Biomedical subjects

Mineo Kurokawa

Publications and source records attributed to Mineo Kurokawa.

At least 19 recordsLinked to original sources

PubMed2007

Early relapse of JAK2 V617F-positive chronic neutrophilic leukemia with central nervous system infiltration after unrelated bone marrow transplantation.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disorder characterized by a proliferation mainly of mature neutrophils. The prognosis is generally poor and an optimal therapeutic strategy remains to be determined. Allogeneic hematopoietic stem cell transplantation (HSCT) is expected to be the only curative therapy so far. We report a 46-year-old male with progressive CNL who underwent bone marrow transplantation from an HLA-matched unrelated donor. After engraftment was achieved on day 35, relapse of CNL was confirmed on day 50. The progression of CNL was very rapid afterward and infiltration to the central nervous system was observed. The Janus Kinase 2 (JAK2) V617F homozygous mutation was detected from the peripheral blood or bone marrow samples throughout the clinical course. From comparison with reports of successful HSCT for CNL in the literature, it was inferred that HSCT should be performed in a stable status before progression. Furthermore, JAK2 V617F-positive CNL may contain an aggressive disease entity in contrast to previous reports. Accumulation of experiences is required to establish a definite role of HSCT in the treatment of CNL and a prognostic significance of JAK2 mutation in CNL.

Antineoplastic Agents, Alkylating↗

PubMed2006

AML1/Runx1 rescues Notch1-null mutation-induced deficiency of para-aortic splanchnopleural hematopoiesis.

The Notch1-RBP-Jkappa and the transcription factor Runx1 pathways have been independently shown to be indispensable for the establishment of definitive hematopoiesis. Importantly, expression of Runx1 is down-regulated in the para-aortic splanchnopleural (P-Sp) region of Notch1- and Rbpsuh-null mice. Here we demonstrate that Notch1 up-regulates Runx1 expression and that the defective hematopoietic potential of Notch1-null P-Sp cells is successfully rescued in the OP9 culture system by retroviral transfer of Runx1. We also show that Hes1, a known effector of Notch signaling, potentiates Runx1-mediated transactivation. Together with the recent findings in zebrafish, Runx1 is postulated to be a cardinal down-stream mediator of Notch signaling in hematopoietic development throughout vertebrates. Our findings also suggest that Notch signaling may modulate both expression and transcriptional activity of Runx1.

Animals↗

PubMed2006

Highly efficient ex vivo expansion of human hematopoietic stem cells using Delta1-Fc chimeric protein.

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, interleukin (IL)-3, and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice, which showed myeloid, B, T, and natural killer cells as well as CD133(+)CD34(+) cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

AC133 Antigen↗

PubMed2006

Treatment results of chemoradiation therapy for localized aggressive lymphomas: A retrospective 20-year study.

In this study we analyzed our cases of localized aggressive lymphoma treated in our institution during the last 20 years to compare the finding of this study with those of previous studies. Forty patients with Ann Arbor stage I-II aggressive lymphoma were treated with 3-6 cycles of a CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisolone) and radiation therapy (30 or 40 Gy with involved field). Between 1985 and 2003, 40 patients with stage I (N = 25) or stage II (N = 15) disease were treated. Chemotherapy mainly preceded radiotherapy, although the sequence of radiotherapy and chemotherapy was determined by individual physicians and patients' choice. Median and mean age was 50.5 and 48.6 years, respectively, at the time of diagnosis, with a male to female ratio of 19:21. Analyses were undertaken to determine (1) response to treatment according to age, international prognostic index (IPI), lactate dehydrogenase (LDH) value, serum interleukin 2 receptor (sIL-2R) value, cell type, stage, extent of maximum local disease, with or without mediastinal lymph nodes, number of sites, anatomic distribution, and irradiation dose, and (2) relapse patterns. Complete follow-up was obtained in all patients. The follow-up period of surviving 33 patients ranged from 24.7 to 180 months with a median of 69 and a mean of 72.7 months. A complete remission (CR) was achieved in 37 patients (93%). A study of relapse patterns after a CR showed that four patients had a first relapse within a radiation field and the other one patient had an extranodal distant relapse. Significant prognostic factors were not identified by multivariate analysis. Combined chemotherapy and radiation therapy is safe, highly effective, and probably curative for most patients with stage I-II aggressive lymphoma.

