MCF7 response to EGF: Difference between revisions
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|TCOverview=ErbB receptor family plays a central role in cellular development, proliferation, differentiation and cell death. There are four members, ErbB1/EGFR, ErbB2, ErbB3 and ErbB4 receptors, belong to this family. Dysregulation of the receptors is highly associated with incidence of variety of cancers [1]. Once activated by growth factor binding, the receptor forms homo- and heterodimer and are trans-activated by tyrosine phosphorylation, transmits the phosphorylation signal to downstream kinase such as ERK and Akt, then those kinases activate/phosphorylate transcription factors such as ELK1, CREB, or SRF in nucleus to initiate gene expression.In human breast cancer MCF-7 cells, the heregulin (HRG; a ErbB3/4 ligand) induces cell differentiation (accumulation of lipid droplets), while epidermal growth factor (EGF; an ErbB1/EGFR ligand) elicits cell proliferation after 5-14 days of the ligand stimulation.These ligand-stimulated cells show similar time-course profiles in terms of signaling activity and immediate early gene (IEG) mRNA expression (up to 1.5 hours)[2,3]. However, analysis of delayed transcription (up to 72 hours) using qRT-PCR showed the significant expression of transcription factors (c-FOS, FRA-1 and FHL2) specific to HRG but not by EGF [4], suggesting the existence of unknown transcription machinery for cell differentiation during this time-course.<br><html><img src=' | |TCOverview=ErbB receptor family plays a central role in cellular development, proliferation, differentiation and cell death. There are four members, ErbB1/EGFR, ErbB2, ErbB3 and ErbB4 receptors, belong to this family. Dysregulation of the receptors is highly associated with incidence of variety of cancers [1]. Once activated by growth factor binding, the receptor forms homo- and heterodimer and are trans-activated by tyrosine phosphorylation, transmits the phosphorylation signal to downstream kinase such as ERK and Akt, then those kinases activate/phosphorylate transcription factors such as ELK1, CREB, or SRF in nucleus to initiate gene expression.In human breast cancer MCF-7 cells, the heregulin (HRG; a ErbB3/4 ligand) induces cell differentiation (accumulation of lipid droplets), while epidermal growth factor (EGF; an ErbB1/EGFR ligand) elicits cell proliferation after 5-14 days of the ligand stimulation.These ligand-stimulated cells show similar time-course profiles in terms of signaling activity and immediate early gene (IEG) mRNA expression (up to 1.5 hours)[2,3]. However, analysis of delayed transcription (up to 72 hours) using qRT-PCR showed the significant expression of transcription factors (c-FOS, FRA-1 and FHL2) specific to HRG but not by EGF [4], suggesting the existence of unknown transcription machinery for cell differentiation during this time-course.<br><html><img src='/resource_browser/images/TC_qc/MCF7_Fig1.png'></html>Figure 1: Approximate expression timing of early, mid, delayed response genes.<br><br>References:<br>[1] Yarden Y1, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2, 127-137, 2001.<br>[2] Nagashima, et al. Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J. Biol. Chem. 282, 4045-4056, 2007.<br>[3] Nakakuki, et al. Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics. Cell 141, 884-896, 2010.<br>[4] Saeki, et al. Ligand-specific sequential regulation of transcription factors for differentiation of MCF-7 cells. BMC Genomics 10, 545, 2009.<br> | ||
|TCQuality_control=<html><img src=' | |TCQuality_control=<html><img src='/resource_browser/images/TC_qc/MCF7_Fig3.png'></html>The mRNA expression of c-FOS, FOSL1 and FHL2 was significantly induced for HRG than EGF shown in the past study (The figures from Saeki et al BMC Genomics 2009[4]).<br><br><html><img src='/resource_browser/images/TC_qc/MCF7_Fig4.png' onclick='javascript:window.open("/resource_browser/images/TC_qc/MCF7_Fig4.png", "imgwindow", "width=736,height=348");' style='width:700px;cursor:pointer'/></html>Time-course patterns the CAGE peaks of c-FOS, FOSL1, FHL2 obtained from current analysis was consistent with the mRNA expression data shown in above.<br><br>References:<br>[4] Saeki, et al. Ligand-specific sequential regulation of transcription factors for differentiation of MCF-7 cells. BMC Genomics 10, 545, 2009.<br> | ||
