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- Results and discussion
We investigated PAX5 expression in childhood B-lineage acute lymphoblastic leukaemia (ALL). Seven of 21 children with B-lineage ALL had multiple PAX5 variants, while 14 children and healthy controls showed full-length (FL) and one variant PAX5. By Western blotting, healthy controls displayed Pax5-FL, while one short Pax5, derived from the deletion of exon 8 (Pax5-ΔE8) was produced in 90% of ALL samples, as well as in ALL cell lines. PAX5-ΔE8 lacked more than 50% of the transactivation domain, indicating that aberrant Pax5 production might lead to the arrest of B-cell differentiation, contributing to the pathogenesis of B-lineage ALL.
B-lineage acute lymphoblastic leukaemia (ALL), which originates from an early B-cell progenitor, is a common subtype of acute leukaemia in children. The transcription factor Pax5 regulates the expression of B-cell-specific genes, including BLNK, CD19, LEF-1, BLK and MB-1 of the immunoglobulin genes, and plays a central role in B-cell development and differentiation (Schebesta et al, 2002). PAX5 is also important for maintaining the identity and function of mature B cells in late B lymphopoiesis (Horcher et al, 2001). In the analysis of various leukaemia subtypes, Pax5 was highly expressed in B-lineage ALL and acute myeloid leukaemia with t(8;21), but not detected in T-lineage ALL (Tiacci et al, 2004). Recently, it has been reported that aberrant PAX5 expression is implicated in the development of a certain type of B-cell lymphomas (Oppezzo et al, 2005). These observations suggest that the alteration of PAX5 function might contribute to leukaemogenesis of B-lineage ALL by aberrantly regulating the differentiation of early B-cell progenitors.
Results and discussion
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- Results and discussion
The expression of PAX5 mRNA was analysed in leukaemic cells from 21 children with B-lineage ALL, including 15 children with CD10− CD19+ ALL (aged 0–12 months, median; 4 months) and six children with CD10+ CD19+ ALL (aged 5 months–8 years, median 3 years). As shown in Fig 1A, seven (33%) of 21 B-lineage ALL samples displayed multiple amplified fragments. Sequencing analysis confirmed that one of these fragments corresponded to the full-length (FL) human PAX5 mRNA (Pax5-FL) and seven fragments to spliced variants with deletions of exon 7 (ΔE7), exon 8 (ΔE8), exons 7 and 8 (ΔE7/8), exons 8 and 9 (ΔE8/9), exons 6, 7 and 8 (ΔE6/7/8), exons 7, 8 and 9 (ΔE7/8/9) and exons 6, 7, 8 and 9 (ΔE6/7/8/9). The remaining 14 samples and all 10 normal controls showed only PAX5-FL and PAX5-ΔE8. No PAX5 expression was detected in other cell types such as T-lineage ALL or myeloid leukaemia (data not shown). The age of ALL patients with multiple variants of PAX5 was younger than that of those without PAX5 variants [10 months (5 months–1 year and 5 months) vs. 2 years (0 month–8 years)], but no significant difference was observed.
Figure 1. (A) PAX5 mRNA expression in representative B-lineage acute lymphoblastic leukaemia (ALL) samples. Multiple PAX5 mRNA variants were produced in seven children with B-lineage ALL (lanes 6–12), whereas only full-length (FL) and one variant were observed in others (lanes 1–5) and normal control (N). By sequencing analysis, these variants include deletion of exon 7 (ΔE7), ΔE8, ΔE7/8, ΔE8/9, ΔE6/7/8, ΔE7/8/9 and ΔE6/7/8/9. (B) Production of Pax5 protein in B-lineage ALL samples. Only Pax5-ΔE8 (46 kDa) was produced in all children with B-lineage ALL (lanes 1–18) except three having Pax5-FL (50 kDa) (lanes 19–21). The 50 and 46 kDa bands were co-migrated with transfected PAX5-FL and PAX5-ΔE8, respectively. N, normal control. (C) Production of BLNK protein in representative B-lineage ALL samples. The production of BLNK protein was suppressed in most of the B-lineage ALL samples with multiple PAX5 variants (lanes 8–12), whereas clear BLNK production was observed in two with PAX5 variants (lanes 6–7), as well as in those with FL and one variant PAX5 (lanes 1–5). The sample in each lane corresponds to that in the lane of (A). N, normal control.