Adult↗

PubMed2006

Antineoplastic effect of a single oral dose of the novel Flt3 inhibitor KRN383 on xenografted human leukemic cells harboring Flt3-activating mutations.

Activating mutations of Fms-like tyrosine kinase 3 (Flt3) are the most common genetic abnormalities found in acute myeloid leukemia (AML) and represent potential therapeutic targets. The novel Flt3 inhibitor KRN383 inhibited the autophosphorylation of Flt3 bearing internal tandem duplications (ITDs) and the Asp835Tyr (D835Y) point mutation with half-maximal inhibitory concentration (IC(50)) values of < or =5.9 and 43 nM, respectively. KRN383 also inhibited the proliferation of the ITD-positive cell lines with IC(50) values of < or =2.9 nM. A single oral administration of 80 mg/kg of KRN383 eradicated ITD-positive xenograft tumors in nude mice and prolonged the survival of SCID mice carrying ITD-positive AML cells. The effectiveness of a single oral dose of KRN383 suggests that it has the potential to be used in a wide variety of clinical regimens, including multicycle and combination therapies.

Administration, Oral↗

PubMed2006

Pharmacokinetics of ganciclovir in haematopoietic stem cell transplantation recipients with or without renal impairment.

OBJECTIVES: We investigated the pharmacokinetics of ganciclovir in 12 haematopoietic stem cell transplantation (HSCT) recipients to evaluate the validity of a 50% reduction in the ganciclovir dosage for mild renal impairment. PATIENTS AND METHODS: Ganciclovir at 5 mg/kg/day was pre-emptively infused in patients with estimated CL(CR) > or = 70 mL/min (Group A), whereas the dose was reduced to 2.5 mg/kg/day in patients with CL(CR) between 50 and 70 mL/min (Group B). RESULTS: The peak concentration was significantly higher in Group A (P < 0.01). However, the decrease in the plasma ganciclovir concentration was slower in Group B (P = 0.09), and the AUC of all patients in both groups was distributed within a narrow range (25.6 +/- 4.77 microg x h/mL), when two patients with exceptionally high AUC values were excluded. CONCLUSIONS: A 50% reduction in ganciclovir appeared to be appropriate for patients with mild renal impairment. Measuring the ganciclovir concentration at 4 h after starting infusion may be adequate for evaluating AUC.

Adult↗

PubMed2006

Pharmacokinetics of alemtuzumab after haploidentical HLA-mismatched hematopoietic stem cell transplantation using in vivo alemtuzumab with or without CD52-positive malignancies.

We recently reported that the addition of in vivo alemtuzumab to the conditioning regimen enables 2- or 3-locus-mismatched hematopoietic stem cell transplantation without an excessive risk of graft rejection or graft-versus-host disease. In a later series of patients, however, one patient with refractory chronic lymphocytic leukemia with large residual tumors at transplantation developed graft rejection. While the peak alemtuzumab concentration in the previous patients without graft rejection was higher than 5 micro g/ml, the peak alemtuzumab concentration in this patient was only 1.44 micro g/ml. We considered that alemtuzumab was bound to the large residual tumors, which resulted in a low blood concentration of alemtuzumab. Therefore, it is important to debulk tumors before the conditioning regimen for patients with refractory CD52-positive hematological malignancies, or the dose of alemtuzumab should be adjusted by monitoring the blood concentration, when alemtuzumab is used for in vivo T-cell depletion in 2- or 3-locus-mismatched transplantation.

Alemtuzumab↗

PubMed2006

Genomewide screening of DNA copy number changes in chronic myelogenous leukemia with the use of high-resolution array-based comparative genomic hybridization.