|TCSample_description=The MCF-7 human breast cancer cell line was obtained from American Type Culture Collection (ATCC) and maintained in DMEM (Gibco BRL, Githersburg, MD) supplemented with 10 % fetal bovine serum. Prior to growth hormone treatment, the cells were serum-starved for 16-24 hours, and then EGF (PeproTech House, London, England) or HRG-176-246 (R&D Systems, Inc., Minneapolis, MN) was added. We prepared the EGF or HRG-stimulated time-course samples at 0 (non-treated), 15min, 30min, 45min, 60min, 80min, 100min, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 5hr, 6hr, 7hr and 8hr, to cover the early phase of cell differentiation and to cover early, mid and delayed gene expression. The all cell samples were snap frozen in liquid nitrogen and provided for RNA extraction for CAGE analysis.<br>MCF-7 cells were incubated in the absence or presence of growth factor in serum-free DMEM. Following cultivation for 14 days, cells were stained with Oil Red O to evaluate differentiation of the cells, which results in lipid droplets accumulation (The figures from Nagashima et al. JBC 2007 [2]).<br><br>References:<br>[2] Nagashima, et al. Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J. Biol. Chem. 282, 4045-4056, 2007.<br> | |TCSample_description=The MCF-7 human breast cancer cell line was obtained from American Type Culture Collection (ATCC) and maintained in DMEM (Gibco BRL, Githersburg, MD) supplemented with 10 % fetal bovine serum. Prior to growth hormone treatment, the cells were serum-starved for 16-24 hours, and then EGF (PeproTech House, London, England) or HRG-176-246 (R&D Systems, Inc., Minneapolis, MN) was added. We prepared the EGF or HRG-stimulated time-course samples at 0 (non-treated), 15min, 30min, 45min, 60min, 80min, 100min, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 5hr, 6hr, 7hr and 8hr, to cover the early phase of cell differentiation and to cover early, mid and delayed gene expression. The all cell samples were snap frozen in liquid nitrogen and provided for RNA extraction for CAGE analysis.<br>MCF-7 cells were incubated in the absence or presence of growth factor in serum-free DMEM. Following cultivation for 14 days, cells were stained with Oil Red O to evaluate differentiation of the cells, which results in lipid droplets accumulation (The figures from Nagashima et al. JBC 2007 [2]).<br><br>References:<br>[2] Nagashima, et al. Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J. Biol. Chem. 282, 4045-4056, 2007.<br> | ||
|Time_Course= | |Time_Course= | ||
Revision as of 20:16, 12 February 2015
| Series: | IN_VITRO DIFFERENTIATION SERIES |
|---|---|
| Species: | Human (Homo sapiens) |
| Genomic View: | Zenbu |
| Expression table: | FILE |
| Link to TET: | TET |
| Sample providers : | Mariko Okada |
| Germ layer: | mesoderm |
| Primary cells or cell line: | cell line |
| Time span: | 8 hours |
| Number of time points: | 16 |
| Overview |
|---|
|
ErbB receptor family plays a central role in cellular development, proliferation, differentiation and cell death. There are four members, ErbB1/EGFR, ErbB2, ErbB3 and ErbB4 receptors, belong to this family. Dysregulation of the receptors is highly associated with incidence of variety of cancers [1]. Once activated by growth factor binding, the receptor forms homo- and heterodimer and are trans-activated by tyrosine phosphorylation, transmits the phosphorylation signal to downstream kinase such as ERK and Akt, then those kinases activate/phosphorylate transcription factors such as ELK1, CREB, or SRF in nucleus to initiate gene expression.In human breast cancer MCF-7 cells, the heregulin (HRG; a ErbB3/4 ligand) induces cell differentiation (accumulation of lipid droplets), while epidermal growth factor (EGF; an ErbB1/EGFR ligand) elicits cell proliferation after 5-14 days of the ligand stimulation.These ligand-stimulated cells show similar time-course profiles in terms of signaling activity and immediate early gene (IEG) mRNA expression (up to 1.5 hours)[2,3]. However, analysis of delayed transcription (up to 72 hours) using qRT-PCR showed the significant expression of transcription factors (c-FOS, FRA-1 and FHL2) specific to HRG but not by EGF [4], suggesting the existence of unknown transcription machinery for cell differentiation during this time-course. |
Figure 1: Approximate expression timing of early, mid, delayed response genes.| Sample description |
|---|
|
The MCF-7 human breast cancer cell line was obtained from American Type Culture Collection (ATCC) and maintained in DMEM (Gibco BRL, Githersburg, MD) supplemented with 10 % fetal bovine serum. Prior to growth hormone treatment, the cells were serum-starved for 16-24 hours, and then EGF (PeproTech House, London, England) or HRG-176-246 (R&D Systems, Inc., Minneapolis, MN) was added. We prepared the EGF or HRG-stimulated time-course samples at 0 (non-treated), 15min, 30min, 45min, 60min, 80min, 100min, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 5hr, 6hr, 7hr and 8hr, to cover the early phase of cell differentiation and to cover early, mid and delayed gene expression. The all cell samples were snap frozen in liquid nitrogen and provided for RNA extraction for CAGE analysis. |
| Quality control |
|---|
|
|
The mRNA expression of c-FOS, FOSL1 and FHL2 was significantly induced for HRG than EGF shown in the past study (The figures from Saeki et al BMC Genomics 2009[4]).
