Biomedical subjects

Shinobu Tsuzuki

Publications and source records attributed to Shinobu Tsuzuki.

14 recordsLinked to original sources

PubMed2006

Genomewide array-based comparative genomic hybridization analysis of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is typically associated with the t(15;17) that generates the PML-RARA fusion protein. Animal models have shown that although the fusion protein is necessary, it is insufficient for the development of APL, implying that additional mechanisms are responsible for full-blown leukemia. The mutation of specific genes has been implicated in leukemogenesis; however, alterations in gene copy number have not been well investigated. Here, we applied the genomewide array-comparative genomic hybridization technique to 30 APL clinical samples and 2 APL cell lines. It was found that (1) approximately half the clinical samples (14 of 30 APL cases) had no detectable chromosomal imbalances; and (2) the remaining 16 cases, including the cell lines, exhibited recurrent chromosomal imbalances, such as loss of 1p36, 2p11, 16p, and 17p, and gain of 8p, 8q, and 13q. These results suggest that chromosomal imbalances are largely absent in APL, although some nonrandom chromosomal imbalances could be linked to the development of APL in a limited number of cases.

Adolescent↗

PubMed2005

MALT1 contains nuclear export signals and regulates cytoplasmic localization of BCL10.

MALT1, BCL10 (B-cell lymphoma 10), and API2 (apoptosis inhibitor 2)-MALT1 are key molecules in mucosa-associated lymphoid tissue (MALT) lymphomagenesis. We previously reported that MALT1 and API2-MALT1 were localized only in cytoplasm, where we suggested that both molecules were likely to be active. In the study presented here, we further examined the localization-determining region by generating various mutants and were able to demonstrate that there were nuclear export signal (NES)-containing domains in the MALT1 C-terminal region. The use of leptomycin B, an NES-specific inhibitor, demonstrated that both MALT1 and API2-MALT1 were predominantly retained in the nuclei, indicating that these molecules were shuttling between nucleus and cytoplasm in an NES-dependent manner. It was also found that MALT1 was involved in the nuclear export of BCL10, which is originally localized in both nucleus and cytoplasm. These results correlate well with the nuclear BCL10 expression pattern in both t(1;14) and t(11;18) MALT lymphomas. The nucleocytoplasmic shuttling of MALT1 and BCL10 complex may indicate that these molecules are involved not only in the nuclear factor kappaB (NF-kappaB) pathway but also in other biologic functions in lymphocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Comparison of genome profiles for identification of distinct subgroups of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) comprises molecularly distinct subgroups such as activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCLs. We previously reported that CD5(+) and CD5(-)CD10(+) DLBCL constitute clinically relevant subgroups. To determine whether these 2 subgroups are related to ABC and GCB DLBCLs, we analyzed the genomic imbalance of 99 cases (36 CD5(+), 19 CD5(-)CD10(+), and 44 CD5(-)CD10(-)) using array-based comparative genomic hybridization (CGH). Forty-six of these cases (22 CD5(+), 7 CD5(-)CD10(+), and 17 CD5(-)CD10(-)) were subsequently subjected to gene-expression profiling, resulting in their division into 28 ABC (19 CD5(+) and 9 CD5(-)CD10(-)) and 18 GCB (3 CD5(+), 7 CD5(-)CD10(+), and 8 CD5(-)CD10(-)) types. A comparison of genome profiles of distinct subgroups of DLBCL demonstrated that (1) ABC DLBCL is characterized by gain of 3q, 18q, and 19q and loss of 6q and 9p21, and GCB DLBCL is characterized by gain of 1q, 2p, 7q, and 12q; (2) the genomic imbalances characteristic of the CD5(+) and CD5(-)CD10(+) groups were similar to those of the ABC and GCB types, respectively. These findings suggest that CD5(+) and CD5(-)CD10(+) subgroups are included, respectively, in the ABC and GCB types. Finally, when searching for genomic imbalances that affect patients' prognosis, we found that 9p21 loss (p16(INK4a) locus) marks the most aggressive type of DLBCL.

Adult↗

PubMed2005

Developmental impact of leukemic fusion genes on stem cell fate.