Chronic myelogenous leukemia (CML) evolves from an indolent chronic phase (CP) characterized by the Philadelphia chromosome. Without effective therapy, it progresses to an accelerated phase (AP) and eventually to a fatal blast crisis (BC). To identify the genes involved in stage progression in CML, we performed a genomewide screening of DNA copy number changes in a total of 55 CML patients in different stages with the use of the high-resolution array-based comparative genomic hybridization (array CGH) technique. We constructed Human 1M arrays that contained 3,151 bacterial artificial chromosome (BAC) DNAs, allowing for an average resolution of 1.0 Mb across the entire genome. In addition to common chromosomal abnormalities, array CGH analysis unveiled a number of novel copy number changes. These alterations included losses in 2q26.2-q37.3, 5q23.1-q23.3, 5q31.2-q32, 7p21.3-p11.2, 7q31.1-q31.33, 8pter-p12(p11.2), 9p, and 22q13.1-q13.31 and gains in 3q26.2-q29, 6p22.3, 7p15.2-p14.3, 8p12, 8p21.3, 8p23.2, 8q24.13-q24.21, 9q, 19p13.2-p12, and 22q13.1-q13.32 and occurred at a higher frequency in AP and BC. Minimal copy number changes affecting even a single BAC locus were also identified. Our data suggests that at least a proportion of CML patients carry still-unknown cryptic genomic alterations that could affect a gene or genes of importance in the disease progression of CML. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Adult↗

PubMed2006

CD1d expression level in tumor cells is an important determinant for anti-tumor immunity by natural killer T cells.

Invariant natural killer T (iNKT) cells are thought to regulate anti-tumor immunity. Human iNKT (i.e. Valpha24+ NKT) cells have been reported to recognize CD1d on target cells and show cytotoxicity directly on the target cells in vitro. However, the anti-tumor effect of mouse iNKT (i.e. Valpha14+ NKT) cells has been repeatedly reported to be dependent on the activity of natural killer (NK) cells via interferon-gamma, with no evidence of direct cytotoxicity. In the present study, we report that in vitro cytolysis of EL-4 mouse lymphoblastic lymphoma cells by Valpha24+ NKT cells and in vivo eradication of these cells are both dependent on the level of CD1d expression on the tumor cell surface. These observations possibly suggest that direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells.

Animals↗

PubMed2006

AML1/Runx1 as a versatile regulator of hematopoiesis: regulation of its function and a role in adult hematopoiesis.

AML1/Runx1, originally identified as a gene located at the breakpoint of the t(8;21) translocation, encodes a transcription factor that is widely expressed in multiple hematopoietic lineages and that regulates the expression of a variety of hematopoietic genes. Numerous studies have shown that AML1 is a critical regulator of hematopoietic development. In addition, AML1 is a frequent target for chromosomal translocation in human leukemia. The activity of AML1 can be modulated by various types of posttranslational modification, including phosphorylation and acetylation. Phosphorylation by extracellular signal-regulated kinase (ERK) is one of the mechanisms that dictate whether AML1 acts as either a transcriptional repressor or an activator of gene expression. Recently, a physiological role for AML1 in adult hematopoiesis was revealed by conditional gene targeting in mice. Remarkably, adult hematopoietic progenitors are maintained even in the absence of AML1, in stark contrast to the total disruption of definitive hematopoiesis during embryogenesis. AML1 is, however, critical for megakaryopoiesis and plays an important role in T-cell and B-cell development in adult mice. Recent analyses engineered to recreate hematopoiesis in vitro revealed that the transcriptional activity of AML1 is closely related with the potential of AML1 to generate hematopoietic cells and support thymocyte development.

Acetylation↗

PubMed2005

Crk-associated substrate lymphocyte type is required for lymphocyte trafficking and marginal zone B cell maintenance.

The lymphocyte-specific Cas family protein Cas-L (Crk-associated substrate lymphocyte type) has been implicated to function in lymphocyte movement, mediated mainly by integrin signaling. However, its physiological role is poorly understood. In this study we analyzed the function of Cas-L in lymphocytes using gene-targeted mice. The mutant mice showed a deficit of marginal zone B (MZB) cells and a decrease of cell number in secondary lymphoid organs. An insufficient chemotactic response and perturbed cell adhesion were observed in Cas-L-deficient lymphocytes, suggesting that the aberrant localization was responsible for the deficit of MZB cells. Moreover, we found that lymphocyte trafficking was altered in Cas-L-deficient mice, which gave a potential reason for contraction of secondary lymphoid tissues. Thus, Cas-L affects homeostasis of MZB cells and peripheral lymphoid organs, which is considered to be relevant to impaired lymphocyte migration and adhesion.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Oligomerization of Evi-1 regulated by the PR domain contributes to recruitment of corepressor CtBP.

