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International Immunology Advance Access originally published online on January 17, 2007
International Immunology 2007 19(3):267-276; doi:10.1093/intimm/dxl143
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Impaired V(D)J recombination and increased apoptosis among B cell precursors in the bone marrow of c-Abl-deficient mice

Queenie Lai Kwan Lam1, Cherry Kam Chun Lo1, Bo-Jian Zheng1, King-Hung Ko1, Dennis G. Osmond2, Gillian E. Wu3, Robert Rottapel4,* and Liwei Lu1,*

1 Department of Pathology and Center of Infection and Immunology, The University of Hong Kong, Hong Kong, China
2 Department of Anatomy and Cell Biology, McGill University, Montreal, Canada
3 Faculty of Pure and Applied Science, York University, Ontario, Canada
4 Ontario Cancer Institute and Department of Immunology, University of Toronto, Ontario, Canada

Correspondence to: L. Lu; E-mail: liweilu{at}hkucc.hku.hk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Previous studies on c-Abl-deficient mice have shown high post-natal mortality and lymphopenia. However, the mechanisms by which c-Abl may influence B lymphopoiesis remain obscure. In this study, we analyzed B cell sub-populations at various differentiation stages in the bone marrow (BM) of c-Abl-deficient mice. Phenotypic analyses revealed that c-Abl–/– pro-B cells were reduced to half of normal incidence and absolute number, while pre-B cells showed an even greater reduction. Both c-Abl–/– pro-B and pre-B cell populations showed considerably elevated apoptosis ex vivo and in short-term culture but their cell cycle progression was not impaired. In contrast, apoptosis of immature IgM+IgD– B lymphocytes remained at normal control levels. Inhibition of c-Abl activity by STI571 in normal BM cultures significantly increased apoptosis in B cell precursors while the survival of immature B cells was not affected. To determine whether c-Abl deficiency affects Ig heavy-chain rearrangement, we found that the frequency of V(D)J recombination was markedly reduced by 15-fold in c-Abl–/– pro-B cells compared with the control values. However, no perturbation in the levels of signal-end recombination intermediates was found. Taken together, we propose that c-Abl mediates a stage-specific anti-apoptotic response in precursor B cells and is required for efficient V(D)J recombination during B cell development.

Keywords: B cell development, bone marrow, c-Abl, V(D)J recombination


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
B cell development is regulated by both cell-autonomous mechanisms and extrinsic signals produced by the bone marrow (BM) microenvironment during post-natal life (1–3). Early B cell progenitors undergo a developmental program involving ordered Ig gene rearrangements at the heavy- (H) and light (L)-chain gene loci and sequential expression of stage-specific intracellular and surface molecular markers (4). Ig H-chain and Ig L-chain gene segments are assembled in a stepwise fashion (5). The earliest committed B cell progenitors in active cell cycle initiate DJ rearrangement, followed by V(D)J rearrangement at the H-chain loci at the pro-B stage. After a productive V(D)J recombination, the cytoplasmic µ heavy chains (cµHCs) are expressed and then paired with surrogate L chains (VpreB and {lambda}5 proteins) to form the pre-B cell receptor (BCR) at the pre-B cell stage. Expression of the pre-BCR on the cell surface initiates a signal that instructs pre-B cells to undergo proliferation and subsequently to initiate VJ recombination at the L-chain loci. Finally, immature B lymphocytes with the surface expression of whole IgM molecules arise (6, 7). Therefore, the steady-state number of developing B cells in the BM represents an intricate balance between cell production by proliferation and cell loss by apoptosis. Modulations of either one of these processes can greatly affect B cell generation in vivo (8). B cell progenitors which have failed to productively recombine their Ig locus or have failed to properly resolve DNA breaks are deleted by apoptosis (2). Mice lacking the RAG-1 or -2 gene abort B cell development at the pro-B stage due to their inability to rearrange Ig H-chain genes (9). This developmental defect can be rescued by the introduction of a µ heavy-chain (µHC) transgene into RAG-deficient mice that results in further differentiation and survival of pro-B cells into the pre-B cell compartment with normal numbers of large cycling and small resting cells (10). Recent evidence indicates that the pre-BCR is a key checkpoint regulator in B cell development. In the absence of the pre-BCR signals, developing pre-B cells do not proliferate and undergo apoptosis (11). Therefore, functional assembly of Ig H chain and surface expression of the pre-BCR are critically required for the survival of B cell progenitors and their progression into the next differentiation stage (4). At the pre-B cell stage, the mechanism that regulates µHC selection involves H-chain pairing with L chains, which is supported by the evidence of selection against dysfunctional µHC that is unable to pair with L chains (4). Thus, functional µHCs that combine well with L chains would be positively selected by inducing further differentiation and proliferation of pre-B cells into B cells.

