Junichiro Fujimoto - Academia.edu (original) (raw)
Papers by Junichiro Fujimoto
Journal of Histochemistry & Cytochemistry, 1990
Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for... more Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for proliferation and differentiation of granulocytes, has been performed up to the present by use of the granulocyte colony-formation assay, because of the lack of a specific anti-G-CSF antibody. This has prevented the advancement of biological investigations of cell dynamics linked to G-CSF, e.g., cell localization of G-CSF and its pathophysiological changes. In the present work, two monoclonal antibodies (MAb), 1E7 and 4A6, against recombinant human G-CSF (rhG-CSF) were developed by cell hybridization between NS-1 myeloma cells and splenocytes from a mouse immunized with rhG-CSF. 1E7 and 4A6 were shown to be reactive with hG-CSF but not with other CSF (hGM-CSF, hIL-3, and mouse GM-CSF) by Western blot analysis. An immunoperoxidase staining method using these MAb was then established. This method was applicable to frozen sections, paraffin-embedded sections, and cells fixed with 4% parafo...
European Respiratory Journal, Sep 1, 2012
Transplantation proceedings, 1995
Blood cancer journal, 2014
CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult... more CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable progn...
Leukemia, 1990
Ig heavy chain (IgH) J (JH) region organization in 83 human acute leukemias was studied. In 31 of... more Ig heavy chain (IgH) J (JH) region organization in 83 human acute leukemias was studied. In 31 of the 35 precursor B acute lymphocytic leukemia (ALL) the rearrangements of the IgH gene involved sites between D regions 5' to DQ52 and JH. The study of the IgH gene organization in 43 T lineage ALL showed nine cases with the rearrangement at the IgJH region, one of which involved the D region 5' to DQ52. When analyzed with another restriction enzyme (BglII), three of the remaining eight showed rearrangement between DQ52 and JH. In the remaining five samples rearrangement of the IgJH region was not shown, but the restriction fragment length polymorphism (RFLP) between MspI sites in 5'DQ52 loci was observed. Thus the RFLP in 5'DQ52 locus was identified and was distinguished from the recombination between DQ52 and JH in non-B lineage leukemias.
Leukemia, 1991
Three Down's syndrome patients with transient myeloproliferative disorder were studied for cl... more Three Down's syndrome patients with transient myeloproliferative disorder were studied for clonality of the proliferating blast cells using the X chromosome-linked polymorphic gene phosphoglycerate kinase, immunoglobulin heavy chain (IgH) gene and T-cell antigen receptor (TCR) (beta, gamma, delta) genes. None of the three cases showed rearrangements of IgH, TCR beta, gamma, or delta genes, indicating the non-lymphoid nature of the proliferating blast cells. The X chromosome inactivation pattern showed that the cells in the blast population in all of the three cases of transient myeloproliferative disorder were clonal. These data suggest that at least some of this disorder can be due to a spontaneously regressing clone of malignant cells.
Protein Expression and Purification, 2001
cytotoxins, consisting of Stx1 and Stx2 subgroups, pro-A new single-step purification method for ... more cytotoxins, consisting of Stx1 and Stx2 subgroups, pro-A new single-step purification method for Shiga duced by Shigella dysenteriae type 1 and Shiga toxintoxin (Stx) was developed using receptor-mediated producing Escherichia coli. In humans, these toxins affinity chromatography, in which Gb3Cer (globotriaohave been shown to be associated with hemorrhagic sylceramide) was conjugated to octyl Sepharose CL-4B colitis and the hemolytic uremic syndrome (HUS) (1, as a carrier. This method achieves high yield and high 2). Stx is composed of one A subunit associated with purity in a small column on which Gb3Cer has been five B subunits (3). The A subunit inhibits protein synimmobilized at high density. Using this affinity column, thesis by catalytic inactivation of the eucaryotic 60S the Stx1 B subunit was purified with homogeneity by ribosomal subunit by RNA N-glycosidase activity (4), a one-step procedure from a crude extract of recombiwhereas the B subunit serves to mediate binding of the nant Stx1 B subunit-producing Escherichia coli. The toxin to the surface receptors on susceptible cells. The purified Stx1 B subunit conserved a natural pentamer cell surface receptor of the B subunit is a membranestructure confirmed by gel filtration and sedimentacomponent glycosphingolipid globotriaosylceramide tion equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer Gb3Cer, also known as CD77 or blood group P k antigen expressed on Burkitt's lymphoma cells. This versatile (5). In addition to its inhibitory activity on protein synpurification method can be used to isolate various thesis, Stx has been shown to induce apoptosis in sevtypes of natural as well as recombinant Stx, facilitating eral different cells or tissues, including Vero cells (6), fundamental studies of human diseases caused by this human renal tubular epithelia-derived ACHN cells (7), toxin. ᭧ 2001 Academic Press and normal human renal tubular epithelial cells (8, 9). Though the Stx1 B subunit alone cannot inhibit protein synthesis, some Burkitt's lymphoma cell lines could reportedly be induced to undergo apoptosis by treatment The Shiga toxins (Stx's) 2 are a family of bacterial with the Stx1 B subunit or antibody against Gb3Cer (10, 11). In addition, treatment of ACHN and Burkitt's