Evi-1 is a transcription factor that is implicated in leukemic transformation of hematopoietic cells. Two distinct alternative forms, Evi-1a and Evi-1c, are generated from the EVI-1 gene. Whereas Evi-1a is widely recognized as an oncoprotein, a role for Evi-1c, which has an additional PR domain in the amino-terminus of Evi-1a, in leukemogenesis, has not been elucidated thus far. Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Here, to study the mechanisms that underlie Evi-1-mediated oncogenesis, we investigated formation of oligomeric complexes by the Evi-1 proteins. We show that Evi-1a forms homo-oligomers, whereas Evi-1c exclusively exists as a monomer in mammalian cells. Remarkably, Evi-1c has lost the ability to interact with CtBP, a transcriptional corepressor that associates with Evi-1a. As a consequence, the ability of Evi-1c to repress transforming growth factor-beta (TGF-beta) signaling is significantly abrogated. These results identify a novel function of a PR domain to regulate oligomerization of transcription factors and suggest that homo-oligomerization may play a critical role in corepressor recruitment by the Evi-1 proteins. In addition, we found that the chimeric oncoprotein acute myelocytic leukemia (AML)1-Evi-1, generated in t(3;21) leukemia, also forms homo-oligomers and hetero-oligomers with Evi-1a, while it did not interact with Evi-1c. Consistent with the results, repression of TGF-beta by AML1-Evi-1 was significantly enhanced by Evi-1a, whereas it was hardly affected by the presence of Evi-1c. These results suggest that oligomerization may contribute to the oncogenic potential of Evi-1-containing proteins.

Alcohol Oxidoreductases↗

PubMed2005

A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays.

We have developed a robust algorithm for copy number analysis of the human genome using high-density oligonucleotide microarrays containing 116,204 single-nucleotide polymorphisms. The advantages of this algorithm include the improvement of signal-to-noise (S/N) ratios and the use of an optimized reference. The raw S/N ratios were improved by accounting for the length and GC content of the PCR products using quadratic regressions. The use of constitutional DNA, when available, gives the lowest SD values (0.16 +/- 0.03) and also enables allele-based copy number detection in cancer genomes, which can unmask otherwise concealed allelic imbalances. In the absence of constitutional DNA, optimized selection of multiple normal references with the highest S/N ratios, in combination with the data regressions, dramatically improves SD values from 0.67 +/- 0.12 to 0.18 +/- 0.03. These improvements allow for highly reliable comparison of data across different experimental conditions, detection of allele-based copy number changes, and more accurate estimations of the range and magnitude of copy number aberrations. This algorithm has been implemented in a software package called Copy Number Analyzer for Affymetrix GeneChip Mapping 100K arrays (CNAG). Overall, these enhancements make CNAG a useful tool for high-resolution detection of copy number alterations which can help in the understanding of the pathogenesis of cancers and other diseases as well as in exploring the complexities of the human genome.

Algorithms↗

PubMed2005

Host-residual invariant NK T cells attenuate graft-versus-host immunity.

Invariant NK T (iNKT) cells have an invariant TCR-alpha chain and are activated in a CD1d-restricted manner. They are thought to regulate immune responses and play important roles in autoimmunity, allergy, infection, and tumor immunity. They also appear to influence immunity after hemopoietic stem cell transplantation. In this study, we examined the role of iNKT cells in graft-vs-host disease (GVHD) and graft rejection in a mouse model of MHC-mismatched bone marrow transplantation, using materials including alpha-galactosylceramide, NKT cells expanded in vitro, and Jalpha18 knockout mice that lack iNKT cells. We found that host-residual iNKT cells constitute effector cells which play a crucial role in reducing the severity of GVHD, and that this reduction is associated with a delayed increase in serum Th2 cytokine levels. Interestingly, we also found that host-residual iNKT cause a delay in engraftment and, under certain conditions, graft rejection. These results indicate that host-residual iNKT cells attenuate graft-vs-host immunity rather than host-vs-graft immunity.

Adoptive Transfer↗

PubMed2005

Functional domains of Runx1 are differentially required for CD4 repression, TCRbeta expression, and CD4/8 double-negative to CD4/8 double-positive transition in thymocyte development.