c-Abl is a proto-oncogene encoding a ubiquitously expressed non-receptor tyrosine kinase, which was initially identified as the cellular homolog of v-Abl, the transforming oncogene of the Abelson murine leukemia virus (A-MuLV) (12–14). The v-Abl gene can induce pre-B cell lymphoma in mice and activation of c-Abl by translocation forming the BCR–ABL fusion gene can generate leukemias in humans (15, 16). Mice homozygous for the c-Abl gene mutation (c-Abl–/–) show multiple defects, including high post-natal mortality, susceptibility to infections and variable reductions of B and T cells in lymphoid organs (17–20). However, the mechanism underlying lymphopenia in c-Abl-deficient mice remains unresolved. Some studies have suggested that c-Abl induces growth arrest while others have demonstrated a role for c-Abl in stimulating cell growth (14, 21–23). Progenitor B cell lines generated from c-Abl–/– mouse BM display normal proliferation in response to IL-7 but manifest an increased sensitivity to apoptotic stimuli (24).

c-Abl appears to be a component of a genotoxic response pathway. The kinase activity of c-Abl is stimulated in irradiated cells or in cells exposed to DNA-damaging agents (25). c-Abl forms a physical complex with DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase involved in sensing and repairing DNA double-stranded breaks (DSBs), which phosphorylates c-Abl in response to ionizing radiation (26). Over-expression of c-Abl can arrest cell growth in a p53-dependent manner (22, 23). v-Abl, the viral transformed version of c-Abl protein, can arrest development of pre-B cells in expanded BM cultures by blocking both transcription and rearrangement of the L-chain gene locus (27), whereas the small molecule Abl kinase inhibitor (STI571) induces differentiation of A-MuLV-transformed pre-B cell lines (28).

To elucidate the role of c-Abl during B cell development, we have analyzed B cell sub-populations, evaluated their cell cycle status and measured the incidence of apoptotic cells at various stages of B cell differentiation in the BM of c-Abl–/– mice. Furthermore, we have analyzed the frequencies of Ig H-chain rearrangements and their signal-end intermediates, indicative of recombinase activity, among pro-B cells in c-Abl–/– mice. The results suggest a novel role for c-Abl as an anti-apoptotic protein operating in a developmentally determined manner to control the efficiency of V to DJ recombination in pro-B cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Homozygous c-Abl-deficient mice (generously provided by S. Goff, Columbia University, NY, USA) and normal control mice were bred in a pathogen-free animal facility at the Ontario Cancer Institute and genotyped as previously described (17). The wild-type and heterozygous littermates of homozygous mutants were used as controls. All mice were used at 9–12 weeks of age and handled according to the guidelines of the Institutional Review Committee.

STI571 preparation
Stock solution of 10 mM STI571 (Gleevec, Novartis, NJ, USA) was prepared by dissolving the reagent in PBS and filtering through a 0.2-µM filter for sterilization.

Cell suspensions and short-term culture
BM cells were collected by flushing femoral shafts with cold MEM supplemented with 10% heat-inactivated FCS (Life Technologies, Grand Island, NY, USA) and 50 µM 2-mercaptoethanol. After lysis of erythrocytes, the number of nucleated cells was determined using either an electronic particle counter (Coulter Electronics, Ontario, Canada) or a hemocytometer. Aliquots of cell samples were either assayed immediately or incubated at 37°C in a humidified atmosphere with 5% CO2, as described (29).

