17β-estradiol Stimulation Research Articles

Abstract 568: Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models

Abstract Introduction HER4, a tyrosine kinase receptor belonging to the Human Epidermal Growth Factor/HER/ErbB-family, has a complex biology that includes four isoforms (JMa-CYT1/2, and JMb-CYT1/2) and tissue specific oncogenic or tumour suppressor roles. In estrogen receptor positive (ER+) breast cancer (BC), HER4 isoforms can either act as a co-regulator of ESR1-mediated gene expression by localising to the nucleus, or activate apoptosis by localising to mitochondria. There is very little known about HER4 function in cutaneous melanoma, despite cutaneous melanoma showing the highest HER4 genomic alteration frequency (17.34%) across different cancers (cBioPortal). Due to the interplay between estrogen signalling and HER4 isoforms in ER+BC, this study investigates the RNA expression of HER4 isoforms along with hormone and nuclear receptors associated with 17β-estradiol stimulation in ER+BC and cutaneous melanoma in vitro models. Material and Methods CAMA1 (ER+BC) and SKMEL24 (cutaneous melanoma) cell lines were serum starved for 4h prior to stimulation with 17β-estradiol (0.5nM, 1nM, 5nM and 10nM). RNA was extracted from cells after 24h using TRIzol, followed by qRT-PCR to assess the expression of HER4 isoforms, the hormone receptors estrogen receptor 1 (ESR1/ER-alpha), estrogen receptor 2 (ESR2/ER-beta), progesterone receptor (PGR/PR), G protein-coupled estrogen receptor 1 (GPER1) and the nuclear receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). Cell proliferation and caspase3/7 apoptosis analysis were performed at 24/48/72h in the 17β-estradiol stimulated cells by using the IncuCyte Live Cell Analysis Instrument (Sartorius). SN-38 (150nM) was used as positive control for apoptosis induction. Two-way ANOVA was used for the statistical analysis, p<0.05 was significant. Results The qRT-PCR analysis showed that upon stimulation of CAMA1 with 1nM of 17β-estradiol there is a numerical shift in the CYT-1/CYT-2 ratio towards CYT-2 (not statistically significant, p>0.05) and a significant increase in PGR (p= 0.0027) and RAR-A (p= 0.0248) expression compared to control. CAMA1 stimulated with any concentration of 17β-estradiol showed an increase in proliferation compared to the control. The stimulation of SKMEL24 with 5nM of 17β-estradiol showed an increase in JM-a (p=0.0075) and CYT-1 (p=0.0271) HER4 isoforms, GPER1 (p= 0.0286), and a dose-dependent increase in RARA (p=0.0011) compared to control, even in the absence of ESR1, ESR2 and PGR. SKMEL24 stimulated with 17β-estradiol showed a decrease in proliferation compared to control, but no apoptosis induction was found. Conclusion These findings suggest potential interplay between 17β-estradiol and HER4 isoforms leading to the activation of intracellular pathways that could influence tumour progression/suppression in melanoma. Further studies are needed to clarify the specific receptor interactions and pathways involved. Citation Format: Marta Valenti, Georg F. Bischof, John P. Crown, Denis M. Collins. Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 568.

PrPC Promotes Endometriosis Progression by Reprogramming Cholesterol Metabolism and Estrogen Biosynthesis of Endometrial Stromal Cells through PPARα Pathway.

Endometriosis (EMs) is characterized as an estrogen-dependent disease. Whereas, the underlying mechanism for activated estrogen biosynthesis in EMs lesions is largely unknown. We analyzed cholesterol metabolism and estrogen biosynthesis condition of EMs lesions by biological information analysis of GEO datasets, and further verified both in vitro and in vivo by constructing EMs models with uterus fragments from donors of PRNP knockout mouse (Prnp-/-, KO119), Octapeptide repeat region of PRNP knockout mouse (KO120) and PRNP transgenic mouse (Tg20). We found that transcriptome of cholesterol metabolism and estrogen-converting enzymes were disturbed in EMs patients, and cellular cholesterol concentration and local estradiol level were substantially increased in EMs lesions, as well as the high level of prion (PrPC, encoded by PRNP). Notably, 17-β estradiol stimulation significantly up-regulated PrPC expression in endometrial stromal cells (ESC) and PrPC promoted the proliferative, migratory and invasive abilities of ESC, and was further verified to accelerate EMs progression in mouse models. More importantly, PrPC promoted cholesterol accumulation and activated estrogen biosynthesis of ESC in a PPARα pathway-dependent manner. Taken together, this study suggests that PrPC-cholesterol metabolism/estrogen biosynthesis contributes to the progression of EMs by negatively regulating PPARα pathway, and could be potential therapeutic targets for EMs intervention.

