Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To clarify the role of c-fos DNA in the activation of human synovial cells, the pH8 expression vector containing human c-fos DNA under the control of murine leukemia virus long terminal repeat was transfected into cultured synovial cells. After G418 selection, the control transfectant clones transfected with pH8 vector not containing c-fos DNA insertion changed their original fibroblastic shape into dendritic cells. They stopped growing at this stage. However, the c-fos DNA transfectant clones continued to grow actively beyond this stage, and regained the fibroblastic appearance. Furthermore, c-fos DNA transfectants adhered to and grew on hyaluronidase treated cartilage surfaces more extensively than control transfectants after 6 days in culture. These findings suggest that c-fos DNA supports active growth of human synovial cells by facilitating transition of synovial dendritic cells into fibroblastic cells.
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PMID:The contribution of human c-fos DNA to cultured synovial cells: a transfection study. 847 46

We constructed a retroviral vector encoding a mutant tRNA(imet) gene followed by a HIV-1 rev-specific antisense sequence in the U3 region of the 3' long terminal repeat (LTR). This Moloney murine leukemia virus (MoMLV)-based double-copy retroviral vector was used to transduce human lymphoblastoid T-cell lines (CEM, Jurkat). In some clonal cell lines the expected short transcript initiated either from the 5' or 3' LTR tRNA-alpha rev gene was not detectable by Northern blot analyses of transduced, G418-resistant cells with an alpha rev-specific oligonucleotide probe. In other clonal cells, neither the short polymerase III transcript nor the full-length genomic polymerase II transcript (containing the 3' LTR tRNA-alpha rev gene) was detectable when compared with the transduced cell pool. Southern blot and DNA-polymerase chain reaction (PCR) analyses specific for the tRNA-alpha rev cassette in the 5' or 3' LTR of the retroviral vector suggested that the transfer of the 3' LTR U3 region to the 5' LTR was incorrect in most proviruses. These data were confirmed by DNA sequence analyses of several clonal lines demonstrating deletions and insertions. In summary, our results indicate that this retroviral vector design with direct repeats flanking the polymerase III transcription unit plus the alpha rev insert is prone to genetic rearrangements and consequently not useful for the development of gene therapy protocols.
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PMID:Genetic instability of a MoMLV-based antisense double-copy retroviral vector designed for HIV-1 gene therapy. 854 53

The construction of a new retroviral vector, pSKV, is described. This vector carries two unique cloning sites, located between two Moloney leukemia virus-derived LTR, into which genes of interest may be introduced. The gene encoding hygromycin resistance (HyR) was subsequently introduced into one of the two sites, producing a second vector (pSKV/HyR) containing a unique SfiI site for the introduction of cDNA clones under the control of the cytomegalovirus (CMV) promoter (P-CMV). The cDNA (mH13), encoding a protein that has been shown to serve as a murine ecotropic retroviral receptor in transient assays, was cloned into the SfiI site (pSKV/HyR/mH13). Both constructs can be packaged into retroviral particles following transfection into an appropriate packaging cell line. Stable transfectants of the human glioblastoma cell line (U118MG) carrying each of these two constructs were generated by transfection and subsequent Hy selection. Clones expressing both the selectable marker and the mH13 gene, but not those expressing only the selectable marker, are shown to be susceptible to infection with murine ecotropic retroviral particles. These cells (HyR and mH13 positive) were then exposed to CRE/Xtk culture supernatant, a packaging cell line producing ecotropic retroviral particles carrying the HSV-TK (Herpes simplex virus-thymidine kinase) and neoR (neomycin-resistance) genes. Selection was in the presence of G418. In vitro growth of the U118MG/HyR/mH13/TK cells, but not that of the U118MG/HyR/mH13 cells, was inhibited by ganciclovir (GCV), indicating the successful transfer of HSV-TK by infection of human cells with murine retroviruses via the mH13 product.
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PMID:Infection of human cells by murine ecotropic viruses: retroviral vectors carrying the hygromycin resistance-encoding gene. 866 55

The relationship between tumorigenicity and expression of MHC class II molecules in a class II-negative murine leukaemia cell line (LBC) was studied. Analysis of structural DNA sequences encoding MHC class II proteins was performed by Southern blot with DNA isolated from both the original LB tumour and LBC cell line, digested with EcoRI, BamHI and HindIII and hybridised with specific probes for I-A alpha d and I-A beta d chains. Similar patterns were obtained for LB, LBC and normal BALB/c lymphocytes. In vitro treatment with IFN-gamma (20 - 1000 IU ml-1) failed to induce the expression of MHC class II antigens in LBC cell line. LBC cells were tri-transfected by a liposome-mediated protocol with I-A alpha d, I-A beta d genes and pSV2neo. Cells were selected for growth in medium containing Geneticin (G418). Surviving transfectants were cloned and three I-A+ clones were obtained after 20 days (LBCT cells). Syngeneic mice inoculated with 1.0 x 10(3) LBCT (I-A+) cells failed to develop a tumour, whereas the DT50 of mice injected with 1.0 x 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). Furthermore, specific CTL response against tumour cells was significantly enhanced upon priming with irradiated LBC-transfected cells (27 +/- 2%) compared with irradiated LBC cells (15 +/- 1.5%) in a 4 h 51Cr-release assay. It is suggested that neoexpression of MHC class II molecules enhances anti-tumour response by transforming tumour cells into professional antigen-presenting cells (APCs), which may be used to improve tumour-specific immunity in the autologous host.
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PMID:Enhancement of anti-tumour immunity in syngeneic mice after MHC class II gene transfection. 868 32

