Biomedical subjects

J Minowada

Publications and source records attributed to J Minowada.

At least 37 records · Page 2Linked to original sources

PubMed1995

Hepatocyte growth factor--pleiotropic cytokine produced by human leukemia cells.

Hepatocyte growth factor (HGF) was identified, purified and molecularly cloned as a potent mitogen for mature rat hepatocytes in primary culture. It is one of the largest cytokines and is composed of disulfide-linked subunits of approximately 60 (heavy chain) and 35 kilodaltons (light chain). Recent observations revealed that HGF is mitogenic to various epithelial cells other than hepatocytes and to endothelial cells, and that it also acts as a motogen, morphogen and tumor-suppressor as well as a mitogen. These various biological activities of HGF are presumably transduced through the same receptor, c-Met, which is a member of the tyrosine kinase receptor family. Although it shows multiple biological activities on cells in culture, HGF is most likely the physiological hepatotrophic factor which triggers liver regeneration. It may also function as a renotrophic and pulmotrophic factor after tissue injury. HGF production in the liver, kidney and lung increases after injury to these organs. An elevated HGF level may act as an inducer of compensatory DNA synthesis. The regulation of HGF production is, therefore, important for the control of organ regeneration. HGF is produced mainly by mesenchymal cells such as fibroblasts and vascular smooth muscle cells. Various types of human leukemia cells also secrete HGF both in vitro and in vivo. Some biological activities of HGF on hematopoietic cells, including co-mitogenic activity on myeloid leukemia cell lines, were recently demonstrated. HGF gene expression and the protein production in leukemia and fibroblast cells are modulated by various cytokines and hormones. Those modulators may indirectly affect organ regeneration and other biological processes by controlling HGF production.

Animals↗

PubMed1995

Molecular characterization of a chromosome translocation breakpoint t(11;14)(p13;q11) from the cell line KOPT-K1.

Recurrent chromosome translocations involving 11p13 and 14q11 are found in 5-10% of cases of T-ALL. The gene involved in the translocation on chromosome 14 is the T cell antigen receptor alpha or delta. The putative oncogene on chromosome 11 is rhombotin 2 (RBTN2)/translocated in T cell gene 2 (ttg-2), a member of the LIM family of proteins. In this paper we characterize a cell line KOPT-K1 that has a t(11;14)(p13;q11). The breakpoint on chromosome 11 involves an Alu-rich region with the break occurring between two Alu sequences on chromosome 11. In addition, approximately 70 bases from the break on chromosome 11 is a tetranucleotide repeat. Whether either of these structures played a role in the translocation is not known. No heptamer or nonamer sequences, implicated in other rearrangements were found near the breakpoint. The breakpoint on chromosome 11 maps more centromeric than previous translocations of this region. Despite this the RBTN2 gene is highly expressed in KOPT-K1. This cell line will be useful for investigating the role of RBTN2 in leukemogenesis and the mechanism by which the translocation alters the expression of RBTN2.

Base Sequence↗

PubMed1994

Characterization of a species of non-specific cross-reacting antigen (NCA) expressed by human monocytic cell lines: structure and expression during cell differentiation.

It has been documented that human monocytes/macrophages are reactive with antibodies directed to carcinoembryonic antigen (CEA) and non-specific cross-reacting antigens (NCAs), a group of glycoproteins antigenically cross-reactive with CEA, yet the molecules responsible for this antigenic activity have not been fully clarified. In the present study, among 7 myelomonocytic cell lines tested, 2 monoblastoid lines, U-937 and THP-1, were found to express NCA-50/90, a glycosylphosphatidylinositol-anchored cell-adhesion molecule chiefly expressed on granulocytes. The 2 cell lines showed a reaction pattern with 5 distinct anti-CEA and anti-NCA monoclonal antibodies, similar to that of CHO transfectants expressing recombinant NCA-50/90. Immunoprecipitation and SDS-PAGE analyses identified glycoproteins of about 95 and 55 kDa in U-937 and THP-1 cells, respectively. Deglycosylation of the 2 antigens with N-glycanase gave the same apparent molecular mass of about 45,000, which was also the same as that of the deglycosylated form of the recombinant NCA-50/90. Upon Northern-blot analysis, only one band of approximately 2.5 kb was detected in both cell lines with a cDNA probe for NCA-50/90, which has a broad specificity to the CEA gene family members. cDNA cloning demonstrated that the 2.5-kb clones encode the peptide of NCA-50/90. The expression of NCA-50/90 by U-937 and THP-1 was down-regulated at both the protein and mRNA levels during cell differentiation from monoblastoid to monocyte/macrophage-like cells induced by stimulation with phorbol 12-myristate 13-acetate. Our observations suggest that NCA-50/90 is a differentiation antigen of cells of the monocyte/macrophage lineage as well as of the granulocyte lineage.

