Down-modulation of CD19,Enhances Migration to CXCL12, and Confers Survival Advantage in pre-BI Cells

PAX5 is a transcription factor essential for B-cell development. Recently, it has been found as a frequent target of aberrancies in childhood acute lymphoblastic leukemia (ALL; 30% of B cell ALL cases), showing monoallelic loss, point mutations, or chromosomal translocations. The role of these aberrancies is still poorly understood. We previously cloned the PAX5/TEL fusion gene in a patient affected by B-cell precursor ALL with a t(9;12) translocation. This is the first report investigating the molecular and functional roles of PAX5/TEL protein in vitro from murine wild-type pre-BI cells. We showed that PAX5/TEL protein acts as an aberrant transcription factor with repressor function, recruiting mSin3A, down-regulating B220, CD19, BLNK, MB-1, FLT3, and mu heavy chain expression, thus suggesting a block on B-cell differentiation. In a PAX5-deficient context, the presence of PAX5/TEL did not replace PAX5 functions. PAX5/TEL protein enhances cell migration towards CXCL12, with the overexpression of CXCR4. Moreover, the presence of the fusion gene overcomes interleukin-7 withdrawal and interferes with transforming growth factor-beta1 pathway, inducing resistance and conferring cells an advantage in proliferation and survival. Thus, in vitro, the PAX5/TEL protein has a dominant effect on wild-type PAX5, interferes with the process of B-cell differentiation and migration, and induces resistance to apoptosis. Taken together, these phenomena likely represent key events in the process of B-cell transformation.


Introduction
The PAX5 gene is emerging as a frequent target of aberrancies in childhood acute lymphoblastic leukemia (ALL; ref. 1).Recently, 30% of B-cell precursor (BCP) cases showed monoallelic loss or point mutations of the PAX5 gene, indicating that these alterations could result in haploinsufficiency or in the generation of hypomorphic PAX5 alleles (2).Moreover, PAX5 is involved in a growing number of chromosomal translocations in BCP-ALL (2).In hematological malignancies, PAX5 was described as being overexpressed in non-Hodgkin's lymphoma carrying the translocation t(9;14) (refs. 3, 4).More recently, after our first description, PAX5 was found to be fused to other partner genes in patients with BCP-ALL, giving rise to PAX5/ELN, PAX5/FOXP1, and PAX5/ EVI3 fusion transcripts (1).However, the role of these aberrancies is still poorly understood (2).
Our group described the generation of the PAX5/TEL fusion gene, as a result of the translocation t(9;12)(q11;p13) in a patient affected by ALL with BCP lineage phenotype (5); a novel fusion transcript was identified, resulting from joining the 5 ¶-end, containing the NH 2 terminal region of the PAX5 gene to the almost whole sequence of the TEL gene.Recent data proposed that PAX5/TEL fusion defines most of the cases with dic(9;12)(p13;p13), a recurrent chromosome abnormality that accounts for f1% of childhood ALL, almost exclusively BCP-ALL (6).
The PAX5 gene belongs to the PAX gene family of transcription factors and it is essential for B lymphoid lineage commitment (7)(8)(9)(10).It recognizes DNA through a highly conserved paired domain (11), retaining a high degree of homology between human and mouse (12).PAX5 functions both as a transcriptional activator and as a repressor on different target genes (13), e.g., it activates CD19 (14), MB-1 (15), and BLNK (16), and it represses MCSFR (8), Notch1 (10) and FLT3 (17).Among them, the most important target is CD19, which is expressed by B lymphocytes starting from pre-BI cells and lasting to mature B cells (8,18).Its expression is directly controlled by the PAX5 gene; indeed, it has been shown that a sequence consensus for the paired domain of PAX5 is present in the CD19 promoter region (14).
The TEL/ETV6 gene belongs to the Ets transcription factor family (19), and it is a sequence-specific transcriptional repressor of Ets binding site-driven transcription (20), associating to histone deacetylase-3 (21) and recruiting corepressors such as mSin3A (22) and NCor (23).Human and murine TEL proteins are particularly homologous within their NH 2 terminal region and their ETS domain (24).It is required for hematopoiesis within the bone marrow (25)(26)(27).As reviewed by Poirel et al. (28) and Bohlander (29), the TEL gene is frequently involved in several translocations occurring in hematological malignancies, leading to many different fusion genes.