British Journal of Cancer - BRIT J CANCER, 1989
We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing&... more We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing's sarcoma (ES). 5C11 specifically reacted with ESs but not with other small round cell tumours in childhood, i.e. neuroblastomas, primitive neuroectodermal tumours (PNETs), rhabdomyosarcomas and malignant lymphomas. 5C11 did not react with any other tumours in children except for hepatoblastomas. No reactivity has been identified in normal tissues with the exception of fetal hepatocytes. Immunoelectron microscopically, 5C11 reactive antigen was located on cell membrane of ES cells. Biochemically, 5C11 immunoprecipitated a cell surface protein having molecular weight of 81,000 Da. 5C11 is the first antibody which can clearly distinguish ES from neurogenic tumours, especially from PNETs which were recently reported to have common features to ESs regarding chromosal abnormality and proto-oncogene expression but show evident differentiation into neurogenic direction. The results strongl...
British journal of …, 1991
We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a ... more We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t(11;22) in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity.
PLoS ONE, 2009
Background: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of ... more Background: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/b-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear. Methodology/Principal Findings: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 59 upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice. Conclusions/Significance: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner.
Pediatric Blood & Cancer, 2006
To determine useful biochemical markers in Langerhans cell histiocytosis (LCH), we analyzed the s... more To determine useful biochemical markers in Langerhans cell histiocytosis (LCH), we analyzed the serum levels of soluble CD154 (sCD154), IL2 receptor (sIL2-R), receptor activator of NF-kappaB ligand (sRANKL), and osteoprotegerin (OPG). Our study included 46 newly diagnosed LCH patients (single-system multi-site (SM type): n = 20, and multi-system multi-site (MM type): n = 26) who were treated with the JLSG-02 protocol between 2002 and 2004. The median age of the patients was 3.8 years old (range 0-18). sCD154, sIL2-R, sRANKL, and OPG were measured by ELISA at diagnosis (n = 46) and after 6-weeks of induction therapy (n = 14). The values of sCD154, sIL-2R, sRANKL, and OPG, and the sRANKL/OPG ratio in sera were significantly higher in patients with LCH compared with controls (1.83 +/- 1.38 vs. 0.22 +/- 0.16 ng/ml, P < 0.001; 1,600 +/- 1,060 vs. 420 +/- 160 pg/ml, P < 0.001; 1.72 +/- 1.20 vs. 1.04 +/- 1.09 pmol/L, P = 0.019; 6.34 +/- 2.94 vs. 3.71 +/- 2.03 pmol/L, P < 0.001; and 0.40 +/- 0.45 vs. 0.16 +/- 0.17, P = 0.023, respectively). Serum levels of sIL-2R were significantly elevated in the MM type compared with the SM type (2,050 +/- 1,060 vs. 870 +/- 340 pg/ml, P < 0.001). Serum OPG levels were also significantly elevated in the MM type compared with the SM type (7.58 +/- 2.72 vs. 5.13 +/- 2.69 pmol/L, P = 0.008) and negatively correlated with the number of bone lesions (r = -0.56, P = 0.007). In contrast, the sRANKL/OPG ratios were significantly higher in the SM type than the MM type (0.57 +/- 0.54 vs. 0.19 +/- 0.14, P = 0.002) and positively correlated with the number of bone lesions (r = 0.34, P = 0.040). In patients who responded to the induction therapy, serum levels of sIL-2R, sRANKL, and OPG, and the sRANKL/OPG ratio decreased significantly after the therapy (1,170 +/- 600 vs. 730 +/- 290 pg/ml, P = 0.029; 2.19 +/- 1.06 vs. 1.24 +/- 0.66 pmol/L, P < 0.001; 6.13 +/- 2.40 vs. 4.72 +/- 2.03 pmol/L, P = 0.040; and 0.57 +/- 0.52 vs. 0.41 +/- 0.37, P = 0.02, respectively). In the three patients who did not respond to the induction therapy, the serum levels of sCD154 increased significantly after the therapy (1.3 +/- 1.1 vs. 2.7 +/- 1.2, P = 0.004). Serum levels of sIL-2R and sCD154 could be useful as indicators of inflammation and sRANKL/OPG ratios as markers of osteolytic activity in LCH patients.
