal microtubules. The mechanisms of the growth inhibition and the interference with microtubules elicited by BPA in AG01522C cells are presently unknown. The formation of rings and loops in the CMTC of AG01522C cells was also observed with two congeners of BPA carrying one and two, respectively, additional methyl groups in ortho-position to the phenolic hydroxyl group at each aromatic ring. However, in contrast to BPA itself, these congeners of BPA behaved "DES-like" by inducing mitotic arrest and kinetochore-positive micronuclei in AG01522C cells.     

Onset of direct 17-beta estradiol effects on proliferation and c-fos expression during oncogenesis of endometrial glandular epithelial cells

In normal endometrial glandular epithelial cells (GEC), 17beta-estradiol (E2) enhances proliferation and c-fos expression only in the presence of growth factors. On the contrary, growth factors are not required for the E2 effects in cancerous cells. Thus, a repression of E2 action could exist in normal cells and be turned off in cancerous cells, allowing a direct estrogen-dependent proliferation. To verify this hypothesis, we established immortalized and transformed cell models, then investigated alterations of E2 effects during oncogenesis. SV40 large T-antigen was used to generate immortalized GEC model (IGEC). After observation of telomerase reactivation, IGEC model was transfected by activated c-Ha-ras to obtain transformed cell lines (TGEC1 and TGEC2). The phenotypic, morphological, and genetic characteristics of these models were determined before studying the E2 effects. In IGEC, the E2 action on proliferation and c-fos expression required the presence of growth factors, as observed in GECs. In TGECs, this action arose in the absence of growth factors. After IGEC transformation, the activation of ras pathway would substitute the priming events required for the release of repression in GEC and IGEC and thus permit direct E2 effects. Our cell models are particularly suitable to investigate alterations of gene regulation by E2 during oncogenesis.     

Direct evidence for the localization of the steroid-binding site of the plasma sex steroid-binding protein (SBP or SHBG) at the interface between the subunits.

Complete dissociation of dimeric plasma sex steroid-binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid-binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid-binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.