Stem and progenitor cells present attractive targets for transformation by leukemia-associated fusion genes generated by chromosomal translocation. The mechanism by which these fusion genes corrupt the transcriptional programs of these cellular compartments remains largely unknown. We have sought to gain insight into these issues through expressing TEL-AML1 and TEL-TRKC fusion genes in murine stem cells and recording effects on cell behavior in a transplant setting.

Animals↗

PubMed2005

[Antiapoptotic functions of the retrovirally transferred API2-MALT1 gene].

t(11 ; 18)(q21 ; q21) is a specific chromosomal aberration in mucosa-associated lymphoid tissue (MALT) lymphoma, and produces chimeric transcript Apoptosis Inhibitor 2 (API2)-MALT1. Although it is known that API2 has an antiapoptotic effect, it is still unclear whether this also applies to API2-MALT1. To investigate its effects against various apoptotic stimuli, API2-MALT1 was expressed by means of retroviral infection on the epithelial cell line HeLa and the murine Pro-B cell line Ba/F3. On both these cell lines, API2-MALT1 was found to cause a significant reduction in UV-induced apoptosis. The apoptosis induced by doxorubicin was also inhibited by API2-MALT1, but not that induced by IL-3 withdrawal from Ba/F3. These findings suggest that API2-MALT1 has an antiapoptotic effect on both epithelial and lymphoid cells and that this effect depends on the apoptotic stimulus.

Animals↗

PubMed2004

Contig array CGH at 3p14.2 points to the FRA3B/FHIT common fragile region as the target gene in diffuse large B-cell lymphoma.

Deletions of the 3p arm have been detected in various solid tumors, but no study to date has investigated this deletion in diffuse large B-cell lymphoma (DLBCL). Recently, we demonstrated that 3p14.2 was deleted in approximately 30% of DLBCL cases by use of a genome-wide array-comparative genomic hybridization (CGH). For a more detailed examination of the genomic losses at 3p14.2, here we made use of contig BAC array for 3p14.2, and found that 12 DLBCL samples displayed losses. All of the deleted regions were located within the fragile histidine triad (FHIT) gene, and the most frequent region of loss was mapped to 0.4 Mbp of the region encompassing the introns 4 and 5 and exon 5 of the FHIT gene. Concomitant analysis of transcripts showed that the FHIT gene was aberrantly transcribed in 31% of the DLBCL samples examined and that the lost exons of the aberrant transcripts were correlated with genomic deletions. These findings indicate that (1) loss of genomic material at 3q14.2 is responsible for exon losses of the FHIT gene, and (2) genomic loss of the FHIT gene is one of the causes of the generation of aberrant transcripts.

Acid Anhydride Hydrolases↗

PubMed2004

Genome-wide array-based comparative genomic hybridization of diffuse large B-cell lymphoma: comparison between CD5-positive and CD5-negative cases.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma and exhibits aggressive and heterogeneous clinical behavior. To genetically characterize DLBCL, we established our own array-based comparative genomic hybridization and analyzed a total of 70 cases [26 CD-positive (CD5+) DLBCL and 44 CD5-negative (CD5-) DLBCL cases]. Regions of genomic aberrations observed in >20% of cases of both the CD5+ and CD5- groups were gains of 1q21-q31, 1q32, 3p25-q29, 5p13, 6p21-p25, 7p22-q31, 8q24, 11q23-q24, 12q13-q21, 16p13, 18, and X and losses of 1p36, 3p14, 6q14-q25, 6q27, 9p21, and 17p11-p13. Because CD5 expression marks a subgroup with poor prognosis, we subsequently analyzed genomic gains and losses of CD5+ DLBCL compared with those of CD5-. Although both groups showed similar genomic patterns of gains and losses, gains of 10p14-p15 and 19q13 and losses of 1q43-q44 and 8p23 were found to be characteristic of CD5+ DLBCL. By focusing on the gain of 13q21-q34 and loss of 1p34-p36, we were also able to identify prognostically distinct subgroups among CD5+ DLBCL cases. These results suggest that array-based comparative genomic hybridization analysis provides a platform of genomic aberrations of DLBCL both common and specific to clinically distinct subgroups.

CD5 Antigens↗

PubMed2004

Modeling first-hit functions of the t(12;21) TEL-AML1 translocation in mice.