The interaction of an exogenous PML/RAR alpha fusion gene associated with acute promyelocytic leukaemia, with all-trans retinoic acid (ATRA) was examined in two lymphoid cell lines. L1210 and MOLT-4 cells were transfected with PML/RAR alpha cDNA in the expression vector pGD and stable transformants (L1210PML/RAR alpha and MOLT-4PML/RAR alpha) were selected with G418. ATRA inhibited the growth of these stable transformants, as assessed by [3H]thymidine incorporation, in a dose-dependent manner, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also induced apoptosis, as assessed by fragmentation of genomic DNA, in L121OPML/RAR alpha and MOLT-4PML/RAR alpha cells but not in control cells. The exogenous PML/RAR alpha fusion gene therefore probably mediates the effects of ATRA on cell growth and apoptosis in these cell lines.
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PMID:Inhibition of growth and induction of apoptosis by all-trans retinoic acid in lymphoid cell lines transfected with the PML/RAR alpha fusion gene. 870 36

We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.
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PMID:Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2. 879 70

Previous studies have suggested that autologous bone marrow or mobilized peripheral blood progenitor cell transplants activated by prior culture of the cells in IL-2 may capture some of the beneficial graft-versus-leukemia effects obtained with unmanipulated allogeneic, but not autologous, transplants. To investigate ways of improving this approach,we have compared the ability of two other immunomodulating cytokines, IL-7 and IL-12, either alone or in combination with IL-2, to stimulate human bone marrow cells (BMC) or peripheral blood cells (PBC) to acquire the potential to lyse K562 or Daudi cells. For these studies, we measured the cytotoxic activity of BMC or PBC both before and at the end of their incubation with various cytokine(s) using a standard 51-chromium release assay. Results suggest that IL-2 at optimal concentration induces cytotoxicity significantly higher than IL-7 or IL-12 when tested alone. At optimal concentration, the combination of IL-2 and IL-12 showed a synergistic effect for BMC. Such a synergistic effect could be observed for PBC only when suboptimal concentrations of IL-2 were used. In addition, the ability of the hematopoietic cells to reduce the number of K562 cells remaining at the end of various culture periods in the presence of the cytokines was measured. This was made possible by the use of a G418-resistant K562 cell line which could, in contrast to normal human BMC or PBC, form colonies that wer detectable after 1 week in methylcellulose cultures containing the neomycin analog G418. Normal human PBC, stimulated by either IL-7 or IL-12 alone effectively suppressed K562 proliferation in both of these assays, whereas no activity could be detected when BMC were incubated under the same conditions. On the other hand, cells from both sources displayed anti-leukemic activity when incubated with IL-2 and IL-12 together, although IL-2/IL-12-activated PBC suppressed the growth of co-cultivated K562-neor cells about eight-fold more efficiently than IL-2/IL-12-activated BMC. Cryopreservation and subsequent stimulation of BMC and PBC with cytokines did not cause a significant decrease in cytotoxicity or their ability to inhibit the growth of co-cultivated K562 cells compared to fresh cells. However, the synergistic effect observed with the combination of IL-2/IL-12 was no longer detectable for BMC. These results suggest that (1) PBC are superior to BMC with respect to developing effective natural killer (NK) activity after culture in cytokines and that, (2) the combination of IL-2 and IL-12 may be more effective than IL-2 alone to inhibit proliferation/growth of K562 cells.
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PMID:Comparison of natural killer activity of human bone marrow and blood cells in cultures containing IL-2, IL-7 and IL-12. 883 97

