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T Naoe

Publications and source records attributed to T Naoe.

At least 109 records · Page 6Linked to original sources
PubMed1997

Mutant AF-2 domain of PML-RARalpha in retinoic acid-resistant NB4 cells: differentiation induced by RA is triggered directly through PML-RARalpha and its down-regulation in acute promyelocytic leukemia.

To study the molecular mechanism of the differentiation induced by retinoic acid (RA) in acute promyelocytic leukemia (APL), we established a new RA-resistant NB4 subline, NB4/RA. The NB4/RA cells were neither differentiated by a single or a combination of RA isoforms, nor by the addition of clotrimazole (P450-inhibitor) or interferon gamma. However, the combination of RA and 8-(4-chlorophenylthio) adenosine cyclic 3',5'-monophosphate (a cAMP analog, 8-CPT-cAMP) induced differentiation. Immunostaining of NB4/RA cells using anti-PML antibody showed a microgranular pattern which was not restored even by the combination of RA and 8-CPT-cAMP, whereas the microgranular pattern in NB4 cells was rapidly restored to the normal speckled pattern by RA. Western blot analysis revealed that RA alone or the combination with 8-CPT-cAMP did not down-regulate PML-RARalpha in NB4/RA cells, which was in contrast to NB4 cells. The PML-RARalpha fusion gene and transcript in NB4/RA cells were conserved as well as the RARalpha gene and transcripts. Sequence analysis of the PML-RARalpha transcript in NB4/RA cells indicated a Pro (CCG) to Leu (CTG) mutation at codon 900 (type L) in AF-2 domain, while the RARalpha transcript had a normal sequence. These data suggest that differentiation of APL by RA is triggered directly through PML-RARalpha, and is associated with its degradation. Furthermore, there might be another mechanism of differentiation which does not require the down-regulation of PML-RARalpha and the restoration of the PML-staining pattern.

Amino Acid Sequence↗
PubMed1997

Interaction of von Willebrand factor with the extracellular matrix and glycocalicin under static conditions.

The binding of human von Willebrand factor (vWF) to a variety of extracellular matrix components immobilized on plates and the binding of vWF to platelet glycoprotein Ib (GPIb) after interacting with these matrix components were examined by means of an enzyme-linked immunosorbent assay. vWF preferably bound to type III collagen, whereas it did not significantly bind to type I, IV, V, or VI collagen, fibronectin, laminin, elastin, or proteoglycans. Soluble type III collagen did not bind to vWF coated on plates and showed a little effect on the vWF binding to the immobilized collagen, suggesting that solid-phase collagen is important for the interaction with vWF. When glycocalicin, the N-terminal carbohydrate-rich extracellular domain of GPIb alpha exhibiting the vWF-binding activity, was added to vWF bound to collagen type III, no significant binding of glycocalicin was observed, but it bound to vWF in the presence of botrocetin, a vWF modulator protein isolated from Bothrops jararaca snake venom. These results indicate that vWF immobilized on collagen can interact with GPIb but that binding of vWF to the collagen matrix alone is insufficient for modulating vWF so that it interacts with GPIb under static conditions. Another unknown physiological modulator functionally mimicking botrocetin or high-shear stress may be involved in the platelet adhesion to extracellular matrix in the early stage of hemostasis.

Antibodies, Monoclonal↗
PubMed1997

Immunoaffinity purification and characterization of CACGTG sequence-binding proteins from cultured mammalian cells using an anti c-Myc monoclonal antibody recognizing the DNA-binding domain.

c-Myc protein contains a basic helix-loop-helix and leucine zipper (bHLHLZ) structure in its carboxyl terminal region, forms heterodimers with Max, and binds to the CACGTG sequence in DNA. A number of bHLHLZ proteins are present in cells, and some of them bind to the same DNA sequence. Using an anti c-Myc monoclonal antibody, MYC5, raised against a bacterially synthesized c-Myc protein, we have carried out immunoaffinity purification of c-Myc proteins from cultured mammalian cells. The immunoaffinity-purified fraction was found to contain not only c-Myc but also other CACGTG sequence-binding proteins, such as Max, Mad, and USF, indicating a wide cross-reaction to CACGTG sequence-binding proteins. The MYC5 antibody recognized the common structural motif in their basic region required for sequence-specific DNA binding and was shown to inhibit their DNA-binding activities. The immunoaffinity-purified N-Myc, Max, Mad, and, presumably, c-Myc were highly phosphorylated, and phosphatase treatment increased the DNA-binding activity of Myc, suggesting that the DNA-binding activity of c-Myc was regulated by phosphorylation in vivo. From these results, we can conclude that the MYC5 antibody constitutes a powerful tool for the purification and characterization of c-Myc and other CACGTG sequence-binding proteins.

