Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.     

A estradiol-17beta receptor in the reproductive system of the female of Octopus vulgaris: characterization and immunolocalization

In this study, for the first time we have identified an estradiol-17beta receptor (ER) in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present in the cytosol, but not in the nuclear extract of the ovary and the oviduct. A steroid specificity competition assay showed that 3H-estradiol-17beta binding activity showed a preference for estradiol-17beta. DNA-cellulose chromatography confirmed the presence of one 3H-estradiol-17beta binding component. By using antibodies anti ER (578-595), we have localized by Western blotting one band of about 70 kDa. ER immunoreactivity has been localized in the nuclei of the follicle cells of the ovary, in the nuclei of the epithelium lining the proximal portion of the oviduct and in the nuclei, and in the cytoplasm of the inner region of the oviducal gland and in the cytoplasm of the outer region of the oviducal gland. These data, taken together, provide evidence that in Octopus vulgaris the ER has biochemical and immunohistochemical characteristics resembling those of ER in vertebrates.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.     

Effect of menstrual cycle phase and hormonal treatments on evaluation of tubal patency in baboons

Background

We evaluated whether menstrual cycle phase influences the assessment of tubal patency by hysterosalpingography (HSG) in baboons.

Methods

Retrospective analysis of baseline tubal patency studies and serum estradiol (E₂) and progesterone (P4) values obtained from female baboons used as models for development of non‐surgical permanent contraception in women. The main outcome measure was bilateral tubal patency (BTP) in relationship with estradiol level.

Results

Female baboons (n = 110) underwent a single (n = 81), two (n = 26), or three (n = 3) HSG examinations. In 33/142 (23%) HSG examinations, one or both tubes showed functional occlusion (FO). The median E₂ in studies with BTP (49 pg/mL) was significantly higher than in those studies with FO (32 pg/mL, P = .005). Among 18 animals with repeat examinations where serum E₂ changed from <60 to ≥ 60 pg/mL, 13 results changed from FO to BTP (P = .0001). No sets showed a change from BTP to FO with an increase in estradiol.

Conclusion

In baboons, functional occlusion of the fallopian tube is associated with low estradiol levels, supporting a role for estrogen‐mediated relaxation of the utero‐tubal junction.     

Brain aromatase, 5α‐reductase,and 5β‐reductase change seasonally in wild male song sparrows: Relationship to aggressive and sexual behavior

In many species, territoriality is expressed only during the breeding season, when plasma testosterone (T) is elevated. In contrast, in song sparrows (Melospiza melodia morphna), males are highly territorial during the breeding (spring) and nonbreeding (autumn) seasons, but not during molt (late summer). In autumn, plasma sex steroids are basal, and castration has no effect on aggression. However, inhibition of aromatase reduces nonbreeding aggression, suggesting that neural steroid metabolism may regulate aggressive behavior. In wild male song sparrows, we examined the neural distribution of aromatase mRNA and seasonal changes in the activities of aromatase, 5α‐, and 5β‐reductase, enzymes that convert T to 17β‐estradiol, 5α‐dihydrotestosterone (5α‐DHT, a potent androgen), or 5β‐DHT (an inactive metabolite), respectively. Enzyme activities were measured in the diencephalon, ventromedial telencephalon (vmTEL, which includes avian amygdala), caudomedial neostriatum (NCM), and the hippocampus of birds captured during spring, molt, or autumn. Aromatase and 5β‐reductase changed seasonally in a region‐specific manner. Aromatase in the diencephalon was higher in spring than in molt and autumn, similar to seasonal changes in male sexual behavior. Aromatase activity in the vmTEL was high in both spring and autumn but significantly reduced at molt, similar to seasonal changes in aggression. 5β‐Reductase was not elevated during molt, suggesting that low aggression during molt is not a result of increased inactivation of androgens. These data highlight the relevance of neural steroid metabolism to the expression of natural behaviors by free‐living animals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 209–221, 2003     

Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala₁₂₂₇ to Ser₁₂₅₁), which contains a single Trp residue (W₁₂₄₆) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala₁₂₂₇ was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring (∼50% α-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp₁₂₄₆ to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including λ_max, lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.