Allogenic hematopoietic stem cell transplantation is associated with a severe complication induced by the T-cells present in the graft: graft-vs-host disease (GVHD). While effectively preventing GVHD, ex vivo T-lymphocyte depletion of the graft unfortunately increases graft rejection and reduces the graft-vs-leukemia (GVL) effect. The ex vivo transfer to the herpes simplex thymidine kinase (HS-tk) suicide gene into T-cells before their infusion with the hematopoietic stem cells should allow for selective in vivo depletion of these T-cells with ganciclovir (GCV) if subsequent GVHD was to occur. In patients not experiencing GVHD, and therefore at a higher risk of relapse, one could preserve the beneficial effects of the donor T-cells on tumor control. Lastly, the early presence of donor T-cells in all patients should contribute to successful engraftment. We have demonstrated that retroviral-mediated transfer of HS-tk and Neomycine resistance genes in T-lymphocytes, followed by G418 selection, results in T-cells specifically inhibited by GCV with no bystander effect. In a phase I study, escalating amounts of HS-tk expressing T-cells will be infused in conjunction with a T-cell depleted marrow graft to allogenic HLA identical recipients. Toxicity, survival, alloreactivity and GCV-sensitivity of the gene-modified cells will be monitored. If successful, such an approach could significantly contribute to expanding the use of alloreactivity as a treatment modality.
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PMID:Gene transfer applied to the modulation of alloreactivity. 893 11

We have previously shown by reverse transcriptase-PCR (rtPCR) that CML CD34+ HLA-DR- cells are enriched for BCR/ABL(-) hematopoietic progenitor cells (HPC) while leukemic HPC reside predominately within CML CD34+ HLA-DR+ cells. We investigated whether the 30/35 kDa fragment of fibronectin (FN) could be used to enhance retroviral-mediated gene transfer (RMGT) in chronic phase CML marrow HPC. CML CD34+ HLA-DR- and CD34+ HLA-DR+ cells were transduced with vector supernate containing the neomycin resistance gene on plates coated with either FN or bovine serum albumin (BSA) as control, then assayed for transduced HPC in progenitor cell assays in the presence or absence of G418. Transduction efficiency of CML CD34+ HLA-DR- cells over BSA ranged from 0.09 to 7.2% (mean 3.3 +/- 1.5%), while that over FN plates ranged from 3.8 to 23% (mean 11.0 +/- 4.5%) (n = 4). Transduction efficiencies of CML CD34+ HLA-DR+ cells ranged from 0.4 to 9.8% (mean 3.7 +/- 1.7%) and 6.0 to 26% (mean 17.3 +/- 4.5%) (n = 5) over BSA and FN, respectively. rtPCR analysis for BCR/ABL mRNA of individual G418-resistant HPC generated from CD34+ HLA-DR- cells revealed that normal BCR/ABL(-) HPC were successfully transduced under these experimental conditions. These results demonstrate the feasibility of transducing normal CML primitive HPC, and illustrate the potential clinical use of FN in the setting of gene therapy for CML, as well as other diseases.
Leukemia 1997 Jan
PMID:The 30/35 kDa chymotryptic fragment of fibronectin enhances retroviral-mediated gene transfer in purified chronic myelogenous leukemia bone marrow progenitors. 900 33

A graft-versus-leukemia (GVL) effect has been considered a major factor responsible for cures in patients with hematologic malignancies undergoing allogeneic bone marrow transplantation; however, associated graft-versus-host disease (GVHD) results in significant morbidity and mortality. T-cell depletion reduces the incidence and severity of GVHD but eliminates, at least partially, the GVL effect. Reinfusion of donor T lymphocytes at relapse posttransplantation can induce a potent antitumor response, but GVHD still occurs in the majority of patients. Prior transduction of T lymphocytes with the suicide gene, the viral thymidine kinase (TK), permits specific cell kill on administration of ganciclovir (GCV). Therefore, infusion of TK-transduced T lymphocytes may induce GVL effect and allow for their subsequent selective elimination in case GVHD develops. To evaluate the efficacy and feasibility of this promising approach, anti-CD3-stimulated primary human lymphocytes cultured in interleukin-2 were TK-transduced by a retroviral vector carrying both TK and neomycin-resistance genes. After selection in G418, more than 90% of the cells contained the TK gene as shown by a semiquantitative polymerase chain reaction. In addition, 1 to 5 days of GCV exposure, at clinically achievable concentrations of 20 to 50 micromol/L, induced > or = 90% killing of G418-selected cells without affecting nontransduced cells. Correlation of the extent of T-cell kill and the proportion of TK-gene-transduced cells is consistent with the absence of a bystander effect. Transduced cells were CD3+ and either CD8+ or CD4+ and retained functional properties of untransduced cells. In vivo administration of GCV prevented tumor development after subcutaneous injection of TK-transduced murine myeloma cells (MOPC-11), whereas such an effect was not observed on injection of untransduced cells into the opposite flank. Our studies provide critical information that (1) adequate numbers of TK-transduced lymphocytes can be selected efficiently with > or = 90% purity, (2) selected cells remain functional, (3) 24 hours of exposure to GCV at clinically achievable concentration effects > or = 90% killing of selected cells, and (4) GCV is effective in vivo in killing TK-transduced cells. Based on these data, a clinical study has been initiated in patients with multiple myeloma with persistent or relapsing disease after T-cell-depleted allogeneic transplants.
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PMID:Thymidine kinase (TK) gene-transduced human lymphocytes can be highly purified, remain fully functional, and are killed efficiently with ganciclovir. 902 56


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