Patient samples and cell lines.
Normal pro–B cells (CD19+ CD34+ μ chain−) and pre–B cells (CD19+ VpreB+ μ chain+) were sorted from human bone marrow (from four healthy donors; Cambrex) by flow cytometry using antibodies from BD Biosciences and a FACSVantage SE cell sorter (BD Biosciences). B1 cells (CD19+ CD5+), naive B cells (CD19+ CD27− IgD+), and memory B cells (CD19+ CD27+ IgD−) as well as plasma cells (CD19+ CD20− CD138+) were sorted from the peripheral blood of 12 healthy donors (buffy coats were obtained from the Institute for Blood Transfusion, Heinrich-Heine-Universität Düsseldorf) by flow cytometry using the same FACS sorter. Human germinal center B cells were isolated from tonsillar resectates provided by T. Hoffmann (Heinrich-Heine-Universität Düsseldorf). To this end, tonsillar B cells were preenriched by MACS using immunomagnetic beads against CD19 (Miltenyi Biotec). Thereafter, CD20+ CD38+ germinal center B cells were isolated by flow cytometry as described above using antibodies from BD Biosciences.
In total, 108 cases of ALL were analyzed for AID expression. 28 cases of Ph+ leukemia, including seven cell lines (BV173, CMLT1, K562, NALM1, SD1, SUP-B15, and TOM1; DSMZ,) and 80 cases of Ph− ALL, including eight cell lines (BEL1, HBP-NULL, KASUMI2, MHH-CALL3, NALM6, REH, RS4;11, and 697; DSMZ) were analyzed by RT-PCR (see below). Ph+ leukemia also included five cases of LBC CML (cases 21–25 in Table S2). In these cases, CD19+ CD34+ B lymphoid leukemia cells were sorted by flow cytometry from leukemic bone marrow samples using antibodies from BD Biosciences.
VH gene rearrangements were amplified and sequenced from 106 cases of ALL, including 46 Ph+ and 60 Ph− cases. For 28 cases (18 Ph+, 10 Ph−), information on both AID expression and VH region gene sequence was available. Cytogenetic data on the patient samples and cell lines studied are given in Tables S1 and S2. Patient samples were provided from the Department of Hematology and Oncology, Universität Frankfurt (W.-K. Hofmann) and the Department of Medical Biosciences, Pathology, Umea University, Umea, Sweden (A. Li) in compliance with Institutional Review Board regulations. Murine Abelson-MuLV–transformed pre–B cells (300–19) were provided by M. Reth (Max-Planck-Institute for Immunobiology, Freiburg, Germany). Human Ph+ ALL cells and v-abl–transformed mouse pre–B cells were cultured in the presence or absence of 10 μmol/l STI571 (human ALL) or 1 μmol/l STI571 (murine pre–B cells), respectively. STI571 was provided by Novartis. Germinal center–derived B cell lines (MHH-PREB1, MN60, Karpas-422, MC116, JEKO-1, and SJO) were obtained from DSMZ.
Induced expression of BCR-ABL1 and ID2.
A murine IL-3–dependent pro–B cell line, TONB210, which carries an inducible BCR-ABL1
transgene under the control of a doxycycline-dependent promoter (provided by G.Q. Daley, Harvard Medical School, Boston, MA), and Ph−
ALL cells transiently transfected with pMIG-GFP or pMIG-GFP/BCR-ABL1 vectors were studied in cell culture experiments as described previously (20
). pMIG-GFP or pMIG-GFP/BCR-ABL1 vectors encode either GFP only or GFP and BCRABL1 and were transfected by electroporation (250 V and 950 μF). For both transfections, GFP+
cells were sorted after 24 h and subjected to further analysis. BV173 and SUP-B15 cells were transduced with the lentiviral vector pCL1 (provided by H. Hanenberg, Heinrich-Heine-Universität Düsseldorf, Germany) encoding GFP or GFP and ID2 as described previously (21
). The coding sequence of the ID2 cDNA (provided by E. Hara, Science University of Tokyo, Noda, Japan) was excised with BamHI and XhoI and subcloned into pIRESEGFP via BglII and XhoI. The expression cassette containing ID2-IRES-EGFP was digested with NheI and BsrGI and cloned into the lentiviral vector pCL1. 10 d after lentiviral transduction, GFP+
cells were sorted by flow cytometry and further analyzed or kept under cell culture conditions.
Sequence analysis of VH and CH region genes and semiquantitative RT-PCR.
To characterize the configuration of VH
region genes, two primer sets were used to amplify the V region alone (using VH
- and JH
-specific primers) or the V region together with the constant region (using VH
- and CH
-specific primers) of Ig heavy chain transcripts as described previously (22
) in two rounds of PCR using the primers listed in Table S4, which is available at http://www.jem.org/cgi/content/full/jem.20062662/DC1
. PCR products were then cloned and sequenced.
Mutation analysis of BCL6 and MYC genes.