Antibodies, Monoclonal↗
PubMed1997

De novo mutation of the platelet glycoprotein Ib alpha gene in a patient with pseudo-von Willebrand disease.

Pseudo (or platelet-type)- von Willebrand disease (vWD) is a very rare autosomal dominant bleeding disorder caused by an abnormal hyper-responsiveness of the platelet membrane glycoprotein (GP) Ib/IX complex, the receptor for von Willebrand factor. We found a heterozygous missense mutation in the GPIb alpha gene in a sporadic case with pseudo-vWD: Met (ATG) to Val (GTG) at residue 239. The mutation was not detected in either parent. Investigation of three variable number of tandem repeat loci, D1S80 (MCT118), vWA and D17S5 (YNZ22), confirmed paternity and the de novo origin of the mutation. Furthermore, we have shown by the TaqI polymorphism analysis, which is located downstream of the GPIb alpha gene, that the mutation occurred in the maternal allele. This is the first description of de novo mutation occurred in pseudo-vWD and/or platelet GPIb alpha gene.

Adult↗
PubMed1997

Molecular analysis of the t(15;17) translocation in de novo and secondary acute promyelocytic leukemia.

To study mechanism of chromosomal translocation, we analyzed the breakpoints (b/p) of the PML and RARA genes in 120 and 5 patients with de novo and secondary (therapy-related) acute promyelocytic leukemia (APL), respectively. In de novo APL, the b/p in the PML gene were clustered in introns 3 (bcr 3; 30%) and around intron 6 (bcr 1 and 2: 70%). The b/p of the RARA gene were widely distributed in intron 2. In studied 8 de novo APL patients, no consensus sequence-motif was found around the b/p, but there were identical stretches of one to seven nucleotides between the PML and RARA genes in the joining regions, suggesting non-selective DNA double strand cleavage followed by single strand base-pairing within identical short stretches as a molecular mechanism of the translocation. In 4 secondary APL patients after chemotherapy including etoposide against Langerhans cell histiocytosis, the b/p of the PML gene were located in intron 6, and those of the RARA gene were in a restricted region within intron 2, 1 kb EcoRI-BamHI fragment, while in an APL patient after chemotherapy without etoposide against breast cancer, the b/p of the PML and RARA genes were located in intron 6 and another region within intron 2, respectively. These data suggest that a different mechanism was associated with the t(15;17) translocation in etoposide-related APL.

Antineoplastic Agents, Phytogenic↗
PubMed1996

In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Apoptosis↗
PubMed1996

Accelerated degradation of PML-retinoic acid receptor alpha (PML-RARA) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway.

Acute promyelocytic leukemia (APL) is associated with a chromosomal translocation t(15;17) and successfully differentiated by all-trans-retinoic acid (ATRA) in vivo as well as in vitro. The PML-retinoic acid receptor alpha (RARA) oncoprotein, which is generated by the translocation, blocks the differentiation, and ATRA is thought to modulate the dominant negative function of PML-RARA. However, the molecular effect of ATRA on PML-RARA is unknown. In this study, we showed by means of immunoblotting that the expression of PML-RARA decreased within 12 h in APL cells treated with ATRA at concentrations greater than 0.1 microM. The decrease of PML-RARA was associated with restoration of the normal subcellular PML localization. PML-RARA transcripts were not down-regulated by ATRA. However, lactacystin, a specific inhibitor of the proteasome, almost completely inhibited the decrease of PML-RARA. These data indicate that the PML-RARA degradation is accelerated by pharmacological concentrations of ATRA, suggesting that ATRA allows APL cells to differentiate by relieving the differentiation block.

Acetylcysteine↗
PubMed1996

cAMP signalling is decisive for recovery of nuclear bodies (PODs) during maturation of RA-resistant t(15;17) promyelocytic leukemia NB4 cells expressing PML-RAR alpha.

