Tohru Miki

BCL6 overexpression prevents increase in reactive oxygen species and inhibits apoptosis induced by chemotherapeutic reagents in B-cell lymphoma cells.

Chromosomal translocations and somatic mutations occurring in the 5′ noncoding region of the BCL6 gene, encoding a transcriptional repressor, are most frequent genetic abnormalities associated with non-Hodgkin B-cell lymphoma and result in deregulated expression of BCL6. However, the significance of deregulated expression of BCL6 in lymphomagenesis and its effect on clinical outcomes of lymphoma patients have remained elusive. In the present study, we established Daudi and Raji B-cell lymphoma cell lines that overexpress BCL6 or its mutant, BCL6-Ala333/343, in which serine residues required for degradation through the proteasome pathway in B-cell receptor-stimulated cells are mutated. BCL6 overexpression did not have any significant effect on cell proliferation, but significantly inhibited apoptosis caused by etoposide, which induced a proteasome-dependent degradation of BCL6. BCL6-Ala333/343 was not degraded after etoposide treatment and strongly inhibited apoptosis. In these lymphoma cell lines, etoposide increased the generation of reactive oxygen species (ROS) and reduced mitochondria membrane potential, both of which were inhibited by the antioxidant N-acetyl-L-cysteine (NAC). NAC also inhibited apoptosis. Furthermore, BCL6 overexpression was found to inhibit the increase in ROS levels and apoptosis in response to etoposide and other chemotherapeutic reagents. These results raise the possibility that deregulated expression of BCL6 may endow lymphoma cells with resistance to chemotherapeutic reagents, most likely by enhancing the antioxidant defense systems.

The role of Bcl6 in mature cardiac myocytes

OBJECTIVE The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle. The objective of this study was to examine the role of Bcl6 in cardiac myocytes. METHOD We developed Bcl6-deficient (Bcl6-/-) mice and histologically examined hearts from these mice. RESULTS Massive myocarditis with eosinophilic infiltration occurred in Bcl6-/- mice after 4-6 weeks of age. Since expression of the Bcl6 gene was induced in normal cardiac myocytes after 2 weeks of age and thereafter detected through adulthood, loss of Bcl6 in mature cardiac myocytes may be related to the induction of eosinophilic myocarditis. To examine the effects of eosinophils from Bcl6-/- mice on normal hearts, bone marrow cells from Bcl6-/- mice were adoptively transferred into sublethally irradiated RAG1-deficient mice. Although massive eosinophilic infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Electron microscopic analysis of cardiac myocytes from Bcl6-/- mice revealed a spectrum of degenerative changes prior to eosinophilic infiltration. CONCLUSION Bcl6 maynot be essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation.

The proto-oncogene Bcl6 inhibits apoptotic cell death in differentiation-induced mouse myogenic cells

The Bcl6 gene is located at chromosomal band 3q27, a breakpoint for translocation that frequently occurs in B cell lymphomas. Bcl6 has been found to be preferentially expressed in germinal center B cells, and expression of this gene has been shown to be essential for germinal center formation in vivo. The physiological function of Bcl6 and its role in lymphomagenesis, however, are not yet known. Since significant expression of Bcl6 has been demonstrated in skeletal muscle, we have utilized a differentiation-inducible mouse myogenic cell line, C2C12, to elucidate the function of the Bcl6 gene product. Expression of Bcl6 mRNA was very low in growing myocytes, but was increased in differentiating myocytes cultured in serum-starved medium. Incubation of these cells with cytokines or chemicals that are known to block differentiation suppressed this increased Bcl6 message abundance, indicating that Bcl6 induction is related to the process of terminal differentiation in muscle cells. While a fraction of myocytes is known to undergo apoptosis after serum-starvation to induce differentiation, adenovirus-mediated overexpression of Bcl6 enhanced the viability of the differentiating cells by preventing the apoptosis. High levels of Bcl6 antisense mRNA expression induced substantial apoptosis during the differentiation of C2C12 cells, but this was effectively prevented by infection with adenovirus that expressed Bcl6 sense mRNA. These results indicate that Bcl6 acts to prevent apoptotic cell death in differentiating myocytes. The deregulation of expression of this anti-apoptotic gene may also contribute to the development of B cell lymphomas.

Identification of negative regulatory regions within the first exon and intron of the BCL6 gene.

