124 | Chapter 5 STI571 treatment led to the loss of C-MYC (Figure 2B) and the induction of a G1 cell cycle arrest (data not shown). The DNA binding activities of p65 and p50 NF-kB transcription factors were modestly but significantly decreased after IKKbi and AKTi treatment (~50% reduction) (SUPPL Figure 3), consistent with a decreased level of phosphorylated inhibitor of kBα (IkBa) and a modest decrease in nuclear p65 (Figure 2C). We also show that AKT is involved in NF-kB signaling because IkBa was increased whereas phosphorylation of IKKα on threonine 23 was decreased by AKTi treatment. Treatment with AKTi resulted in increased phosphorylation of AKT on serine 473 (Figure 2B) as a result of disturbed negative feedback regulation, which was previously described.29-31 AKT and NF-kB inhibit endogenous Igk recombination To determine whether endogenous Igk recombination was regulated by NF-kB, we assessed Vk6-23 to Jk1 coding joins by semiquantitative PCR. Coding joins were observed in Abl cells treated with STI571 and also in cells treated with IKKbi, AKTi, or IKKbi and AKTi combined (Figure 3A). In addition, cytoplasmic Igk light chain expression was significantly induced in Abl cells treated with IKKbi and AKTi, exceeding induction by STI571 (Figure 3B). Cytoplasmic k light chain expression upon treatment with STI571 was accompanied by the transition from large to small pre-B cells, as shown by forward scatter (FSC) analysis. However, a sizeable fraction of IKKbi- and AKTi-treated cells expressing cytoplasmic k light chain remained large, suggesting that these cells did not exit the cell cycle (Figure 3C), which is in agreement with our finding that C-MYC is still detectable under these conditions (Figure 2B). In addition, a considerable portion of the cells (15%) incorporated BrdU. Although BrdU incorporation in the IKKbi- and AKTi-treated cultures was diminished compared with that in untreated cells (15% vs 61%), it was clearly higher than that in STI571-treated cells (15% vs 5%) (Figure 3D). Figure 3. Inhibition of AKT and NF-kB signaling results in recombination of the endogenous Igk locus in mouse Abl pre-B cells. (A) Semiquantitative PCR analysis of Vk6-23 to Jk1 coding joins in mouse Abl pre-B cells treated with 2.5 mM IKKβi, 2.5 mM AKTi, or 10 mM STI571 for 72 hours. Semiquantitative PCR analysis for Gapdh was performed as a loading control. (B) Representative fluorescence-activated cell sorter (FACS) plots showing cytoplasmic Igk expression vs forward scatter (FSC) in mouse Abl pre-B cells stimulated as in (A). Numbers below outlined gates indicate percentage of cells. Graph depicts percentages of cytoplasmic Igκ-positive cells in mouse Abl pre-B cell cultures stimulated for 72 hours. A representative example of 2 independent experiments is shown, 4 replicate measurements were performed per experiment, and error bars represent means ± SD. Statistical significances between groups were determined by 1-way analysis of variance (ANOVA) using a Bonferroni’s posttest. (C) Representative FACS plot showing overlay FSC histograms of cytoplasmic Igk-positive mouse Abl pre-B cells treated as indicated. (D) Representative FACS plots showing BrdU incorporation vs DNA content as measured by propidium iodide (PI) staining in mouse Abl pre-B cells treated for 48 hours as indicated above FACS plots. Numbers above outlined gates indicate percentage of cells. *P < .05; ***P < .001.
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