According to the sequence of the PAX5/TEL gene (5), and the physiologic role of both partner genes (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), it is reasonable to hypothesize that the PAX5/TEL protein could act as an aberrant transcription factor, retaining the PAX5 DNA-binding domain and both the dimerization and DNA-binding domains of TEL.This is the first report investigating the molecular and functional roles of PAX5/TEL protein in vitro.For this purpose, the study was done in an in vitro hematopoietic context, characterized by murine pre-BI cells of fetal liver origin (30).Those cells were derived from a wild-type mouse and were positive for B220, cKIT, and CD19 antigens.Thus, in vitro, we showed that the PAX5/TEL protein has a dominant effect on wild-type PAX5, interferes with the process of B-cell differentiation and migration, and allows the cells to resist apoptosis.Taken together, these phenomena likely represent key events in the process of B-cell transformation.

Materials and Methods
Retroviral vectors.The full-length PAX5/TEL cDNA was cloned from an ALL patient carrying a t(9;12)(q11;p13) translocation (5).A FLAG synthetic sequence tag (Sigma-Aldrich Co.) was fused at the 5 ¶-end and the FLAG full-length fusion transcript was cloned into the retroviral vector pMSCV-IRES-GFP (MIGR), a bicistronic vector which allows the expression of PAX5/TEL and GFP under the control of the long terminal repeat promoter (kindly provided by Prof. M. Busslinger, Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria).
Retroviral transduction.The retroviral supernatant was obtained by calcium-phosphate transfection of Phoenix packaging cell line following the standard protocol by Dr. G.P. Nolan. 4 The transduction of cells was done by spinning with viral supernatant and only for Ba/F3 cells in the presence of Retronectin-CH296 (Takara, Cambrex Corporation).On day +3 from transduction, cell sorting for GFP fluorescence was done by the FACS Aria instrument (BD Biosciences).
Immunoprecipitation and Western blot analysis.Ba/F3 cells were lysed in radioimmunoprecipitation assay buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) to recover total proteins, in the presence of protease inhibitor cocktail (Sigma-Aldrich).Protein concentration was determined by Bradford assay (Sigma-Aldrich).Western blot analysis was done following standard protocols, using polyvinylidene difluoride membrane (Bio-Rad), enhanced chemiluminescence reagents (GE Healthcare), and Kodak image station (Kodak SpA).The quality of protein extracts was checked using anti-ACTIN antibody (AC-15, Sigma-Aldrich).The expression of PAX5/TEL was analyzed by anti-FLAG antibody (M2, Sigma-Aldrich).The expression of mSin3A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed, respectively, by anti-mSin3A antibody (K20) and by anti-GAPDH (6C5) purchased from Santa Cruz Biotechnology.The coimmunoprecipitation assay was performed using Catch and Release Reversible Immunoprecipitation System (Upstate), following the manufacturer's protocol.The stripping procedure was performed at 50jC, in solution with 2% SDS, Tris-HCl (pH 6.7) and 100 mmol/L of mercaptoethanol.
Antibodies and flow cytometry.Phycoerythrin-conjugated antibodies anti-CD117 (2B8), anti-CD19 (MB19-1), and anti-CD115/MCSFR (AFS98); and allophycocyanin-conjugated antibodies against B220 (A7R34) and IgM/A heavy chain (II/41) were purchased from e-Bioscience.Staining of cells was performed and they were analyzed in the presence of propidium iodide (PI) to exclude nonliving cells.Intrastaining of cells was performed using Fix&Perm Cell Permeabilization Reagents (Caltag Lab, Invitrogen Corporation), following the manufacturer's protocol.Flow cytometry was performed using FACSCalibur (BD Biosciences); data were analyzed using the CellQuest Software (BD Biosciences) and expressed as percentages of positive cells or as a ratio between mean fluorescence intensity (MFI) of the antigen expression on PAX5/TEL cells over MIGR-GFP cells.
