Yuichi Nakamura

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

Expression of the NUP98/HOXA9 fusion transcript in the blast crisis of Philadelphia chromosome‐positive chronic myelogenous leukaemia with t(7;11)(p15;p15)

The t(7;11)(p15;p15) translocation is a recurrent aberration observed in acute myeloblastic leukaemia (AML) and chronic myelogenous leukaemia (CML). It has been shown that the NUP98 gene at 11p15 is fused with the HOXA9 gene at 7p15 in AML with t(7;11). We report the first case with CML expressing the NUP98/HOXA9 fusion transcript. A 27‐year‐old Japanese man was initially diagnosed as in the chronic phase of Philadelphia‐positive CML. At the diagnosis of myeloid blast crisis, the karyotype evolved to 46, XY, t(7;11)(p15;p15), t(9;22)(q34;q11). Reverse transcriptase polymerase chain reaction identified the NUP98/HOXA9 transcript, suggesting that the NUP98/HOXA9 fusion protein could play a critical role in the progression to blast crisis.

Functional regulation of TEL by p38-induced phosphorylation

TEL is a nuclear phosphoprotein that belongs to a member of the ETS family transcription factors. TEL acts as a tumor suppressor and is essential for establishing hematopoiesis in neonatal bone marrow. Because TEL possesses multiple putative mitogen-activated protein (MAP) kinase phosphorylation sites, we here investigated functional regulation of TEL via stress signaling pathways. We showed that TEL becomes phosphorylated in vivo by activated p38 but not by JNK1. The constitutive and inducible phosphorylation sites were found to be Ser(22) and Ser(257), respectively. TEL bound to p38 and was directly phosphorylated in vitro by p38. In vivo p38-dependent phosphorylation reduced trans-repressional abilities of TEL through ETS-binding consensus site. These data indicate that TELs functions are potentially regulated by p38 which is activated by various kinds of stresses. TEL could be a constituent downstream of the specific MAP kinase in the signal transduction system.

Leukemia-related transcription factor TEL accelerates differentiation of Friend erythroleukemia cells.

TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific δ-aminolevulinate synthase and β-globin mRNAs. TEL bound to a corepressor mSin3A through the helix–loop–helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.

Serum levels of Thl/Th2 cytokines, angiogenic growth factors, and other prognostic factors in young adult patients with hemophagocytic syndrome.

Serum levels of T helper 1 (Th1)/T helper 2 (Th2) cytokines, angiogenic growth factors, and other prognostic factors were measured in 5 young adult patients with virus-associated hemophagocytic syndrome (HPS). Levels of 2 Th1 cytokines (interleukin [IL]-18 and tumor necrosis factor-alpha), 2 Th2 cytokines (IL-10 and IL-6), and 2 angiogenic growth factors (soluble intercellular adhesion molecule-1 and hepatocyte growth factor) were high in all of the patients examined, whereas those of Th1 cytokines such as IL-12 and macrophage inflammatory protein-1a were normal or low. Levels of IL-18 and IL-10 were highest in case 2, with a fatal outcome, and were lowest in case 4, with rapid recovery within 1 month. Although IFN-gamma levels were not elevated in 2 patients (cases 3 and 5), IL-18 levels were markedly high in both of these cases and the IL-6 level was highest in case 3. In contrast with the marked increase in the level of IL-10, the levels of IL-6, sIL-2R, and ferritin decreased rapidly and returned to normal within 2 months after therapy in case 3. The IL-18 level decreased somewhat, but remained elevated for 6 months, and the patient achieved a complete response within 11 months. Taken together, our findings suggest that both IL-18 and IL-10, but not IL-12, may play important roles in young adult patients with HPS via enhancing and suppressing Th1 immune responses, respectively.

Cloning and Characterization of the Novel Chimeric Gene TEL/PTPRR in Acute Myelogenous Leukemia with inv(12)(p13q13)

We have cloned a novel TEL/protein tyrosine phosphatase receptor-type R (PTPRR) chimeric gene generated by inv(12)(p13q13). PTPRR is the first protein tyrosine phosphatase identified as a fusion partner of TEL. The chimeric gene fused exon 4 of the TEL gene with exon 7 of the PTPRR gene, and produced 10 isoforms through alternative splicing. Two isoforms that were expressed at the highest level in the leukemic cells could have been translated into COOH-terminally truncated TEL protein possessing the helix-loop-helix domain (tTEL) and TEL/PTPRR chimeric protein linking the helix-loop-helix domain of TEL to the catalytic domain of PTPRR. These two mutant proteins exerted a dominant-negative effect over transcriptional repression mediated by wild-type TEL, although they themselves did not show any transcriptional activity. Heterodimerization with wild-type TEL might be an underlying mechanism in this effect. TEL/PTPRR did not exhibit any tyrosine phosphatase activity. Importantly, overexpression of TEL/PTPRR in granulocyte macrophage colony-stimulating factor-dependent UT7/GM cells resulted in their factor-independent proliferation, whereas overexpression of tTEL did not. After cytokine depletion, phosphorylated signal transducers and activators of transcription 3 (STAT3) significantly declined in mock cells, but remained in both tTEL- and TEL/PTPRR-overexpressing cells. Loss of tumor suppressive function of wild-type TEL and maintenance of STAT3-mediated signal could at least partly contribute to the leukemogenesis caused by inv(12)(p13q13).

