DrugBank:EXPT01586 - FACTA Search

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Query: DrugBank:EXPT01586 (G418)
2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Umbilical cord blood is rich in hematopoietic stem and progenitor cells and has recently been used successfully in the clinic as an alternative source of engrafting and marrow repopulating cells. With the likelihood that cord blood stem/progenitor cells will be used for gene therapy to correct genetic disorders, we evaluated if a TK-neo gene could be directly transduced in a stable manner into single isolated subsets of purified immature hematopoietic cells that demonstrate self-renewed ability as estimated by colony replating capacity. Sorted CD34(3+) cells from cord blood were prestimulated with erythropoietin (Epo), steel factor (SLF), interleukin (IL)-3, and granulocyte-macrophage colony stimulating factor (GM-CSF) and transduced with the gene in two ways. CD34(3+) cells were incubated with retroviral-containing supernatant from TK-neo vector-producing cells, washed, and plated directly or resorted as CD34(3+) cells into single wells containing a single cell or 10 cells. Alternatively, CD34(3+) cells were sorted as a single cell/well and then incubated with viral supernatant. These cells were cultured with Epo, SLF, IL-3, and GM-CSF +/- G418. The TK-neo gene was introduced at very high efficiency into low numbers of or isolated single purified CD34(3+) immature hematopoietic cells without stromal cells as a source of virus or accessory cells. Proviral integration was detected in primary G418-resistant(R) colonies derived from single immature hematopoietic cells, and in cells from replated colonies derived from G418R-colony forming unit-granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) and -high proliferative potential colony forming cells (HPP-CFC). This demonstrates stable expression of the transduced gene into single purified stem/progenitor cells with replating capacity, results that should be applicable for future clinical studies that may utilize selected subsets of stem/progenitor cells for gene therapy.
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PMID:High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34(3+) hematopoietic stem/progenitor cells from human umbilical cord blood. 750 56

The human osteosarcoma cell line, MG63, responds both to GM-CSF and to G-CSF in vitro. To assess the significance of these observations to tumor growth in vivo, MG63 cells were engineered by retroviral infection to produce human GM-CSF or G-CSF. These retrovirally infected cells become autostimulatory as measured by increased [3H]-thymidine incorporation (3- to 7-fold) and anchorage-independent colony formation (7- to 10-fold) as compared with uninfected MG63 cells or cells infected with control (neor) retrovirus. The increased proliferation induced by exogenous GM-CSF or G-CSF on uninfected MG63 cells in both assays could be completely inhibited by anti-GM-CSF or anti-G-CSF antibodies, while the same antibodies only partially abrogated proliferation by the growth-factor-producing cells. None of 34 nude or SCID mice developed tumors when injected s.c. with uninfected or neor-virus-infected cells. In contrast, all 30 mice injected with GM-CSF- or G-CSF-producing MG63 cells developed tumors which were G418-resistant and factor-producing. Tumor cell DNA showed a polyclonal retroviral integration pattern indistinguishable from that in the DNA of cells injected into mice. Tumors that formed following injection of a mixture of G418-resistant, GM-CSF-producing cells and cells infected with virus containing only the hygror gene contained hygromycin-resistant cells in the same proportion as was present in the original cell mixture. These data indicate that GM-CSF and G-CSF can support the growth of an osteosarcoma cell line both in vitro and in vivo whether the factor is supplied by autocrine production or from exogenous sources.
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PMID:The effect of GM-CSF and G-CSF on the growth of human osteosarcoma cells in vitro and in vivo. 750 89

We previously demonstrated stable integration of a transduced thymidine kinase (TK)-neo gene into immature and replatable stem and progenitor cells, as assessed by the presence of the gene in second-generation colonies. To evaluate whether this integration was still present in third- and fourth-generation colonies, nonadherent low-density T-lymphocyte-depleted (NALT-) cells from human umbilical cord blood were prestimulated with recombinant human (rhu) erythropoietin (Epo), steel factor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-stimulating factor (CSF), and granulocyte (G)-CSF. Prestimulated NALT- cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony formation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introduced into hematopoietic progenitor cells without stromal cells as a source of virus. As previously reported, proviral integration was detected in primary G418R-colonies, and in second-generation replated colonies derived from G418R granulocyte erythroid macrophage megakaryocyte colony-forming units and high-proliferative potential colony-forming cells (HPP-CFCs). Moreover, we now document that proviral integration was apparent in cells from colonies derived from third- and fourth-generation replated HPP-CFC, suggesting a high degree of stable integration of the transduced gene.
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PMID:Stable integration of retrovirally transduced genes into human umbilical cord blood high-proliferative potential colony-forming cells (HPP-CFC) as assessed after multiple HPP-CFC colony replatings in vitro. 774 19