Reverse transcription-PCR and real-time quantitative-PCR assays.RNA extraction was performed using TRIZOL reagent (Invitrogen Corporation), following the manufacturer's protocol.Superscript II enzyme (Invitrogen Corporation) was used for cDNA synthesis, whereas Platinum Taq DNA polymerase (Invitrogen Corporation) was used for PCR reaction.The HPRT gene was analyzed as a constitutive gene with the following PCR primers: forward, 5 ¶-GGGGGCTATAAGTTCTTTGC-3 ¶ and reverse, 5 ¶-TCCAACACTTCGAGAGGTCC-3 ¶; the CD19-specific PCR primers were: forward, 5 ¶-CCCAGTCATGAAGAAGATGCA-3 ¶ and reverse 5 ¶-GCAGCACTT-GAGTAGGTTCAC-3 ¶.PCR reactions were performed at 60jC annealing temperature for 35 cycles.Real-time analysis was performed on Light Cycler 480 (Roche Diagnostics; F. Hoffmann-La Roche Ltd.) with Universal Probe Master system; primers and probes for HPRT, PAX5/TEL, GFP, BLNK, MB-1, FLT3, MCSFR , and CXCR4 genes were selected according to the Software Probe Finder (Roche Diagnostics).Data were expressed using the comparative Ct method (35); both t test and SD values referred to are triplicates of a single experiment.
Migration assay.Transwell plates (5.0 Am) were used; 5 Â 10 5 cells were loaded in the upper well suspended in 100 AL of standard medium, and in the lower well, 600 AL of standard medium in presence or not of 100 ng/mL of hCXCL12 (Peprotech Inc.).After 4 h, the cells in the lower well were collected and counted by fluorescence-activated cell sorting (FACS).
IL-7 starvation assay and IgM/M heavy chain analysis.Cells were washed out by IL-7 and plated in IMDM + 2% FBS on OP9 stroma.Every 24 h, from 0 h up to 96 h, GFP+ cells were counted by FACS with PI exclusion, stained for surface CD19 and for intracellular IgM/A heavy chain.
Transforming growth factor-B1 culture.Cells were grown in standard medium in presence or not of 10 ng/mL of human transforming growth factor-h1 (TGFh1; Peprotech, Inc.), on OP9 stromal cells.After 96 h of culture, cells were counted by FACS with PI exclusion.

Results
PAX5/TEL is a nuclear protein.Immunofluorescence analysis using anti-FLAG showed a specific nuclear localization of the PAX5/TEL fusion protein in the NIH3T3 cell line transduced by pMSCV-PAX5/TEL-IRES-GFP vector (Supplementary Fig. S1).This evidence supports the possibility that PAX5/TEL functions as a transcription factor.
PAX5/TEL protein recruits the corepressor complex.The PAX5/TEL fusion protein retains the TEL sequences responsible for the recruitment of repressor cofactors such as mSin3A, NCoR, and HDAC (20)(21)(22)(23)36).Considering this feature, we addressed the question of whether PAX5/TEL could act as a repressor of transcription.Figure 1 shows a representative result of the coimmunoprecipitation assay of PAX5/TEL with mSin3a in transduced Ba/F3 cells.As indicated in Fig. 1A (top), mSin3A was immunoprecipitated using anti-mSin3A antibody and its specific band of 140 kDa was detected in the lanes (4-6) corresponding to the immunoprecipitated samples, whereas in the flow-through lanes (1-3), the mSin3A signal was completely absent.On the same membrane (Fig. 1A, middle), PAX5/TEL protein was evident as a band of 70 kDa in lane 6 of the immunoprecipitated MIGR-PAX5/TEL cells following anti-FLAG detection (middle).The signal was completely absent in the immunoprecipitated control cells and MIGR-GFP cells (Fig. 1A, middle, lanes 4-5).The specificity of the mSin3A-PAX5/TEL coimmunoprecipitation was proved on the same membrane by the anti-GAPDH antibody detection.In fact, GAPDH, a protein not related to the transcription complex, was detected only in the flow-through samples (lanes 1-3), but was completely absent in all the immunoprecipitated samples (Fig. 1A, bottom), thus confirming the specificity of the coimmunoprecipitation and interaction of PAX5/TEL with mSin3A.In total, protein lysatespecific bands for PAX5/TEL and actin proteins were detected by specific antibodies (Fig. 1B).
