Expression data from healthy postmenopausal women on 4 different types of hormone therapy, the RET study
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE24859 | GEO | 2012/01/17
SECONDARY ACCESSION(S): PRJNA132113
REPOSITORIES: GEO
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Project description:Postmenopausal hormone therapy (HT) is associated with many diseases and conditions, but the underlying molecular mechanisms involved are incompletely understood. The aim of the current study was to investigate the effect of 4 types of HT on gene transcription. 24 women (6 women in 4 treatment groups) received 2 mg 17M-NM-2-estradiol combined with 1 mg noresthisterone acetate (NETA), 1 mg 17M-NM-2-estradiol combined with 0.5 mg NETA, tibolone, or raloxifene hydrochloride. RNA was isolated from whole blood before treatment (baseline) and after 6 weeks on treatment. The changes in mRNA from baseline to 6 weeks were assessed with a microarray chip. 4 treatment groups with 6 women in each group were blood sampled before treatment (baseline) and after 6 weeks on treatment, that is a total of 48 samples. The gene expression data at 6 weeks were compared to the expression data at baseline for each treatment.
2012-01-17 | E-GEOD-24859 | biostudies-arrayexpress
Project description:Five years of tamoxifen reduces breast cancer risk by nearly 50% but is associated with significant side-effects and toxicities. A better understanding of the direct and indirect effects of tamoxifen in benign breast tissue could elucidate new mechanisms of breast carcinogenesis, suggest novel chemoprevention targets, and provide relevant early response biomarkers for Phase II prevention trials. Seventy-three women at increased risk for breast cancer were randomized to tamoxifen (20 mg daily) or placebo for three months. Blood and breast tissue samples were collected at baseline and post-treatment. Sixty-nine women completed all study activities (37 tamoxifen and 32 placebo). The selected biomarkers focused on estradiol and IGFs in the blood, DNA methylation and cytology in random periareolar fine needle aspirates, and tissue morphometry, proliferation, apoptosis, and gene expression (microarray and RT-PCR) in the tissue core samples. Tamoxifen downregulated ets-oncogene family members ETV4 and ETV5 and reduced breast epithelial cell proliferation independent of CYP2D6 genotypes or effects on estradiol, ESR1 or IGFs. Reduction in proliferation was correlated with downregulation of ETV4 and DNAJC12. Tamoxifen also modulated expression of RAB GTPases, and several genes involved in epithelial-stromal interaction, and reduced tumor suppressor gene methylation. Three months of tamoxifen did not affect breast tissue composition, cytological atypia, preneoplasia or apoptosis. Tamoxifen may durably reduce breast cancer risk through downregulation of ETV4 and ETV5 which could deplete mammary progenitor populations. This pathway has the potential to provide novel targets and early response biomarkers for phase II prevention trials. Randomized prospective double blinded placebo-controlled trial of tamoxifen (20 mg daily) versus placeo in women at increased risk for breast cancer. Gene expression was assessed in whole breast tissue cores obtained at baseline and after three months of treatment for 35 women. Core biopsies were obtained in the late luteal phase (day 28 +/- 2) for premenopausal women (N = 19). Breast lobules from the baseline and post-treatment cores were microdissected for 5 tamoxifen subjects.
2011-07-21 | E-GEOD-29338 | biostudies-arrayexpress
Project description:Estrogen is a reproductive steroid hormone that has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Since we still have an incomplete picture of the gene targets of estrogen in the vasculature, and our understanding of SERM gene targets in the vasculature is even less well-developed, it is important to broaden the available information on this subject. The present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation. Goal of the experiment: To examine the effects of three formulations of estrogen (ethinyl estradiol, equilin, and estradiol benzoate) and of the selective estrogen receptor modulators, tamoxifen and raloxifene, on gene expression in the mesenteric arteries of the rat. Keywords: rat, artery, gene expression, mesentery, estrogen, tamoxifen, raloxifene Experimental factors: hormone treatment, drug treatment Experimental design: Prepubertal female Sprague Dawley rats (Harlan, Indianapolis, IN) were ovariectomized at 4 weeks of age, prior to the initiation of regular ovarian cycles. The animals were allowed to recover for 2 weeks before the experimental protocol was initiated. The rats were randomly divided into 6 groups (n=3-4/group) and treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After 3 weeks of treatment, the rats were euthanized and the mesentery obtained. Total RNA was extracted from each individual sample and used for the synthesis of labeled cRNA for microarray analysis. After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated and harvested and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Mesenteric artery RNA was extracted and purified using an RNeasy Mini Kit (Qiagen). Any potentially contaminating DNA was removed by treatment with RNase-free DNase (Qiagen) on the RNeasy column. Total RNA was quantified and RNA integrity was assessed using the Agilent Bioanalyzer (Santa Clara, CA). Following analysis, the RNA was stored at -80ºC until it was used for gene expression studies. DNA microarray: CodeLink Whole Rat Genome Bioarrays (Applied Microarrays, Tempe, AZ) were used. Five μg total RNA was used from each mesenteric arterial sample for the microarray analysis. Complementary RNA was synthesized per company protocol (Ambion). A set of bacterial RNAs was added to each total RNA sample and carried through the synthesis and labeling reactions as controls and for nor¬malization of the data. The microarrays were scanned with the GenePix 4000B (Axon Instruments, Union City, CA) with GenePix Pro 5.0 software (Axon Instruments). Analysis of the microar¬rays was performed with the CodeLink Expression Analysis software (Applied Microarrays) and Acuity 3.1 software (Axon), and statistical analysis was performed with GeneSpring 7.1 software (Agilent, Santa Clara, CA). A sample of n=3 was completed with the microarrays for each treatment. Quality control steps: The cRNA that was synthesized from each mesenteric artery treatment sample was used for hybridization to a single CodeLink (Applied Microarrays) whole rat genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were used. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were isolated mesenteric arteries from individual Sprague Dawley rats that had been treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). Manipulations of biological samples and protocols used: Ovariectomized female rats were treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated, harvested, and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Experimental factor: hormone treatment, drug treatment Technical protocols: The mesenteric arteries were minced with scalpels and TRI reagent (MRC) was added. The tissue was homogenized with a Polytron homogenizer. Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labeled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 5.0 microgram of rat mesenteric artery total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides (CodeLink Whole Rat Genome Bioarrays), and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
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2011-04-12 | E-GEOD-28543 | biostudies-arrayexpress
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Project description:The NEWEST (Neoadjuvant Endocrine Therapy for Women with Estrogen-Sensitive Tumours) trial compared the clinical and biological activity of fulvestrant 500 mg vs 250 mg in the neoadjuvant setting. In this multi-centre phase II study, post-menopausal women with operable, locally advanced (T2, 3, 4b; N0-3; M0) ER-positive breast tumours were randomised to receive neoadjuvant treatment with either dose of fulvestrant for 16 weeks before surgery. Tumour core biopsies were obtained at baseline, 4 weeks and at surgery for assessment of changes in biomarker expression. Tumour volumes were measured by 3-D ultrasound at the same timepoints. In this trial, the percentage of patients who showed a reduction in tumour volume or stabilisation of disease (using RECIST criteria) after treatment with fulvestrant 500 mg was 36% (26 out of 69 patients). Therefore, within a population of endocrine-therapy naive patients whose tumours were confirmed as being ER-positive at the time of study entry, there is a subgroup who gained particular clinical benefit from fulvestrant treatment. These clinical response data together with the availability of biological response information and frozen tumour tissue from participants makes the NEWEST trial an attractive setting in which to investigate the potential of new markers of response to fulvestrant. 42 samples
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