Leukemia Research, 2014
Upon analyzing 696 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, we id... more Upon analyzing 696 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, we identified the characteristics of CD66c expression. In addition to the confirmation of strong correlation with BCR-ABL positivity and hyperdiploid, we further observed that CD66c is frequently expressed in CRLF2-positive (11/15, p<0.01 against chimeric gene-negative) as well as hypodiploid cases (3/4), whereas it is never expressed in ETV6-RUNX1, MLL-AF4, MLL-AF9, MLL-ENL, and E2A-PBX1-positive cases. Although the expression of CD66c itself is not directly linked to the prognosis, the accompanying genetic abnormalities are important prognostic factors for BCP-ALL, indicating the importance of CD66c expression in the initial diagnosis of BCP-ALL.
Journal of Proteomics, 2011
Lung cancer, COPD and cardiovascular diseases are highlighted as some of the most common disease ... more Lung cancer, COPD and cardiovascular diseases are highlighted as some of the most common disease that cause mortality, and for that reason are the most active areas for drug development. This perspective paper overviews the urgent need to develop a health care system for a rapidly growing patient population in Japan, including forthcoming demands on clinical care, expecting outcomes, and economics. There is an increasing requirement to build on the strengths of the current health care system, thereby delivering urgent solutions for the future. There is also a declaration from the Ministry of Health, Labour and Welfare (MHLW), to develop new biomarker diagnostics, which is intended for patient stratification, aiding in diagnostic phenotype selection for responders to drug treatment of Japanese patients. This perspective was written by the panel in order to introduce novel technologies and diagnostic capabilities with successful implementation. The next generation of personalized drugs for targeted and stratified patient treatment will soon be available in major disease areas such as, lifestyle-related cancers, especially lung cancers with the highest mortality including a predisposing disorder chronic obstructive pulmonary disease, cardiovascular disease, and other diseases. Mass spectrometric technologies can provide the "phenotypic fingerprint" required for the concept of Personalized Medicine. Mass spectrometry-driven target biomarker diagnoses in combination with high resolution computed tomography can provide a critical pathway initiative facilitated by a fully integrated e-Health infrastructure system. We strongly recommend integrating validated biomarkers based on clinical proteomics, medical imaging with clinical care supported by e-Health model to support personalized treatment paradigms to reduce mortality and healthcare costs of chronic and co-morbid diseases in the elderly population of Japan.
Journal of Cellular Biochemistry, 2001
Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many d... more Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1mediated cytotoxicity, we observed that multiple caspases, including caspase-3,-7, and-8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase-8 is located upstream of both caspase-3 and-7, suggesting that Stx1-mediated apoptosis utilizes a similar caspase cascade to that involved in Fas-mediated apoptosis. Neither anti-Fas mAb nor TNF-a, however, affected the Stx1-mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1-mediated activation of caspase-8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with speci®c antibody is suf®cient to induce activation of caspase-8. Our ®ndings should provide new insight into the understanding of the molecular basis of Stx1-mediated cell injury.
Infection and …, 1999
A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. ... more A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1.1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producing E. coli infection.
International Journal of Cancer, 1989
The biochemical characterization of human yolk-sac tumor (YST) antigen 2GI0, detected by monoclon... more The biochemical characterization of human yolk-sac tumor (YST) antigen 2GI0, detected by monoclonal antibody (MAb) MPI2-2GI0, was studied. Previous results indicated that glycolipids having a non-reducing terminal N-acetyllactosamine structure were the epitope of 2G 10 on human erythrocytes. In this study, the glycoprotein nature of 2GIO on the infantile embryonal carcinoma line, MTE, was investigated. 2GlO activity, measured by a new enzyme-linked immunosorbent assay (2G 10-ELISA), was recovered in residual fractions of MTE from which glycolipids were removed. Chromatographically, 3H-galactose-labelled 2G 10 on MTE had a molecular weight (mw) of about 580 kDa, which decreased after pronase or alkaline-borohydride treatment. Our results indicate the glycoprotein nature of ZG I 0 on MTE. Furthermore, 2G 10, both on erythrocytes and on MTE, was sensitive to galactosidase but not to neuraminidase and fucosidase, suggesting that terminal galactose is involved in the antigenic structure. It was also found by 2G 10-ELISA that 2G I0 sheds from tumor cells. Shedding occurs in nude mice transplanted with MTE as well as in patients with germ-cell tumors (GCTs). The serum level o f 2G I0 in non-tumor patients was low, but high levels were detected in patients with YSTs and with GCTs having YST components. Immunohistochemically, the presence of ZG 10positive YST components was shown in patients who had high serum levels of 2GIO. Sera from other urogenital and childhood solid tumors did not have elevated 2GIO. The mw of shed 2G I0 was lower than that of 2G I0 on the cell surfaces. Our results clearly indicate the usefulness of serum 2G 10 as a tumor marker for GCTs having YST components. 'To whom requests for reprints should be addressed.