In conclusion, the PAX5/TEL protein recruits the mSin3A cofactor, which is known to mediate the repression of the transcription, thus suggesting a role of PAX5/TEL protein as a repressor of transcription.
Both the observations of nuclear localization and recruitment on mSin3A prompted us to further explore which genes PAX5/TEL might regulate, in particular, PAX5 target genes such as CD19, BLNK, and MB-1-genes known to be important for controlling B-cell differentiation (1,(13)(14)(15)(16).
Because reverse transcription-PCR (RT-PCR) analysis indicated that Ba/F3 cells do not express any of these genes (data not shown), we considered a more physiologic in vitro model, such as wild-type pre-BI cells, which are primary cells purified from mouse fetal liver as B220+/cKIT+/CD19+ (30); these cells grow on the OP9 stroma layer and show a stable phenotype in culture.
In order to exclude the role of genetic background or the influence of a specific setting, all the experiments, carried out in wild-type pre-BI cells, were performed in two independent wildtype pre-BI cell cultures derived from different mouse donors, with overlapping results, thus excluding the influence of a particular genetic background and minimizing the contextspecific bias.All the results reported in this article were each performed in different pre-BI cell cultures from at least three independent experiments.PAX5/TEL mediates B220 and CD19 down-modulation in wild-type pre-BI cells.We transduced wild-type pre-BI cells with either MIGR-PAX5/TEL or MIGR-GFP, and after 3 days, cells were sorted for GFP.After sorting, cell phenotype was analyzed, focusing on lineage-specific BCP antigens.We show a representative phenotype analysis.PAX5/TEL transduced wild-type pre-BI showed a time-dependent trend to decreased expression of B220 (Fig. 2A).At day +3 from sorting, PAX5/TEL cells expressed 20% lower levels (in independent experiments: range, 20-37%) of the antigen compared with MIGR-GFP cells (expressed as MFI ratio); at day +13, its expression was 36% lower (expressed as MFI ratio, in independent experiments: range, 30-50%) and a fraction of PAX5/ TEL cells, equal to 55%, were completely negative for B220.At day +19, a predominantly B220-negative population was clearly distinguishable among PAX5/TEL cells, whereas only 17% of the cells were still positive for B220, with a down-regulation of B220 expression equal to 63% (in independent experiments: range, 61-66%).
The lower CD19 expression consistently had a different intensity in many independent transduction experiments, revealing an inverse correlation between the GFP and the CD19 expression levels on PAX5/TEL cells: higher GFP levels (e.g., higher PAX5/TEL protein expression) corresponded to lower CD19 level; in contrast with MIGR-GFP cells, in which CD19 was indeed expressed at a constant level (Fig. 2C).In addition, in PAX5/TELexpressing cells, using real-time quantitative-PCR (RQ-PCR), we showed a tight correspondence between PAX5/TEL and GFP mRNA levels, correlating with GFP levels analyzed by FACS (Supplementary Fig. S2).
PAX5/TEL represses BLNK, MB-1, and FLT3 expression and does not activate MCSFR expression in wild-type pre-BI cells.We further investigated the effect of PAX5/TEL on genes regulated by PAX5 (1).The PAX5/TEL presence affected both BLNK (16) and MB-1 (15) expression; indeed, using RQ-PCR analysis, we observed a down-regulation of expression of 66% and 43%, respectively, in PAX5/TEL cells compared with MIGR-GFP cells (Fig. 2D, P < 0.01 and P < 0.05, respectively).We analyzed FLT3 and MCSFR genes, Figure 2. Phenotypic and PAX5 target gene analysis of wild-type pre-BI cells transduced by MIGR-PAX5/TEL or MIGR-GFP vectors.Days indicated refer to the sorting day, and all analyses refer to gated GFP-positive cells.A, B220 expression decreases in time in PAX5/TEL cells.B, a decrease in CD19 expression (67% expressed as MFI ratio) was observed in PAX5/TEL cells at day +3.C, correlation between CD19 and GFP expression at day 3: higher GFP levels correspond to lower CD19 levels in PAX5/TEL cells, whereas CD19 is expressed at constant levels in MIGR-GFP cells; in particular, low GFP cells gated on R5 and high GFP cells gated on R6 expressed, respectively, 34% and 50% less CD19 than MIGR-GFP cells (expressed as MFI ratio).D, RQ-PCR analysis of BLNK, MB-1 , and FLT3 expression at day +3; the transcript level of the gene is normalized for HPRT constitutive gene expression.t test: *, P < 0.05; **, P < 0.01; 2 ÀDDCt = the RQ-PCR data are reported as a difference between DCt of PAX5/TEL and MIGR-GFP cells, where DCt is defined as the difference between the cycle threshold (Ct) of the target gene and the HPRT constitutive gene.