The effect of oestrogen on mandibular condylar cartilage via hypoxia-inducible factor-2α during osteoarthritis development.

Oestrogen and hypoxia inducible factor-2α (HIF2α) are key regulators in the pathogenesis of osteoarthritis (OA). However, the cellular interaction between oestrogen and HIF2α in articular cartilage during OA process remains unknown. Our previous study has revealed that high-physiological level of oestrogen aggravates the degradation of condylar cartilage in the early stage of temporomandibular joint osteoarthritis (TMJ OA). Here, we hypothesize that HIF2α involves the effect of oestrogen on mandibular condylar cartilage in the progression of TMJ OA. Our experiment in vivo found that the degeneration of condylar cartilage caused by unilateral anterior crossbite (UAC) model, characterized by obvious degenerative morphology, loss of cartilage extracellular matrix, up-regulation of TNF-α, HIF2α and its' down-stream OA-related cytokines (MMP-13, VEGF and Col X), could be alleviated by lack of oestrogen while aggravated by high level of oestrogen in rats. Meanwhile, our in vitro study found that 17β-estradiol stimulation resulted in the loss of extracellular matrix, increased expression of TNF-α, IL-1, HIF2α and its' down-stream OA-related cytokines (MMP-13, VEGF and Col X) in primary condylar chondrocytes via oestrogen receptor beta (ERβ), which could be reversed by ER antagonist, selective estrogen receptor modulators (SERMs) and HIF2α translation inhibitor. Our results reveal that high level of oestrogen can aggravate the degenerative changes of mandibular condylar cartilage, while lack of oestrogen can alleviate it via oestrogen-ERβ-HIF2α pathway during TMJ OA progression.

Estrogen prevents articular cartilage destruction in a mouse model of AMPK deficiency via ERK-mTOR pathway.

To investigate the mechanism underlying the chondroprotective effect of estrogen in AMP-activated protein kinase (AMPK) deficiency mice. Female cartilage-specific AMPKα double knockout (AMPKα cDKO) mice were generated and subjected to ovariectomy (OVX). The model of osteoarthritis (OA) was induced by destabilization of medial meniscus (DMM). Histopathological changes were evaluated by using OARSI scoring systems. Autophagy changes were analyzed by immunofluorescence staining. Human chondrocytes were subjected to mechanical stress to mimic OA development. and incubated in presence of or absence of 17β-estradiol or/and compound C (AMPK inhibitor) or/and U0126 (ERK inhibitor). The expression levels of ERK1/2 phosphorylation, p70S6K phosphorylation and light chain 3 (LC3) were detected by Western blot. Compared with in OVX-sham AMPKα cDKO and OVX-sham WT mice, DMM-induced OA is more severe, and significantly low level of LC3 was observed in articular cartilage in OVX AMPK cDKO mice. Both mechanical stress and compound C were shown to induce an increase in phosphorylation of p70S6K, respectively. 17β-estradiol stimulation led to a reduction in the basal level of p70S6K phosphorylation as well as in the compound C or mechanical stress-induced level of p70S6K phosphorylation. 17β-estradiol stimulation not only led to an increase in LC3 conversion but also overrode the inhibitory effect of compound C on LC3 conversion. The effects of 17β-estradiol were abrogated by blocking ERK signaling pathway. Our findings suggest that estrogen can protect articular cartilage from damage during OA development by promoting chondrocyte autophagy via ERK-mammalian target of rapamycin (mTOR) signaling, and give new insight into the mechanism of the chondroprotective effect of estrogen.