Immunostaining and flow cytometric analysis
BM cell samples were incubated with PE–anti-mouse B220 (clone RA3-6B2) or PE–anti-mouse IgD (clone IA6-2) and FITC–anti-mouse IgM (clone II/41) mAbs (BD Biosciences, San Jose, CA, USA). To examine total µHC expression, anti-B220-stained cells were ethanol fixed and incubated with FITC–anti-mouse µHC mAb, as described (30). Immunostained cells were acquired with a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software. A minimum of 15 000 events per sample were collected from various B cell sub-populations of defined phenotypes. Cell debris and clumps were excluded by setting a gate on forward scatter versus side scatter.

Cell cycle analysis and apoptosis detection
Phenotypically labeled BM B cell sub-populations including B220+µ– pro-B cells and large cµHC+sIgM– pre-B cells were sorting-purified and then fixed with cold 70% ethanol (30). The cell fractions were re-suspended in 50 µg ml–1 RNase (Boehringer Mannheim Corp., Mannheim, Germany) and stained with 50 µg ml–1 propidium iodide (Sigma Chemical Co., St Louis, MO, USA) in PBS and kept on ice in the dark until flow cytometric analysis. For cell cycle analysis, cells with DNA content located at the S + G2/M phase were defined as the cycling cell population. Accordingly, apoptotic cells were identified in the hypodiploid region of DNA content profiles, as described (29). To determine the direct effect of STI571 inhibition on the survival of B cells, freshly prepared BM cell suspensions from normal mice were cultured with serum-free media in the absence and presence of STI571 for 6–10 h and then immunolabeled with PE–anti-mouse B220 or PE–anti-mouse IgD and FITC–anti-mouse IgM mAbs. After washing, cell samples were re-suspended in the Annexin V binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2) and incubated with 2 µl of Cy-5–Annexin V (BD Biosciences) for 15 min in the dark at room temperature before flow cytometric analysis (29).

Cell purification by FACS sorting
To analyze pro-B cells, BM samples were stained with biotinylated anti-mouse CD19 (clone 6D5) mAb (BD Biosciences), revealed by streptavidin–Quantum Red (Sigma) and further stained with FITC–CD25 (clone M-A251) and PE–anti-mouse CD43 (clone S7) mAbs (BD Biosciences). To purify viable pro-B cells for the recombination assays, CD19+CD43+CD25– cells in BM from c-Abl–/– and c-Abl+/– mice were purified using a dual-laser FACStar sorter (BD Biosciences). Collected cell samples were stained with FITC–Annexin V and further sorted for Annexin V– viable cell populations. Collected cell fractions were routinely >98% pure.

DNA preparation
FACS-sorted CD19+CD43+CD25– pro-B cells from the BM of c-Abl–/– and c-Abl+/– mice were washed twice in PBS and then lysed at a density of 10 000 cells µl–1 in PCR lysis buffer [10 mM Tris (pH 8.3), 50 mM KCl, 1.8 mM MgCl2, 0.45% Nonidet P-40 and 0.45% Tween 20] containing 60 µg ml–1 proteinase K (Boehringer Mannheim Corp.). The samples were then incubated for 1 h at 56°C and heated to 90°C for 15 min.

PCR amplification
To detect the V(D)J rearrangements, a VH-specific primer, VHall, and a primer specific for a sequence immediately 3' of JH4, JH4-3', were used (see Table 1 for all oligonucleotide and probe sequences). PCRs contained 10 mM Tris (pH 8.3), 50 mM KCl, 1.8 mM MgCl2, 0.5% Triton X-100, 100 µg ml–1 BSA, 250 nM of each of deoxynucleoside triphosphate (Boehringer Mannheim Corp.) and 0.5-µM concentration of each primer. The amplification was conducted for 30 cycles with 30 s at 94°C, 1 min at 60°C and 2 min at 72°C. After 30 cycles, a 10-min 72°C primer extension step was performed.


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  		Table 1. Sequences for oligonucleotides and probes used in recombination and ligation-mediated PCR

 
To detect the DJ rearrangements, a degenerate primer for the D region, DSF-Dgen, and the JH4-3' primer were used. The amplification was conducted for 30 cycles of 30 s at 94°C, 30 s at 62°C and 1 min at 72°C. After 30 cycles, a 10-min 72°C primer extension step was added. As a control, actin was amplified using two {alpha}-actin-specific primers, actin-5' and actin-3'. For the actin amplification, DNA samples were diluted 1:100 to be used in the PCR. Samples were amplified for 30 cycles of 30 s at 94°C, 1 min at 60°C and 2 min at 72°C, followed by a final 10-min extension step at 72°C. Titration of the amount of DNA templates and optimization of temperature and PCR cycle numbers for all amplifications have been performed before Southern blot analysis in order to achieve the linear range of each reaction as we previously described (31).