We analysed the expression of retinoid receptors and PML in relation to the morphology of PML-containing nuclear bodies (PODs) in maturation sensitive (NB4) and resistant subclones (NB4-R1 and R2) of the promyelocytic NB4 cell line. The basal level of RARalpha, RXRalpha and PML mRNA and protein were roughly the same in the three cell lines. While NB4 and NB4-R1 cells express comparable amounts of PM-RARalpha mRNA and 120 kDa protein, NB4-R2 cells despite normal mRNA levels the 120 kDa protein was not detectable. In NB4-R2 cells however, two novel PML-related entities of 65 kDa and 85 kDA were detected with a anti-PML antibody, in addition to the two PML isoforms of 78 and 97 kDa found in any NB4 cells. Despite the 120 kDa PML-RARalpha defect, NB4-R2 cells show micropunctuated nuclear bodies typical of APL cells. Contrasting with NB4 cells, neither NB4-R1 cells which express PML-RARalpha, nor NB4-R2 cells lacking the 120 kDa PML-RARalpha reorganised nuclear bodies (PODs) in response to RA. Importantly, in RA-primed NB4-R1 cells, a secondary event triggered by cAMP restored PODs, concomitant to maturation. This indicates that the recovery of nuclear bodies in APL is dissociated from the early action of RA in cell maturation. Finally, the key finding of this work is that cAMP signalling ultimately determines the recovery of nuclear bodies associated to cell maturation.

Cell Cycle↗
PubMed1996

Different T-cell receptor repertoires between lesions and peripheral blood in acute graft-versus-host disease after allogeneic bone marrow transplantation.

From the viewpoint of T-cell receptor (TCR) repertoire, we studied the role of T cells in acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) from an HLA-identical sibling. By means of inverse polymerase chain reaction method and DNA sequencing, we analyzed TCR-alpha and -beta transcripts from GVHD lesions and peripheral blood (PB) in a patient with typical GVHD together with PB from donor. At the initial onset of GVHD, V alpha-7 and -19 subfamilies were oligoclonally expanded in the PB compared with those in the oral mucosal lesions. At the second onset, V alpha-2, and V beta-6 subfamilies were more frequently detected in the cutaneous lesion than in the PB. Some TCR transcripts were recurrently found either in the mucosal or cutaneous lesions (or in both) and not in the PB. Furthermore, some of recurrent TCR transcripts in the lesions shared V gene segments and common motifs of complementarity determining region-3. These findings suggested that T cells infiltrating the GVHD lesions recognized a limited kind of antigens presented by patient's tissues with GVHD, and that T-cell repertoire in the GVHD lesions was different from that in the PB.

Acute Disease↗
PubMed1996

Feasibility of salvage chemotherapy for refractory or relapsed non-Hodgkin's lymphoma with two topoisomerase II inhibitors, MST-16 and VP-16. MST-16 Study Group.

A feasibility study was carried out on the treatment for refractory and relapsed non-Hodgkin's lymphomas with a combination of two oral topoisomerase II inhibitors, MST-16 and VP-16. On the basis of the synergistic activity in preclinical studies and the schedule dependency in these drugs, low-dose and long-term administration was planned. For the anticipated myelosuppression, two different regimens were designed as an open label trial in this study. In Regimen I, 400 mg of MST-16 combined with 25 mg of VP-16 was administered daily. With this regimen, the response rate (RR)/median time to tumor progression (TTP) in all evaluable patients was 50% (2/4)8.5 months in low grade (indolent) lymphoma and 60% (6/10)/5.2 months in intermediate/high grade (aggressive) lymphomas. In Regimen II, 400 mg of MST-16 combined with 25 mg of VP-16 was administered intermittently (3 days a week or every other day). With this regimen, there was an RR/median TTP of 60% (3/5)/7.0 months in indolent lymphoma and 33.3% (4/12)/1.1 months in aggressive lymphoma. A major side effect in both of these regimens was myelosuppression, with the incidence of grades 3 and 4 toxicity being higher in Regimen I than in Regimen II. The other side effects were uncommon and not severe. These findings indicated that two regimens were tolerated well and were promising for refractory and relapsed aggressive non-Hodgkin's lymphomas. To define the anti-tumor activity and safety of these regimens precisely, large-scale prospective randomized trials are necessary.