Single-Cell Transcriptome Analysis Reveals Estrogen Signaling Coordinately Augments One-Carbon, Polyamine, and Purine Synthesis in Breast Cancer

Estrogen drives breast cancer (BCa) progression by directly activating estrogen receptor α (ERα). However, because of the stochastic nature of gene transcription, it is important to study the estrogen signaling pathway at the single-cell level to fully understand how ERα regulates transcription. Here, we performed single-cell transcriptome analysis on ERα-positive BCa cells following 17β-estradiol stimulation and reconstructed the dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Notably, differentially expressed genes show an estrogen-stimulated metabolic switch that favors biosynthesis but reduces estrogen degradation. Moreover, folate-mediated one-carbon metabolism is reprogrammed through the mitochondrial folate pathway and polyamine andpurine synthesis are upregulated coordinately. Finally, we show AZIN1 and PPAT are direct ERα targets that are essential for BCa cell survival and growth. In summary, our study highlights the dynamic transcriptional heterogeneity in ERα-positive BCa cells upon estrogen stimulation and uncovers a mechanism of estrogen-mediated metabolic switch.

Transcriptional regulation of the sodium-coupled neutral amino acid transporter (SNAT2) by 17β-estradiol.

The sodium-coupled neutral amino acid transporter 2 (SNAT2) translocates small neutral amino acids into the mammary gland to promote cell proliferation during gestation. It is known that SNAT2 expression increases during pregnancy, and in vitro studies indicate that this transporter is induced by 17β-estradiol. In this study, we elucidated the mechanism by which 17β-estradiol regulates the transcription of SNAT2. In silico analysis revealed the presence of a potential estrogen response element (ERE) in the SNAT2 promoter. Reporter assays showed an increase in SNAT2 promoter activity when cotransfected with estrogen receptor alpha (ER-α) after 17β-estradiol stimulation. Deletion of the ERE reduced estradiol-induced promoter activity by 63%. Additionally, EMSAs and supershift assays showed that ER-α binds to the SNAT2 ERE and that this binding competes with the interaction of ER-α with its consensus ERE. An in vivo ChIP assay demonstrated that the binding of ER-α to the SNAT2 promoter gradually increased in the mammary gland during gestation and that maximal binding occurred at the highest 17β-estradiol serum concentration. Liquid chromatography-elevated energy mass spectrometry and Western blot analysis revealed that the SNAT2 ER-α-ERE complex contained poly(ADP-ribose) polymerase 1, Lupus Ku autoantigen protein p70, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) proteins and that the silencing of each of these proteins nearly abolished 17β-estradiol-stimulated SNAT2 promoter activity. Nuclear levels of GAPDH increased progressively during gestation in the mammary gland, and GAPDH binding was nucleotide-specific for the SNAT2 ERE. Thus, this study provides new insights into how the mammary epithelium adapts to control amino acid uptake through the transcriptional regulation of the SNAT2 transporter via 17β-estradiol.

Differential Expression of MicroRNAs in Omental Adipose Tissue From Gestational Diabetes Mellitus Subjects Reveals miR-222 as a Regulator of ERα Expression in Estrogen-Induced Insulin Resistance

Omental adipose tissue plays a central role in insulin resistance in gestational diabetes mellitus (GDM), and the molecular mechanisms leading to GDM remains vague. Evidence demonstrates that maternal hormones, such as estradiol, contribute to insulin resistance in GDM. In this study we determined the differential expression patterns of microRNAs (miRNAs) in omental adipose tissues from GDM patients and pregnant women with normal glucose tolerance using AFFX miRNA expression chips. MiR-222, 1 of 17 identified differentially expressed miRNAs, was found to be significantly up-regulated in GDM by quantitative real-time PCR (P < .01), and its expression was closely related with serum estradiol level (P < .05). Furthermore, miR-222 expression was significantly increased in 3T3-L1 adipocytes with a high concentration of 17β-estradiol stimulation (P < .01), whereas the expressions of estrogen receptor (ER)-α protein and insulin-sensitive membrane transporter glucose transporter 4 (GLUT4) protein (P < .01) were markedly reduced. In addition, ERα was shown to be a direct target of miR-222 in 3T3-L1 adipocytes by using the luciferase assay. Finally, antisense oligonucleotides of miR-222 transfection was used to silence miR-222 in 3T3-L1 adipocytes. The results showed that the expressions of ERα and GLUT4, the insulin-stimulated translocation of GLUT4 from the cytoplasm to the cell membrane and glucose uptake in mature adipocytes were dramatically increased (P < .01). In conclusion, miR-222 is a potential regulator of ERα expression in estrogen-induced insulin resistance in GDM and might be a candidate biomarker and therapeutic target for GDM.

