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)

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication was demonstrated by using tat- and rev-directed antisense oligoribonucleotides 68 and 69 nucleotides in length. In this study, human T-lymphoid cells were transduced with a murine amphotropic retroviral vector containing a polymerase III-driven chimeric gene consisting of the human tRNA(imet) sequence and the short tat- and rev-directed antisense sequences that had been shown before to inhibit HIV-1 replication. Pools of transduced, G418-resistant human T-lymphoid Jurkat or CEM cells showed reduced replication of HIV-1 in the presence of antisense-containing chimeric transcripts, but not with sense sequence-containing transcripts. These results demonstrate that short inhibitory antisense RNA transcripts can be stably expressed endogenously using polymerase III promoters, which can reduce replication of HIV-1. The approach described in this work combines the advantages of short and, usually, synthetic oligonucleotides with the stable intracellular expression of inhibitory genes for HIV-1 in target cells. Considering the small size of the described chimeric polymerase III genes, it appears feasible to combine multiple antiviral genes with the currently available retroviral vectors as gene delivery systems.
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PMID:Reduction in replication of the human immunodeficiency virus type 1 in human T cell lines by polymerase III-driven transcription of chimeric tRNA-antisense RNA genes. 784 87

We have studied effects of ferric transferrin (FeTF), ferric lactoferrin (FeLF), ferric complexes of pyridoxal- or salicylaldehyde-isonicotinoyl hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on expression of protein kinase C (PKC) mRNA transcripts in a variety of cultured cell lines. FeTF supported an increase of PKC-beta mRNA transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B-lymphoblastoid (Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte (U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an increase of PKC-beta mRNA transcripts in any of these cell lines. Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in HL-60, K562, and U937 cells only. Preincubation of cells with desferrioxamine (DF), a cell-permeable iron chelator, abolished the increments of PKC-beta mRNA observed in response to FeTF or FAC. In contrast to results with PKC-beta, neither FeTF nor FAC caused an increase of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA regulatory elements of the PKC-beta gene, we prepared genetic constructs containing various portions of the human PKC-beta 5'-flanking DNA linked to the firefly luciferase gene. Constructs were cotransfected with the neomycin resistance plasmid, Pwl-neo, into HRE H9 cells, and stable transfectants were selected in G418. Treatment with FeTF of transfectants bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking region increased luciferase activity and mRNA transcripts 2.5-fold. This increase was blocked by DF. Neither luciferase activity nor mRNA increased with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp of the PKC-beta 5'-flanking region. These data provide direct confirmation that iron is involved in regulation of PKC-beta but not PKC-alpha gene expression in many cell lines. The form in which iron is presented to these cell lines appears to affect its availability for this function, and cells vary in their capabilities to use nontransferrin iron to support PKC-beta gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF is mediated by DNA sequences located between -2200 bp and -587 bp in the 5'-flanking region of the human PKC-beta gene.
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PMID:Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'-flanking region of the PKC-beta gene in the response to ferric transferrin. 794 5

We have previously reported that chimeric neomycin phosphotransferase (neo)-Rev response element (RRE) transcripts suppress the function of the human immunodeficiency virus type 1 (HIV-1) Rev trans-activator protein in HeLa cells. In an extension of these experiments, human CD4+ CEM cells (G418-resistant cell populations and clonal isolates) stably expressing chimeric neo-RRE genes (2, 3, or 6 RRE copies) were generated using retroviral-mediated gene transfer. The transduced CEM clones were infected with the HIV-1 HTLVIIIB isolate and the following three phenotypes were observed: (i) the transduced CEM cells were readily infected with HIV-1 indistinguishable from the control CEM cells; (ii) the appearance of HIV-1 replication markers was significantly delayed; (iii) no signs of HIV-1 replication were detectable although proviral HIV-1 DNA sequences could be detected in these cells. Furthermore, HIV antigen expression was limited in neo-resistant CEM cell populations inoculated with the HIV-1 HTLVIIIB isolate. Only 10% of the CEM-pX17-3xRRE cells and 20% of the CEM-pX17-2xRRE cells displayed HIV-1 antigens 43 days after challenge and had retained CD4 surface expression on 47% and 64% of the cells, respectively. In sharp contrast, 80% of the CEM-pX17 or the CEM-pX17-6xRRE cells expressed HIV-1 antigens but no CD4 antigens were detectable in these cultures. These results clearly indicate that RRE decoys could be developed into an effective somatic gene therapy approach against HIV-1 induced acquired immunodeficiency syndrome (AIDS).
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PMID:Constitutive expression of chimeric neo-Rev response element transcripts suppresses HIV-1 replication in human CD4+ T lymphocytes. 818 99

