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- MATERIALS AND METHODS
The PAX5 gene encodes the BSAP (B-cell-specific activator protein) which is a key regulator of B-cell development and differentiation. A recurring translocation t(9;14)(p13;q32) in non-Hodgkin's lymphoma moves the PAX5 on 9p13 within close proximity of the immunoglobulin heavy chain gene (IGH). KIS-1 cell line was established from a patient with diffuse large cell lymphoma of B-cell type carrying t(9;14). We analysed PAX5/BSAP expression by Northern and Western blotting in a panel of haematological tumour cell lines with other chromosome abnormalities in comparison with that of KIS-1. PAX5 mRNA and BSAP expression were detected in all B-cell lines tested, and the high level in KIS-1 was confirmed. However, a diffuse large B-cell lymphoma cell line and an acute B-lymphoid/myeloid leukaemia cell line expressed the PAX5/BSAP at levels comparable with KIS-1. PAX5 transcripts were readily detectable in clinical materials with a wide variety of B-cell neoplasms by reverse transcriptase-mediated polymerase chain reaction (PCR). Thus, PAX5/BSAP activation in haematological tumour cells is not necessarily associated with t(9;14). Although binding sites for BSAP have been identified in the promoters of CD19, this study failed to find clear correlation between the level of PAX5/BSAP expression and that of CD19. In contrast to KIS-1 in which the Eμ enhancer of IGH was juxtaposed to PAX5, cloning of t(9;14) from another case by long-distance PCR revealed that the PAX5 promoter was linked to a Cγ constant region in divergent orientation, suggesting that the mechanism of PAX5 activation through recombination with IGH varies among individual cases. Breakpoints on 9p13 of the two translocations were clustered upstream of PAX5, leaving the PAX5 coding region intact.
Non-Hodgkin's lymphoma of B-cell type (B-NHL) represents multiple diseases with diverse morphological and clinical characteristics. The vast majority of cases with B-NHL exhibit chromosomal abnormalities including many types of reciprocal translocations. Some of the recurring translocations are closely correlated with specific histopathological phenotypes ( Yunis et al, 1984 ), and molecular genetic techniques have demonstrated that oncogenes which are located at the breakpoints of the translocations play an integral role in the development of particular subtypes of B-NHL ( Rabbitts, 1994). t(8;14)(q24;q32) in Burkitt's lymphoma/leukaemia, t(11;14)(q13;q32) in mantle cell lymphoma and t(14;18)(q32;q21) in follicular lymphoma, involving the c-MYC, BCL1/PRAD1 and BCL2 oncogenes, respectively, are well-characterized examples of translocations observed in B-NHL; these translocations consistently result in the juxtaposition of oncogenes to the immunoglobulin heavy chain gene (IGH). In contrast, the BCL6 gene is rearranged in a number of translocations involving chromosomal band 3q27, and there is no clear consistent association of the 3q27 translocation with a specific subtype of B-NHL ( Ohno & Fukuhara, 1997).
Close association of another recurring translocation, t(9;14)(p13;q32), with small lymphocytic lymphoma with plasmacytoid differentiation was demonstrated in a large series of karyotypically analysed NHL ( Offit et al, 1992 ). The lymphoma cells are composed of small lymphoid cells and show maturation to plasma cells which express immunoglobulins in the cytoplasm as well as on the cell surface. Although t(9;14) was first reported in the CD30-positive diffuse large cell lymphoma cell line KIS-1 ( Kamesaki et al, 1988 ; Ohno et al, 1990 ), a small percentage of the cells expressed cytoplasmic λ immunoglobulin and the same type of immunoglobulin was detected in the culture medium ( Kamesaki et al, 1988 ), suggesting plasmacytoid features of KIS-1. Thus, this potential subtype of B-NHL shows an indolent clinical presentation followed by progression to large cell lymphoma ( Iida et al, 1996 ; Offit et al, 1992 ).
Initial molecular analysis of t(9;14) suggested the presence of a gene on 9p13 ( Ohno et al, 1990 ), and the PAX5 gene has recently been identified as the responsible gene by two independent groups ( Busslinger et al, 1996 ; Iida et al, 1996 ). The PAX gene family are nuclear transcription factors which share a highly homologous region known as the paired box domain ( Adams et al, 1992 ). The PAX5 gene encodes the BSAP (B-cell-specific activator protein) which is a key regulator of B-cell development and differentiation ( Busslinger & Urbánek, 1995; Neurath et al, 1995 ). KIS-1 cells carrying t(9;14) show rearrangement of PAX5 with the IGH gene, and express abundant PAX5 messages ( Busslinger et al, 1996 ; Iida et al, 1996 ). In this study we analysed expression of PAX5/BSAP and its potential target genes in a panel of haematological tumour cells as compared with KIS-1, and cloned a t(9;14) junctional area of another case to elucidate the general mechanism of PAX5 activation through chromosomal translocation.
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- MATERIALS AND METHODS
Chromosomal translocations in B-NHL result in juxtaposition of cellular oncogenes to the immunoglobulin genes (IGs) or equivalent loci. In general, coding regions of the oncogenes are not interrupted by the translocation. Therefore the overall consequences of the translocation appear to be deregulated expression of the gene product ( Rabbitts, 1994); the gene may be expressed at inappropriate levels compared with normal lymphocytes of the equivalent stage of B-cell differentiation and/or at inappropriate times when unaffected genes are usually switched off. However, the gene products have been found normally in lymphoid tissues at considerable levels, and, more importantly, currently available methods have failed to demonstrate clear differences in the level of expression of a particular gene between lymphoma tissues carrying a translocation involving the relevant gene and those lacking the gene rearrangement ( Akagi et al, 1994 ; Pezzella et al, 1990 ). For instance, Bcl-6 protein is expressed in germinal centre B cells as well as in a variety of germinal-centre-derived lymphoma tissues independently of BCL6 gene rearrangement ( Onizuka et al, 1995 ).