Dual-specificity phosphatase 6 (Dusp6), a negative regulator of FGF2/ERK1/2 signaling, enhances 17β-estrodial-induced cell growth in endometrial adenocarcinoma cell

Dual-specificity phosphatase 6 (Dusp6) is a negative feedback mechanism of fibroblast growth factors (FGFs)/mitogen-activated protein kinase (MAPK)/ERK1/2 signaling. The aim of this study was to explore the expression of Dusp6 in human endometrial adenocarcinomas and the role of Dusp6 expression in the growth regulation of endometrial adenocarcinoma cell. We found that Dusp6 was over-expressed in human endometrial adenocarcinomas. In Ishikawa cells, plasmid-driven Dusp6 expression efficiently blocked the activity of FGF2-induced MAPK/ERK1/2 signaling. Unexpectedly, Dusp6 expression significantly enhanced the growth of Ishikawa cells. In Dusp6 forced-expression cells, 17β-estradiol stimulation increased the cell growth by all most threefolds. In addition, progesterone treatment reduced the cell growth to about half both in Ishikawa cells with and without forced-Dusp6-expression. Dusp6 over-expression is involved in the pathogenesis and development of human endometrial adenocarcinomas. Dusp6 functions as a negative regulator of FGF2/ERK1/2 signaling but enhances the growth and 17β-estradiol-induced cell growth in endometrial adenocarcinoma cell.

Estrogen is increased in male cholangiocarcinoma patients’ serum and stimulates invasion in cholangiocarcinoma cell lines in vitro

Cholangiocarcinoma is defined as a chronic liver disease with altered estrogen metabolism and could result in estrogen retention. Estrogenic response was known as a promoting factor in progression of some cancer. In this study, we determined the significant increase of estrogen level in cholangiocarcinoma patients' sera. The estrogen levels in cholangiocarcinoma patients' sera were measured and correlated with clinical presentations. Estrogen receptor-α expressions in cholangiocarcinoma tissues were detected by immunohistochemistry method. KKU-100 and KKU-M213 cholangiocarcinoma cell lines were treated with 17β-estradiol and tested the proliferative and invasive effects. The estrogen levels showed positive correlations with serum bilirubin and alkaline phosphatase and a negative correlation with albumin. This study also showed an association with shorter survival times when patients with low and high serum estrogen levels were compared. In vitro studies demonstrated the effect of estrogen on cell proliferation and invasion in dose-dependent manners, which could be inhibited by tamoxifen, a clinical used estrogen antagonist. Invasion showed an association with the TFF1 gene expression and could be inhibited by small interfering RNA against TFF1 gene. Estrogen receptor-α was the main estrogen receptor that response to 17β-estradiol stimulation. TFF1 trefoil protein could be one of the effectors for estrogen-induced invasion in cholangiocarcinoma via the estrogen receptor-α. These findings could lead to an understanding of the mechanism of cholangiocarcinoma progression.

Estrogen receptors mediate rapid activation of phospholipase C pathway in the rat endometrium

The aim of the present study was to investigate the activation of rapid signaling events by 17β-estradiol in the rat uterus. 17β-Estradiol induced a rapid increase of total [ 3H]-inositol phosphate accumulation in the whole uterus and endometrium, but not in the myometrium. The effect of 17β-estradiol in the endometrium was blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [ 3H]-inositol phosphate accumulation in the endometrium. The G-1 effects were blocked by GPER antagonist (G-15). 17β-Estradiol and G-1 promoted an additive effect on total [ 3H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17β-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormone stimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.

