Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Analysis of Id-1 and Twist-1 Regulation in Bone Development Anna E. Muñoz Cal State University, Los Angeles-City of Hope Cancer Collaborative April 7, 2008 Outline o Introduction o Helix-Loop-Helix proteins o Id-1 and Twist-1 o Human stem cells o Study model system o Significance of Study o Cell line preliminary results o Collaborative research project o Project overview o Acknowledgements Helix Loop Helix Proteins o Various helix-loop-helix (HLH) proteins play a key role in the regulation of cellular growth and differentiation o Basic HLH, bHLH, proteins include a basic DNA binding domain o MyoD (directs muscle development) and TWIST-1 o HLH proteins lack the basic domain o Id proteins do not bind DNA Id-1 o o o o o Belongs to the Id protein family (Id-1, 2, 3, 4) HLH protein Inhibitor of differentiation Preferentially dimerizes with bHLH proteins Acts in a dominant negative fashion o Prevents bHLH proteins from forming dimers with other bHLH proteins o Prevents bHLH proteins from binding DNA o Is differentially regulated during differentiation of mesenchymal stem cells to different cell types Twist-1 o bHLH transcription factor o Homodimer or heterodimer with other bHLH proteins (i.e. E proteins) o Regulates cell movement and mesoderm development during early embryogenesis (i.e. bone and muscle) o Twist-1 has both positive and negative functions regulating mesenchymal cell differentiation o Binds to a conserved E-box sequence (CANNTG) on the promoter region that activates or inhibits transcription of a target gene HLH Proteins Id bHLH bHLH +1 E-Box Stem Cells o Unspecialized cells o ability to self regenerate o ability to differentiate into other cells http://stemcells.nih.gov/info/scireport/chapter5.asp Mesenchymal Stem Cells o Also known as “bone marrow stromal cells” o Capacity to differentiate along myogenic, chondrogenic, osteogenic, and adipogenic lineages www.worldhealthspecialists.org/stemCellBasics.asp Study’s Model System o Normal human cells undergo a limited number of cell divisions in culture o Enter senescence, a non-dividing state o Telomere shortening has been linked to cellular senescence o Retroviral transduction of human telomerase reverse transcriptase (hTERT) o Maintains telomere length o Extends life span o Dr. Glackin’s lab at COH created a human fetal mesenchymal stem cell line that has been immortalized by the hTERT gene, hfMSC-SK-hTERT cell line Mol Biol Cell, 2005, 16:1491-1499 Significance of Study o Different members of the Id family are overexpressed in different tumor types o Abnormally high expression of Twist-1 in cancer cells has been associated with metastasis o Invasive breast cancer o Twist-1 overexpression prevents normal bone and muscle development o The molecular basis of mechanisms that induce the differentiated osteoblastic phenotype is poorly understood CELL LINE PRELIMINARY RESULTS Experimental Methods o Cell culture experiment performed by Dr. Glackin in 2007 o To determine the expression of Id-1, Id-2, Twist-1, Dermo-1 and bone markers during the differentiation of hfMSC-SK-hTERT cell line to bone. Experimental Methods o Cells were grown in expansion medium o Alpha-Minimal Essential Medium supplemented with Fetal bovine serum, penicillin, streptomycin, L-glutamine, and ascorbic acid 2- phosphate. o Differentiation was induced by changing medium conditions. o Expansion medium was supplemented with dexamethasone, sodium pyruvate, hepes, and inorganic phosphate to induce differentiation to bone. o Differentiation was carried out for 28 days o RNA was collected at days 2, 4, 7, 14, 21, and 28 o Expression of the genes listed above along with bone marker genes was measured by real time RT PCR Experimental Methods DAY 4 DAY 2 OSTEOGENIC ADIPOGENIC MYOGENIC OSTEOGENIC ADIPOGENIC DAY 7 MYOGENIC OSTEOGENIC ADIPOGENIC MYOGENIC Diff Media Control Media DAY 14 OSTEOGENIC Diff Media Control Media ADIPOGENIC DAY 21 MYOGENIC OSTEOGENIC ADIPOGENIC DAY 28 MYOGENIC OSTEOGENIC ADIPOGENIC MYOGENIC MSC differentiation to bone hfMSC Osteogenic Timecourse 35 30 25 Fold Change 20 15 10 5 0 -5 -10 Day2 Day 4 Week 1 Week 2 Week 3 Week 4 Unpublished preliminary data collected by Dr. Glackin, 2007 Twist-1 and Id-1 Expression in Osteogenic Differentiation of MSCs Id-1 TwitstMSC Osteoprogenitor 1 Preosteoblast Osteocyte Osteoblast Bone Cell Lining CANCER COLLABORATIVE RESEARCH PROJECT Research Goals o To compare the regulation of Id-1 and Twist-1 in the hfMSC-hTERT cell line throughout its osteogenic differentiation. o To identify and analyze the regulatory features of the Id-1 and Twist-1 promoters that contribute to the development of MSCs to osteoblasts. Id-1 and Twist-1 Regulation in MSC-hTERT line o To compare Id-1 and Twist-1 regulation Grow cells in maintenance medium Maintain cells at 70%80% confluency Change medium every 3 days Obtain mRNA from cells at various time points Introduce osteogenic medium to promote differentiation Perform Quantitative PCR Analyze Id-1 and Twist-1 expression Human Id-1 and Twist-1 promoter constructs o Make Id-1 and Twist-1 promoter/reporter constructs o To study transcriptional regulation of Twist-1 and Id-1 in differentiating MSCs Bioinformatic analysis of human Id-1 and Twist-1 Isolate genomic DNA from fhMSC-SK-hTERT cells Clone Id-1 and Twist-1 upstream regions via PCR Design primers for human Id-1 and Twist-1 upstream regions Perform luciferase assays Grow cells and differentiate Grow and isolate sufficient quantity luciferase reporter vector Ligate promoters into vectors Transform fhMSC-SKhTERT cells Acknowledgements o CSULA-COH Cancer Collaborative Program o NIH grant o Dr. Sharp, Cal State LA o Laura Martinez, Sharp Lab o Dr. Glackin, City of Hope o Shan Li, Glackin Lab o Joyce Ho, Cal State LA Collaborating student Thank You