118 | Chapter 5 pre-B cells, and IL7-dependent mouse pre-B cell cultures representing tractable models to study the regulation of RAG expression in (transformed) pre-B cells because inhibition and/ or abrogation of BCR-ABL, Abl, or IL7 signaling induces differentiation that is accompanied by RAG expression and Igl recombination.18,19 In addition, we studied RAG expression in BCR-ABL-negative primary human B-ALL samples. We report the unexpected finding that nuclear factor kappa B (NF-kB) and AKT signaling suppresses RAG expression and activity in cycling-transformed mouse pre-B cells and in human B-ALL cells and show that inhibition of NF-kB and AKT signaling results in RAG-dependent DNA damage. Materials and methods Cell culture and small molecule inhibitors Abl-transformed mouse pre-B cell lines generated from wild-type (WT) and RAG2−/− mice carrying an Em-Bcl2 transgene were kindly provided by Dr Craig Bassing (University of Pennsylvania School of Medicine, Philadelphia, PA). The human BCR-ABL-positive B-ALL cell lines BV173 and SUP-B15 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Cells were treated with the following small molecule inhibitors at 106 cells per milliliter as indicated: STI571 (imatinib methanesulfonate, LC Laboratories, Woburn, MA), BMS-345541 (Sigma Aldrich), GSK-690693 (Selleckchem, Houston, TX), MLN120B (MCE MedChem Express, Princeton, NJ), CAL-101 (Idelalisib; Selleckchem), and PD-0332991 (Palbociclib; Selleckchem). Immunoblotting Protocols for immunoblotting experiments are available in the supplemental Data available at the Blood Web site. Flow cytometry Intracellular, intranuclear, and 5-bromo-2’-deoxyuridine (BrdU) stainings were done as previously described.20,21 Detailed protocols are available in the supplemental Data. PCR analysis and real-time reverse transcription PCR Vk6-23 to Jk1 coding joins were determined in mouse Abl cells by semiquantitative polymerase chain reaction (PCR) by using previously published primers.22 PCR was performed on 100, 25, and 6.25 ng of DNA. For expression analysis by quantitative real-time reverse transcription PCR, RNA was isolated by using TRI Reagent (Sigma Aldrich), and equal amounts of RNA (0.5 mg) were first-strand transcribed into complementary DNA by using random primers (Promega Corp., Fitchburg, WI) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). Gene expression levels were normalized by using RPLP0 (ribosomal protein, large, P0) and 18S ribosomal RNA (rRNA) housekeeping
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