Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lymphocytes have a finite and predictable proliferative life span in culture similar to that observed in fibroblasts. In general, the senescence of human fibroblasts is inevitable and irreversible, but their proliferative life span can be extended by certain DNA tumor virus oncogenes, such as the large T antigen of the SV40 virus. Here, we show that human T lymphocytes (HTL) can be stably transfected with SV40 large T and that expression of T antigen extended the life span of T cell cultures. PHA-stimulated HTL were transfected with pSV3neo, an expression vector containing the SV40 early region and the neomycin resistance gene. Transfectants were selected for neomycin (G418) resistance. Control HTL, either mock transfected or transfected with pSV2neo (containing the neomycin resistance gene only), ceased proliferation after about 17 population doublings. In contrast, HTL transfected with pSV3neo underwent more than 170 doublings. pSV3neo-transfected cells expressed SV40 large T RNA, detectable by in situ hybridization, and SV40 T antigen, detectable by immunofluorescence. Greater than 95% of the transfected cells were CD4 positive. These results clearly show that SV40 large T enables HTL to escape senescence. Transfection with SV40 large T may be a valuable method for obtaining long term human T cell lines for studies of both aging and immunology.
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PMID:Extension of lifespan of human T lymphocytes by transfection with SV40 large T antigen. 154 79

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.
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PMID:Construction and characterization of retroviral vectors expressing biologically active human interleukin-12. 771 Nov 42

Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus-leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV-induced T-cell depletion in allogeneic BMT recipients.
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PMID:Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation? 804 49

Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.
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PMID:Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy. 1063 78

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (<5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.
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PMID:T lymphocyte transduction with herpes simplex virus-thymidine kinase (HSV-tk) gene: comparison of four different infection protocols. 1064 72

The human interleukin-17F(hIL-17F) gene was amplified by RT-PCR from PHA-activated human peripheral blood mononuclear cells (PBMCs). It was then subcloned into the retrovirus vector pSIV-1. The pSIV-1/hIL-17F together with its two-helper virus vectors pHIT456 and pHIT60 cotransfected into the package cell 293T by lipofectin to produce mature recombinant retrovirus, which was then used to infect SMMC-7721 hepatocarcinoma cells (HCCs), and the cells were selected in the presence of G418. The integration, transcription, expression of hIL-17F gene in SMMC-7721 cells was identified by PCR, RT-PCR and Western blot respectively. MTT and FCM showed that hIL-17F couldn't alter the proliferation and cell cycle of SMMC-7721 cells, but ELISA showed that it could down-regulate IL-6, IL-8 and VEGF expression. The effect of rhIL-17F supernatant on growth suppressing of ECV304 cells was observed by MTT. The experiment of human hepatocarcinoma xenograft tumor in nude mice showed that the formation and growth rates of hIL-17F-transgenic SMMC-7721 showed an obvious decline, and VEGF and CD34 expression and angiogenesis of the transgenic neoplasms was also evidently defined. hIL-17F can markedly inhibit the growth of human hepatocarcinoma xenograft tumor in nude mice by antiangiogenesis. This study provided an experimental evidence for further conducting tumor gene therapy by targeting vascularity and exploiting antiangiogenic novel medicine related to hIL-17F.
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PMID:[The effect of human IL-17F on growth of human hepatocarcinoma xenograft tumor in nude mice]. 1703