Antigens, Differentiation, Myelomonocytic↗

PubMed1994

Natural human interferon-alpha augments apoptosis in activated T cell line.

We have previously shown that interferon-alpha (IFN-alpha) augments interleukin-2 (IL-2) production in mitogen-activated but not unstimulated T cells (1). Here, we studied the effect of IFN-alpha on activation-driven cell death (apoptosis) using a human leukemia T cell line, MOLT-16, as a T cell activation model. IFN-alpha alone had no effect on either the IL-2 production or apoptosis of MOLT-16 cells, but significantly increased both the IL-2 production and apoptosis in the MOLT-16 cells after phytohemagglutinin (PHA) stimulation as determined by CTLL-2 assay and by flow cytometric analysis using propidium iodide (PI) staining. Since IL-2 has been shown to induce apoptosis in some systems, we next evaluated whether the apoptosis in MOLT-16 cells was due to endogenous IL-2 production upon PHA stimulation. However, the addition of exogenous IL-2 to unstimulated cultures of MOLT-16 did not induce DNA fragmentation, a characteristic feature of apoptosis, as determined by DNA electrophoresis and by flow cytometric analysis with PI-stained cells. Furthermore, anti-IL-2 antibody did not prevent PHA-induced DNA fragmentation in MOLT-16 cells. Thus, we conclude that both PHA-induced IL-2 production and apoptosis are outcomes of T cell activation and that IFN-alpha may exert immunoregulatory effects on T cell activation by augmenting both processes.

Apoptosis↗

PubMed1994

Interleukin-1 and interferon-alpha augment interleukin-2 (IL-2) production by distinct mechanisms at the IL-2 mRNA level.

The production of interleukin-2 (IL-2) by phytohemaglutinin (PHA)-stimulated cells of human leukemia T cell line MOLT-16 can be significantly increased by interleukin-1 (IL-1) or interferon-alpha (IFN-alpha). The enhancing effect of IL-1 and IFN-alpha on IL-2 production was studied at the IL-2 mRNA level. We show that IL-1 enhances considerably and transiently, with the maximum level between 1 and 2 hr after stimulation, the expression of IL-2 mRNA in the PHA-stimulated cells. The level of IL-2 mRNA declined rapidly within 4 to 6 hr after stimulation in both PHA- and PHA plus IL-1-stimulated cell cultures. On the contrary, IFN-alpha does not elevate the level of IL-2 mRNA above the level in PHA-stimulated cultures, but maintains an enhanced level of IL-2 mRNA in the activated cells for more than 6 hr after stimulation. These observations correlate well with the kinetics of IL-2 protein production into the culture media. The results thus suggest that IL-1 and IFN-alpha may exert an enhancing effect on IL-2 production by distinct mechanisms. In addition, none of the five other lymphokines tested (i.e., IL-2, IL-3, IL-4, IL-5, and IL-6) had any significant effect on IL-2 mRNA expression in the activated MOLT-16 cells.

Cell Line↗

PubMed1994

The phylogeny of proteinase 3/myeloblastin, the autoantigen in Wegener's granulomatosis, and myeloperoxidase as shown by immunohistochemical studies on human leukemic cell lines.