Gene therapy for chronic myelogenous leukaemia (CML) may provide a therapeutic option for patients who are ineligible for bone marrow transplantation. To determine the feasibility of such an approach we evaluated the transduction efficiency of CML progenitor colonies from seven patients in chronic phase. Vector transduction was optimized using the CML-derived K562 cell line and applied to CML mononuclear cells. After vector exposure, optimal gene transfer was noted when CML mononuclear cell cultures contained stem cell factor, IL-3, GM-CSF and erythropoietin. The addition of IL-6 to this combination decreased transduction efficiency. Using these conditions, 20.4% +/- 2.4 (SE) of erythroid colonies (CFU-GEMM and BFU-E) and 20.2% +/- 4.7 of CFU-GM colonies were G418 resistant. This compares with a transduction efficiency of 5.9% +/- 1.1 and 6.4% +/- 1.5, respectively, for erythroid and CFU-GM colonies using marrow obtained from normal donors. Only a modest increase in gene transfer was noted when CML cells were stimulated with cytokines for the 24 h preceding vector exposure. Vector DNA in colonies expressing the BCR/ABL transcript was documented by performing PCR analysis on individual colonies. The relatively high gene transfer rate in CML suggests that this disease might be very suitable for gene therapy.
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PMID:Retroviral mediated gene transfer in chronic myelogenous leukaemia. 794 72

Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3' untranslated region (3'UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony-stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3'UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12-0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3'UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3'UTR had only an approximately twofold to threefold increase in accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Number and location of AUUUA motifs: role in regulating transiently expressed RNAs. 819 53

Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34 cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34 granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, which could potentially have been induced by IL-12 from CD34 cells. TNF-alpha protein release was significantly increased in CD34 cells incubated with IL-12. No detectable levels of IFN-gamma were noted. Anti-TNF-alpha, but not anti-IFN-gamma, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-alpha, but not IFN-gamma, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-alpha.
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PMID:Inhibitory effects of interleukin 12 on retroviral gene transduction into CD34 cord blood myeloid progenitors mediated by induction of tumor necrosis factor-alpha. 872 96

We have analyzed the relative level of gene expression and viral titer from different types of retroviral vectors used for gene therapy, the LTR-based MFG vector and the internal promoter-containing vectors, LNCX, LNSX and LXSN. The CAT gene was used for comparison of retroviral vector gene expression in both transfected and transduced cells, while the neo gene was used to evaluate viral liter. In transfected cells, MFG-CAT expressed higher levels of CAT then the other vectors, LNC-CAT was next, while L-CAT-SN and LNS-CAT produced much lower levels. CAT expression from MFG-CAT was particularly high in the human T lymphoid cell lines CEM-SS and H9. In nonselected transduced cells. CAT expression from MFG was 10- to 50-fold higher than with the other vectors. Similar observations were made with retroviral constructs expressing human EPO and murine GM-CSF. In transient transfection assays, the titer of MFG was at least five-fold higher than the other vectors as determined by Southern analysis and G418 resistance. Analysis of the steady-state RNAs produced after transfection of the packaging cell lines showed that MFG expressed a significantly higher level of genomic RNA, which contains the packaging signal, than the other vectors while still expressing a high level of the subgenomic RNA encoding CAT. The high level of genomic RNA most likely contributes directly to the higher titer of MFG. We also compared viral titers from subcloned PA317 producer lines containing LNC-CAT and MFG-CAT-Neo, and confirmed that the titer of the MFG virus was higher than that of the LNCX. In selected subcloned transduced NIH3T3 cells, average levels of CAT activity were nine-fold higher from MFG-based vector. Our results suggest that there are significant differences in both the titer and the level of gene expression between retroviral vectors which are currently being used in gene therapy clinical trials.
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PMID:Analysis of the relative level of gene expression from different retroviral vectors used for gene therapy. 887 26