Adolescent↗
PubMed1996

Molecular cloning of the breakpoint of t(11;22) (q23;q11) chromosome translocation in an adult acute myelomonocytic leukaemia.

Southern blot analysis with a cDNA probe of MLL indicated that the breakpoint is in a BamHI 8.3 kb fragment which carries the exon 5-11 of MLL gene in DNA from an adult acute myelomonocytic leukaemia with a t(11;22) (q23;q11) translocation. The structural analysis of the rearranged MLL locus demonstrated that the breakpoint is localized between exon 8 and 9 of MLL locus. The normal counterpart fused to the MLL locus was proved to be derived from chromosome 22q11(AF-22) by somatic cell hybrids analysis and FISH. By FISH, AF-22 was localized to the region more centromeric to the BCR gene.

Adult↗
PubMed1996

Comparison of the immunoglobulin heavy-chain complementarity determining region-3 structure among the DNA sequences and the mu- and gamma-transcripts in human B-lineage cells.

To study the recombinational significance of the (immunoglobulin heavy) IgH chain gene in human B-cell development, we compared the complementarity determining region (CDR)-3 sequences of the DNA and the mu-transcripts from human normal pre-B cells and mature B cells, and the gamma-transcripts from bone marrow cells. The CDR-3 sequences were longer in the DNA than in the mu- and gamma-transcripts, and this was independent of whether or not the rearrangement was productive. The DLR family genes were less frequently used in the mu- and gamma-transcripts. When translated into amino acids, all CDR-3 sequences from the mu- and gamma-transcripts were productive, although 26.2% of the DNA sequences had stop codons in the D element and/or frameshifts of the JH gene segments. The CDR-3 of the productive DNA sequences in pre-B cells frequently (26.6%) contained at least three continuous hydrophobic amino acids, which were mainly coded by the DLR and DXP family genes at the third reading frame. However, such motifs were rate in the mu-transcripts of pre-B (7.7%) and mature B cells (3.9%), and in the gamma-transcripts of bone marrow cells (1.1%) as well as in the DNA of mature B cells (10.4%). These findings suggested that the length and/or hydrophobicity of the IgH CDR-3 might play a role in the selection mechanisms of B-cell development.

Adult↗
PubMed1996

Serum thrombopoietin and plasma glycocalicin concentrations as useful diagnostic markers in thrombocytopenic disorders.

Using enzyme-linked immunosorbent assays, we measured the concentrations of serum thrombopoietin (TPO) and plasma glycocalicin, a proteolytic fragment of platelet glycoprotien Ib alpha, in 13 patients with myelodysplastic syndrome (MDS), aplastic anaemia (AA) or idiopathic thrombocytopenic purpura (ITP). In the patients with AA or MDS, the TPO concentrations were remarkably increased, and their glycocalicin concentrations were decreased compared with the normal control individuals. In the patients with ITP, however, the TPO and glycocalicin levels were not changed as much as in the AA/MDS patients in spite of the same degree of thrombocytopenia. During immunosuppressive treatment of ITP patients, there was an inverse relationship between the level of TPO and the platelet count. Thus, measurements of TPO and glycocalicin levels are useful for the diagnosis of thrombocytopenia, and our results from ITP patients did not support the model which suggested the simple feedback regulation of TPO in thrombocytopenia.

Adult↗
PubMed1996

Increased but highly dispersed levels of plasma glycocalicin in patients with disseminated intravascular coagulation.

Thrombocytopenia is one of the most common laboratory manifestations of disseminated intravascular coagulation (DIC). To investigate whether thrombocytopenia in DIC is indeed due to platelet consumption, we measured the plasma levels of glycocalicin, a proteolytic fragment of the platelet membrane glycoprotein (GP) Ib alpha, a component of the GPIb/IX complex, in patients with solid tumors either with DIC(n=18) or without DIC (n=18). Patients with DIC had significantly elevated levels of glycocalicin(2.53 +/- 1.59 mg/1;n=18,p<0.05) compared to those without DIC (1.50 +/- 0.41 mg/1;n=18), indicating enhanced platelet consumption, or destruction, in patients with DIC. However, glycocalicin concentrations had a wide range (0.18-5.74 mg/1), and 3 DIC patients had levels lower than the normal lower limit. These findings suggest that, in patients with DIC, thrombocytopenia is not always due to increased platelet consumption, but it may be due to decreased production of platelets. Determination of plasma glycocalicin concentrations is an easy way to identify thrombocytopenia, due to bone marrow insufficiency in these patients.