Runx1 (AML1) has multiple functions in thymocyte development, including CD4 repression in immature thymocytes, expression of TCRbeta, and efficient beta-selection. To determine the functional domains of Runx1 important for thymocyte development, we cultured Runx1-deficient murine fetal liver (FL) cells on OP9-Delta-like 1 murine stromal cells, which express Delta-like 1 and support thymocyte development in vitro, and introduced Runx1 or C-terminal-deletion mutants of Runx1 into the FL cells by retrovirus infection. In this system, Runx1-deficient FL cells failed to follow normal thymocyte development, whereas the introduction of Runx1 into the cells was sufficient to produce thymocyte development that was indistinguishable from that in wild-type FL cells. In contrast, Runx1 mutants that lacked the activation domain necessary for initiating gene transcription did not fully restore thymocyte differentiation, in that it neither repressed CD4 expression nor promoted the CD4/8 double-negative to CD4/8 double-positive transition. Although the C-terminal VWRPY motif-deficient mutant of Runx1, which cannot interact with the transcriptional corepressor Transducin-like enhancer of split (TLE), promoted the double-negative to double-positive transition, it did not efficiently repress CD4 expression. These results suggest that the activation domain is essential for Runx1 to establish thymocyte development and that Runx1 has both TLE-dependent and TLE-independent functions in thymocyte development.

Amino Acid Motifs↗

PubMed2005

Identification of a SRC-like tyrosine kinase gene, FRK, fused with ETV6 in a patient with acute myelogenous leukemia carrying a t(6;12)(q21;p13) translocation.

The SRC family of kinases is rarely mutated in primary human tumors. We report the identification of a SRC-like tyrosine kinase gene, FRK (Fyn-related kinase), fused with ETV6 in a patient with acute myelogenous leukemia carrying t(6;12)(q21;p13). Both reciprocal fusion transcripts, ETV6/FRK and FRK/ETV6, were expressed. In ETV6/FRK, exon 4 of ETV6 was fused in-frame to exon 3 of FRK, producing a chimeric protein consisting of the entire oligomerization domain of ETV6 and the kinase domain of FRK. The ETV6/FRK protein was shown to be constitutively autophosphorylated on its tyrosine residues. ETV6/FRK phosphorylated histones H2B and H4 in vitro to a greater extent than did FRK, suggesting it had elevated kinase activity. ETV6/FRK could transform both Ba/F3 cells and NIH3T3 cells, which depended on its kinase activity. Moreover, ETV6/FRK inhibited ETV6-mediated transcriptional repression in a dominant-negative manner. This report provides the first evidence that a SRC-like kinase gene, FRK fused with ETV6, could directly contribute to leukemogenesis by producing an oncoprotein, ETV6/FRK, with dual functions: constitutive activation of the ETV6/FRK tyrosine kinase and dominant-negative modulation of ETV6-mediated transcriptional repression.

Aged↗

PubMed2005

Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery.

OBJECTIVE: For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs. MATERIALS AND METHODS: Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice. RESULTS: The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse. CONCLUSION: In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.

Animals↗

PubMed2005

Notch1 oncoprotein antagonizes TGF-beta/Smad-mediated cell growth suppression via sequestration of coactivator p300.

The Notch proteins constitute a family of transmembrane receptors that play a pivotal role in cellular differentiation, proliferation and apoptosis. Although it has been recognized that excess Notch signaling is potentially tumorigenic, little is known about precise mechanisms through which dysregulated Notch signaling induces neoplastic transformation. Here we demonstrate that Notch signaling has a transcriptional cross-talk with transforming growth factor-beta (TGF-beta) signaling, which is well characterized by its antiproliferative effects. TGF-beta-mediated transcriptional responses are suppressed by constitutively active Notch1, and this inhibitory effect is canceled by introduction of transcriptional coactivator p300. We further show that this blockade of TGF-beta signaling is executed by the sequestration of p300 from Smad3. Moreover, in a human cervical carcinoma cell line, CaSki, in which Notch1 is spontaneously activated, suppression of Notch1 expression with small interfering RNA significantly restores the responsiveness to TGF-beta. Taken together, we propose that Notch oncoproteins promote cell growth and cancer development partly by suppressing the growth inhibitory effects of TGF-beta through sequestrating p300 from Smad3.

Animals↗