For mutation analysis of BCL6
genes, genomic fragments were amplified and sequenced as described previously (3
) using PFU DNA polymerase. For each PCR product, both DNA strands were sequenced and mutations were only counted if they were found both in the forward and reverse sequence. PCR primers used for amplification of BCL6
fragments are listed in Table S4.
Mutation analysis of TCRB and TCRG V region genes.
TCRB and TCRG gene rearrangements were amplified from multiple leukemia samples, including Ph+ ALL cell lines, primary leukemia cells from Ph+ LBC CML (CD19+ CD34+ B lymphoid cells were sorted from leukemic bone marrow samples), and Ph− ALL cell lines. As controls, TCRB and TCRG gene rearrangements were amplified from normal CD3+ T cells (purified by CD3+ MACS from peripheral blood) using the primers listed in Table S4.
Affymetrix GeneChip analysis and semiquantitative RT-PCR.
Total RNA from cells used for microarray or RT-PCR analysis was isolated by RNeasy (QIAGEN) purification. For microarray analysis, two human Ph+ ALL cell lines (BV173, SUP-B15) were cultured for 16 h in the presence or absence of 10 μmol/l STI571 (Novartis). Double-strand cDNA was generated from 5 μg of total RNA using a poly(dT) oligonucleotide that contains a T7 RNA polymerase initiation site and the SuperScript III Reverse Transcriptase (Invitrogen). Biotinylated cRNA was generated and fragmented according to the Affymetrix protocol and hybridized to U133A 2.0 microarrays (Affymetrix). After scanning (scanner from Affymetrix), the expression values for the genes were determined using Affymetrix GeneChip software. For semiquantitative RT-PCR analysis of human AID, ID2, GAPDH, and BCR-ABL1 and for RT-PCR analysis of murine Aid, Oct2, Obf1, and Hprt transcripts, PCR primers are listed in Table S4.
Western blotting and flow cytometry.
For the detection of AID by Western blot, an antibody against human AID (L7E7; Cell Signaling Technology) was used together with the WesternBreeze immunodetection system (Invitrogen). Detection of EIF4e was used as a loading control (Santa Cruz Biotechnology, Inc.). For analysis of AID and ID2 expression by flow cytometry, antibodies against ID2 (rabbit anti–human ID2 IgG; C-20; Santa Cruz Biotechnology, Inc.) and AID (mouse anti–human AID IgG1; L7E7; Cell Signaling Technology) were used together with secondary antibodies (goat anti–rabbit IgG Cy2 and goat anti–mouse IgG Cy3; Jackson ImmunoResearch Laboratories). Before staining, cells were fixed with 0.4% paraformaldehyde and incubated for 10 min in 90% methanol on ice.
Silencing of AID mRNA expression by RNA interference.
For silencing of AID mRNA expression, one previously validated siRNA (23
) and a nontargeting siRNA duplex were used. All siRNA duplexes (for sequences see Table S4) were labeled with fluorescein using an siRNA labeling kit (Ambion) according to the manufacturer's protocol. Fluorochrome-labeled siRNA duplexes were transfected into Ph+
ALL cell lines (BV173, Nalm1, SD1) by nucleofection according to the manufacturer's protocol (Amaxa). Transfection was repeated after 48 h and transfected fluorescein+
cells were sorted by FACS after 72 h as described previously (24
). RNA interference–mediated knockdown of AID mRNA expression was verified by RT-PCR.
Ligation-mediated PCR (LM-PCR) for detection of DNA-SSB.
Genomic DNA from 2.5 × 106
cells containing a nick on the lower strand was denatured for 10 min at 95°C. Thereafter, a gene-specific primer (Table S4) was hybridized and extended to the position of the nick as described previously (first strand extension; reference 25
), leaving a blunt end using Vent DNA polymerase (New England Biolabs, Inc.). Next, a double-stranded linker was ligated to the newly created blunt end using T4 DNA ligase (Invitrogen) at 14°C overnight. The linker was constructed by annealing of the oligonucleotides 5′- TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACATG 3′ and 3′ amino (C7)- GACGAGCTTAAGTTCGAAGATTGCTACATGCCCCT-5′, and protruding 3′ overhangs were removed by 3′→5′ exonuclease activity of the Klenow fragment of Escherichia coli
DNA polymerase I (Invitrogen). LM-PCR (26
) was performed with modifications as described previously (27
). In two semi-nested rounds of amplification at an annealing temperature of 59°C, linker-ligated intermediates of DNA-SSB within various genes were amplified using gene-specific primers together with two linker-specific primers (Table S4).
Online supplemental material.
Fig. S1 shows the morphology measurement of mRNA levels of Aid in normal mouse splenocytes that were stimulated with IL-4 and LPS and treated with or without STI571. Fig. S2 shows mRNA levels in human B cell lymphoma cells that constitutively express AID after treatment with or without STI571. Tables S1–S4 and Figs. S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20062662/DC1