Chromosomal translocations involving BCL6 gene are frequent in human B-cell lymphomas. Chromosomal breaks preferentially occur within a 3-kb region containing the first exon and intron. Recent reports have revealed that internal deletions or point mutations also are common in this region, suggesting that structural alteration of this region may be a crucial event in the development of lymphomas. In this study, we identified two regions in the BCL6 gene that negatively regulate BCL6 expression. One region, ES, is located within the first exon between nucleotides +472 and +543, and a second region, IS, is located between +783 and +918 of the first intron. A consensus nucleotide sequence for the binding of the BCL6 protein itself was found within the ES region. An electrophoretic mobility shift assay and a co-transfection experiment using a BCL6 expression vector showed that transcription of the BCL6 gene was negatively regulated by the BCL6 gene product. The IS region which is included in the regions commonly deleted in B-cell lymphomas had a silencer activity. Structural alterations of these two regions may play roles in the deregulated expression of the BCL6 gene in B-cell lymphomas.

p38 MAP kinase plays a role in G2 checkpoint activation and inhibits apoptosis of human B cell lymphoma cells treated with etoposide.

Abstractp38 MAPK is mainly activated by stress stimuli and mediates signals that regulate various cellular responses, including cell-cycle progression and apoptosis, depending on cell types and stimuli. Here we examine the role of p38 in regulation of apoptosis and cell cycle checkpoint in Daudi B-cell lymphoma cells treated with the topoisomerase II inhibitor etoposide. Etoposide activated p38, inhibited the G2/M transition with the persistent inhibitory phosphorylation of Cdc2 on Tyr15, and caused apoptosis of Daudi cells. Inducible expression of a dominant negative p38α mutant in Daudi cells reduced the inhibition of Cdc2 as well as G2/M arrest and augmented apoptosis induced by etoposide. SB203580, a specific inhibitor of p38α and p38β, similarly reduced the inhibitory phosphorylation of Cdc2 as well as G2/M arrest and augmented apoptosis of Daudi cells treated with etoposide. These results suggest that p38 plays a role in G2/M checkpoint activation through induction of the persistent inhibitory phosphorylation of Cdc2 and, thereby, inhibits apoptosis of Daudi cells treated with etoposide. The present study, thus, raises the possibility that p38 may represent a new target for sensitization of lymphoma cells to DNA-damaging chemotherapeutic agents.

A variant-type MLL/SEPT9 fusion transcript in adult de novo acute monocytic leukemia (M5b) with t(11;17)(q23;q25)

As a result of recurrent chromosomal translocations in acute leukemias, the mixed-lineage-leukemia (MLL) gene fuses with a variety of partner genes, which include several members of the septin gene family. SEPT9 is a very rare but recurrent fusion partner of MLL, and has recently been implicated in the oncogenesis of various malignancies. Herein, we report a case of de novo acute monocytic leukemia (M5b) with t(11;17)(q23;q25). MLL involvement was revealed by fluorescent in situ hybridization (FISH) analysis, and an MLL/SEP9 fusion transcript was detected by RT–PCR. Sequencing analysis further showed that, in contrast to originally reported cases, MLL exon 8 was fused not with SEPT9 exon 3 but with exon 2, which codes for the unique N-terminal region of the SEPT9_v1 isoform, the region implicated in the regulation of gene expression and cell proliferation. We did not detect any mutation of FLT3, which was expressed at a relatively low level in the leukemic cells. Relapsing after a very short complete remission, the leukemia progressed rapidly and became fatal in spite of intensive therapies including hematopoietic stem cell transplantation. It is thus suggested that, in common with the original MLL/SEPT9 cases, monocytic differentiation and a poor prognosis may also be associated with acute myeloid leukemia with the variant MLL/SEPT9 fusion transcript.

T-cell prolymphocytic leukemia with der(11)t(1;11)(q21;q23) and ATM deficiency

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell malignancy and is similar to a mature T-cell leukemia seen in some patients with ataxia telangiectasia, which is a recessive hereditary chromosomal instability syndrome caused by mutations of the ataxia telangiectasia mutated (ATM) gene located on 11q23. Intriguingly, recent studies have strongly implicated ATM in the pathogenesis of T-PLL as a tumor suppressor gene, because biallelic inactivation of ATM is frequently observed in this disease; however, translocations involving 11q23 have rarely been reported in T-PLL. We report here a case of T-PLL with der(11)t(1;11)(q21;q23). Southern blot analysis did not reveal any abnormality of ATM, nor of MLL, which is also located on 11q23 and is involved in t(1;11)(q21;q23) in acute myelomonocytic leukemia. Northern blot analysis further showed that the ATM transcript of normal size is expressed in the leukemic cells at a level higher than that in normal peripheral blood lymphocytes. Western blot analysis, however, revealed that expression of ATM in the leukemic cells is much lower than that in normal lymphocytes. These results imply that translation of the ATM transcript is impaired or that the ATM protein is highly unstable in the leukemic cells, thus suggesting the presence of nucleotide changes in both alleles.