Ligation-mediated PCR assay
DNA from 30 µl of cell lysate was ligated to the BW linker in a final volume of 70 µl, containing 20 pM linker, 1 mM ATP, 1 mM dithiothreitol, 5 mM MgCl2, 66 mM Tris (pH 7.5) and 2.5 U T4 DNA ligase (Boehinger Mannheim Corp.) for 16 h at 16°C before inactivation at 95°C for 15 min (32). All the following experimental conditions were optimized from our recent studies (32, 33). To detect the signal-end intermediates, 20 µl of ligated DNA mixture was used in a 50-µl reaction containing the PCR mixture and 0.5-µM concentration of the linker primer BW-1H and 0.5 mM of a locus-specific primer for VH signal-end intermediates, VHJ558 or VH81x. The reaction was performed for 10 cycles of 30 s at 94°C, 30 s at 61°C and 1 min at 72°C, followed by 10 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C. Finally, another 10 cycles of 30 s at 94°C, 30 s at 59°C and 1 min at 72°C were followed by a 10-min extension step at 72°C.

Southern blot analysis
PCR products were separated on 1% agarose gels and transferred onto Hybond filter (Amersham Pharmacia Biotech, Buckinghamshire, UK) as we previously described (32). Membranes were hybridized with probes labeled with 32P by the T4 polynucleotide kinase (Canadian Life Technologies Co.) according to the manufacturer's protocols. For the detection of V(D)J and DJ rearrangments, JH4-IN probe was used. For the detection of VHJ558 and VH81x ligation-mediated PCR, VHJ558 spacer and VH81x spacer were used as probes, respectively. {alpha}-actin probe was used to detect the amplified {alpha}-actin product as control. The hybridized filters were exposed to PhosphorImager plates and analyzed using the PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

Recombination and signal-end frequency analyses
To compare the relative frequencies of Ig rearrangements, the signal intensity for all four structures of the V(D)J and DJ rearrangements as well as their {alpha}-actin bands were quantified. The mean values based on three experiments of the recombination ratios were then calculated among the experimental samples using the following equation:

Formula
To quantitate the levels of signal-end intermediates, the band intensities from the ligation-mediated PCR products were normalized to those from the {alpha}-actin amplification as described above.

Statistical analysis
Student's t-test was performed where appropriate. Data are presented as the mean ± SEM. Values of P < 0.05 were considered statistically significant among various groups.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Impaired B lymphopoiesis in c-Abl–/– mouse BM
The total BM cellularity recovered from each femoral shaft was reduced from control values of 19.2 x 106 ± 0.7 x 106 cells to 16.4 x 106 ± 0.6 x 106 cells in adult c-Abl–/– mice (Table 2). Flow cytometric analysis revealed a significant reduction in the incidence of B lineage cells to levels less than one half the control value, indicating that c-Abl–/– mice displayed impaired B cell development in the BM (Fig. 1). Whereas B220+µHC– pro-B cells were reduced to about half of normal incidence and absolute number, pre-B cells that express cµHC but lacking surface IgM (sIgM) were more severely decreased to levels at ~25% of normal values. Immature sIgM+IgD– B lymphocytes were reduced to ~30% of control values (Table 2). Furthermore, the reduction of BM cellularity in c-Abl–/– mice was accounted for completely by the decreased number of B lineage cells (Table 2). These results indicate that c-Abl deficiency affects B cell development in a stage-specific manner, which is consistent with previous findings (19).


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  		Table 2. Reduced B lineage cell populations in BM of c-Abl–/– and control micea

 

Figure 1
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  		Fig. 1. Reduced frequencies of B lineage cells in the BM of c-Abl–/– mice. BM cells from c-Abl–/– and control c-Abl+/– mice were analyzed for their surface B220 and µHC expression by flow cytometry. Representative data from one of eight separate experiments are shown.

