The amplification at 13q31-q32 has been reported in not only hematopoietic malignancies but also in other solid tumors. We identified previously frequent amplification of chromosomal band 13q31-q32 in 70 cases of diffuse large B-cell lymphoma patients by conventional comparative genomic hybridization analysis. In an attempt to identify a candidate gene within this region, we used array comparative genomic hybridization and fluorescent in situ hybridization to map the 13q31-q32 amplicon. We then screened the 65 expressed sequence tags and Glypican 5 (GPC5) by reverse transcription-PCR and Northern blotting. As a result, we identified a novel gene, designated Chromosome 13 open reading frame 25 (C13orf25), which was overexpressed in B-cell lymphoma cell lines and diffuse large B-cell lymphoma patients with 13q31-q32 amplifications. However, GPC5, which has been reported to be a target gene for 13q31-q32 amplification, was truncated in one cell line, Rec1, possessing the amplification, and its expression in various cell lines with amplification at 13q31-q32 was not significantly different from that in other cell lines without amplification, suggesting that GPC5 is not likely to be the candidate gene. Additional analysis identified two major transcripts in the C13orf25 gene. The two transcripts A and B predicted open reading frames of 32 and 70-amino acid polypeptides, respectively. The former has been reported as bA121J7.2, which is conserved among species. Transcript-B also contained seven mature microRNAs in its untranslated region. These results suggest that the C13orf25 gene is the most likely candidate gene for the 13q31-q32 amplicon found in hematopoietic malignancies.

Mantle cell lymphoma (MCL) is characterized by 11q13 chromosomal translocation and CCND1 overexpression, but additional genomic changes are also important for lymphomagenesis. To identify the genomic aberrations of MCL at higher resolutions, we analysed 29 patient samples and seven cell lines using array-based comparative genomic hybridization (array CGH) consisting of 2348 artificial chromosome clones, which cover the whole genome at a 1.3 mega base resolution. The incidence of identified genomic aberrations was generally higher than that determined with chromosomal CGH. The most frequent imbalances detected by array CGH were gains of chromosomes 3q26 (48%), 7p21 (34%), 6p25 (24%), 8q24 (24%), 10p12 (21%) and 17q23 (17%), and losses of chromosomes 2p11 (83%), 11q22 (59%), 13q21 (55%), 1p21-p22 (52%), 13q34 (52%), 9q22 (45%), 17p13 (45%), 9p21 (41%), 9p24 (41%), 6q23-q24 (38%), 1p36 (31%), 8p23 (34%), 10p14 (31%), 19p13 (28%), 5q21 (21%), 22q12 (21%), 1q42 (17%) and 2q13 (17%). Our analyses also detected several novel recurrent regions of loss located at 1p36, 1q42.2-q43, 2p11.2, 2q13, 17p13.3 and 19p13.2-p13.3, as well as recurrent regions of homozygous loss such as 2p11 (Igj), 2q13 and 9p21.3-p24.1 (INK4a/ARF). Of the latter, we investigated the 2q13 loss, which led to identification of homozygous deletions of the proapoptotic gene BIM. The highresolution array CGH technology allowed for the precise identification of genomic aberrations and identification of BIM as a novel candidate tumor suppressor gene in MCL.

The constitutive activation of nuclear factor-B (NF-B) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-B pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in EpsteinBarr virus-infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-B activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-B pathways may be advantageous for treatment.

IntroductionMicroRNAs (miRNAs) are small (20-24 nt's), noncoding RNAs that function as key regulators of gene expression. By pairing with the transcripts of protein-coding genes, they mediate cleavage of the targeted mRNAs or repression of their productive translation. [1][2][3] Notably, miRNAs exhibit dynamic temporal and spatial expression patterns, disruption of which may be associated with tumorigenesis. [4][5][6][7] The C13orf25/miR-17-92 cluster encodes 6 miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92). The miR-17-92 cluster is known to be overexpressed in a variety of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, follicular lymphoma, and mantle cell lymphoma, 5,[8][9][10][11][12][13] all of which arise through various primary and additional genetic changes, including genomic translocations, losses, and amplifications. [8][9][10][11][12][13] Recurrent overexpression of miR-17-92 in B-cell lymphomas suggests the polycistron possesses tumorigenic potential. Consistent with that idea, He et al demonstrated that forced expression of the miR-17-19b-1 (a miR-17-92 variant) in the Eu-myc transgenic mouse model of B-cell lymphoma accelerated disease onset and progression. 5 This suggests dysregulation of the miR-17-92 polycistron contributes to lymphomagenesis by repressing tumor suppressor gene(s). The most likely target of miR-17-92 in lymphomagenesis is the proapoptotic protein Bim, 14 which is known to be a tumor suppressor. 15 Recently, Xiao et al generated a mouse line that selectively overexpressed miR-17-92 in their lymphocytes. 16 As a result, these mice developed lymphoproliferative disease and died prematurely. Following activation, B and T cells from these transgenic mice showed increased proliferation and survival as a result of dysregulation of the proapoptotic gene Bcl2l11/Bim. In addition, Ventura et al established a strain of miR-17-92 knockout mice, 17 which enabled the researchers to demonstrate that the miR-17-92 cluster is essential for the survival signal that mediates progression from the pro-B-to the pre-B-cell stage. The absence of miR-17-92 led to elevated Bim protein levels and inhibition of B-cell development. Finally, Koralov et al ablated Dicer in early B-cell progenitors by conditionally deleting dcr-1 in the B-cell lineage of mice. The resulting phenotype was similar to that seen with miR-17-92 deletion, which was characterized by a developmental block at the transition from the pro-B-to the pre-B-cell stage. The researchers also found that cells lacking Dicer showed increased Bim transcription and Bim protein production. 18 All of these reports suggest that down-regulation of Bim mRNA and protein induced by miR-17-92 overexpression could contribute lymphomagenesis via antiapoptotic activity. Nonetheless, Bim is an unlikely target of miR-17-92 in some aggressive B-cell lymphomas, despite overexpression of the polycistron. For instance, Jeko-1 cells, which are derived from an aggressive B-cell lymphoma, exhibit both homozygous...

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