E-GEOD-5463 - Transcription profiling of mouse double-positive (CD4+CD8+) thymocytes following dexamethasone treatmen
Submitted on 7 August 2006, released on 13 June 2008, last updated on 4 May 2014
Glucocorticoids play a role in regulation of T lymphocytes homeostasis and development. In particular, glucocorticoid treatment induces massive apoptosis of CD4+CD8+ double positive (DP) thymocytes. This effect is due to many mechanisms, mainly driven by modulation of gene transcription. To find out which genes are modulated, we analyzed DP thymocytes treated for 3 hours with dexamethasone or medium alone, by global gene expression profiling using the Affymetrix technology (MGU74Av2 GeneChip). The data were analyzed with MAS 5.0 imposing a cut off of 1.7 fold in up regulation and 1.35 fold in down regulation. To further filter results we also used the statistical software, SAM (d 0.88 see figure 1s in supplementary data of the above cited manuscript). Results indicate modulation of 156 genes, also confirmed by either RNAse protection assay or Real Time PCR. For data mining we also used Go Miner to explore the Gene Ontology data bank (see tables 1-3 of the above cited manuscript). The overall results demonstrated that dexamethasone caused the down-regulation of genes promoting survival of DP thymocytes (e.g. Notch1, Socs1 and Id3) or the modulation of genes activating cell death through the ceramide pathway (Ugcg, Sgpp1, Degs1 and Gpr65) or through the mithocondrial machinery. Among the latter, there are Bcl-2 family members (Bim, Bfl-1, Bcl-xL and Bcl-xbeta), genes involved in the control of redox status (thioredoxin reductase, TXNIP and idh2) and genes belonging to Tis11 family which are involved in mRNA stability. Our study suggests that dexamethasone treatment of DP thymocytes modulates several genes belonging to apoptosis-related systems that can contribute to their apoptosis. Experiment Overall Design: Thymi from 4 week old C3H/HeN mice were teased in culture medium, CD4+CD8+ double positive (DP) thymocytes were sorted with microbeads Miltenyi Biotech methods and were plated in 6 well plates with 3 ml of the culture medium at a concentration of 5.0E+06 cell/ml together with 1E-07 M Dexamethasone or a corresponding volume of PBS. DP thymocytes were kept at 37°C, 5% CO2 in a humidified atmosphere for 3 hours. Total RNA was extracted and analyzed. We used 2 technical replicate-extracts.
transcription profiling by array, compound treatment
Modulation of pro- and antiapoptotic molecules in double-positive (CD4+CD8+) thymocytes following dexamethasone treatment. Rodolfo Bianchini, Giuseppe Nocentini, Ludovic Tibor Krausz, Katia Fettucciari, Stefano Coaccioli, Simona Ronchetti, Carlo Riccardi. J Pharmacol Exp Ther 319(2):887-97 (2006)