Experimental Hematology, 2000
Objective. In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene trans... more Objective. In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene transcription, we characterized the 5 Ј-flanking region of the mouse GPV gene. Materials and Methods. The promotor activity of a Ϫ 481 րϩ 22 5 Ј-fragment of the mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, luciferase and green fluorescence protein (GFP). Results. When a DNA construct consisting of this fragment and a GFP reporter gene were transiently expressed in thrombopoietin-supported mouse BMC culture, GFP was identified only in megakaryocytes. The same construct expressed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full Ϫ 481 րϩ 22 fragment could drive variable promoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA. A deletion and point mutation study indicated that GATA and Ets motifs, typical cis-acting elements for platelet-specific genes, located of Ϫ 75 and Ϫ 46, respectively, were essential for promoter function. Conclusion. The GPV promoter has the general characteristics found in platelet-specific genes, and the mechanism for megakaryocyte-specific, maturation-dependent regulation of GPV gene transcription is highly conserved between mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturational process of megakaryocytes.
European Journal of Pediatrics, 2005
We report a 2-year-old Japanese boy without bone marrow involvement who developed a primary granu... more We report a 2-year-old Japanese boy without bone marrow involvement who developed a primary granulocytic sarcoma in his spinal canal. Tumour cells were positive for myeloperoxidase, MIC2, CD56 and, CD68 on formalin-fixed, paraffin-embedded tissue sections and CD13, CD33, CD45, and CD64 on acetone-fixed fresh frozen sections. Nine months after the initiation of treatment, the tumour had significantly regressed and the patient was able to walk with help. Conclusion: Our patient is the youngest case of granulocytic sarcoma of the spine without bone marrow involvement. Immunohistochemical methods are very helpful in establishing a diagnosis of granulocytic sarcoma. Keywords Granulocytic sarcoma AE Myeloid sarcoma AE Spinal cord compression AE Without bone marrow involvement
European Journal of Immunology, 2012
We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in... more We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in Burkitt's lymphoma (BL) among hematologic malignancies. Here, we investigated ZNF385B expression in healthy B cells in a variety of hematological tissues by RT-PCR and immunohistochemistry. ZNF385B expression was found to be limited to a subset of GC B cells, the healthy counterpart to BL B cells. To elucidate the function of ZNF385B in healthy B cells, we established a tetracycline-controlled protein-inducible system in B-cell lines and observed that ectopic expression of the longest transcript variant of ZNF385B, possessing four ZF domains, induced upregulation of PERP and FAS/CD95, a downstream target of p53, and activation of caspase, resulting in apoptosis induction. However, a ZNF385B deletion mutant with three ZF domains corresponding to shorter isoforms, did not induce upregulation; rather it inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. The direct binding of ZNF385B with p53 has suggested the involvement of ZNF385B in B-cell apoptosis via modulation of p53 transactivation; our data indicate that ZNF385B characteristically expressed in GC B cells has both proapoptotic and antiapoptotic activities depending on the type of isoform and should be a novel player in GC B-cell selection.
