Effect of tamoxifen on early gene expression in T47D cells
Ontology highlight
ABSTRACT: Tamoxifen is the treatment of choice in estrogen receptor alpha breast cancer patients. However, ~50% of ERM-NM-1-positive tumors exhibit intrinsic or rapidly acquire resistance to endocrine treatment, requiring chemotherapy. M-NM-^Yt has been difficult to predict de novo resistance to endocrine therapy and/or assess the likelihood of early relapse, while no concrete mechanism regulating the acquisition and the maintenance of endocrine resistance has been identified. We have performed a whole transcriptome analysis of an ER-positive (T47D) and a triple-negative (MDA-MBA-231) breast cancer cell line exposed to tamoxifen for a short time frame (hours) in order to study resistance mechanisms that are initiated early after initiation of tamoxifen treatment. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with vehicle, E2 (10-6M) or tamoxifen (10-6M) in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturerM-bM-^@M-^Ys instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:Tamoxifen is the treatment of choice in estrogen receptor alpha breast cancer patients. However, ~50% of ERM-NM-1-positive tumors exhibit intrinsic or rapidly acquire resistance to endocrine treatment, requiring chemotherapy. M-NM-^Yt has been difficult to predict de novo resistance to endocrine therapy and/or assess the likelihood of early relapse, while no concrete mechanism regulating the acquisition and the maintenance of endocrine resistance has been identified. We have performed a whole transcriptome analysis of an ER-positive (T47D) and a triple-negative (MDA-MBA-231) breast cancer cell line exposed to tamoxifen for a short time frame (hours) in order to study resistance mechanisms that are initiated early after initiation of tamoxifen treatment. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with vehicle,E2 (10-6M) or tamoxifen (10-6M) in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturerM-bM-^@M-^Ys instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:ERM-NM-117p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERM-NM-1) and initially synthesized to mimic its calmodulin binding site. ERM-NM-117p was subsequently found to elicit estrogenic responses in E2-deprived ERM-NM-1-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERM-NM-117p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERM-NM-117p induces a massive early (3h) transcriptional activity in breast cancer cell line T47D. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2 (10-6M) or ERa17p in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturerM-bM-^@M-^Ys instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:Estrogens have been reported to activate several processes via membrane binding to either classic estrogen receptors (ERs) or GPR30. We have used either estradiol or BSA-conjugated estradiol in order to initiate membrane-initiated actions and ICI 172,780 (ICI) or G15 to explore ER- and GPR30-related transcription. Our results show that the majority of G15-inhibited transcription is depending on ERs, as it is also inhibited by ICI. However, a small number of transcripts, related to specific actions/pathways is either exclusively inhibited by G15, providing evidence about a specific GPR30 signature, or not inhibited by ICI or G15 suggesting the existence of another, yet unidentified estrogen receptor. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2-BSA (10-6M) in the presence or absence of specific antagonists, in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturer’s instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:ERM-NM-117p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERM-NM-1) and initially synthesized to mimic its calmodulin binding site. ERM-NM-117p was subsequently found to elicit estrogenic responses in E2-deprived ERM-NM-1-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERM-NM-117p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERM-NM-117p induces a massive early (3h) transcriptional activity in breast cancer cell line MDA-MB-231. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2 (10-6M) or ERa17p in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturerM-bM-^@M-^Ys instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:Tamoxifen Resistant (TR) gene profile from Breast cancer cell lines T47D and ZR75-1 with their oestrogen-deprieved conterparts were analysed for gene associated with TR. We used Microarray Affymetrix HU133plus 2.0 chips for gene expression of TR cell lines, normalised them against GEO data available for normal T47D (GSM70667) and ZR75-1 (GSM70668). We grew parental breast cancer cell lines in tamoxifen containing media (0.1 microM) for 6 months and labelled them tamoxifen resistant (TR). Oestrogen-Deprieved cells were grown in charcoal-stripped media for 6 months then tamoxifen (0.1 microM) was added to the media and cells maintained a further 6 months and termed Oestrogen deprieved-tamoxifen resistant (ODTR) .
Project description:Estrogens have been reported to activate several processes via membrane binding to either classic estrogen receptors (ERs) or GPR30. We have used either estradiol or BSA-conjugated estradiol in order to initiate membrane-initiated actions and ICI 172,780 (ICI) or G15 to explore ER- and GPR30-related transcription. Our results show that the majority of G15-inhibited transcription is depending on ERs, as it is also inhibited by ICI. However, a small number of transcripts, related to specific actions/pathways is either exclusively inhibited by G15, providing evidence about a specific GPR30 signature, or not inhibited by ICI or G15 suggesting the existence of another, yet unidentified estrogen receptor. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2-BSA (10-6M) in the presence or absence of specific antagonists, in McCoys's 5A medium supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturer’s instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:Estrogens have been reported to activate several processes via membrane binding to either classic estrogen receptors (ERs) or GPR30. We have used either estradiol or BSA-conjugated estradiol in order to initiate membrane-initiated actions and ICI 172,780 (ICI) or G15 to explore ER- and GPR30-related transcription. Our results show that the majority of G15-inhibited transcription is depending on ERs, as it is also inhibited by ICI. However, a small number of transcripts, related to specific actions/pathways is either exclusively inhibited by G15, providing evidence about a specific GPR30 signature, or not inhibited by ICI or G15 suggesting the existence of another, yet unidentified estrogen receptor. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2-BSA (10-6M) in the presence or absence of specific antagonists, in DMEM/F12 (1:1) supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturer’s instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:Estrogens have been reported to activate several processes via membrane binding to either classic estrogen receptors (ERs) or GPR30. We have used either estradiol or BSA-conjugated estradiol in order to initiate membrane-initiated actions and ICI 172,780 (ICI) or G15 to explore ER- and GPR30-related transcription. Our results show that the majority of G15-inhibited transcription is depending on ERs, as it is also inhibited by ICI. However, a small number of transcripts, related to specific actions/pathways is either exclusively inhibited by G15, providing evidence about a specific GPR30 signature, or not inhibited by ICI or G15 suggesting the existence of another, yet unidentified estrogen receptor. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2-BSA (10-6M) in the presence or absence of specific antagonists, in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturer’s instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:This study aims to identify transcriptional differences between parental ER+ breast cancer cell lines and those that are resistant to several common endocrine therapies, including tamoxifen, fulvestrant, and aromatase inhibition. To mimic aromatase inhibitors, cells were grown in estrogen withdrawl (EWD) conditions, using phenol red free media and charcoal-stripped serum. Cells were kept in their respective treatments until RNA was procured.