REARRANGEMENTS OF THE BCL6 GENE AND CHROMOSOME ABERRATIONS AFFECTING 3Q27 IN 54 PATIENTS WITH NON-HODGKIN'S LYMPHOMA

Chromosome aberrations affecting 3q27 are among the most frequent non-random abnormalities in non-Hodgkins lymphomas (NHL), especially the diffuse, large cell type. Recently, an association between BCL6 rearrangement and frequent extranodal lesions, rare bone marrow infiltration and a favorable clinical outcome was reported. We performed molecular studies of the BCL6 gene in 54 patients with NHL. Twelve patients (22%) with rearranged BCL6 genes were selected for histological, clinical, molecular, and cytogenetic studies. Ten of these cases were diffuse, large cell type lymphoma, one a follicular lymphoma, and one a mantle cell lymphoma (MCL). All cases were of the B-cell type and this is the first time a rearranged BCL6 gene has been found in an MCL. Cytogenetic data for 10 cases were available and the partner sites of the 3q27 translocation were determined in 7 of 10 patients. These locations were variable, including 6p21.3, 9p22, and 14q11 in addition to the immunoglobulin loci 14q32 (IGH), 2p12 (IGK), and 22q11 (IGL). The heterogeneity in partner sites is distinct from other lymphoma subgroups and may suggest that the genetic events are not uniform among patients with BCL6 rearrangements.

Interphase Detection of BCL6/IgH Fusion Gene in Non-Hodgkin Lymphoma by Fluorescence In Situ Hybridization

We characterized a t(3;14)(q27;q32) translocation in nine patients with B-cell, non-Hodgkin lymphoma (B-NHL) by fluorescence in situ hybridization (FISH). Fluorescence in situ hybridization with immunoglobulin heavy chain (IgH) and BCL6 gene probes detected t(3;14) rapidly and accurately, including complex t(3;14) in three patients; one with t(3;12;8;14)(q27;p13;q24.1;q32) and two with t(3;?;14)(q27;?;q32). Among these nine patients, seven escaped from cytogenetic detection by our G-banding analysis. Double-color FISH with IgH (Y6) and BCL6 (cosB5-1) showed fusion of BCL6 and IgH genes on der(3)t(3;14) in all nine patients, suggesting that der(3) may play a critical role in the development of lymphoma carrying complex as well as standard t(3;14) translocations. BCL6/IgH fusion gene was also demonstrated in interphase nuclei at a frequency of 23% to 91.5% over the cut-off value in control studies (9.0 +/- 2.76%). The breakpoints assessed by FISH with two cosmid clones containing BCL6 probes, cosB5-1 and cosB5-2, were within the cluster region in seven patients including one with complex type, but were not evaluated in two patients with t(3;?;14), because of the loss of partner chromosome. Using double-color FISH with these two BCL6-specific probes, none of an additional 32 patients in whom mitotic spreads were available showed 3q27 translocations. Fluorescence in situ hybridization with IgH and BCL6 gene probes is a rapid and sensitive method to detect t(3;14) in routine cytogenetic studies.

Clonal identification of Burkitt's lymphoma arising from lymphocyte-predominant Hodgkin's disease

The occurrence of large cell lymphomas subsequent to, or concurrent with, lymphocyte predominant Hodgkins disease (LPHD) is a well‐documented phenomenon. We present a case of Burkitts lymphoma of the bladder, occurring after the successful treatment of LPHD of a cervical lymph node. To evaluate the clonal relationship of the two tumours, we amplified the complementarity‐determining‐region 3 of two samples from paraffin‐embedded slides, using the polymerase chain reaction (PCR). The sequences of the PCR products showed 96% homology to each other. These results indicate that the malignant clone of Burkitts lymphoma arose from the corresponding LPHD.