… and cellular biology, 2008
Yoshitaka Miyagawa, 1 Hajime Okita, 1* Hideki Nakaijima, 1 Yasuomi Horiuchi, 1 Ban Sato, 1 Tomoko... more Yoshitaka Miyagawa, 1 Hajime Okita, 1* Hideki Nakaijima, 1 Yasuomi Horiuchi, 1 Ban Sato, 1 Tomoko Taguchi, 1 Masashi Toyoda, 3 Yohko U. Katagiri, 1 Junichiro Fujimoto, 2 Jun-ichi Hata, 1 Akihiro Umezawa, 3, and Nobutaka Kiyokawa 1
Journal of Histochemistry & Cytochemistry, 1990
Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for... more Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for proliferation and differentiation of granulocytes, has been performed up to the present by use of the granulocyte colony-formation assay, because of the lack of a specific anti-G-CSF antibody. This has prevented the advancement of biological investigations of cell dynamics linked to G-CSF, e.g., cell localization of G-CSF and its pathophysiological changes. In the present work, two monoclonal antibodies (MAb), 1E7 and 4A6, against recombinant human G-CSF (rhG-CSF) were developed by cell hybridization between NS-1 myeloma cells and splenocytes from a mouse immunized with rhG-CSF. 1E7 and 4A6 were shown to be reactive with hG-CSF but not with other CSF (hGM-CSF, hIL-3, and mouse GM-CSF) by Western blot analysis. An immunoperoxidase staining method using these MAb was then established. This method was applicable to frozen sections, paraffin-embedded sections, and cells fixed with 4% parafo...
European Respiratory Journal, Sep 1, 2012
Transplantation proceedings, 1995
Blood cancer journal, 2014
CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult... more CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable progn...
Leukemia, 1990
Ig heavy chain (IgH) J (JH) region organization in 83 human acute leukemias was studied. In 31 of... more Ig heavy chain (IgH) J (JH) region organization in 83 human acute leukemias was studied. In 31 of the 35 precursor B acute lymphocytic leukemia (ALL) the rearrangements of the IgH gene involved sites between D regions 5' to DQ52 and JH. The study of the IgH gene organization in 43 T lineage ALL showed nine cases with the rearrangement at the IgJH region, one of which involved the D region 5' to DQ52. When analyzed with another restriction enzyme (BglII), three of the remaining eight showed rearrangement between DQ52 and JH. In the remaining five samples rearrangement of the IgJH region was not shown, but the restriction fragment length polymorphism (RFLP) between MspI sites in 5'DQ52 loci was observed. Thus the RFLP in 5'DQ52 locus was identified and was distinguished from the recombination between DQ52 and JH in non-B lineage leukemias.
Leukemia, 1991
Three Down's syndrome patients with transient myeloproliferative disorder were studied for cl... more Three Down's syndrome patients with transient myeloproliferative disorder were studied for clonality of the proliferating blast cells using the X chromosome-linked polymorphic gene phosphoglycerate kinase, immunoglobulin heavy chain (IgH) gene and T-cell antigen receptor (TCR) (beta, gamma, delta) genes. None of the three cases showed rearrangements of IgH, TCR beta, gamma, or delta genes, indicating the non-lymphoid nature of the proliferating blast cells. The X chromosome inactivation pattern showed that the cells in the blast population in all of the three cases of transient myeloproliferative disorder were clonal. These data suggest that at least some of this disorder can be due to a spontaneously regressing clone of malignant cells.
Protein Expression and Purification, 2001
cytotoxins, consisting of Stx1 and Stx2 subgroups, pro-A new single-step purification method for ... more cytotoxins, consisting of Stx1 and Stx2 subgroups, pro-A new single-step purification method for Shiga duced by Shigella dysenteriae type 1 and Shiga toxintoxin (Stx) was developed using receptor-mediated producing Escherichia coli. In humans, these toxins affinity chromatography, in which Gb3Cer (globotriaohave been shown to be associated with hemorrhagic sylceramide) was conjugated to octyl Sepharose CL-4B colitis and the hemolytic uremic syndrome (HUS) (1, as a carrier. This method achieves high yield and high 2). Stx is composed of one A subunit associated with purity in a small column on which Gb3Cer has been five B subunits (3). The A subunit inhibits protein synimmobilized at high density. Using this affinity column, thesis by catalytic inactivation of the eucaryotic 60S the Stx1 B subunit was purified with homogeneity by ribosomal subunit by RNA N-glycosidase activity (4), a one-step procedure from a crude extract of recombiwhereas the B subunit serves to mediate binding of the nant Stx1 B subunit-producing Escherichia coli. The toxin to the surface receptors on susceptible cells. The purified Stx1 B subunit conserved a natural pentamer cell surface receptor of the B subunit is a membranestructure confirmed by gel filtration and sedimentacomponent glycosphingolipid globotriaosylceramide tion equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer Gb3Cer, also known as CD77 or blood group