 
Normal cell cycle progression in c-Abl–/– B cell precursors
To determine whether the diminished numbers of B cell precursors in the c-Abl-deficient mice were due to perturbations in proliferation, cell cycle analysis was performed on freshly prepared BM samples. As shown in Fig. 2(A), the DNA cell content profiles of B220+µHC– pro-B cells from c-Abl–/– mice showed no differences in cell cycle characteristics compared with c-Abl+/– control mice. Although large cµHC+sIgM– pre-B cells from c-Abl–/– mice showed a slight reduction in the percentage of cycling cells, the difference was not statistically significant (Fig. 2B). Consistent with our previous findings (29), small cµHC+sIgM– pre-B cells and immature sIgM+IgD– B cells were post-mitotic populations (data not shown).


Figure 2
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  		Fig. 2. Normal cell cycle progression in c-Abl–/– BM B cell precursors. (A) Cell cycle analysis by nuclear staining of B220+µHC– pro-B cells and large cµHC+sIgM– pre-B populations in the BM of c-Abl–/– and control c-Abl+/– mice. Representative histograms from six separate experiments are shown. (B) The percentages of indicated B cell precursors in the S + G2/M phase as determined by cell cycle analysis. Data are derived from six separate experiments (mean ± SEM).

 
Increased apoptosis in c-Abl–/– B cell precursors
To determine whether increased frequency of apoptosis contributed to the reduced B cell compartments in c-Abl-deficient mice, flow cytometric analysis of hypodiploid DNA content was performed (30). The incidence of hypodiploid DNA in B220+ B cells from freshly prepared BM cell suspensions was significantly higher in c-Abl–/– mice compared with controls (3.8 ± 0.6% versus 1.4 ± 0.3%, P < 0.01). Analysis of B cell precursor sub-populations revealed that the frequency of apoptosis among c-Abl–/– pro-B cells and pre-B cells was elevated 3-fold and 6-fold, respectively (Fig. 3). In contrast, the apoptotic incidence among sIgM+IgD– B lymphocytes remained at control levels (Fig. 3).


Figure 3
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  		Fig. 3. Increased apoptosis among c-Abl–/– BM B cell precursors. BM B cell populations from the c-Abl+/– (open bar) and c-Abl–/– (hatched bar) mice were analyzed for their apoptotic cells at sub-G1 phase at 0 h and after 4 h of culture. (A) Representative histograms showing apoptotic B cells at defined phenotypic stages in BM suspensions from c-Abl–/– and control mice after 4 h incubation. Hypodiploid incidences are indicated, representative of four separate experiments. (B) Data shown are the mean hypodiploid incidences of BM B cells at various differentiation stages both ex vivo and after 4 h of culture (*P < 0.05 and **P < 0.01).

 
To confirm the observation of increased spontaneous apoptosis among c-Abl–/– B cell precursors, BM cells were assayed in short-term cultures where apoptotic cells accumulate in the absence of phagocytosis (29). After 4 h incubation, the incidence of hypodiploid B220+ B cells from c-Abl–/– mouse BM was significantly elevated compared with controls (21.0 ± 1.2% versus 12.3 ± 1.4%, P < 0.05; Fig. 3A). The percentages of apoptotic cells from c-Abl–/– pro-B cells and pre-B cells were greatly increased compared with controls (25.2 ± 1.9% versus 10.2 ± 1.2% and 28.9 ± 1.1% versus 17.5 ± 1.3%, P < 0.01, respectively; Fig. 3B), whereas the apoptotic incidence of c-Abl–/– sIgM+IgD– B lymphocytes showed only a small increase (16.6 ± 2.9% versus 13.2 ± 1.7%, P > 0.05; Fig. 3B). These hypodiploid incidences evaluated by nuclear staining with propidium iodide were consistent with the apoptotic frequencies detected by Annexin-5-labeling technique (data not shown). These findings confirm that c-Abl is required for the normal development and survival of B cell precursors in a stage-specific manner.

