Biomedical subjects
Publications and source records attributed to N Emi.
Explore the source record for details and available documents.
We have established a novel human megakaryoblastic cell line, designated as MEG-A2, from a patient with megakaryoblastic crisis of Philadelphia (Ph) chromosome positive chronic myelogenous leukemia. MEG-A2 cells showed positive phenotypes for periodic acid Schiff and alpha-naphthylbutyrate esterase reactions, but were negative for myeloperoxidase and naphthol ASD chloroacetate esterase reactions. Flow cytometric analyses of cell surface markers revealed that MEG-A2 cells had a low level of GP IIb/IIIa expression as well as apparent expressions of CD4, CD7, CD13, CD33 and CD34 antigens, but no expression of GP Ib nor glycophorin A. Stimulation with phorbol 12-myristate 13-acetate (PMA) dramatically increased the expression of megakaryocyte-related markers such as HPL-3, J15, Pit-1, Y2/51 and AN51 in MEG-A2 cells. The PMA-stimulation also induced expression of platelet peroxidase (PPO) in MEG-A2 cells on electromicroscopic observation. Proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or erythropoietin were observed, and the expression of GP IIb/IIIa was increased by stimulation with GM-CSF, IL-3, erythropoietin and interleukin-6 (IL-6). Protein S mRNA expression was seen in cultured cells on Northern blot analysis. Expression of platelet factor 4 mRNA was induced in PMA-stimulated cells, and a marked accumulation of protein was observed in the culture medium. In conclusion, a new cell line, MEG-A2, belongs to the relatively immature megakaryocytic lineage and has markedly increased megakaryocytic characteristics with PMA stimulation.
The effect of granulocyte colony-stimulating factor (G-CSF) on neutropenia and infection was studied in a randomized trial in patients receiving intensive chemotherapy for acute leukemia. Fifty seven patients with acute leukemia (35 cases of refractory acute myeloid leukemia, 19 cases of acute lymphoblastic leukemia and 3 cases of blast crisis of chronic myeloid leukemia) were given G-CSF under either of the following two conditions; 1) Group A: starting G-CSF (200 micrograms/m2iv) administration 24 hrs after chemotherapy. 2) Group B: the same dose of G-CSF administration after a febrile episode of 38 degrees C with neutropenia (less than 1,000/microliters). Five patients were excluded from the study. Group A (27 patients) showed a shorter febrile period (2.15 +/- 2.98 days) than the 25 patients of Group B (3.40 +/- 4.78 days), but the difference was not statistically significant. Compared to Group B, Group A showed significantly early recovery of neutrophil counts as well as early recovery from documented infections. There was no evidence that early administration of G-CSF accelerates the growth of leukemic cells nor causes early relapse of acute leukemia.
Tumor necrosis factor-alpha receptors (TNFR-55 and TNFR-75) were inserted into retrovirus derived vector. They were transfected into a packaging cell line. The high titer of transfectants, which produced virus containing TNFR-55 or TNFR-75, was obtained. TNFR-55 and TNFR-75 negative human colon cancer cells were infected with this virus as a model experiment of gene therapy. Although the original colon cancer cell line did not express TNFR-55 or TNFR-75, the colon cancer cells, which were infected by recombinant virus, expressed a high level of TNFR-55 or TNFR-75. TNFR-55 or TNFR-75 transformed colon cancer cells were killed by recombinant TNF.
A 37-year-old man was found to have an inherited abnormal factor VII (FVII) (designated FVIINagoya) that has a lower FVII procoagulant activity when tested in a one-stage assay using rabbit, simian, or human tissue factor (TF), but surprisingly has a nearly normal activity using bovine TF. DNA sequence analysis of the propositus' FVII gene revealed a C to T mutation that results in a novel amino acid substitution of Arg304 to Trp. Restriction enzyme analysis of the amplified fragment obtained from each family member demonstrated that the patient is homozygous for the point mutation, and the mother and sister of the patient are heterozygous for this mutation. To elucidate the abnormality in FVIINagoya, we compared the recombinant mutant FVII with normal FVII. In the presence of human TF, kinetic analyses demonstrated that the apparent Km values of FVIINagoya for factor Xa-mediated FVII activation and for FVIIa-mediated factor X (FX) activation were 3.6- and 4.5-fold higher than those of normal FVII, respectively. The ligand binding assay to human TF also showed that FVIINagoya had a 5.2-fold larger apparent Kd than normal. In the presence of bovine TF, however, these values showed no difference between normal and mutant FVII. These findings indicate that the major abnormality in FVIINagoya is not a catalytic dysfunction but an impaired complex-formation of human TF.FVII.FX, suggesting the importance of Arg304 in the interaction with human TF.