PAX5/TEL does not replace PAX5 in PAX5 À/À pre-BI cells.PAX5 À/À cells are hematopoietic multipotent pre-BI cells blocked at this stage of B-cell differentiation, without any possibility of maturing into B cells, even in an in vivo context; the extensive in vivo self-renewal, the long-term reconstitution capacity of PAX5deficient precursor B-cells are fully described (31,32,37).These cells are B220+/cKIT+ but CD19À due to the absence of the PAX5 gene.We transduced PAX5 À/À pre-BI cells to investigate whether PAX5/TEL was able to switch-on CD19 expression without any influence by the endogenous PAX5.
PAX5/TEL wild-type pre-BI cells migrate towards CXCL12.B-cell progenitors require CXCL12 or stroma-derived factor 1 to proliferate (38); moreover, the involvement of CXCR4 in several hematologic malignancies has previously been described (39).For these reasons, we investigated the ability of PAX5/TEL wild-type pre-BI cells to migrate towards CXCL12.In a 4-h migration assay using the transwell system, we observed an increased migration rate of PAX5/TEL cells specific towards CXCL12 and not due to better migratory properties per se; indeed, in the absence of the chemokine, the basal level of migration is comparable in both cell populations.In Fig. 4A, a representative experiment is shown.In independent experiments (n = 4), we evaluated the migration index (MI), defined as the ratio between the number of migrated cells in the presence and in the absence of the chemokine stimulus.PAX5/ TEL cells showed a median MI equal to 82.48 (range, 31.62-132.69),whereas MIGR-GFP cells had a median MI equal to 40.37 (range, 14.28-53.29),corresponding to 2.09 times higher MI in PAX5/ TEL cells (t test analysis, P < 0.05).Moreover, we observed an upregulation of expression of the chemokine receptor CXCR4 in PAX5/ TEL cells following RQ-PCR analysis (Fig. 4B, P < 0.01).
PAX5/TEL wild-type pre-BI cells resist IL-7 starvation without acquiring long-term IL-7 independence.Wild-type pre-BI cells were grown for 4 days in the presence or absence of IL-7, with the purpose of testing whether PAX5/TEL protein would be able to confer cytokine-independent cell growth.After 24 h, pre-BI cells started to suffer and die and GFP-positive living cells  (PI negative) were counted by FACS and compared with the number of cells at 0 h.At 24 and 48 h, PAX5/TEL cells were surviving, whereas MIGR-GFP cells progressively died (10% and 30%, respectively).At 72 h of IL-7 starvation, PAX5/TEL cell numbers reached the highest value in culture compared with MIGR-GFP cells (ratio between the number of PAX5/TEL over MIGR-GFP cells was equal to 3).At 96 h, a significant number of living cells were still present in culture with a prevalence of PAX5/ TEL-positive cells with a correspondent ratio of 1.9 (Fig. 5A).
Indeed, a relative enrichment in highly expressing PAX5/TELÀ GFP-positive cells was observed both at 0 h of starvation and at 96 h in the presence of IL-7, with a peak at 72 to 96 hours; in MIGR-GFP cells, this enrichment was not observed (Supplementary Fig. S4).Overall, these data showed that under IL-7 starvation conditions, PAX5/TEL gives a survival advantage, although it does not confer IL-7 independence to pre-BI cells for prolonged periods.
PAX5/TEL impairs M heavy chain expression in wild-type pre-BI cells.In the absence of IL-7, pre-BI cells receive the stimulus to continue on the B-cell differentiation pathway to the pre-BII stage (12,40), characterized by the expression of IgM, and in particular, it is possible to detect A heavy chain in the cytoplasm.However, the in vitro stimulus alone is not enough to sustain differentiation and proliferation, implying that, as described above, cells start to suffer but still express the A heavy chain at a cytoplasmic level, with the highest levels at 72 to 96 h.As shown in Fig. 5B, after 96 h of IL-7 starvation, PAX5/TEL cells did not express significant levels of cytoplasmic A heavy chain (8.96% A/CD19positive cells) compared with MIGR-GFP cells (26.05% A/CD19positive cells).