To determine whether c-Abl kinase activity is required for the survival of developing B cells, we cultured freshly prepared BM cell suspensions from normal mice in serum-free medium in the absence or presence of STI571, a specific inhibitor of c-Abl kinase (28). Consistent with our results from c-Abl-deficient mice, B220+IgM– B cell precursors displayed a significantly increased incidence of apoptotic cells after treatment with STI571 in a dose- and time-dependent manner (Fig. 4A and B). Interestingly, STI571 had no apparent effect on inducing apoptosis among IgM+IgD– immature B cells (Fig. 4A). These data suggest that the survival function of c-Abl in B cell precursors requires its kinase activity.


Figure 4
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  		Fig. 4. Elevated apoptosis among BM B cell precursors upon inhibition of c-Abl kinase. BM cells from normal mice were incubated with serum-free media in the absence or presence of STI571 at different concentrations for 6–10 h before staining with Annexin V and flow cytometric analysis. (A) Representative histograms from one of four experiments showing apoptotic incidences among B220+IgM– B cell precursors and IgM+IgD– immature B cells after treatment with indicated concentrations of STI571 for 6 h. (B) Incidences of Annexin V+ apoptotic B220+IgM– B cell precursors upon treatment with 10 µM of ST1571 in short-term culture. Data are derived from four independent experiments (mean ± SEM; **P < 0.01).

 
Decreased frequency of V(D)J rearrangements in c-Abl–/– pro-B cells
It is known that c-Abl is a component of a multi-protein complex involved in sensing or repairing DNA DSBs following irradiation or exposure to DNA toxic agents. We investigated whether c-Abl plays a role in mediating DSB repair during Ig H-chain recombination in B cell progenitors. To this aim, we first sought to determine whether the c-Abl deficiency affects Ig H-chain rearrangement in pro-B cells. CD19, a specific B lineage marker, was used in place of B220 to exclude the contamination of B220+ non-B lineage cells (6, 7). Therefore, the recombination assays were performed using DNA samples purified from viable CD19+CD43+CD25– pro-B cells. Frequencies of recombined Ig locus in the pro-B cell population were assessed with the DSF-Dgen/JH4 and Vall/JH4 primer pairs to detect the DJ and V(D)J recombinations, respectively (31). This assay provides a semi-quantitative measurement of the relative frequencies of Ig H-chain rearrangement events (31). The relative frequencies of DJ and V(D)J rearrangements were quantitated and normalized to {alpha}-actin levels. The levels of DJ rearrangements in c-Abl–/– pro-B cells were only slightly lower than the control level, whereas the VDJ rearrangements were markedly reduced by ~15-fold (Fig. 5A and B). Although the possibility that the decreased frequency of pro-B cells might partially be attributed to decreased survival of pro-B cells with V(D)J rearrangement cannot be excluded, our findings demonstrate that c-Abl deficiency severely affect the efficiency of V(D)J rearrangements in pro-B cells.


Figure 5
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  		Fig. 5. Markedly reduced V(D)J rearrangement products in c-Abl–/– pro-B cells. DNA prepared from sorting-purified viable CD19+CD43+CD25– pro-B cells was amplified with PCR primers specific for DJ and V(D)J rearranged alleles or the germ-line {alpha}-actin gene. Southern blot analysis was performed with the PCR products and appropriate 32P-labeled oligonucleotide probes were used to detect the rearranged Ig locus or {alpha}-actin gene, as described in Methods. (A) Representative Southern blots showing amplified PCR products of DSF-Dgen-JH4-3', Vall-JH4-3'' and their corresponding {alpha}-actin in the pro-B cells after hybridization with appropriate 32P-labeled probes. (B) The intensities of radioactive signals were quantitated and relative frequencies of DJ and V(D)J rearrangements normalized to {alpha}-actin were expressed as arbitrary units. Data represent three independent experiments (Mean ± SEM; **P < 0.01).