Flt3/flk-2 ligand (Flt3-L) co-stimulates and synergizes with cytokines such as granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin in the proliferation of bone marrow and cord blood hematopoietic stem and progenitor cells. To study the biological effects of Flt3-L on the Flt3-L responsive AML5 cell line, the retroviral vector L(Flt3-L)SN was constructed based on the vector LXSN, but containing the human Flt3-L cDNA transcriptionally regulated by the Mo-MLV LTR. High-titer amphotropic producer cells that generated 10(6) cfu/mL after shuttle packaging through ecotropic packaging cells were isolated. AML5 cells were cultured overnight with L(Flt3-L)SN retroviral supernatant, 8 micrograms/mL polybrene, and 100 U/mL G-CSF, and expanded 1 week in medium with G-CSF. Transduced cells were selected in medium containing 0.4 mg/mL G418 and then in medium with 1.0 mg/mL G418. Retroviral mediated gene transfer in G418-resistant cells was confirmed after amplification by PCR of neo-specific sequences in genomic DNA. Northern blot analysis demonstrated L(Flt3-L)SN mRNA expression. Soluble Flt3-L was undetectable (< 100 pg/mL) by ELISA assay of conditioned medium from transduced cells, but Flt3-L was detected on the surface of AML5 cells by FACS analysis. Cells were plated in colony assay with and without 100 ng/mL Flt3-L, 100 U/mL G-CSF, and the combination. Gene transfer or growth factor treatment increased somewhat the clonogenicity of the nontransduced AML5 cells. More strikingly, L(Flt3-L)SN and each growth factor combination greatly increased the size of the resultant colonies such that the size of colonies from AML5/Flt3-L cells without added growth factor approximated that of the AML5 cells stimulated by exogenous soluble Flt3-L. Moreover, MAP kinase activity in L(Flt3-L)SN-transduced cells cultured without soluble Flt3-L was increased to the level induced in control cells by soluble Flt3-L. These results indicate that retroviral mediated gene transfer and autologous expression of the Flt3-L enhances growth and intracellular signaling of AML5 cells, information that should be of value for studying the effects of Flt3-L on immature subsets of primary hematopoietic stem and progenitor cells.
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PMID:Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells. 898 7

Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9-induced erythroid cell proliferation and differentiation.
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PMID:Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation. 1073 74

The effects of different cytokines on growth of human cord blood CD34 cells was studied by performing long-term culture (LTC) with primary human stromal cells transduced with genes for either Flt3-ligand (L) (human transmembrane, murine soluble or murine membrane-bound forms), human interleukin 3 (IL-3) or human GM-CSF. Molecular analysis of genomic DNA from transduced stromal cells using neo-specific polymerase chain reaction demonstrated gene transfer of G418-selected stromal cell populations. Enzyme-linked immunosorbent assay and biological assays of conditioned media from transduced stromal cells indicated expression and release of soluble cytokines. Numbers of both immature and more mature progenitors (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GEMM, BFU-E, CFU-GM) were increased threefold compared to control in the Flt3-L (transmembrane) LTC throughout five weeks of culture. IL-3 and GM-CSF feeders increased progenitor cell output also, but these effects were significantly lower than Flt3-L feeders. The two Flt3-L isoform engineered feeders, Ex6 (soluble isoform) and 5H (membrane-bound isoform), showed a decreased effect compared to the transmembrane Flt3-L feeders and, in particular, Ex6 feeders were similar to control feeders and 5H feeders were comparable to Flt3-L feeders only in the first two weeks of LTC. These results were apparent also by limiting dilution assays that showed a higher frequency of pre-CFU in the transmembrane Flt3-L feeders compared to control and the other cytokine feeders. Exogenous addition of soluble growth factors to suspension cultures without feeder layers, while superior to stromal feeders for short-term expansion of early progenitors, were inferior to the long-term maintenance/output on stromal feeders. Pre-CFU analysis supported these data. These results may be of some significance to understanding the actions of Flt3-L on blood cell production.
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PMID:Comparative effects of retroviral-mediated gene transfer into primary human stromal cells of Flt3-ligand, interleukin 3 and GM-CSF on production of cord blood progenitor cells in long-term culture. 1101 47

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