The present study showed that the most recently identified t(9;14) shares these common features of translocations in B-NHL, i.e. (1) the translocation occurs upstream of the PAX5 pomoter region and therefore the translocated allele obviously encodes the wild-type BSAP protein, and (2) PAX5/BSAP activation in haematological tumour cells is not necessarily associated with t(9;14); we found no clear quantitative differences in the levels of PAX5/BSAP expression between haematological diseases with t(9;14) and those lacking the translocation. Thus, to elucidate the ultimate implications of t(9;14) for the pathogenesis of B-NHL, studies of alternative targets of BSAP may be more straightforward than those of the gene itself. CD19 is a key member of a B-cell surface signal transduction system, and its activation initiates multiple intracellular signal transduction cascades ( Tedder et al, 1994 ). The present study, however, failed to identify any clear correlations in haematological tumour cell lines between the level of PAX5/BSAP expression and that of CD19, which could lead to tumour development. Further analysis of these cell lines, including KIS-1 cells, should enable us to identify specific regulatory defects that can alter CD19 expression.
The variability in the chromosomal breakpoints on IGH makes it difficult to draw conclusions regarding the general mechanism of PAX5 activation through the fusion with IGH. Although the PAX5/Eμ fusion in KIS-1 cells has been emphasized, PAX5 in other t(9;14) including 895 presented here, was linked to IGH at a point that did not allow the Eμ to exert an effect on PAX5 ( Iida et al, 1996 ). Analogous molecular features were observed in the Burkitt's lymphoma Manca cell line, in which the c-MYC coding exons 2 and 3 are linked to Eμ in head-to-head orientation and the c-MYC on the translocated allele was expressed at markedly high levels ( Hayday et al, 1984 ). However, this characteristic molecular structure is exceptional; in the majority of Burkitt's lymphoma cases, the c-MYC gene is recombined with IGH within the switch regions resulting in deletion of Eμ at the junction and c-MYC expression is only moderately elevated ( Cory, 1986; Wiman et al, 1984 ). These observations suggest that the absolute level of oncogene expression may not be important in the development of B-NHL.
Within the haemopoietic system, BSAP expression is normally restricted to the B-cell lineage and occurs in B cells at all developmental stages from pro-B cells to mature B cells, but not in terminally differentiated plasma cells ( Busslinger & Urbánek, 1995; Neurath et al, 1995 ). PAX5-deficient mice failed to produce pre-B, B and plasma cells owing to a complete arrest of B-cell development at an early precursor stage ( Nutt et al, 1997 ; Urbánek et al, 1994 ). It is notable that the B-lymphoid/myeloid leukaemia cell line TA-1 showed significant levels of PAX5/BSAP expression which could be responsible for its CD19 expression. The TA-1 cells examined in the present study revealed that the cells were positive for cytoplasmic μ chain but lacked light chain expression, suggesting that the TA-1 were in pre-B-cell stage of B-cell differentiation. Thus, the B-lymphoid/myeloid bilineage phenotype in TA-1 cells may reflect peculiar features of leukaemic cells associated with malignant transformation. Alternatively, assuming that any leukaemic phenotype would represent a normal counterpart in the haemopoietic system, TA-1 cells could correspond to multipotent stem cells capable of differentiating along both myeloid and lymphoid pathways, and PAX5/BSAP could be expressed at more primitive stages of haemopoietic differentiation. An analogous myeloid/lymphoid phenotype has been observed in M2 acute myeloid leukaemia carrying t(8;21) translocation in which CD19 expression is a characteristic feature at the cell surface ( Kita et al, 1994 ). PAX5/BSAP expression in this particular type of acute leukaemia is currently under investigation in our laboratory.
Distinct disease entities within low-grade B-NHL have been recognized. Small lymphocytic lymphoma with plasmacytoid differentiation described by Offit et al (1992 ) corresponds to lymphoplasmacytoid immunocytoma of the Kiel classification ( Lennert & Feller, 1992), although the international lymphoma study group proposed that these tumours not be given a separate diagnostic category, but rather be regarded as a variant of B-cell chronic lymphocytic leukaemia ( Harris et al, 1994 ). However, it is evident that there are cases which are indistinguishable from other B-cell tumours with features of plasmacytes, e.g. lymphoplasmacytic type of immunocytoma ( Lennert & Feller, 1992) and lymphoplasmacytoid lymphoma/immunocytoma ( Harris et al, 1994 ). Thus, t(9;14) could be a molecular marker to discriminate this particular type of lymphoma within the generic group of low-grade B-NHL. Since adequate metaphases are sometimes difficult to obtain in low-grade lymphoma, and since distant breakpoints on PAX5 locus were reported ( Pellet et al, 1989 ), interphase fluorescence in situ chromosomal hybridization is the most comprehensive method to detect this translocation ( Iida et al, 1996 ). The LD-PCR method used here has many advantages for the rapid and sensitive detection of t(9;14) in which breakage occurs within the upstream promoter region. Studies of large numbers of clinical materials using these methods should further clarify the clinical features of B-NHL carrying t(9;14)(p13;q32).