P k antigen expressed on Burkitt's lymphoma cells. This versatile (5). In addition to its inhibitory activity on protein synpurification method can be used to isolate various thesis, Stx has been shown to induce apoptosis in sevtypes of natural as well as recombinant Stx, facilitating eral different cells or tissues, including Vero cells (6), fundamental studies of human diseases caused by this human renal tubular epithelia-derived ACHN cells (7), toxin. ᭧ 2001 Academic Press and normal human renal tubular epithelial cells (8, 9). Though the Stx1 B subunit alone cannot inhibit protein synthesis, some Burkitt's lymphoma cell lines could reportedly be induced to undergo apoptosis by treatment The Shiga toxins (Stx's) 2 are a family of bacterial with the Stx1 B subunit or antibody against Gb3Cer (10, 11). In addition, treatment of ACHN and Burkitt's
British Journal of Cancer - BRIT J CANCER, 1989
We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing&... more We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing's sarcoma (ES). 5C11 specifically reacted with ESs but not with other small round cell tumours in childhood, i.e. neuroblastomas, primitive neuroectodermal tumours (PNETs), rhabdomyosarcomas and malignant lymphomas. 5C11 did not react with any other tumours in children except for hepatoblastomas. No reactivity has been identified in normal tissues with the exception of fetal hepatocytes. Immunoelectron microscopically, 5C11 reactive antigen was located on cell membrane of ES cells. Biochemically, 5C11 immunoprecipitated a cell surface protein having molecular weight of 81,000 Da. 5C11 is the first antibody which can clearly distinguish ES from neurogenic tumours, especially from PNETs which were recently reported to have common features to ESs regarding chromosal abnormality and proto-oncogene expression but show evident differentiation into neurogenic direction. The results strongl...
British journal of …, 1991
We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a ... more We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t(11;22) in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity.
PLoS ONE, 2009
Background: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of ... more Background: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/b-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear. Methodology/Principal Findings: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 59 upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice. Conclusions/Significance: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner.
Pediatric Blood & Cancer, 2006
To determine useful biochemical markers in Langerhans cell histiocytosis (LCH), we analyzed the s... more To determine useful biochemical markers in Langerhans cell histiocytosis (LCH), we analyzed the serum levels of soluble CD154 (sCD154), IL2 receptor (sIL2-R), receptor activator of NF-kappaB ligand (sRANKL), and osteoprotegerin (OPG). Our study included 46 newly diagnosed LCH patients (single-system multi-site (SM type): n = 20, and multi-system multi-site (MM type): n = 26) who were treated with the JLSG-02 protocol between 2002 and 2004. The median age of the patients was 3.8 years old (range 0-18). sCD154, sIL2-R, sRANKL, and OPG were measured by ELISA at diagnosis (n = 46) and after 6-weeks of induction therapy (n = 14). The values of sCD154, sIL-2R, sRANKL, and OPG, and the sRANKL/OPG ratio in sera were significantly higher in patients with LCH compared with controls (1.83 +/- 1.38 vs. 0.22 +/- 0.16 ng/ml, P < 0.001; 1,600 +/- 1,060 vs. 420 +/- 160 pg/ml, P < 0.001; 1.72 +/- 1.20 vs. 1.04 +/- 1.09 pmol/L, P = 0.019; 6.34 +/- 2.94 vs. 3.71 +/- 2.03 pmol/L, P < 0.001; and 0.40 +/- 0.45 vs. 0.16 +/- 0.17, P = 0.023, respectively). Serum levels of sIL-2R were significantly elevated in the MM type compared with the SM type (2,050 +/- 1,060 vs. 870 +/- 340 pg/ml, P < 0.001). Serum OPG levels were also significantly elevated in the MM type compared with the SM type (7.58 +/- 2.72 vs. 5.13 +/- 2.69 pmol/L, P = 0.008) and negatively correlated with the number of bone lesions (r = -0.56, P = 0.007). In contrast, the sRANKL/OPG ratios were significantly higher in the SM type than the MM type (0.57 +/- 0.54 vs. 0.19 +/- 0.14, P = 0.002) and positively correlated with the number of bone lesions (r = 0.34, P = 0.040). In patients who responded to the induction therapy, serum levels of sIL-2R, sRANKL, and OPG, and the sRANKL/OPG ratio decreased significantly after the therapy (1,170 +/- 600 vs. 730 +/- 290 pg/ml, P = 0.029; 2.19 +/- 1.06 vs. 1.24 +/- 0.66 pmol/L, P < 0.001; 6.13 +/- 2.40 vs. 4.72 +/- 2.03 pmol/L, P = 0.040; and 0.57 +/- 0.52 vs. 0.41 +/- 0.37, P = 0.02, respectively). In the three patients who did not respond to the induction therapy, the serum levels of sCD154 increased significantly after the therapy (1.3 +/- 1.1 vs. 2.7 +/- 1.2, P = 0.004). Serum levels of sIL-2R and sCD154 could be useful as indicators of inflammation and sRANKL/OPG ratios as markers of osteolytic activity in LCH patients.