Time-course patterns the CAGE peaks of c-FOS, FOSL1, FHL2 obtained from current analysis was consistent with the mRNA expression data shown in above.Profiled time course samples
Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples
| 13032-139E8 | MCF7 breast cancer cell line response to EGF1 | 00hr15min | biol_rep1 |
| 13033-139E9 | MCF7 breast cancer cell line response to EGF1 | 00hr30min | biol_rep1 |
| 13034-139F1 | MCF7 breast cancer cell line response to EGF1 | 00hr45min | biol_rep1 |
| 13035-139F2 | MCF7 breast cancer cell line response to EGF1 | 01hr00min | biol_rep1 |
| 13036-139F3 | MCF7 breast cancer cell line response to EGF1 | 01hr20min | biol_rep1 |
| 13037-139F4 | MCF7 breast cancer cell line response to EGF1 | 01hr40min | biol_rep1 |
| 13038-139F5 | MCF7 breast cancer cell line response to EGF1 | 02hr00min | biol_rep1 |
| 13039-139F6 | MCF7 breast cancer cell line response to EGF1 | 02hr30min | biol_rep1 |
| 13040-139F7 | MCF7 breast cancer cell line response to EGF1 | 03hr00min | biol_rep1 |
| 13041-139F8 | MCF7 breast cancer cell line response to EGF1 | 03hr30min | biol_rep1 |
| 13042-139F9 | MCF7 breast cancer cell line response to EGF1 | 04hr | biol_rep1 |
| 13043-139G1 | MCF7 breast cancer cell line response to EGF1 | 05hr | biol_rep1 |
| 13044-139G2 | MCF7 breast cancer cell line response to EGF1 | 06hr | biol_rep1 |
| 13045-139G3 | MCF7 breast cancer cell line response to EGF1 | 07hr | biol_rep1 |
| 13046-139G4 | MCF7 breast cancer cell line response to EGF1 | 08hr | biol_rep1 |
| 13097-140D1 | MCF7 breast cancer cell line response to EGF1 | 00hr00min | biol_rep2 |
| 13098-140D2 | MCF7 breast cancer cell line response to EGF1 | 00hr15min | biol_rep2 |
| 13099-140D3 | MCF7 breast cancer cell line response to EGF1 | 00hr30min | biol_rep2 |
| 13100-140D4 | MCF7 breast cancer cell line response to EGF1 | 00hr45min | biol_rep2 |
| 13101-140D5 | MCF7 breast cancer cell line response to EGF1 | 01hr00min | biol_rep2 |
| 13102-140D6 | MCF7 breast cancer cell line response to EGF1 | 01hr20min | biol_rep2 |
| 13103-140D7 | MCF7 breast cancer cell line response to EGF1 | 01hr40min | biol_rep2 |
| 13104-140D8 | MCF7 breast cancer cell line response to EGF1 | 02hr00min | biol_rep2 |
| 13105-140D9 | MCF7 breast cancer cell line response to EGF1 | 02hr30min | biol_rep2 |
| 13106-140E1 | MCF7 breast cancer cell line response to EGF1 | 03hr00min | biol_rep2 |
| 13107-140E2 | MCF7 breast cancer cell line response to EGF1 | 03hr30min | biol_rep2 |
| 13108-140E3 | MCF7 breast cancer cell line response to EGF1 | 04hr | biol_rep2 |
| 13109-140E4 | MCF7 breast cancer cell line response to EGF1 | 05hr | biol_rep2 |
| 13110-140E5 | MCF7 breast cancer cell line response to EGF1 | 06hr | biol_rep2 |
| 13111-140E6 | MCF7 breast cancer cell line response to EGF1 | 07hr | biol_rep2 |
| 13112-140E7 | MCF7 breast cancer cell line response to EGF1 | 08hr | biol_rep2 |
| 13163-141B4 | MCF7 breast cancer cell line response to EGF1 | 00hr00min | biol_rep3 |
| 13164-141B5 | MCF7 breast cancer cell line response to EGF1 | 00hr15min | biol_rep3 |
| 13165-141B6 | MCF7 breast cancer cell line response to EGF1 | 00hr30min | biol_rep3 |
| 13166-141B7 | MCF7 breast cancer cell line response to EGF1 | 00hr45min | biol_rep3 |
| 13167-141B8 | MCF7 breast cancer cell line response to EGF1 | 01hr00min | biol_rep3 |
| 13168-141B9 | MCF7 breast cancer cell line response to EGF1 | 01hr20min | biol_rep3 |
| 13169-141C1 | MCF7 breast cancer cell line response to EGF1 | 01hr40min | biol_rep3 |
| 13170-141C2 | MCF7 breast cancer cell line response to EGF1 | 02hr00min | biol_rep3 |
| 13171-141C3 | MCF7 breast cancer cell line response to EGF1 | 02hr30min | biol_rep3 |
| 13172-141C4 | MCF7 breast cancer cell line response to EGF1 | 03hr00min | biol_rep3 |
| 13173-141C5 | MCF7 breast cancer cell line response to EGF1 | 03hr30min | biol_rep3 |
| 13174-141C6 | MCF7 breast cancer cell line response to EGF1 | 04hr | biol_rep3 |
| 13175-141C7 | MCF7 breast cancer cell line response to EGF1 | 05hr | biol_rep3 |
| 13176-141C8 | MCF7 breast cancer cell line response to EGF1 | 06hr | biol_rep3 |
| 13177-141C9 | MCF7 breast cancer cell line response to EGF1 | 07hr | biol_rep3 |
| 13178-141D1 | MCF7 breast cancer cell line response to EGF1 | 08hr | biol_rep3 |