A modulated empirical Bayes model for identifying topological and temporal estrogen receptor α regulatory networks in breast cancer

BackgroundEstrogens regulate diverse physiological processes in various tissues through genomic and non-genomic mechanisms that result in activation or repression of gene expression. Transcription regulation upon estrogen stimulation is a critical biological process underlying the onset and progress of the majority of breast cancer. Dynamic gene expression changes have been shown to characterize the breast cancer cell response to estrogens, the every molecular mechanism of which is still not well understood.ResultsWe developed a modulated empirical Bayes model, and constructed a novel topological and temporal transcription factor (TF) regulatory network in MCF7 breast cancer cell line upon stimulation by 17β-estradiol stimulation. In the network, significant TF genomic hubs were identified including ER-alpha and AP-1; significant non-genomic hubs include ZFP161, TFDP1, NRF1, TFAP2A, EGR1, E2F1, and PITX2. Although the early and late networks were distinct (<5% overlap of ERα target genes between the 4 and 24 h time points), all nine hubs were significantly represented in both networks. In MCF7 cells with acquired resistance to tamoxifen, the ERα regulatory network was unresponsive to 17β-estradiol stimulation. The significant loss of hormone responsiveness was associated with marked epigenomic changes, including hyper- or hypo-methylation of promoter CpG islands and repressive histone methylations.ConclusionsWe identified a number of estrogen regulated target genes and established estrogen-regulated network that distinguishes the genomic and non-genomic actions of estrogen receptor. Many gene targets of this network were not active anymore in anti-estrogen resistant cell lines, possibly because their DNA methylation and histone acetylation patterns have changed.

Study of the activity of Estrogen Response Elements present on Pax‐5 promoter and their potential role on regulating Pax‐5 expression in breast cancer

Pax‐5 is a transcription factor playing a crucial role in B lymphopoïesis and its deregulation has been associated to certain malignancies, namely non‐Hodgkin lymphomas and acute lymphoblastic leukemia. Although Pax‐5 is mainly known to be expressed in B cells, there is a body of preliminary data generated in our laboratory suggesting its expression in some breast cancer cell lines and primary breast tumors.In an attempt to explain Pax‐5 expression in these breast cancer cases, we analyzed its promoter and found a number of Estrogen Receptor Elements (EREs) and Estrogen Related Receptors sites (ERRs), suggesting a role for estrogen receptors in Pax‐5 expression in breast cancer. Therefore, electrophoretic mobility shift assays (EMSA) were performed to assess the binding capacity of estrogen receptors ERα and ERβ on those predicted sites, which were also cloned in the Luciferase reporter vector pGL3‐Promoter to evaluate the receptors activity by dual luciferase reporter assays.We report here the specific binding of estrogen receptors on some of the EREs located on Pax‐5 promoter and their differential activation under 17β‐estradiol stimulation in MCF‐7 cells. These results suggest a role for estrogen receptors in the expression of the oncogene Pax‐5 in breast cancer and suggest a mechanism that contributes to breast carcinogenesis.This work has been supported by an ''Atlantic Innovation Fund'' grant.

Sexual Dimorphism in Mouse Metanephroi Exposed to 17β-Estradiol in vitro

17β-Estradiol, the most potent circulating estrogen, has been shown to greatly impact on the development and formation of tissues of the urogenital tract. The adult kidney has previously been shown to be highly responsive to 17β-estradiol stimulation. However, the direct effect of 17β-estradiol on kidney development remains unclear. Aim: To investigate the direct effect of 17β-estradiol on male and female metanephric kidney development. Methods: Whole embryonic-day-12.5 (E12.5) C57Bl/6 male and female mouse metanephroi were cultured in the presence of varying concentrations of 17β-estradiol (0.1–5.0 nM) for 72 h. Metanephric development was assessed using immunofluorescence labeling techniques. The real-time polymerase chain reaction was used to investigate estrogen receptor-α (ERα), glial-cell-line-derived neurotrophic factor (GDNF) and its associated receptor cRET, transforming growth factor-β (TGFβ1), TGFβ2 and TGFβ3 mRNA expression levels. Results:ERα was present in developing metanephroi at E12.5; however, ERβ was absent. No significant sex difference in ERα mRNA expression was observed. Significant increases in the number of ureteric branch points, terminal tips and developing glomeruli were observed in female metanephroi cultured in the presence of 1.0 and 5.0 nM 17β-estradiol. Conversely, no significant effect was observed in male metanephroi cultured with 17β-estradiol. GDNF and cRET mRNA expression was increased in both male and female metanephroi, whilst TGFβ1 and TGFβ2 mRNA expression was decreased following culture in the presence of 17β-estradiol. Conclusion: This study is the first to establish that the mouse metanephros displays a sexual dimorphism in response to specific concentrations of estrogens.

