H., N. mutations. (3), (4), and gene as the most frequent target of somatic mutations in childhood and adult B-progenitor acute lymphoblastic leukemia (ALL), being altered in 38.9% Rabbit polyclonal to TP53INP1 and 34% of cases, respectively (7, 8), and these KL-1 findings further emphasized the essential role of PAX5 in the proper development of B cells. Somatic mutations consist of partial or complete hemizygous deletions, homozygous deletions, partial or complete amplifications, point mutations, or fusion genes (7). These aberrations in the gene are considered to impair PAX5 function and play a role in blocking B cell differentiation. PAX5 fusion proteins such as PAX5-TEL, PAX5-ENL, PAX5-PML, and PAX5-C20S have been shown previously to have dominant-negative effects on PAX5 transcriptional activity and have been suggested to be mainly responsible for KL-1 the differentiation disorder of ALL with these fusion genes (9,C12). Consistently, a previous study has reported that PAX5 haploinsufficiency cooperated with the constitutive activation of STAT5 to initiate ALL in mice (13). However, the oncogenicity of PAX5 mutations, including fusion genes, has yet to be demonstrated. PML is usually a potent growth suppressor and proapoptotic factor (14, 15). In normal cells, the PML protein is usually localized in discrete subnuclear compartments called KL-1 PML nuclear bodies (NBs) (16). In PML NBs, PML co-accumulates with more than 70 proteins that are involved in tumor suppression, apoptosis, regulation of gene expression, anti-viral responses, and DNA repair. KL-1 PML has been suggested to exert its effects by regulating the functions of binding partners at the core of PML NBs (17). PML NBs have been found previously to be disrupted in human acute promyelocytic leukemia (APL) by PML-RAR, an oncogenic fusion protein of PML and retinoic acid receptor (RAR) , which is considered to be the underlying mechanism responsible for the anti-apoptotic effects of PML-RAR (18,C20). Arsenic trioxide (ATO), a chemotherapeutic agent used clinically in the treatment of APL, reportedly induced the restoration of disrupted PML NBs and apoptosis in APL cells, resulting in prolonged remission of this disease (21,C24). These findings emphasize the importance of the integrity of PML NBs in tumor suppression. The fusion gene has been detected in two cases of B-progenitor ALL with chromosomal translocation t(9;15)(p13;q24) (25). We have exhibited previously that PAX5-PML dominant-negatively inhibited PAX5 transcriptional activity in a luciferase reporter assay and suppressed the expression of PAX5 transactivation targets when expressed in a B lymphoid cell line. Furthermore, we have shown that this expression of PAX5-PML in a non-hematological tumor cell line induced the disruption of PML NBs and resistance to apoptosis and that ATO treatment induced the reconstitution of PML NBs and abrogation of apoptosis resistance. These findings suggested the possible involvement of this fusion protein in the leukemogenesis of B-ALL in a dual dominant-negative manner and the potential of ATO therapy for this type of ALL (11). In this study, we exhibited the leukemogenicity of PAX5-PML by introducing it into normal mouse pro-B cells and showed selective BLNK repression among the transactivation targets of PAX5 in leukemia cells. We also showed that PML NBs were intact in leukemia cells, indicating that the disruption of PML NBs was not required for the PAX5-PML-induced onset of leukemia. These results provide novel insights into the molecular mechanisms underlying the onset of leukemia by PAX5 mutations. Experimental Procedures Antibodies and Reagents The anti-PML antibody (H-238); anti-PAX5 N antibody (N-19); anti-CD19 antibody (4G7), phycoerythrin-conjugated; and arsenic trioxide have been described previously (11). The KL-1 anti-CD43 antibody, phycoerythrin-conjugated; anti-B220 antibody, allophycocyanin-Cy7-conjugated; anti-IgM antibody, allophycocyanin-conjugated; and anti-human CD8 antibody, V450-conjugated were purchased from BD Biosciences, BioLegend (San Diego, CA), and Beckman Coulter (Miami, FL), respectively. The anti-mouse PML antibody for immunostaining was from LSbio (Seattle, WA). Plasmids PAX5-PML/pCDNA has been described previously (11). PAX5-PML/MigRI was constructed by subcloning PAX5-PML cDNA fragments into MigRI. MigRI is usually a bicistronic retroviral vector using GFP as.