PAX5/TEL wild-type pre-BI cells are resistant to the TGFB1.To investigate the ability to resist the apoptotic stimulus as evidenced by the IL-7 starvation assay, we grew pre-BI cells in the presence of TGFh1, a cytokine with antiproliferative and proapoptotic effects.Moreover, its homeostatic effect was shown in association with genetic lesions in many different types of hematological malignancies (41).
The response of PAX5/TEL pre-BI cells to the effects of TGFh1 was analyzed after 96 h of culture, counting the PI-negative and GFP+ cells by FACS.Although in the absence of TGFh1, PAX5/TEL cells were slightly slower growing.In the presence of TGFh1, PAX5/ TEL-transduced pre-BI cells showed increased proliferation and survival compared with MIGR-GFP pre-BI cells, which were completely inhibited in growth (Fig. 6A).More specifically, the resistance index (RI, defined as the ratio between the number of cells in the presence or absence of the cytokine) was two times higher in PAX5/TEL cells (RI, 0.40) than in MIGR-GFP cells (RI, 0.18).The specific effect of TGFh1 on PAX5/TEL-positive cells was further reinforced by the observation that the GFP-negative fraction of cells, present in culture together with the PAX5/TELÀ GFP-positive cells as a residue of sorting, were inhibited by TGFh1 to the same extent of MIGR-GFP cells; in line with this finding, we observed a relative enrichment of PAX5/TELÀ GFP-positive cells, especially in highly expressing PAX5/TELÀ GFPÀ cells, thus positively selected by TGFh1 (Supplementary Fig. S5).These data indicate that PAX5/TEL pre-BI cells are resistant to the TGFh1 antiproliferative and proapoptotic effects.

Discussion
This is the first study investigating the oncogenic role of the PAX5/TEL fusion protein in vitro.Our group previously described an ALL patient carrying a t(9;12) translocation as the first report on PAX5 involvement in chromosomal translocations generating a fusion protein (5).In addition, PAX5 has been reported as fused to other partner genes in ALL, such as FOXP1, EVI3, and ELN (1, 2).
Similarly to other fusions involving TEL or PAX family genes, PAX5/TEL potentially constitutes an aberrant transcription factor, resulting from the joining of DNA-binding paired domain sequence of PAX5 to almost the whole TEL, a transcription factor whose functional domains are completely conserved in the fusion protein.Indeed, the demonstration that PAX5/TEL localizes exclusively in the nucleus, like both PAX5 and TEL proteins do (19), highly supports the possibility that PAX5/TEL acts as a transcription factor.In the fusion event, differently from PAX5/ELN transcript (42), the nuclear localization sequence of PAX5 is lost, thus suggesting that the nuclear localization sequence of the fusion protein is driven by the TEL signal sequence.Evidence of the potential molecular mechanism of action of the fusion protein as a transcription factor is provided by the specific molecular interaction of PAX5/TEL and mSin3A, clearly demonstrating the association of fusion protein with the transcriptional complex, and thus supporting an active role for PAX5/TEL as a repressor.This finding is in agreement with the observation that PAX5 activator/ inhibitory domains are lost, whereas the retained TEL regulatory domains are known to recruit transcriptional cofactors such as mSin3A, NCoR, and HDAC3 leading to transcription repression of target genes (21)(22)(23).