GEMS (Gene Expression Metasignatures), a Web Resource for Querying Meta-analysis of Expression Microarray Datasets: 17β-Estradiol in MCF-7 Cells

With large amounts of public expression microrray data being generated by multiple laboratories, it is a significant task for the bench researcher to routinely identify available datasets, and then to evaluate the collective evidence across these datasets for regulation of a specific gene in a given system. 17beta-Estradiol stimulation of MCF-7 cells is a widely used model in the growth of breast cancer. Although myriad independent studies have profiled the global effects of this hormone on gene expression in these cells, disparate experimental variables and the limited power of the individual studies have combined to restrict the agreement between them as to the specific gene expression signature elicited by this hormone. To address these issues, we have developed a freely accessible Web resource, Gene Expression MetaSignatures (GEMS) that provides the user a consensus for each gene in the system. We conducted a weighted meta-analysis encompassing over 13,000 genes across 10 independent published datasets addressing the effect of 17beta-estradiol on MCF-7 cells at early (3-4 hours) and late (24 hours) time points. In a literature survey of 58 genes previously shown to be regulated by 17beta-estradiol in MCF-7 cells, the meta-analysis combined the statistical power of the underlying datasets to call regulation of these genes with nearly 85% accuracy (false discovery rate-corrected P < 0.05). We anticipate that with future expression microarray dataset contributions from investigators, GEMS will evolve into an important resource for the cancer and nuclear receptor signaling communities.

Indole-3-Carbinol Selectively Uncouples Expression and Activity of Estrogen Receptor Subtypes in Human Breast Cancer Cells

Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)-alpha (ERalpha) and ERbeta. Indole-3-carbinol (I3C) strongly down-regulated ERalpha protein and transcript levels, without altering the level of ERbeta protein, in both cell lines. In cells transfected with the ERalpha promoter linked to a luciferase gene reporter, I3C ablated ERalpha promoter activity. Propyl pyrazole triol (PPT) is a highly selective ERalpha agonist, whereas, 17beta-estradiol activates both ERalpha and ERbeta. I3C treatment inhibited the PPT- and 17beta-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17beta-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ERalpha and stimulated the level of ERE binding ERbeta even though the protein levels of this receptor remained constant. In ERalpha-/ERbeta+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERbeta to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERbeta is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ERalpha and ERbeta can be uncoupled by I3C with a key cellular consequence being a significantly higher ERbeta:ERalpha ratio that is generally highly associated with antiproliferative status of human breast cancer cells.

Myometrial maxi-K channel β1 subunit modulation during pregnancy and after 17β-estradiol stimulation

Myometrial maxi-K channels are modulated by β subunits. We aimed to determine whether β subunits are modulated to affect uterine excitability during gestation. RNase protection analyses revealed that mouse β1 subunit transcripts are regulated during gestation with peak expression at day 14 of pregnancy. Immunohistochemical analysis indicates an increase of this subunit during gestation. Upregulation of the β1 transcript occurs with 4-day exposure to 17β-estradiol but not progesterone, and acute estradiol exposure has no effect on β1 transcript expression. These findings verify that β1 subunit transcript is regulated in mouse myometrium during gestation and estrogens may contribute to this increase.