The t(12;21) translocation, which generates the TEL-AML1 (ETV6-RUNX1) fusion gene, is the most common structural chromosome change in childhood cancer and is exclusively associated with the common B cell precursor subset of acute lymphoblastic leukemia (ALL). Evidence suggests that the translocation usually occurs in utero during fetal hemopoiesis and most probably constitutes an initiating or first-hit mutation that is necessary but insufficient for the development of overt, clinical leukemia. The mechanism by which TEL-AML1 contributes to this early stage of leukemogenesis is unknown. To address this question we have analyzed hemopoiesis in mice syngeneically transplanted with TEL-AML1-transduced bone marrow stem cells. TEL-AML1 expression was associated with an accumulation/expansion of primitive c-kit-positive multipotent progenitors and a modest increase in myeloid colony-forming cells. TEL-AML1 expression was, however, permissive for myeloid differentiation. Analysis of B lymphopoiesis revealed an increase in early, pro-B cells but a differentiation deficit beyond that stage, resulting in reduced B cell production in the marrow. TEL-AML1-positive B cell progenitors exhibited reduced expression of the surrogate light-chain component lambda5 and the IL-7 receptor, both of which may contribute to impedance of differentiation in vivo and account for their reduced in vitro clonogenicity in IL-7. A selective differentiation deficit of B lineage progenitors (i) is consistent with the phenotype of TEL-AML1-associated leukemia in children and (ii) provides a potential mechanism for the protracted preleukemic state that often precedes ALL. These results provide mechanistic insight into the role of the t(12;21) translocation in the initiation of common B cell precursor ALL.

Animals↗

PubMed2004

Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma.

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.

Amino Acid Sequence↗

PubMed2004

Involvement of mitogen-activated protein kinase in the cytokine-regulated phosphorylation of transcription factor GATA-1.

Gene-targeting experiments in transgenic mice have revealed an essential role for GATA-1 in the normal differentiation and development of erythroid cells. GATA-1 is phosphorylated in vivo on seven of its serine residues; the regulation and function of GATA-1 phosphorylation, however, is not understood. Here we demonstrate a role for MAP kinase (MAPK) signalling in the control of GATA-1 phosphorylation. We show that EGF-induced MAPK signalling results in the phosphorylation of ectopically expressed GATA-1 in COS cells. This phosphorylation can be positively or negatively regulated by genetic manipulation of the MAPK pathway through expression of constitutively activated, or dominant-negative, mutants of MAPK kinase (MAPKK), an upstream regulator of MAPK activity. In vitro phosphorylation experiments using purified MAPK and either recombinant GATA-1 or synthetic GATA-1 peptides suggest that GATA-1 is a MAPK substrate with MAPK phosphorylation occurring primarily on Ser26 and Ser178. We also show that GATA-1 is phosphorylated in factor-dependent haemopoietic progenitor cells in response to cytokine-induced signalling. Through the further use of a dominant-negative MAPKK mutant as well as chemical inhibitors of specific MAPKs, we identify ERK as an in vivo GATA-1 kinase. Finally, we demonstrate that mutation of serines 26 and 178 compromises the ability of GATA-1 to interact with the LIM-only protein LMO2 when both proteins are expressed in COS cells. These data implicate receptor-mediated signalling through the MAPK pathway as a control point in the regulation of transcription factor GATA-1.

Amino Acid Substitution↗

PubMed2004

Comparison of genetic aberrations in CD10+ diffused large B-cell lymphoma and follicular lymphoma by comparative genomic hybridization and tissue-fluorescence in situ hybridization.

CD10 is one of the hallmarks of germinal center B-cells where follicular lymphomas (FL) originate. It has not been clearly established, however, whether CD10(+) diffuse B-cell lymphomas (DLBCL) are genetically similar to FL. We therefore examined 19 CD10(+) DLBCL and 40 FL by means of comparative genomic hybridization (CGH) and tissue-fluorescence in situ hybridization (T-FISH). Chromosomal imbalance was more frequently detected in CD10(+) DLBCLs (19/19) than in FLs (24/40). Significant differences were found in eight frequently imbalanced regions, namely those with gains of chromosomes 7q and 12 and those with losses of chromosomes 1p, 4p, 6q, 15q, 16p and 17. Amplification of the 3q region where BCL6 is located is reported to occur frequently in DLBCL, but it was only found in one of the 19 CD10(+) DLBCL cases we examined. The involvement of t(14;18) in CD10(+)+ DLBCL (31%) and in FL (73%) was significantly different (P = 0.0064). The CGH pattern of CD10(+) DLBCL with t(14;18) was also different from that of FL with t(14;18). Taken together, our results indicate that CD10(+) DLBCL constitutes a unique subtype entity with genetic characteristics significantly different from those of FL and DLBCL.