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

A hairpin ribozyme targeting the 3' LTR region (9456) of SIVmac238 was cloned into a murine retroviral vector. This target sequence is conserved among various SIV, as well as most HIV-2, strains. The ribozyme cassette is driven from a polymerase III promoter, that of the human tRNAval gene. Hybrid human B-/T-cell lines (CEM/174) were transduced with the retroviral constructs and selected for G418 resistance. Cells stably expressing the 9456 ribozyme exhibited long-term resistance to infection by a pathogenic molecular clone of SIV and two strains of HIV-2. The ribozyme was also able to effectively reduce the proviral DNA burden. Its efficient protection against SIV/HIV-2 infection constitutes an important step toward evaluating ribozyme gene therapy in a primate model.
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PMID:Intracellular immunization against SIVmac utilizing a hairpin ribozyme. 861 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

The gene encoding for p16ink4a, a negative regulator of transition between G1 and S phase, is homozygously deleted in a large proportion of acute lymphoblastic leukaemias (ALL). Transfer of p16ink4a gene in several solid tumour cell lines with functional pRb and lacking both p16ink4a alleles has resulted in a dramatic reduction of cell proliferation, and the aim of this work was to confirm this effect in leukaemic (especially ALL) cell lines. We tested the proliferation in liquid medium and in soft agar after transfer of p16ink4a gene by a retroviral vector in leukaemic cell lines with homozygous p16ink4a gene deletion (K562, CEM, Jurkat cell lines) or with p16ink4a gene hemizygous deletion and a point mutation inactivating the remaining allele (HL60 cell line). The viral titre obtained after transfection of PA317 amphotropic packaging cell line, which has a p16ink4a gene homozygous deletion, was low, suggesting that p16ink4a gene expression could impair viral production of retroviral packaging cell lines derived from the NIH3T3 cell line. After retroviral transfer of p16ink4a in cell lines and G418 selection in liquid medium, a strong cell proliferation inhibition was observed for K562, CEM and Jurkat, but no inhibition was seen for HL60. A strong growth reduction in soft agar was also observed with p16ink4a-transduced CEM, Jurkat and K562 cells, with a moderate growth reduction in the HL60 cell line. The growth inhibition in liquid culture, of K562 and Jurkat cell lines, was confirmed by electroporation transfer of the p16ink4a gene. Our findings show that p16ink4a gene transfer has a growth-inhibitory effect in leukaemic cell lines with p16ink4a gene homozygous deletion. These data suggest that p16 could be a suitable gene for gene therapy in ALL.
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PMID:Transfer of p16inka/CDKN2 gene in leukaemic cell lines inhibits cell proliferation. 890 84

Human immunodeficiency virus type 1 Vpr is a 96-amino-acid virion-associated protein that arrests cells in the G2 phase of the cell cycle in peripheral blood lymphocytes, HeLa, 293, 293T, A549, Jurkat, CEM, SupT1, CV-1 and COS1 cells. When we transfected Vpr expression vector into mouse NIH3T3 and then cultured it in the presence of G418, NIH3T3 cells were the drug resistant cells yielded. The surviving colonies, however, exhibited a degenerating morphology up to 8 approximately 20-fold smaller than the control vector colonies. In addition, the growth of NIH3T3 cells transiently transfected with Vpr expression vector declined dramatically compared with that of transfectants with control vector, suggesting that Vpr significantly interferes with cell proliferation of NIH3T3 cells. Cell cycle characterization by flow cytometry indicated that expression of Vpr did not induce G2 cessation in NIH3T3. These findings strongly suggest that Vpr has a novel pathway to retard cell growth independently and arrests the G2 phase of the cell cycle.
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PMID:Human immunodeficiency virus type 1 Vpr gene product prevents cell proliferation on mouse NIH3T3 cells without the G2 arrest of the cell cycle. 912 20

Expression of certain variants of dihydrofolate reductase (DHFR) in mammalian cells protects them from methotrexate. Retroviral transfer of the gene for such a variant DHFR into hematopoietic cells might permit selection of modified cells in vivo by antifolate administration or alleviate antifolate-induced myelosuppression in patients receiving antifolate therapy. We examined protection of cells of the human lymphoblastoid line, CCRF-CEM, transduced with variants of mouse DHFR. In transduced cells selected with G418 but not with antifolate, the variant that had arginine substituted for leucine 22 did not protect against either methotrexate or trimetrexate; however, four other variants did offer protection, with the best having leucine 22 changed to tyrosine. Polyclonal cultures transduced with the different variants express DHFR at about the same level, but clones within each polyclonal population differ in DHFR expression levels and in resistance. These differences in expression were shown to reflect different integration sites for proviral DNA. Exposure to trimetrexate selects highly resistant clones, with high expression due to both high copy number and integration sites that are favorable for expression. Differences in the resistance of cultures expressing different variants at the same level are due to differences in the catalytic activity of the expressed DHFR, its affinity for antifolates, and its stability.
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PMID:Protection of CCRF-CEM human lymphoid cells from antifolates by retroviral gene transfer of variants of murine dihydrofolate reductase. 969 74