Adult↗
PubMed1996

Randomized trials between behenoyl cytarabine and cytarabine in combination induction and consolidation therapy, and with or without ubenimex after maintenance/intensification therapy in adult acute myeloid leukemia. The Japan Leukemia Study Group.

PURPOSE: We analyzed complete remission (CR), disease-free survival (DFS), and event-free survival (EFS) rates in two groups of patients treated with either N4-behenoyl-1-beta-D-arabinosylcytosine (BHAC) or cytarabine, and analyzed DFS with or without ubenimex, a biologic response modifier. PATIENTS AND METHODS: Newly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either BHAC or cytarabine as remission-induction combination chemotherapy and two courses of consolidation therapy. After maintenance/intensification therapy, patients in CR were randomized to receive either ubenimex and no drug. RESULTS: Of 341 patients registered, 326 were assessable. The age of assessable patients ranged from 15 to 82 years (median, 48). The overall CR rate was 77%: 72% in the BHAC group and 81% in the cytarabine group, and there was a significant difference between the two groups (P = .035, chi 2 test). The predicted 55-month EFS rate of all patients was 30%: 23% in the BHAC group and 35% in the cytarabine group, with a significant difference between groups (P = .0253). The predicted 55-month DFS rate of all CR patients was 38% and that of CR patients less than 50 years of age was 47%. There was no significant difference in DFS between the ubenimex group and the group that did not receive ubenimex. CONCLUSION: Analyses of our clinical trial showed that the use of BHAC in remission-induction therapy and in consolidation therapy resulted in poorer CR and EFS rates in adult AML patients compared with the use of cytarabine at the doses and schedules tested. Immunotherapy with ubenimex after the end of all chemotherapy did not improve DFS.

Acute Disease↗
PubMed1996

[Detection of minimal residual leukemia using polymerase chain reaction method].

Until recently, it was impossible to identify leukemia cells making up less than 1% of a bone marrow sample, which is designated as minimal residual disease (MRD). Owing to the development of the polymerase chain reaction (PCR) MRD can be detected at the 10(-5) level by amplifying the leukemia-specific DNA rearrangement (BCR/ABL, PML/RARA etc.) or clone-specific DNA sequences (IgH chain CDR-III etc.). Here, our studies on MRD of acute lymphoblastic leukemia and acute promyelocytic leukemia are presented, and their technical problems and clinical significance are discussed.

DNA, Neoplasm↗
PubMed1996

Comparable gene structure of the immunoglobulin heavy chain variable region between multiple myeloma and normal bone marrow lymphocytes.

To characterize multiple myeloma (MM) from the viewpoint of the immunoglobulin (Ig) gene structure, we compared the transcripts of the Ig heavy chain variable region from 23 MM samples with 221 clones of the gamma, alpha and mu chain transcripts amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) from normal bone marrow (BM) cells. The usage of D and JH gene segments and the length of the N regions were the same between MM and the normal gamma, alpha and mu transcripts. Compared with the known germline VH genes, the frame work regions (FWRs) and complementarity determining regions (CDRs) of the VH segments mutated at rates of 8.3 +/- 4.7% and 15.9 +/- 7.7%, respectively, which were the same as the normal gamma and alpha (gamma/alpha) transcripts and higher than the normal mu transcripts. The replacement/silent (R/S) ratios of the mutations in FWRs and CDRs were 1.9 +/- 1.3 and 2.7 +/- 1.8, respectively, which were the same as the gamma/alpha and mu transcripts. On the other hand, we detected the clone-specific mu transcripts by RT-PCR using the primers corresponding to the each respective CDR-III and the constant region of the mu chain in three of the studied six MM samples, suggesting the involvement of a pre-switched B cell in some cases of MM. These findings suggested that the cellular origin of MM is heterogeneous, but that the Ig structure in MM reflects normal B cell maturation to plasma cell through mutation and selection.

Adult↗