126 | Chapter 5 AKT and NF-kB signaling prevents RAG-dependent DNA damage in cyclingtransformed pre-B cells Flow cytometric analysis of phosphorylated H2AX (gH2AX) in WT and RAG2−/− Abl cells revealed that treatment with IKKbi and AKTi resulted in significantly higher levels of intracellular gH2AX in RAG-proficient cells, indicating that these cells experienced RAG-dependent DNA damage. RAG-dependent DNA damage was also increased in cells treated with IKKbi alone, but not with AKTi (Figure 4A). Strikingly, RAG-dependent gH2AX staining was exclusively observed in the large cells (FSC high), suggesting that the cycling cells sustained DNA damage (Figure 4B). In cycling cells, RAG2 protein expression is restricted by cyclin A/ CDK2-mediated phosphorylation, whereas RAG1 is not regulated in a cell cycle–dependent manner.32,33 Interestingly, the levels of cyclin and CDK2 mRNA expression were significantly decreased in IKKbi- and AKTi-treated Abl cells at a level comparable to that in STI571-treated cells, whereas the level of cyclin D1 was unaffected in contrast to STI571-treated cells (Figure 4C). The low levels of cyclin A/CDK2 could provide a window for RAG2 protein expression and RAG activity in cycling cells. To provide additional evidence that RAG is active in cycling cells under these conditions, we performed Jk2-RSS signal-end LM-PCR on IKKbi- and AKTi-treated WT and RAG2−/− mouse Abl pre-B cells sorted according the G1 and S phase of the cell cycle. This approach detects transient RAG-dependent DNA breaks at the Jk2-RSS upstream of the Jk2 coding region. Upon visual inspection, Jk2-RSS breaks appeared most prevalent in the G1 phase but were also detectable in S phase cells (Figure 4D). Cloning and sequencing of the PCR products obtained from G1 phase cells showed that 3 of 7 clones harbored the correct insert, whereas 2 of 7 clones obtained from S phase cells contained Jk2-RSS linker fragments, indicating RAG activity in S phase. Negative clones harbored PCR products caused by promiscuity of the Jk2-ko5 LM-PCR forward primer (data not shown). Jk2-RSS LM-PCR products were not detected in RAG2−/− cells, confirming their RAG-dependence. Moreover, Jk2-RSS breaks were not detected in untreated WT mouse Abl pre-B cells, consistent with the absence of endogenous Igk recombinations in untreated cells as shown in Figure 3A. Expression of NF-kB superrepressor stimulates RAG activity To provide further evidence that NF-kB signaling suppresses RAG activity, we introduced the NF-kB superrepressor IkBaSR-IRES-YFP cassette together with the RAG-reporter construct into the WT mouse Abl pre-B cell line. NF-kB signaling was effectively repressed as shown by diminished lipopolysaccharide-induced CD69 expression in IkBaSR-transduced cells (SUPPL Figure 4). We found that IkBαSR-expressing cells (YFP+RFP+) had ∼10-fold higher RAG activity compared with the control cell population (YFP–RFP+) (mean of 24% vs 2% GFP+ cells) (Figure 5A). In agreement, Rag1 mRNA expression was ∼10-fold higher in
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