Leukemia Research, 2014
Upon analyzing 696 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, we id... more Upon analyzing 696 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, we identified the characteristics of CD66c expression. In addition to the confirmation of strong correlation with BCR-ABL positivity and hyperdiploid, we further observed that CD66c is frequently expressed in CRLF2-positive (11/15, p<0.01 against chimeric gene-negative) as well as hypodiploid cases (3/4), whereas it is never expressed in ETV6-RUNX1, MLL-AF4, MLL-AF9, MLL-ENL, and E2A-PBX1-positive cases. Although the expression of CD66c itself is not directly linked to the prognosis, the accompanying genetic abnormalities are important prognostic factors for BCP-ALL, indicating the importance of CD66c expression in the initial diagnosis of BCP-ALL.
Journal of Proteomics, 2011
Lung cancer, COPD and cardiovascular diseases are highlighted as some of the most common disease ... more Lung cancer, COPD and cardiovascular diseases are highlighted as some of the most common disease that cause mortality, and for that reason are the most active areas for drug development. This perspective paper overviews the urgent need to develop a health care system for a rapidly growing patient population in Japan, including forthcoming demands on clinical care, expecting outcomes, and economics. There is an increasing requirement to build on the strengths of the current health care system, thereby delivering urgent solutions for the future. There is also a declaration from the Ministry of Health, Labour and Welfare (MHLW), to develop new biomarker diagnostics, which is intended for patient stratification, aiding in diagnostic phenotype selection for responders to drug treatment of Japanese patients. This perspective was written by the panel in order to introduce novel technologies and diagnostic capabilities with successful implementation. The next generation of personalized drugs for targeted and stratified patient treatment will soon be available in major disease areas such as, lifestyle-related cancers, especially lung cancers with the highest mortality including a predisposing disorder chronic obstructive pulmonary disease, cardiovascular disease, and other diseases. Mass spectrometric technologies can provide the "phenotypic fingerprint" required for the concept of Personalized Medicine. Mass spectrometry-driven target biomarker diagnoses in combination with high resolution computed tomography can provide a critical pathway initiative facilitated by a fully integrated e-Health infrastructure system. We strongly recommend integrating validated biomarkers based on clinical proteomics, medical imaging with clinical care supported by e-Health model to support personalized treatment paradigms to reduce mortality and healthcare costs of chronic and co-morbid diseases in the elderly population of Japan.
Journal of Cellular Biochemistry, 2001
Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many d... more Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1mediated cytotoxicity, we observed that multiple caspases, including caspase-3,-7, and-8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase-8 is located upstream of both caspase-3 and-7, suggesting that Stx1-mediated apoptosis utilizes a similar caspase cascade to that involved in Fas-mediated apoptosis. Neither anti-Fas mAb nor TNF-a, however, affected the Stx1-mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1-mediated activation of caspase-8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with speci®c antibody is suf®cient to induce activation of caspase-8. Our ®ndings should provide new insight into the understanding of the molecular basis of Stx1-mediated cell injury.
Infection and …, 1999
A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. ... more A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1.1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producing E. coli infection.
International Journal of Cancer, 1989
The biochemical characterization of human yolk-sac tumor (YST) antigen 2GI0, detected by monoclon... more The biochemical characterization of human yolk-sac tumor (YST) antigen 2GI0, detected by monoclonal antibody (MAb) MPI2-2GI0, was studied. Previous results indicated that glycolipids having a non-reducing terminal N-acetyllactosamine structure were the epitope of 2G 10 on human erythrocytes. In this study, the glycoprotein nature of 2GIO on the infantile embryonal carcinoma line, MTE, was investigated. 2GlO activity, measured by a new enzyme-linked immunosorbent assay (2G 10-ELISA), was recovered in residual fractions of MTE from which glycolipids were removed. Chromatographically, 3H-galactose-labelled 2G 10 on MTE had a molecular weight (mw) of about 580 kDa, which decreased after pronase or alkaline-borohydride treatment. Our results indicate the glycoprotein nature of ZG I 0 on MTE. Furthermore, 2G 10, both on erythrocytes and on MTE, was sensitive to galactosidase but not to neuraminidase and fucosidase, suggesting that terminal galactose is involved in the antigenic structure. It was also found by 2G 10-ELISA that 2G I0 sheds from tumor cells. Shedding occurs in nude mice transplanted with MTE as well as in patients with germ-cell tumors (GCTs). The serum level o f 2G I0 in non-tumor patients was low, but high levels were detected in patients with YSTs and with GCTs having YST components. Immunohistochemically, the presence of ZG 10positive YST components was shown in patients who had high serum levels of 2GIO. Sera from other urogenital and childhood solid tumors did not have elevated 2GIO. The mw of shed 2G I0 was lower than that of 2G I0 on the cell surfaces. Our results clearly indicate the usefulness of serum 2G 10 as a tumor marker for GCTs having YST components. 'To whom requests for reprints should be addressed.