Proteinase 3/myeloblastin (Pr3/MBN) is a serine protease found in primary granules of neutrophilic granulocytes and monocytes in man. This enzyme has been identified as the main autoantigen in Wegener's granulomatosis. Pr3/MBN was earlier identified in the promyelocytic cell line HL-60 and was assumed to be at least partly responsible for controlling proliferation and differentiation within the granulocytic-monocytic system. We have investigated 98 leukemic cell lines representing all lineages of the hemopoietic system. Pr3/MBN was found to appear in mature myeloblastic precursors and in CD34+ immature monoblastic cells and was restricted to the granulocytic and monocytic lineage. Myeloperoxidase (MPO) was found to be expressed earlier in development in the granulocytic lineage, but appeared later than Pr3/MBN in the monocytic lineage. The identification of cell lines which contained Pr3 only or MPO only may make them useful for demonstration of anti-neutrophil cytoplasmic antibodies in primary vasculitides.

Autoantigens↗

PubMed1994

Production of hepatocyte growth factor by human haematopoietic cell lines.

Hepatocyte growth factor (HGF) is a multi-functional molecule characterized as a mitogen, a motogen, a morphogen and a tumour suppressor. Little is known about cell types which produce HGF, so we analysed HGF production from cultured cell lines of haematopoietic cell lineage. A total of 138 human leukemia and virus-transformed cell lines were studied and the levels of HGF were measured by ELISA. A significant amount of HGF was detected in a variety of cell lines, including one T, four B, five non-T non-B, eight myeloid one erythroid and two EBV-transformed B cell lines. The amount of HGF spontaneously produced by three of the myeloid cell lines, KCL-22 (33.48 ng/ml), KG-1A (26.21 ng/ml), and KG-1 (18.81 ng/ml), is comparable to the amount produced by human embryonic lung fibroblast cells, known as high HGF-producers. Biological assays together with Western blot analyses verified that the immunoreactive HGF detected in the culture supernatant of haematopoietic cell lines had the same properties as authentic HGF. Moreover, HGF mRNA was detected in high HGF producers by Northern blot analysis. Our findings that lymphoid and myeloid cells function as a source of HGF may provide significant evidence for the involvement of haematopoietic cells in HGF-related morphogenesis and cell growth.

Blotting, Northern↗

PubMed1994

Effects of direct current on T cell activity: modulation of interleukin-2 production.

The production of interleukin-2 (IL-2) by mitogen-stimulated human peripheral blood leukocytes (PBL) was significantly enhanced by a short pretreatment of PBL with direct current (DC) at low intensities. Exposure of PBL to DC of 1 mA for more than 10 min resulted in inhibition of IL-2 production which correlated with decreased proliferation capacity of the treated cells. DC had similar enhancing and inhibitory effects on IL-2 production by the mitogen-stimulated human leukemia T cell line MOLT-13. However, DC itself did not stimulate IL-2 production in PBL or in MOLT-13 cells. In two additional T cell lines tested, MOLT-16 and H-9, DC did not enhance mitogen-stimulated IL-2 production, but suppressed it when applied at 1 mA for more than 10 min. Both the mitogen-stimulated and the interleukin-1 (IL-1)-enhanced IL-2 production were completely inhibited by 1 mA of DC applied for 20 min. The observed effects of DC were due to a direct action of DC on the cells and not to possible effects of the secondary by-products of electrolysis. These results show that pretreatment of cells with DC can enhance or suppress IL-2 production depending on the intensity of DC and the type of IL-2 producing cell clones.

Electricity↗

PubMed1994

Recombination hotspot associated factors specifically recognize novel target sequences at the site of interchromosomal rearrangements in T-ALL patients with t(8;14)(q24;q11) and t(1;14)(p32;q11).