In addition to this mechanism of direct transcription regulation, the PAX5/TEL protein could potentially act as a repressor of function of the endogenous PAX5 alleles by a competition mechanism, as described for the PAX5/ELN fusion protein (42).For these reasons, we extensively analyzed the influence of PAX5/ TEL on the PAX5 physiologic role, investigating the potential role of the fusion protein on B-cell differentiation in a more physiologic context, closer to the potential target of transformation of BCP-ALL (43,44).We considered the in vitro model of pre-BI cells (30), primary cells derived from a wild-type mouse fetal liver and characterized as being positive for CD19 (direct target of PAX5), B220, and cKIT.These cells can be grown in vitro, on the stroma layer, and in the presence of IL-7.The role of the stromal cells is 2-fold: first, to specifically interact with precursor B-cells, and second, to provide them with growth factors, the global effect being to sustain cell proliferation by maintaining their original phenotype (43)(44)(45).This model is well characterized and it offers the possibility of investigating the role of a translocation in vitro, in a wild-type B cell context, thus avoiding the influence of confounding events such as genetic abnormalities or previous transformation events.In this setting, our findings provide an important way to understand the role of the PAX5/TEL oncogenic protein in the process of B-cell transformation.
First, we focused our interest on the analysis of B-cell differentiation.Although B220 is expressed throughout B-cell development from pro-B cells to the antibody-secreting plasma cells (12,40,46), herein, we showed that PAX5/TEL is responsible for the progressive down-regulation of B220 until the main population became negative.The effect on CD19 antigen is more important.In normal B lymphocytes, CD19 is expressed from pre-BI cell stage to mature B cells (12,40), as well as in all BCP ALLs (44,47).Its expression was strongly down-regulated on PAX5/TEL cell surface.Interestingly, patients carrying either the t(9;12) or the dic(9;12) translocations (2,5,6), as well as patients with PAX5 hemizygous loss (2), displayed a phenotype positive for this marker.These evidences are not in contrast with our results because in pre-BI cells, PAX5/TEL should compete with the expression of the two endogenous PAX5 alleles, causing the down-regulation of CD19 but not the complete block of its expression.In analogy with CD19 expression, PAX5/TEL affected the expression of other B-cell genes physiologically controlled by PAX5, such as MB-1 (15) and BLNK (16); and in fact, both genes were significantly repressed in PAX5/ TEL cells compared with MIGR-GFP cells.Moreover, FLT3 gene expression, physiologically repressed by PAX5, is even more repressed in the presence of PAX5/TEL protein.The fusion protein is unable to switch-on MCSFR expression.This finding suggested that the fusion protein seems to function as a dominant repressor of transcription in many PAX5-target genes.Recently, it was proposed that a fusion protein consistent in PAX5 fused with the elastin (ELN) gene was able to down-regulate CD19 mRNA in a luciferase reporter gene system by a mechanism of competition with endogenous PAX5 (42).In contrast, no mRNA downregulation effect was detected in a Burkitt's lymphoma cell line transfected with PAX5/ELN.It could be argued that the competition mechanism was not sufficient to block endogenous PAX5 action, but a reinforcing role of the repressive domains of the TEL protein is required to block the transcription process.
The contemporary down-modulation of CD19 protein, the complete block of B220 antigen, and the down-regulation of BLNK and MB-1 gene expression by PAX5/TEL could indicate a reversion of pre-BI cells into a previous B-cell differentiation stage, e.g., pro-B cells (16).Indeed, this issue is further supported by the results obtained in PAX5-deficient cells, which are CD19-negative cells, blocked in their differentiation pathway.In this context, CD19 expression was not restored by PAX5/TEL protein, neither at the mRNA nor at the protein level.These features suggest that PAX5/ TEL is not able to supply the PAX5 function, thus confirming that PAX5/TEL protein acts as a repressor on CD19 gene and its function is not context-dependent (13,48).
Our results in wild-type pre-BI cells and in PAX5 À/À cells reinforce the issue that PAX5/TEL is an aberrant transcription factor with dominant-negative functions on PAX5, and consequently, on B-cell differentiation, in agreement with the precursor-B phenotype of leukemia patients.Although in the presence of PAX5, the endogenous PAX5 and PAX5/TEL can compete, in the absence of PAX5, the fusion protein cannot activate PAX5 targets.
We further investigated the migratory abilities of wild-type pre-BI cells towards CXCL12.The migration advantage of PAX5/TEL cells compared with control cells, in response to the CXCL12 gradient, suggests the bone marrow as well as various CXCL12secreting organs as a home for these cells, where they find a microenvironment favorable to tumor growth and survival (49,50).Moreover, the up-regulation of CXCR4 and the enhanced migration in PAX5/TEL cells can be interpreted in two manners: first, that PAX5/TEL cells acquired features of more immature cells, which are more sensitive to CXCL12 stimulation (38).Second, in agreement with the review by Burger and Burkle (50), it can indicate that PAX5/TEL cells, such as leukemia cells, are able to access niches that are normally restricted to progenitor cells, and thereby reside in a microenvironment that favors their growth and survival; however, this finding has to be proven in vivo.
