120 | Chapter 5 Gene expression analysis Data from a previously published study in which the gene expression profiles of 207 untreated B-ALL patients were determined26, 27 were reanalyzed using the R2 microarray analysis and visualization platform developed in our institute and publicly available (http:// r2.amc.nl). Statistics The GraphPad Prism software package (GraphPad Software, La Jolla, CA) was used for statistical testing. Results AKT and NF-kB suppress RAG activity in Abl mouse pre-B cells RAG activity in mouse Abl pre-B cells was assessed by using a green fluorescence protein (GFP)-based RAG activity reporter.25 Abl-transformed pre-B cells can be induced to undergo differentiation toward small pre-B cells with the Abl kinase inhibitor STI571.18, 22 As expected, STI571 induced GFP expression in WT but not in RAG2−/− Abl cells (Figure 1A). The PI3K p110d inhibitor CAL-101 (PI3Ki) and AKTi GSK-690693 increased RAG-reporter activity, showing that RAG activity is suppressed by PI3K and AKT (Figure 1B). Treatment with IKKbi BMS-345541 resulted in a modest but consistent increase in RAG activity. Strikingly, combined treatment with IKKbi and PI3Ki or AKTi synergistically increased RAG activity (Figure 1B-C). A structurally unrelated IKKbi (MLN120B) yielded similar results, demonstrating the specificity of this effect (SUPPL Figure 1). Figure 1. AKT and NF-κB regulate RAG activity in mouse Abl pre-B cells. (A) Representative fluorescence-activated cell sorter plots of the WT and RAG2−/− mouse Abl pre-B cells transduced with the retroviral RAG-reporter construct consisting of an antisense GFP complementary DNA flanked by a 12-bp spacer RSS and a 23-bp spacer RSS, followed by an IRES-RFP cassette. RAG activity mediates inversional recombination of the antisense GFP gene, which can be quantified by flow cytometry. WT and RAG2−/− mouse Abl pre-B cells were treated for 96 hours with 10 mM STI571. (B) WT mouse Abl pre-B cells transduced with the RAG-reporter construct stimulated with 5 mM PI3Ki, 2.5 mM AKTi, 2.5 mM IKKbi, or untreated (dimethylsulfoxide [DMSO] vehicle) for 96 hours. GFP histograms (RAG-reporter activity) of transduced cells are shown by gating on RFP+ cells. (C) Titration curve for IKKbi and for IKKbi plus 2.5 mM AKTi. RAG-reporter activity (y-axis) of WT mouse Abl pre-B cells is plotted against the concentration of IKKbi (x-axis). RAG-reporter activity of mouse Abl pre-B cells treated with 5 mM STI571 is shown. Cells were treated for 72 hours. A representative example of 3 independent experiments is shown, 4 replicate measurements were performed per experiment, and error bars show means ± standard deviation (SD).
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