17β-Estradiol upregulates distinct maxi-K channel transcripts in mouse uterus

The mouse maxi-K channel transcript undergoes alternative splicing to produce isoforms differing in sensitivity to intracellular regulators. We hypothesized that 17β-estradiol could induce myometrial maxi-K channel transcripts to differentially splice. Polymerase chain reaction demonstrated two products at site D in mice injected with either 8.5 μg of 17β-estradiol for 4 days or a vehicle control. Splicing of site D is known to modulate the sensitivity of the maxi-K channel to calcium and voltage. RNase protection analyses revealed that the α subunit transcript, and an exon encoding 59 amino acids at site D that enhances Ca 2+- and voltage-sensitivity, are upregulated ∼1.4-fold after 17β-estradiol stimulation however, the insertless isoform of this transcript is enhanced ∼5-fold. Immunoblotting demonstrates that the total maxi-K channel α subunit expression mimics transcript regulation. These findings verify that maxi-K channel transcripts are differentially spliced by 17β-estradiol, which may contribute to stoichiometric changes in isoform expression during pregnancy.

Immunohistochemical localization of vitellogenin in rainbow trout ( Oncorhynchus mykiss) hepatocytes using immunofluorescence

In fish, vitellogenin, the egg yolk protein precursor of any oviparous species, is specifically synthesized in the liver of females in response to circulating estrogens. It is released into the blood, taken up by the ovary and used to form the yolk in the eggs. In order to localize vitellogenin in hepatocytes of rainbow trout ( Oncorhynchus mykiss) after external stimulation, both male and female fish were given a single intraperitoneal injection of 17β-estradiol at 1 mg/kg. Whereas in control male liver vitellogenin could not be demonstrated, both sexes produced vitellogenin after 17β-estradiol stimulation. Induced male fish displayed the same pattern of antibody binding as female individuals. However, in males the staining intensity was lower than in females. Under the light microscope, antibodies primarily bound to perinuclear regions, which contain, among other organelles, rough endoplasmic reticulum and Golgi fields, where vitellogenin is synthesized. Under the electron microscope, these organelles could be confirmed as the sites of vitellogenin synthesis by means of specific antibody labeling.

Selection of a highly tumorigenic breast cancer cell line sensitive to estradiol to evidence in vivo the tumor-inhibitory effect of butyrate derivative monobut-3

To increase butyric acid mean residence time in vivo, we have produced a stable butyric acid derivative, Monobut-3. Recently, we have described that Monobut-3 is able to induce phenotypic changes in human mammary tumor cells in vitro. In this study, we explore the in vivo effect of Monobut-3. Human breast tumor cell-lines did not easily produce in vivo xenografts, thus, MCF-7 cells required exogenous 17β-estradiol to grow and to form in vivo xenografts. To evaluate in vivo anti-tumor effects of monobut-3 without exogenous 17β-estradiol addition, we have established MCF-7 variant cells, highly tumorigenic MCF-7vht, in which transfection of rats oncogene induced a bypass of estrogen requirement but did not delete the presence of functional estrogen receptor (ER). Monobut-3 inhibited growth of this variant by about 90% at 4 mM and reduced 17β-estradiol cell growth stimulation. In vivo, in absence of 17β-estradiol, 2 mg per mouse monobut-3 decreased tumor take by about 25% and tumor growth by about 50% in nude mice. This is the first experimental demonstration of an in vivo antitumoral effect of a butyric acid derivative alone on a solid human tumor. These data suggest that this compound does not only act by reducing of 17β-estradiol stimulation but it also has an 17β-estradiol-independent effect. Absence of toxicity and its antiproliferative effects could suggest its use in clinical treatment of well differentiated carcinoma.

Estrogen-Controlled Gene Expression: Induction of Two Estrogen-Responsive Genes in the Liver of Rainbow Trout (Salmo ǵairdneri)

Administration of 17β-estradiol to reproductively active male rainbow trout results in the induction of synthesis of two estrogen-responsive mRNA, vitellogenin (VG) and ULER2, to high levels. The complementary DNA (cDNA) of these two mRNA were prepared by molecular cloning. Two genomic DNA fragments generated by EcoRI digestion of trout genomic DNA showed hybridization to VG cDNA. A quantitative RNA dot blot hybridization method was adopted for the determination of levels of estrogen-responsive mRNA in livers of male trout after 17β-estradiol stimulation. This assay was used to determine the dose-response relationship and the time course of estrogen-responsive mRNA accumulation in juvenile fish.