90 | Chapter 4 fold in NCS-treated cells compared to DMSO-treated control cells (Figure 1B). Induction of DNA damage by NCS resulted in the rapid down-regulation of RAG1 protein in BV173 cells (Figure 1A). Similar results were obtained using a different human pre-B cell line, SUP-B15 (SUPPL Figure 1B-C). To determine the effect of DNA damage on RAG1 protein stability and turnover we blocked protein synthesis using cycloheximide (CHX) in STI571-treated BV173 cells. In accordance with previous reports29,30 we demonstrate that the RAG1 protein has a short half-life of about 30’-60’ (Figure 1C, compare DMSO-treated to CHX-treated samples), which is decreased by NCS treatment (Figure 1C, compare CHX-treated to CHX- +NCS-treated samples). These results indicate that DNA damage negatively affected RAG1 protein stability. Interestingly, we found RAG1 mRNA transcripts also being significantly down-regulated following the NCS treatment in BV173 cells: 59% of DMSO-treated controls (p=0.01), and in A70 (WT mouse v-Abl) pre-B cells: 10% of DMSO-treated controls (p<0.001), (Figures 1D and 1E). Furthermore, DNA damage induction by NCS treatment caused a comparable down-regulation (16% of untreated controls, p=0.01 as compared to untreated controls) of Rag1 mRNA in primary mouse WT pre-B cells (Figure 1F), and of Rag2 mRNA in A70 cells (Figure 1G). ATM kinase is involved in DNA damage-mediated regulation of RAG1/2 Inhibition of ATM kinase activity using the selective inhibitor KU55933 prevented NCS-induced down-regulation of RAG1 protein. It also effectively reduced ATM Ser1981 autophosphorylation in BV173 cells (Figure 1A). Moreover, KU55933 treatment prevented the DNA Figure 1. DNA damage down-regulates RAG1 protein and mRNA expression. (A) Western blotting analysis of RAG1 expression in STI571-treated BV173 BCR-ABL-positive B-ALL cells. DNA damage induction by 2h treatment with 50 ng/mL NCS resulted in a rapid downregulation of RAG1 protein expression, which was prevented by 2h pretreatment with 5 mM KU55933. Phospho-p53 (Ser15) and phospho-ATM (Ser1981) blots were used to show the induction of the ATMdependent DNA damage response. The b-actin blot was used to control for loading. (B) Realtime quantitative RT-PCR analysis of CDKN1A (p21) mRNA expression in BV173 cells treated as indicated in (A). Means ± SEM are shown (n=4, * p<0.05, one-sample t test). (C) Western blot analysis of RAG1 protein stability after cycloheximide (CHX) treatment in BV173 cells treated overnight with 5 mM STI571. Cells were treated for the indicated timepoints with DMSO (vehicle control), 50 ng/mL NCS, 20 mg/mL CHX, or the combination of both. The b-actin blot was used to control for loading. (D) Realtime quantitative RT-PCR analysis of RAG1 mRNA expression in BV173 cells treated as indicated in (A), means ± SEM are shown (n=4, * p=0.01, one-sample t test). (E) Realtime quantitative RT-PCR analysis of Rag1 mRNA expression in A70 WT mouse v-Abl cells treated as indicated in (A), means ± SEM are shown (n=4, *** p<0.001, one-sample t test). (F) Realtime quantitative RT-PCR analysis of Rag1 mRNA expression in WT mouse IL-7 dependent pre-B cell cultures, cells were treated as indicated in (B) 48h after IL7 withdrawal. Cultures from 3 mice were analyzed independently, means ± SEM are shown (n=3, * p=0.01, one-sample t test). (G) Realtime quantitative RT-PCR analysis of Rag2 mRNA expression in A70 cells
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