A DNA binding protein was identified which binds to two novel target-like sequences: (i) at the 5' flanking site of the breakpoint junction of chromosome 8 in a patient with T-acute lymphoblastic leukemia (ALL) carrying the t(8;14)(q24;q11) rearrangement and (ii) on chromosome 1 in three of five T-ALL patients with the t(1;14)(p32;q11) rearrangement. This protein [provisionally called recombination hotspot associated factor (ReHF-1)] was also found to bind to a similar target sequence that is present immediately at the 3' end of the human V delta 3 gene segment. In a small number of lines tested, the ReHF-1 protein was expressed in gamma delta lineage T cells and in a number of B cell precursor ALLs whose TCR delta locus has been rearranged. The molecular weight of ReHF-1 protein was determined to be approximately 30 kDa by UV cross-linking analysis. Gel filtration chromatography and sedimentation velocity centrifugation analyses indicate that the ReHF-1 protein exists as a multimeric protein in its native form. These data might suggest a possible role for this protein in the rearrangement of the TCR delta locus. Furthermore, another protein, ReHF-2, that appears to have strict sequence specificity was found to bind only to the complementary single strand of the target sequence. The interaction of these proteins with a conserved target sequence at the chromosomal breakpoint junction might suggest that they are involved in a novel enzymatic mechanism reminiscent of the general features of DNA recombination or replication events in Escherichia coli or Saccharomyces cerevisiae.

Base Sequence↗

PubMed1994

Significant amount of hepatocyte growth factor detected in blood and bone marrow plasma of leukaemia patients.

Hepatocyte growth factor (HGF) has been known as a versatile functional molecule, and as being involved in the colony formation of haemopoietic progenitor cells. Clinically, an elevated HGF level in the blood has been associated with liver diseases such as fulminant hepatic failure and acute hepatitis. We have found a high level of HGF in blood and bone marrow plasma from patients with various types of leukaemia and lymphoma. In particular, 21/31 acute myeloblastic leukaemia (AML) patients showed a significant level of HGF (> 0.40 ng/ml) in their blood or bone marrow plasma. The mean value of HGF in the plasma of AML patients was 2.03 ng/ml, which was higher than that in the serum of patients with acute hepatitis. This demonstrates, for the first time, evidence of frequent association of increased levels of HGF in non-lymphocytic leukaemias, though its significance in the disease remains unknown.

Bone Marrow↗

PubMed1994

Establishment and characterization of a novel immature T-ALL cell line, UHKT-42, with TCR beta/delta expression.

A novel immature human T-ALL cell line, UHKT-42, was established from a 12 year old male patient with acute undifferentiated leukemia. The cell line expressed surface CD7, CD5 and cytoplasmic CD3 antigens. All other T-lymphocytic antigens were undetectable on the surface or in the cytoplasm of cultured cells. Expression of the T-cell receptor (TCR) beta, TCR delta, CD3 delta and CD3 epsilon genes was detected by Northern blotting in total cellular RNA extracts, however, the expression of TCR alpha and TCR gamma was undetectable. After stimulation by TPA for 3 days, only the appearance of CD25 (Tac antigen) was detected by immunofluorescence and flow cytometry. Secretion of interleukin-2 (IL-2) into the culture media was also detected after stimulation by PHA or TPA, but not in unstimulated cells. These results suggest that UHKT-42 cells are early precursors of T cells, with TCR beta/delta expression.

Antigens, CD↗

PubMed1994

Expression of Bcl-2 protein and Fas antigen in non-Hodgkin's lymphomas.

Expression of Bcl-2 protein and Fas antigens was analyzed in 12 cases of follicular lymphoma and 32 cases of diffuse lymphoma, including 22 B-cell and 10 T-cell lymphomas. It was shown that 75% of follicular lymphomas had clear expression of both Bcl-2 protein and Fas antigen. Thus, follicular lymphomas may have a growth advantage due to their high expression of Bcl-2 protein, which tended to impede apoptosis mediated by Fas antigen. On the other hand, diffuse lymphomas showed various patterns; 28% were double positive, 16% were only Bcl-2 protein-positive, 28% were only Fas antigen-positive, and 28% were double negative or equivocal. Cytocidal assay of seven leukemia/lymphoma cell lines using anti-human Fas monoclonal antibody revealed that overexpression of Bcl-2 protein tended to impede apoptosis mediated by Fas antigen. However, this inhibitory effect of Bcl-2 protein was incomplete and its effect might be dependent upon cell type.