Experimental Hematology, 2000
Objective. In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene trans... more Objective. In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene transcription, we characterized the 5 Ј-flanking region of the mouse GPV gene. Materials and Methods. The promotor activity of a Ϫ 481 րϩ 22 5 Ј-fragment of the mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, luciferase and green fluorescence protein (GFP). Results. When a DNA construct consisting of this fragment and a GFP reporter gene were transiently expressed in thrombopoietin-supported mouse BMC culture, GFP was identified only in megakaryocytes. The same construct expressed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full Ϫ 481 րϩ 22 fragment could drive variable promoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA. A deletion and point mutation study indicated that GATA and Ets motifs, typical cis-acting elements for platelet-specific genes, located of Ϫ 75 and Ϫ 46, respectively, were essential for promoter function. Conclusion. The GPV promoter has the general characteristics found in platelet-specific genes, and the mechanism for megakaryocyte-specific, maturation-dependent regulation of GPV gene transcription is highly conserved between mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturational process of megakaryocytes.
European Journal of Pediatrics, 2005
We report a 2-year-old Japanese boy without bone marrow involvement who developed a primary granu... more We report a 2-year-old Japanese boy without bone marrow involvement who developed a primary granulocytic sarcoma in his spinal canal. Tumour cells were positive for myeloperoxidase, MIC2, CD56 and, CD68 on formalin-fixed, paraffin-embedded tissue sections and CD13, CD33, CD45, and CD64 on acetone-fixed fresh frozen sections. Nine months after the initiation of treatment, the tumour had significantly regressed and the patient was able to walk with help. Conclusion: Our patient is the youngest case of granulocytic sarcoma of the spine without bone marrow involvement. Immunohistochemical methods are very helpful in establishing a diagnosis of granulocytic sarcoma. Keywords Granulocytic sarcoma AE Myeloid sarcoma AE Spinal cord compression AE Without bone marrow involvement
European Journal of Immunology, 2012
We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in... more We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in Burkitt's lymphoma (BL) among hematologic malignancies. Here, we investigated ZNF385B expression in healthy B cells in a variety of hematological tissues by RT-PCR and immunohistochemistry. ZNF385B expression was found to be limited to a subset of GC B cells, the healthy counterpart to BL B cells. To elucidate the function of ZNF385B in healthy B cells, we established a tetracycline-controlled protein-inducible system in B-cell lines and observed that ectopic expression of the longest transcript variant of ZNF385B, possessing four ZF domains, induced upregulation of PERP and FAS/CD95, a downstream target of p53, and activation of caspase, resulting in apoptosis induction. However, a ZNF385B deletion mutant with three ZF domains corresponding to shorter isoforms, did not induce upregulation; rather it inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. The direct binding of ZNF385B with p53 has suggested the involvement of ZNF385B in B-cell apoptosis via modulation of p53 transactivation; our data indicate that ZNF385B characteristically expressed in GC B cells has both proapoptotic and antiapoptotic activities depending on the type of isoform and should be a novel player in GC B-cell selection.
… and cellular biology, 2008
Yoshitaka Miyagawa, 1 Hajime Okita, 1* Hideki Nakaijima, 1 Yasuomi Horiuchi, 1 Ban Sato, 1 Tomoko... more Yoshitaka Miyagawa, 1 Hajime Okita, 1* Hideki Nakaijima, 1 Yasuomi Horiuchi, 1 Ban Sato, 1 Tomoko Taguchi, 1 Masashi Toyoda, 3 Yohko U. Katagiri, 1 Junichiro Fujimoto, 2 Jun-ichi Hata, 1 Akihiro Umezawa, 3, and Nobutaka Kiyokawa 1