122 | Chapter 5 AKT and NF-kB regulate RAG transcription by inhibiting FOXO1 Combined treatment with IKKbi and AKTi induced RAG1 and RAG2 messenger RNA (mRNA) expression in Abl mouse pre-B cells and in two human BCR-ABL-positive B-ALL cell lines (BV173 and SUP-B15) at levels comparable to those observed after STI571 treatment (Figure 2A and SUPPL Figure 2); RAG1 protein expression was correspondingly induced (Figure 2B). We next studied the expression of FOXO1 and FOXO3a, which are known regulators of RAG transcription.6,7 Nuclear FOXO1 levels were increased upon AKTi treatment and upon combined AKTi and IKKbi treatment, whereas FOXO3a levels remained unchanged (Figure 2C). Concomitantly, phosphorylation of FOXO1 on serine 256 and serine 329, which negatively regulate its stability, was decreased in cells treated with IKKbi and AKTi (Figure 2B). Recently, it was demonstrated that cyclin-dependent kinase 4 (CDK4) suppresses RAG expression in Myc-driven lymphomas by phosphorylating FOXO1 at serine 329.28 In line with this, we found that CDK4 protein and mRNA expression was decreased in IKKbi- and AKTi-treated cells (Figure 2D-E). Moreover, treatment with the CDK4-specific inhibitor PD-0332991 (CDK4i) increased RAG activity to a degree comparable to that with IKKbi and AKTi treatment (Figure 2F). AKT and NF-kB inhibition does not interfere with Abl signaling In Abl-transformed pre-B cells, the Abl kinase activity results in constitutive STAT5 phosphorylation, which is inhibited by STI571 (Figure 2B). However, phospho-STAT5 remained unchanged in cells treated with IKKbi and AKTi, showing that Abl kinase signaling was not abrogated. In addition, C-MYC was detectable in IKKβi- and AKTi-treated cells, whereas Figure 2. Transcriptional regulation of RAG1 by the AKT and NF-kB pathways. (A) Real-time reverse transcription PCR (RT-PCR) analysis of RAG1 mRNA in the human BCR-ABLpositive B-ALL cell lines BV173 (left) and SUP-B15 (middle) and Rag1 mRNA in the WT mouse Abl pre-B cell line (right). Cells were treated with 2.5 mM IKKbi, 2.5 mM AKTi, or 10 mM STI571 for 48 hours. (B) Immunoblot analysis of whole-cell extracts from BV173 and SUP-B15 cells treated as in (A). (C) Immunoblot analysis of nuclear and cytoplasmic extracts from BV173 cells treated as in (A). (D) Immunoblot analysis of CDK4 cells treated as in (A). b-actin was used as loading control in (B) and (D). KU70 is expressed in the nucleus and the cytoplasm and was used as loading control in (C). (E) Cdk4 real-time RT-PCR analysis of the WT mouse Abl pre-B cell line; cells were treated as in (A), and results were normalized to those in untreated cells (DMSO vehicle). Real-time RT-PCR results are presented relative to the expression of the housekeeping genes RPLPO (for human cells) and 18S ribosomal RNA (rRNA) (mouse cells); PCRs were performed at least in duplicate, and error bars show means ± SD of 3 independent experiments. (F) Titration curve of CDK4i. RAG-reporter activity of WT mouse Abl pre-B cells treated with 5 mM STI571 is plotted against the concentration of CDK4i. Cells were stimulated for 96 hours. A representative example of 3 independent experiments is shown, 4 replicate measurements were performed per experiment, and error bars represent means ± SD. *P < .05, as determined by the 1-sample Student t test.
RkJQdWJsaXNoZXIy MTk4NDMw