Antibodies, Monoclonal↗

PubMed1994

Establishment of multiple leukemia cell lines with diverse myeloid and/or megakaryoblastoid characteristics from a single Ph1 positive chronic myelogenous leukemia blood sample.

Seven cell lines, MOLM-6, -7, -8, -9, -10, -11 and -12, were established from a single blood sample from a patient with chronic myelogenous leukemia (CML) in blastic phase having the Ph1 chromosome abnormality. Based on immunophenotyping, two of these seven cell lines, MOLM-7 and -11, represented the megakaryoblastoid lineage, and the other five cell lines represented two different maturation stages of the myeloid lineage.

Adult↗

PubMed1994

Nuclear proteins binding to a novel target sequence within the recombination hotspot regions of Bcl-2 and the immunoglobulin DH gene family.

The chromosomal breakpoints of follicular lymphomas carrying the t(14;18)(q32;q21) are known to be clustered within a 150-bp region in the major breakpoint region (mbr) of the Bcl-2 oncogene. We have demonstrated that nuclear proteins specifically bind to a novel target sequence within this 150-bp region and a region of Dxp genes, members of the immunoglobulin (Ig) diversity (DH) gene family. One protein, designated BCLF-1, appears to be specifically expressed in lymphoid lineage cells. Two other proteins, BCLF-2 and -3, bind only to the complementary single strand of the target sequence. The manner in which these proteins interact with the target sequence is similar to the interaction of the ReHF-1 and -2 proteins to the signal-like sequence at the chromosomal breakpoint junctions in patients with the t(8;14)(q24;q11) and t(1;14)(p32;q11) translocations. It was further suggested that the BCLF-1 is quite similar to or identical to the ReHF-1. It is therefore hypothesized that these conserved target sequences found in recombination hotspot regions may define novel sequence motifs recognized by two classes of DNA binding proteins. One class of DNA binding proteins is specifically expressed in lymphoid cells while the other class binds to the complementary single strand DNA. These binding activities may play a crucial role in chromosomal translocation in lymphoid neoplasms.

Base Sequence↗

PubMed1993

Reversal of methotrexate-induced folate pool depletion by thymidine in a human leukemia cell line in vitro.

In an in vitro study using a human monocytic leukemia cell line, U-937, the effects of interferon-gamma (IFN-gamma) in combination with the antifolate methotrexate and the role of thymidine introduced as a biochemical modulator were investigated. Methotrexate alone or in combination with INF-gamma was found to enhance the induction of morphologic and functional monocytic differentiation in the U-937 cell line. Various cellular effects with the addition of thymidine to the medium with methotrexate and IFN-gamma were studied. Enhanced inhibition of cell growth and perturbation of the cell cycle were noted when methotrexate and IFN-gamma were used in combination, but not when methotrexate was used alone. The reduction of cellular folate by methotrexate was also enhanced in combination with IFN-gamma. Cell cycle delay, resulting in cell growth inhibition of folate depletion, caused the induction of differentiation in U-937 cells, which was found to be greater with methotrexate + IFN-gamma than with methotrexate alone. Cellular differentiation, as assessed by nitroblue tetrazolium reduction assay, indirect immunofluorescence and morphology, showed better effects towards the differentiation of U-937 cells when the agents were used in combination. However, addition of thymidine to the medium was found to cancel all the aforementioned effects. The addition of thymidine to the medium also caused reversal of the inhibitory effect of methotrexate and IFN-gamma on cell growth and repletion of the endogenous folate level. Repletion of the folate level by exogenous thymidine is a new possibility for the role of the thymidine in cellular growth.

Antibodies, Monoclonal↗

PubMed1993

Interleukin-2 production by T cells: a study of the immunoregulatory actions of interferon-alpha, interferon-gamma, and tumor necrosis factor-alpha in phenotypically different T cell clones.