Detection of chimaeric transcripts of the immunoglobulin heavy chain and BCL6 genes by reverse‐transcriptase polymerase chain reaction in B‐cell non‐Hodgkin's lymphomas

T(3;14)(q27;q32) is frequently detected in B‐cell non‐Hodgkins lymphomas, especially the diffuse large cell type and the follicular type. The BCL6 gene encoding a putative transcriptional factor which resides on 3q27 rearranges to the immunoglobulin heavy chain (IgH) gene on 14q32 in this chromosomal translocation. The upstream regulatory region of the BCL6 gene is replaced by the IgH gene. Deregulation of the BCL6 gene may contribute to tumourigenesis of these diseases. The rearrangement between the IgH and BCL6 genes generates chimaeric transcripts in which the joining (J) region of the IgH gene fuses to exon 3 of the BCL6 gene. We established a method to detect these chimaeric transcripts by reverse transcriptase polymerase chain reaction (RT‐PCR) using the consensus sequence of the J region and the sequence of exon 3 of the BCL6 gene as primers. Using the semi‐nested RT‐PCR method and a cell line carrying t(3;14)(q27;q32), we detected one lymphoma cell among 10 000 background cells. We detected these chimaeric transcripts in two out of 13 clinical samples by this method. This method can detect t(3;14)(q27;q32) easily, whereas this alteration is frequently overlooked by routine karyotype analysis. Since this technique is sensitive enough to detect a small number of lymphoma cells with this genetic abnormality, it could be employed to detect contaminating lymphoma cells in bone marrow and peripheral blood and minimal residual diseases.

Biallelic DNA rearrangements and deletions within the BCL-6 gene in B-cell non-Hodgkin's lymphoma

Summary. Chromosomal translocations involving band 3q27 are recently described common specific cytogenetic abnormalities in B‐cell neoplasms, and the BCL‐6 gene, identified on 3q27, was shown to be disrupted and over‐expressed in lymphoma cells having these chromosomal translocations. In the present study we found rearrangements within the BCL‐6 gene in seven out of 3 5 cases with B‐cell non‐Hodgkins lymphoma (NHL). Further analysis revealed that three of these patients with BCL‐6 abnormality had multiple rearranged bands hybridized with probes from a single restriction fragment within the major translocation cluster (MTC). suggesting that independent DNA rearrangements would occur on both alleles. Additionally, Southern blot analysis indicated that three patients carry deletions encompassing the area containing the first exon of the BCL‐6 gene. Our results suggest that biallelic DNA rearrangements and deletions would occasionally occur in NHL patients with BCL‐6 abnormality.

Diffuse Nodular Lymphoid Hyperplasia of the Large Bowel Without Hypogammaglobulinemia or Malabsorption Syndrome A Case Report and Literature Review

Diffuse nodular lymphoid hyperplasia (DNLH) of the intestine is an extremely rare lymphoproliferative disorder occasionally associated with non-Hodgkin lymphomas. We report an unusual case of DNLH of the entire colon, which resembled malignant lymphoma (particularly low-grade B-cell lymphoma) both on clinical and pathologic grounds. The patient was a 62-year-old Japanese woman who was found to have multiple polypoid lesions along the entire large intestine by colonoscopy. Abdominal computed tomography revealed hepatosplenomegaly and multiple mesenteric lymphadenopathy. Histologically, the lesion was characterized by numerous lymphoid follicles with active germinal centers and a diffuse infiltrate of lymphoid cells in the mucosa and submucosa. The present case appears to be an essentially benign condition bearing a resemblance, both clinically and histologically, to malignant lymphoma, and from which it can be distinguished by use of immunohistochemical or molecular analysis.

Internal Deletions with in the BCL6 Gene in B-Cell Non-Hodgkin's Lymphoma

The BCL6 gene, encoding a POZ/Zinc finger protein which acts as a transcriptional repressor, is frequently altered at its 5′ non-coding region by 3q27 chromosomal translocations in B-cell non-Hodgkins lymphoma (NHL). BCL6 rearrangement is one of the most common genetic abnormalities in NHL. As a result of translocations, the regulatory region of the BCL6 gene is replaced by an heterologous reciprocal partner such as the immunoglobulin (IG) genes. Pro-motor substitution leads to deregulation of the BCL6 expression, which may be associated with lymphomagenesis. Recent studies have shown that the 5′ non-coding region of the BCL6 gene is also subject to somatic hypermutation physiologically operating in germinal center (GC) B-cells in a similar pattern to that of the IG genes. There is little evidence to show that structural alterations of the BCL6 gene may be caused by mechanisms other than chromosomal translocations. To date, five cases with NHL exhibiting gross 5CL6 deletions of the 1.5-2.4 kb have been reported. These deletions occurred in the same region as translocational breakpoints and the somatic hypermutations cluster, but independently of chromosomal rearrangements. The deletions overlapped at the 270 bp region and this region contains a putative protein-binding sequence which may play a role in the regulation of the BCL6 expression. Small separated deletions of 22-101bp, which may also contain protein-binding sequences, were evident in another NHL case. In contrast to the TALI deletion in T-cell acute lymphoblastic leukemia (ALL), the BCL6 deletion is considered to be mediated by a mechanism other than aberrant activity of the IG recombinase. Internal deletion within the BCL6 gene is a recurrent molecular abnormality in B-cell NHL, which is sometimes indistinguishable from rearrangements by chromosomal translocations. At present, the mechanism of DNA recombination and its role in lymphomagenesis remain unknown.