 
Normal levels of signal-end recombination intermediates in c-Abl–/– pro-B cells
To provide greater insight into the mechanism underlying reduced V(D)J recombination frequencies in c-Abl-deficient pro-B cells, we analyzed the generation of signal-end cleavage intermediates in c-Abl–/– pro-B cells. During the process leading to V(D)J recombination, DSBs produce two intermediate cleavage products, a double-stranded, 5' phosphorylated signal end and a hair-pinned coding end (5). Detection of the levels of signal-end intermediates can be used as a means to reflect the overall activity of DNA cleavage at the target loci (32, 34). Using a ligation-mediated PCR assay, we were able to capture the VH signal-end intermediates present in purified pro-B cells (32, 34). We chose to assay signal-end intermediates generated from VH81x (the most proximal VH segment) and VHJ588 (the largest and most distal VH segment) that are frequently utilized in B cell precursors (31). We found that the level of signal-end intermediates generated from either of the VH segments in c-Abl–/– pro-B cells was comparable to the control values (Fig. 6). Furthermore, DNA fragments were cloned and sequenced, and confirmed to be true signal-end intermediates (data not shown). These results indicate that c-Abl deficiency does not impair the cleavage event within the Ig H locus per se as measured by the production of signal-end intermediates during the V(D)J recombination, but rather suggests a possible defect at the stage of joining.


Figure 6
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  		Fig. 6. Normal levels of signal-end intermediates in c-Abl–/– pro-B cells. Signal-end intermediates generated from recombination at the VH81x and VHJ558 segments in viable CD19+CD43+CD25– pro-B cells from c-Abl–/– and c-Abl+/– mouse BM were detected by ligation-mediated PCR (LM-PCR) and Southern blotting. Appropriate 32P-labeled oligonucleotide probes were used to detect the signal-end recombination intermediates at the specific segments of the Ig locus or {alpha}-actin gene. (A) Schematic diagram showing coding end (light shaded block), signal end (triangle), linker (dark shaded block) and locations of primers (arrow) for LM-PCR. (B) Representative Southern blots showing amplified LM-PCR products of DSF-Dgen-JH4-3', Vall-JH4-3' and their corresponding {alpha}-actin in the pro-B cells after hybridization with appropriate 32P-labeled probes. (C) The intensities of radioactive signals were quantitated and relative frequencies of VH81x and VHJ558 signal-end intermediates normalized to {alpha}-actin were expressed as arbitrary units. Data represent three independent experiments (mean ± SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Previous studies have shown that c-Abl-deficient mice display defects in lymphopoiesis, bone and skin development (17, 18, 35). In this study, we have revealed that mice lacking c-Abl manifest a severe impairment of B cell development, accompanied by enhanced apoptosis among B cell precursors in BM. Moreover, we have demonstrated that V(D)J gene rearrangement is markedly reduced whereas the DNA cleavage process as measured by the frequencies of signal-end intermediates remains unperturbed in c-Abl–/– pro-B cells. These data suggest a novel role for c-Abl during the B cell developmental epoch when V(D)J recombination occurs within the H-chain locus required for productive Ig rearrangement.

During the course of V(D)J recombination, RAG-1 and RAG-2 proteins introduce DSBs at precise recombination-targeting signals that flank segments of the IgR coding sequence. Resolution of DSBs through non-homologous end joining requires a repair complex composed of ATM, DNA-PK, Ku, XRCC4 and ligase IV (36). Although it is currently unclear how c-Abl regulates these events, there is increasing evidence that c-Abl is a component of a multi-protein complex involved in sensing and repair of DNA DSBs. c-Abl has been reported to be in physical complex with ATM (37, 38), RAD51(39, 40), DNA-PK (41, 42) and BRCA1(43). Among them, ATM is mutated in ataxia telangiectasia, a disease that is associated with chromosomal instability, predisposition to lymphoid malignancies and increased radiosensitivity (44). ATM phosphorylates a number of substrates including BRCA1, Nbs1, Chk2 and p53, which are involved in the DNA damage response pathway controlling the cell cycle checkpoints and DNA repair (45). ATM is required for resistance to DNA DSB-inducing agents (46), and it binds to and phosphorylates c-Abl in response to ionizing radiation which is associated with increased c-Abl kinase activity (37, 38). These findings indicate that ATM has a surveillance function in monitoring V(D)J recombination intermediates to protect against aberrant recombination when repair fails. Thus, c-Abl may be a component of the signaling apparatus lying downstream of ATM that is involved in this surveillance process to suppress apoptosis or inhibit cell cycle progression until DSBs are properly resolved.