The A15, L6, and S5.7(TAPA1) proteins are members of the transmembrane 4 superfamily (TM4SF). The A15 is expressed in immature human T cells and in the human brain. The MXS1(CCG-B7) gene which codes for A15 contains triplet nucleotide repeats which have been associated with neuropsychiatric diseases such as Huntington's chorea, fragile X syndrome, and myotonic dystrophy. The L6 antigen is mainly expressed in lung, breast, colon, ovarian carcinomas, and healthy epithelial tissue in humans. The S5.7(TAPA1) antigen is expressed in most human cell lines and is shown to be associated with B-cell surface molecules CD19 and Leu-13. In this study we have used interspecies specific somatic cell hybrids and human-specific cDNA probes to localize the A15 (MXS1), L6 (M3S1), and TAPA1 genes to Xq11, 3q21-25, and 11p15.5, respectively.
An immunization procedure with naked DNA that generates both humoral and cellular immune responses is described. A mammalian expression vector encoding human factor IX as an example of immunogen was injected into mice three times at 10-day intervals. Three of the 4 mice in which the DNA was injected intramuscularly and 5 of the 9 mice injected subcutaneously produced antibodies to human factor IX. Spleen cells from inoculated mice also showed significant cytotoxic T lymphocyte response to target cells expressing human factor IX. The titers of anti-single-stranded (ss)DNA antibodies were significantly higher in the subsets of genetically inoculated mice than in the control mice, but anti-double-stranded (ds)DNA antibodies were not detected in any serum samples. Thus, intramuscular and subcutaneous injection of plasmid DNA can induce immune responses against the encoded protein without an exposure to virus particles, and this approach may serve as the basis for immunotherapy in the treatment of cancer and infectious diseases in humans.
A novel immunization procedure for eliciting murine monoclonal antibodies (mAb) is described. A murine leukemia virus (MLV)-based retroviral vector, designed as a marker protein for the expression of human factor IX (FIX), was constructed. Direct injections of mice with 0.8-1.3 x 10(4) retroviruses were carried out three times at 4-week intervals. Three out of 4 mice that had the viruses injected subcutaneously produced the antibodies against FIX, and 2 out of 3 mice injected intraperitoneally produced the antibodies. From 1 of the mice with the antibodies, an anti-human FIX murine monoclonal antibody, designated ACTIX (IgMk type), was produced. The immunization of mice by direct injection of viruses facilitated mAb production in many instances in which cDNAs are available.
The findings of morphologically dysplastic features in haemopoietic cells in de novo acute myeloid leukaemia (AML) has been named AML with trilineage myelodysplasia (AML/TMDS). We analysed the clinical data, karyotypes, and treatment outcomes of 230 de novo AML patients treated with the Japan Adult Leukaemia Study Group AML-87 protocol. 40 (17%) patients had AML/TMDS. Platelet count was significantly higher (P = 0.006) and bone marrow blasts were fewer (P = 0.01) in the AML/TMDS group than in the AML without TMDS. Abnormal karyotype was shown in 12/30 patients (40%) analysed. The complete remission (CR) rate for AML/TMDS was significantly lower than AML without TMDS (63% v 81%) (P = 0.01). The overall survival curves showed that the 40 patients with TMDS had a significantly worse survival than the 190 without TMDS (P = 0.0005). AML/TMDS also showed significantly worse disease-free survival (DFS) (P = 0.0001). Multivariate analysis revealed that the absence of TMDS in AML was the most significant factor in obtaining CR (P = 0.01) and a significant factor in predicting longer DFS (P = 0.04). Our data suggest that AML/TMDS responds poorly to intensive chemotherapy. Further study is required to determine the best treatment strategy for AML/TMDS and the biological differences between AML/TMDS and other types of AML.
The thymidine kinase (tk) gene of herpes simplex virus type 1 (HSV-1) was transduced into three glioma cell lines (T98, U251MG, T9) using a retrovirus vector. The supernatants of viral producer cell line PA317/LTRNL was used for infection and three transduced cell lines (T98/LTRNL, U251MG/LTRNL, T9/LTRNL) were established. The toxicities of the anti-herpetic drugs, acyclovir and gancyclovir, were evaluated in vitro. The cytotoxicities of acyclovir and gancyclovir to the HSV-1 tk gene-transduced cells increased 100-1000 fold compared to the non-transduced parental cell lines. The cytotoxic effect of gancyclovir was higher than that of acyclovir, requiring a concentration of less than 0.31 microM to obtain 50% inhibition. Deoxyribonucleic acid analysis of the gancyclovir-treated cells demonstrated fragmentation, suggesting that apoptosis is involved in the mechanism of cell death. The HSV-1 tk gene-transduced cells were co-cultured with parental cells and treated with gancyclovir. More than 95% of cells were killed in a mixture ratio of 1:1, suggesting that the "bystander effect" operated in this system. Selective transduction of HSV-1 tk gene into glioma cells using a retroviral vector and treatment with gancyclovir is a promising therapy for patients with malignant glioma.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Retrovirus-mediated gene transfer into human hematopoietic stem cells has been proposed as a means of therapy for various inherited diseases and as a method of gene marking. The transduction efficiency of an amphotropic retroviral vector (PA317/HyTK) containing a hygromycin phosphotransferase-thymidine kinase fusion gene was examined with human CD34+ bone marrow cells in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor. Transduction efficiencies determined from the ability of transduced granulocyte-macrophage colony forming units (CFU-GM) to grow in hygromycin B and from polymerase chain reaction analysis of individual transduced CFU-GM growing in the presence of hygromycin B were 0.3-3.0% (mean +/- S.D., 1.1 +/- 0.9%) and 0.1-1.2% (mean +/- S.D., 0.5 +/- 0.4%), respectively. Ganciclovir at a dose of approximately 1 microM reduced the number of CFU-GM derived from vector-infected CD34+ cells by 50%. These findings demonstrate that human hematopoietic stem cells infected with this retroviral vector are susceptible to ganciclovir, offering the potential to control transduced gene expression in vivo.