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To elucidate the translation of these PAX5 variants, we performed Western blotting. As shown in Fig 1B, healthy donors displayed a single band at 50 kDa, whereas all B-lineage ALL samples except three samples revealed one protein band at 46 kDa. The 50 and 46 kDa molecular bands were co-migrated with transfected PAX5-FL and PAX5-ΔE8, indicating that these 50 and 46 kDa molecular mass proteins correspond to the product of the PAX5-FL or PAX5-ΔE8 transcripts, respectively. The age and subtype of three ALL samples with Pax5-FL were 0, 1 and 6 months of age with CD10− CD19+ ALL. The production of Pax5-ΔE8 was also observed in all three B-lineage ALL cell lines, regardless of the different karyotypes (Fig 2A).
Figure 2. (A) Production of Pax5 protein in B-lineage acute lymphoblastic leukaemia (ALL) cell lines. Production of Pax5-ΔE8 was observed in all B-lineage ALL cell lines. Lane 1, KOCL-69 with t(4;11); lane 2, KOPN-36 with t(1;19); lane 3, KOPN-41 with t(12;21); N, normal control. (B) Production of Pax5 protein in B lymphocytes from different age groups. The dominant production of Pax5-ΔE8 was detected in neonates (lanes 1–2) and infants (lanes 3–4), whereas dominant Pax5-FL protein with no or faint Pax5-ΔE8 was observed in children (lanes 5–6), as well as in adults. The results are shown from representative samples of each age group. (C) Structure of Pax5-FL and Pax5-ΔE8 proteins. Pax5-ΔE8 lacks most of the transactivation domain. PD, paired domain; O, octapeptide; HD, homeo domain; TD, transactivation domain; ID, inhibition domain.
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We analysed the production of Pax5-FL and Pax5-ΔE8 proteins in B lymphocytes from three different age groups of children. The dominant production of Pax5-ΔE8 protein was detected in B lymphocytes from all neonates and infants, whereas dominant Pax5-FL protein with no or faint Pax5-ΔE8 was observed in children and adults (Fig 2B).
Pax5 regulates the BLNK gene, which endows B lymphocytes with the ability to respond to B-cell-specific signals, and this control is mediated by PAX5 binding to promoter region of BLNK (Schebesta et al, 2002). Hence, we examined BLNK expression and protein production in B-lineage ALL cells. Seven of 18 B-lineage ALL samples (39%) either lacked or weakly expressed BLNK (Fig 1C). In particular, all leukaemic cells with multiple PAX5 variants showed low or negative expression of BLNK.
Recently, Borson et al (2002) observed multiple PAX5 variants in patients with multiple myeloma. In contrast, most of the patients with B-cell lymphoma displayed a single predominant PAX5-FL, whereas only a few cases expressed the sole variant, PAX5-ΔE8 (Robichaud et al, 2004). B cells from chronic lymphocytic leukaemia displayed two major amplified fragments, PAX5-FL and PAX5-ΔE8 (Oppezzo et al, 2005). In our study, seven of 21 ALL samples revealed multiple PAX5 variants, while 14 patients and all healthy controls showed only PAX5-FL and one PAX5 variant. By Western blotting, all ALL samples except three showed only Pax5ΔE8 protein, compared with healthy controls having Pax5-FL. We also analysed the Pax5 protein in different age groups of children, because B-cell differentiation is characterised by a commitment event that requires expression of PAX5, and B lymphocytes are a more primitive lineage in neonates and infants than in adults (Sanz et al, 2003). The dominant production of Pax5-ΔE8 protein was detected in neonates and infants, whereas only Pax5-FL was observed in children and adults. These results indicate that Pax5-ΔE8 participates in the early stage of B-cell differentiation predominantly detected in very young children.
PAX5-ΔE8 lacks more than 50% of the sequence as a ‘minimal transactivation domain’ of PAX5, and remains in the inhibitory domain located in exons 9 and 10 (Dorfler & Busslinger, 1996). In contrast, transient co-transfection reporter assays demonstrated that PAX5-ΔE8 has transactivation properties nearly equal to those of PAX5-FL (Robichaud et al, 2004). In our study, low or negative expression of BLNK was not associated with Pax5-ΔE8 protein but with multiple PAX5 variants. Although the data concerning the expression and putative role of PAX5-ΔE8 are scarce, Pax5-ΔE8 protein might be potential to repress Pax5-regulated genes and disrupt normal cell development.
In conclusion, altered Pax5 expression might disrupt B-cell haematopoiesis because of the absence or reduction of Pax5-regulated genes and cause the arrest at the stage of early B-cell differentiation and an abnormal expansion of immature B cells in B-lineage ALL in children.