The production of interleukin 2 (IL-2) by phytohemagglutinin (PHA)-stimulated human leukemia T cell lines MOLT-13 and MOLT-14, the T cell receptor (TcR) gamma delta-bearing clones, was significantly increased by adding human natural interferon-gamma (IFN-gamma) to the cell cultures. In addition, the IL-2 production by PHA-stimulated MOLT-13 and MOLT-14 cells was also augmented by human natural tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner but was inhibited by IFN-alpha even at low concentrations (10 IU/ml). This pattern of reactivity is different from the responsiveness of the MOLT-16 cells, a TcR alpha beta-bearing cell line, which respond to IFN-alpha and TNF-alpha, but not to IFN-gamma, by augmentation of IL-2 production. These results suggest that (1) the addition of IFN-gamma has a direct effect on PHA-stimulated MOLT-13 and MOLT-14, resulting in an augmentation of IL-2 production; (2) phenotypically different T cell lines vary in their susceptibility to IFN-alpha and IFN-gamma regulation; and (3) the augmentation of IL-2 production by TNF-alpha appears to have no correlation with phenotypically different T cell clones.

Clone Cells↗

PubMed1993

Human recombination activating gene-1 in leukemia/lymphoma cells: expression depends on stage of lymphoid differentiation defined by phenotype and genotype.

The recombination activating gene, RAG-1, which is supposed to encode a molecule regulating V(D)J recombination, has been isolated. In the current study, the distribution of RAG-1 expression in human neoplastic hematopoietic cells was compared with the phenotypic and genotypic status of differentiation. Thirty-one hematopoietic cell lines (16 B-lineage, 9 T-lineage, 2 Hodgkin's disease, and 4 nonlymphoid cell lines) were investigated for the expression of human RAG-1 using the reverse-transcriptase polymerase chain reaction (RT-PCR). RAG-1 was not expressed in nonlymphoid, Hodgkin's disease, or mature-stage lymphoid cell lines, but was present in some acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL) cell lines. The investigation was extended to 45 cases of fresh ALL/LBL cells. The patterns of RAG-1 expression found in the cell lines and fresh ALL/LBL cells were similar. In B-lineage cells, the product of RAG-1 RT-PCR was detected in CD19+ CD10- CD20- CD5- stage (stage II, Nadler's classification) and was at the highest level in CD19+ CD10+ CD20- CD5- stage (stage III), but was absent or limited in CD19+ CD10+ CD20-+ CD5- (stage IV) or CD19+ CD10+ (or CD10-) CD5+. In stage II, monoclonal gene rearrangements of only the immunoglobulin heavy chain (IgH) were found, whereas monoclonal gene rearrangements of both IgH and T-cell receptor (TCR)-beta chain were frequently noted in stages III and IV. The expression of CD20 or CD5 antigen apparently correlated with the decline of RAG-1 expression. In T-lineage cells, RAG-1 was highly expressed in CD3- CD4+ CD8+/CD3+ CD4+ CD8+ thymic stages, but was negative or only weakly expressed in the CD3- CD4- CD8- prothymic or early thymic stage, in which the TCR-beta gene was often germline, or the CD3+ CD4+ CD8- mature thymic stage. The relative levels of RAG-1 mRNA give an additional delineating frame to the schemes of lymphoid differentiation based on phenotypic and genotypic status. RAG-1 is exhibited by cells of the thymic stage capable of synthesizing TCR or expressing it on the cell surface. The weak or absent expression of RAG-1 in the prothymic or early thymic stage suggests that the contribution of RAG-1 to the gene rearrangement may differ quantitatively between TCR-delta/TCR-gamma and TCR-beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1993

Effects of natural interferon alpha, natural tumor necrosis factor alpha and their combination on human mesothelioma xenografts in nude mice.

Effects of human natural interferon alpha (nIFN) alone, human natural tumor necrosis factor alpha (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day -12. nIFN was given intraperitoneally (i.p.) at a dose of 2 x 10(7) or 2 x 10(8) IU kg-1 day-1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 x 10(7) or 2 x 10(8) U kg-1 day-1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.

Animals↗