c-Abl has also been shown to interact with and regulate another member of the PI3K family, DNA-PK. Mice that lack DNA-PK (47–49) or its non-catalytic subunit Ku suffer from SCID and are deficient in V(D)J recombination (50–52). In SCID mouse BM, pro-B cells are able to generate signal-end recombination intermediates while failing to repair DSBs associated with coding joints. It is known that c-Abl forms a physical complex with the catalytic subunit of DNA-PK and is phosphorylated by it in the presence of DSBs by ionizing radiation. DNA-PK can also be reciprocally phosphorylated by c-Abl which inhibits the kinase activity of the DNA-PK catalytic subunit in an apparent negative feedback loop (41, 42). These findings are of considerable interest as c-Abl deficiency partially phenocopies the severe defect observed in SCID mice. We observed a 15-fold reduction in V(D)J recombination in c-Abl-deficient pro-B cells while the level of signal-end intermediates, a surrogate indicator of RAG-mediated DNA cleavage events, is normal. Our data suggest that c-Abl does not participate in the cleavage event during the process of V(D)J recombination but is involved in joining the coding ends, which is in line with its functional interactions with DNA-PK. However, further studies are warranted to reconcile this notion with the previous findings that v-Abl blocks L-chain rearrangement and that STI571 promotes differentiation of A-MuLV cell lines (27, 28). c-Abl deficiency in vivo does not create a definitive block in V(D)J recombination and B cell development but rather may modulate the activity of DNA-PK which in turn reduces the efficiency of V to DJ segment joining in developing B cells. Thus, B cell precursors with impaired V(D)J rearrangements are presumably more susceptible to apoptosis, contributing to the observed lymphopenic phenotype in the c-Abl–/– mice.

The previous findings that c-Abl-deficient embryonic fibroblasts are resistant to cispaltin or ionizing radiation-induced apoptosis suggest that c-Abl is a component in the genotoxicity-induced cell death pathway (26, 53, 54). The capacity of c-Abl to induce apoptosis may be coupled to its interaction and phosphorylation with p53/p73 family members (53, 54) or stress-induced kinases (23, 26, 55). However, the findings from c-Abl-deleted DT40 cells (a chicken B cell line) demonstrate similar sensitivity to ionizing radiation compared with wild-type controls suggesting that the apoptotic function of c-Abl may be cell-type specific (56, 57). We found that c-Abl–/– primary B cell progenitors were more susceptible to spontaneous apoptosis than c-Abl+/– controls, which is consistent with the previous finding of increased sensitivity of c-Abl-deficient B cell lines to apoptosis (24). Importantly, our data that developing B cell precursors do not require c-Abl for cell cycle progression support the notion that c-Abl functions as a survival factor in this cellular context. On the other hand, the increased frequency of apoptotic B cell precursors observed in the c-Abl–/– mice might result from the role that c-Abl serves in controlling cell morphogenesis and chemotaxis by modulating the assembly of the actin cytoskeleton (58–60). Thus, it remains to be determined whether c-Abl deficiency may impair the migration and interaction of B cell precursors in the BM microenvironment with growth factors such as IL-7, Kit ligand and Flt3L.

In summary, while the precise mechanism of the impaired Ig gene rearrangement and increased apoptosis among c-Abl–/– B cell precursors remains to be elucidated, our present work indicates that the c-Abl tyrosine kinase plays an important role in the quality control of B cell precursors during early development in the BM. Further studies are required to determine the precise mechanism by which c-Abl regulates recombination repair during B cell development.


   Acknowledgements
 
This work was supported by grants from the Research Grants Council of Hong Kong (HKU7447/03M), National Key Basic Research Program of China (2001CB510002), the Canadian Institutes of Health Research, Arthritis Society of Canada and the National Cancer Institute of Canada.


   Abbreviations
 
A-MuLV, Abelson murine leukemia virus
BCR, B cell receptor
BM, bone marrow
cµHC, cytoplasmic µ heavy chain
DNA-PK, DNA-dependent protein kinase
DSB, double-stranded break
H, heavy
µHC, µ heavy chain
L, light
sIgM, surface IgM

   Notes
 
* Equal contributions. Back

Transmitting editor: C. Paige

Received 29 August 2006, accepted 18 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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