Aged↗

PubMed2004

Cross talk between retinoic acid signaling and transcription factor GATA-2.

All-trans-retinoic acid (RA) stimulates differentiation of normal hematopoietic progenitors and acute myeloid leukemia cells. GATA-2 is a transcription factor expressed in early progenitor cells and implicated in the control of the fate of hematopoietic stem cells and progenitor cells. We have investigated the possibility that the GATA and nuclear hormone receptor pathways are functionally linked through direct protein-protein interaction. Here we demonstrate that in human myeloid KG1 cells, RA receptor alpha (RARalpha), the major RAR expressed in hematopoietic cells, associates with GATA-2. This association is mediated by the zinc fingers of GATA-2 and the DNA-binding domain of RARalpha. As a consequence of this interaction, RARalpha is tethered to the DNA sites that are recognized and bound by GATA-2, and the transcriptional activity of GATA-2 becomes RA responsive. The RA responsiveness of GATA-dependent transcription is eliminated by expression of either a dominant negative form of RARalpha or a GATA-2 mutant that fails to interact with RARalpha. Overexpression of RXRalpha inhibits RARalpha binding to the GATA-2-DNA complex, thus resulting in attenuation of the effects of RARalpha on GATA-2 activity. In addition, inhibition by RA of GATA-2-dependent hematopoietic colony formation in an embryonic stem cell model of hematopoietic differentiation provided biological evidence for functional cross talk between RA and GATA-2-dependent pathways.

Amino Acid Motifs↗

PubMed2003

Stability and subcellular localization of API2-MALT1 chimeric protein involved in t(11;18) (q21;q21) MALT lymphoma.

t(11; 18) (q21; q21) is a chromosomal aberration specific to low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, and generates the chimeric product apoptosis inhibitor 2 (API2)-MALT1, which has been suggested to play an important role in MALT lymphomagenesis. However, little is known about the characteristics of API2, MALT1, and API2-MALT1 proteins. We therefore investigated the subcellular localization and stability of these products. API2 was localized in the nucleus and the cytoplasm, and MALT1 and API2-MALT1 in the cytoplasm only. Western blot analysis showed that the products of API2 and MALT1 were unstable, while the API2-MALT1 product was stable. The API2 deletion mutants at the end of the C-terminal and the MALT1 deletion mutants at the end of the N-terminal were stable compared with the full-length products. These results indicate that the domains responsible for protein instability are located in the end of the C-terminal of API2 and in that of the N-terminal of MALT1, and also that API2-MALT1 became stable because it lacks these domains. It has been suggested that NF-kappaB activation plays an important role in the tumorigenesis of MALT lymphoma. Our findings further suggest that the stabilized expression of API2-MALT1 products may continuously stimulate the NF-kappaB activating pathway, thus leading to MALT lymphomagenesis.

Animals↗

PubMed2002

Interactions of GATA-2 with the promyelocytic leukemia zinc finger (PLZF) protein, its homologue FAZF, and the t(11;17)-generated PLZF-retinoic acid receptor alpha oncoprotein.

Transcription factor GATA-2 is implicated in the survival and growth of multipotential progenitors. Here we report that the promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA-2 and can modify its transactivation capacity. Fanconi anemia zinc finger (FAZF), a PLZF-homologous protein that has been variously described as ROG (repressor of GATA), and TZFP (testis zinc finger protein) also interact with GATA-2. The zinc finger region of GATA-2 is required for binding to PLZF and FAZF, but distinct interfaces on the PLZF and FAZF molecules mediate the interaction, suggesting that GATA-2 activity is controlled by these 2 homologous proteins through distinct mechanisms. GATA-2 can also physically associate with the PLZF-RARalpha fusion protein generated by the t(11;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). Functional experiments showed that this interaction has the capacity to render GATA-dependent transcription responsive to treatment with a combination of all-trans retinoic acid and the histone deacetylase inhibitor trichostatin A (TSA). This combination of drugs has been shown to stimulate the terminal differentiation of leukemic t(11;17)-associated APL blasts, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.

Binding Sites↗