We then focused our attention to cytokine growth independence as suggestive of resistance to apoptotic stimuli and transformed activity.In starvation assays using IL-7, we found that PAX5/TEL confers a survival advantage to apoptosis in short-term culture but it does not render pre-BI cells independent by IL-7 in long-term culture.This finding is not surprising because several examples of fusion genes are described involving TEL with a transcription factor as a partner, as not sufficient to overcome cytokine dependence on growth, whereas it is possible when the TEL partner is a kinase (29).
During the IL-7 starvation experiment, it was possible to appreciate a significant level of A heavy chain expression in control cells compared with the very low level in PAX5/TEL cells.A reduced IgM expression was also shown in murine plasmacytoma 558LAM cell line by Mullighan et al. (2) when PAX5/TEL was expressed together with the wild-type PAX5.
As reviewed by Dong and Blobe (41), many different types of hematological malignancies develop resistance to the homeostatic effects of TGFh1, an antiproliferative and proapoptotic cytokine.Interestingly, PAX5/TEL showed a remarkable resistance to TGFh1, continuing to actively proliferate in the presence of the cytokine, although to a lower extent than without TGFh1.It is possible to conclude that PAX5/TEL pre-BI cells are resistant to TGFh1 antiproliferative and proapoptotic effects, although the specific mechanism of PAX5/TEL disruption of TGFh1 signaling pathway remains to be investigated.
Finally, we showed that PAX5/TEL protein recruits mSin3A, has a dominant effect on wild-type PAX5, down-regulating B220, CD19, BLNK, MB-1, and FLT3 expressions and not activating MCSFR , interferes with the process of B-cell differentiation and causes the failure to express A heavy chain.Thus, we can speculate a potential block in B-cell development by PAX5/TEL, the importance of which has to be investigated in vivo.
In a PAX5-deficient contest, the presence of PAX5/TEL does not replace PAX5 functions.Moreover, the presence of PAX5/TEL protein determines an advantage in migration to CXCL12 with the overexpression of CXCR4, and it allows the cells to resist apoptotic stimuli.
In conclusion, this is the first report formally demonstrating the functional role of PAX5/TEL protein in vitro, as an aberrant transcription factor with repressor function.PAX5/TEL potentially blocks the B-cell differentiation process, enhances migratory ability, and induces increased survival.Taken together, these phenomena likely represent key events in the process of B-cell transformation.

Figure 3 .
Figure3.Analysis of PAX5 À/À pre-BI cells transduced by MIGR-PAX5/TEL or MIGR-GFP vectors.A, RT-PCR analysis did not reveal any CD19 expression.HPRT was tested as a constitutive gene.B, CD19 protein is absent in both MIGR-GFP and MIGR-PAX5/TEL cells; day indicated refers to the sorting day, and all analyses refer to gated GFP-positive cells.

Figure 4 .
Figure 4. Migration of wild-type pre-BI cells towards the CXCL12.A, absolute number of cells which migrated in the absence or presence of CXCL12.B, RQ-PCR analysis of CXCR4 expression; the transcript level of the gene is normalized for HPRT constitutive gene expression.t test: *, P < 0.05; **, P < 0.01; 2 ÀDDCt = the RQ-PCR data are reported as a difference between DCt of PAX5/TEL and MIGR-GFP cells, where DCt is defined as the difference between the cycle threshold (Ct) of the target CXCR4 gene and the HPRT constitutive gene.

Figure 5 .
Figure 5. Culture of wild-type pre-BI cells in the absence of IL-7.A, number of surviving cells in culture evaluated as fold increase of all GFP-positive cells.B, left, IgM expression evaluated as cytoplasmic A heavy chain expression; analysis every 24 h and percentages of double-positive A/CD19 cells are indicated.Right, PAX5/ TEL cells presented a lower cytoplasmic A heavy chain expression at 96 h.