We constructed a new factor IX expression vector containing a full length factor IX cDNA. The Moloney Murine Leukemia Virus (MoMLV)-based retroviral vector pLRNL was cloned with the entire coding region of human factor IX down stream of the promoter of Rous sarcoma virus, giving rise to the construct pL9RNL. The GP+E 86 packaging cell was transfected with pL9RNL and two mouse fibroblast cell lines (PA317 and NIH3T3) were infected with the virus generated from PA317 cell. During the 24 hr culture period, the maximum 1.2 micrograms of factor IX was secreted into the medium from 10(6) of GP+E 86 cells. Factor IX in the culture media had the relatively normal procoagulant activity and was barium-citrate precipitated. Western blotting analysis of the barium-citrate precipitates revealed that a single chain 50Kd protein indistinguishable from the plasma-derived factor IX was produced in these three cells. The retroviral expression system that we utilized herein may contribute to the study of recombinant wild type or various mutant factor IX, and to the basic study for the future gene therapy.
A novel cDNA clone, A15, was isolated by the differential screening of a cDNA library of an immature T cell line, HPB-ALL using radioactive cDNA probes from the mRNA of either HPB-ALL or peripheral blood lymphocytes. It hybridized to a single mRNA species of about 2.0 kilobases which is expressed in HPB-ALL cell line, but not in the PBL or a promyelocytic leukemia cell line, HL-60. The A15 gene codes for a protein of 244 amino acids which contains four potential transmembrane domains and four possible N-linked glycosylation sites. A computer-aided comparison showed a marked similarity to several other membrane proteins: CD9, CD37, CD53, TAPA-1, Sm23, CO-029, and ME491/CD63.
One of the recent strategies for gene therapy as a cancer control is the targeted introduction of a drug-sensitivity gene into tumor cells. We investigated the gene transfer of herpes simplex virus type I thymidine kinase (HSV-TK) gene as a drug-sensitivity gene into human lung cancer cell lines. We used a recombinant retroviral vector derived from Moloney murine leukemia virus (MuLV) as one of potential vectors for gene therapy. The amphotropic retroviral vector consisted of the HSV-TK gene and the neomycin-resistant gene under Rous sarcoma virus (RSV) promoter control. The antiherpes drugs, acyclovir (ACV) and ganciclovir (GCV), were chosen for testing the activity of HSV-TK that was transferred into human lung cancer cell lines. ACV and GCV are nucleoside analogs specifically converted by HSV-TK to a toxic form capable of inhibiting DNA synthesis. The cytotoxicity was determined by using a tetrazolium-based colorimetric assay (MTT assay). The results obtained from our experiments demonstrated that the retroviral vector-mediated HSV-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, which were both small cell carcinoma and non-small cell carcinoma established from human specimens. These findings suggest that the gene transfer of HSV-TK gene into tumor cells would be one of the models for the use of gene therapy to control lung cancer.
To investigate the possibility of killing tumor cells by the expression of an exogenously introduced toxic gene, we have constructed a novel retroviral vector (LTRNL) which has the polyA signal deleted herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. The vector becomes toxic by treating cells expressing HSV1-tk with the antiherpetic drugs acyclovir or ganciclovir (GCV). Cells of the human leukemia lines (K562, MEG-01) were infected with this vector and two transduced cell lines (K562/LTRNL, MEG-01/LTRNL) were established. Southern blot analysis confirmed the integration of the HSV1-tk transgene in these cells and Northern blot analysis exhibited the expression of 4.8-kb viral mRNA containing the HSV1-tk gene. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the in vitro cytotoxic effects of GCV to these cells demonstrated that concentrations of about 2.5 microM for K562/LTRNL and 1.25 microM for MEG-01/LTRNL cells resulted in 50% inhibition of cell growth after 72 hr. Subcutaneous tumors of MEG-01/LTRNL in KSN nude mice, but not those of uninfected MEG-01 cells, showed durable regressions after exposure of the mice to 40 mg/kg of GCV given subcutaneously once a day for 15 days. This study indicates that the LTRNL-infected human leukemia cells exhibit inducible susceptibility to GCV.