No abstract available.
Humans ; Ulcer* ; Zidovudine*

No abstract available.
HIV* ; Humans ; Humans* ; Lymphocytes* ; Zidovudine*

No abstract available.
Genes, pol* ; HIV-1 ; Humans* ; RNA-Directed DNA Polymerase ; Zidovudine*

Background : Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs (suicide genes) into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase (HSV-tk) gene followed by administration of the antiviral drug ganciclovir. METHODS: The LNC/tK vector was transfered in vitro into mouse Neuro 2a cell lines (ATCC) and the transduced cell lines were selected in G-418, 500microgram/ml, for 14 days. Onex104 cells were cultured in 96 well culture plates in increasing concentrations of ganciclovir for 72 hours. The sesitivity to ganciclivir of these HSV-tk transduced, G-418 selected cells was measured with MTT assay RESULTS: The survival of HSV-tk transduced 1x104 neuro 2a cell lines is 103+/-3.5%, 68+/-4.2%, 54+/-3.8%, 17+/-2.6%, 13+/-3.1% at the concentration of 0, 0.1, 1.0, 10, 20microgram/ml ganciclovir, respectively. And the survival of HSV-tk not transduced 1x104 neuro 2a cell lines is 100+/-4.5%, 97+/-5.6%, 104+/-3.5%, 106+/-3.8%, 101+/-4.2%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Animals ; Cell Line* ; DNA ; Ganciclovir ; Mice ; Neuroblastoma* ; Phosphotransferases ; Retroviridae ; Zidovudine*

Background : Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs (suicide genes) into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase (HSV-tk) gene followed by administration of the antiviral drug ganciclovir. METHODS: The LNC/tK vector was transfered in vitro into mouse Neuro 2a cell lines (ATCC) and the transduced cell lines were selected in G-418, 500microgram/ml, for 14 days. Onex104 cells were cultured in 96 well culture plates in increasing concentrations of ganciclovir for 72 hours. The sesitivity to ganciclivir of these HSV-tk transduced, G-418 selected cells was measured with MTT assay RESULTS: The survival of HSV-tk transduced 1x104 neuro 2a cell lines is 103+/-3.5%, 68+/-4.2%, 54+/-3.8%, 17+/-2.6%, 13+/-3.1% at the concentration of 0, 0.1, 1.0, 10, 20microgram/ml ganciclovir, respectively. And the survival of HSV-tk not transduced 1x104 neuro 2a cell lines is 100+/-4.5%, 97+/-5.6%, 104+/-3.5%, 106+/-3.8%, 101+/-4.2%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Animals ; Cell Line* ; DNA ; Ganciclovir ; Mice ; Neuroblastoma* ; Phosphotransferases ; Retroviridae ; Zidovudine*

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are. focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer, To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of leo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.
Cell Line ; Eukaryotic Cells ; Helper Viruses ; Product Packaging ; Retroviridae ; Transfection ; Zidovudine*

BACKGROUND: The CCAAT/enhancer binding protein epsilon (C/EBPepsilon), one of the transcription factors, plays an important role in granulopoiesis. We examined an essential site of C/EBP epsilon for granulopoiesis. METHODS: 32Dcl3 cells and #1111 cells were transduced with retroviral constructs of C/EBP epsilon and C/EBP epsilon(R211A) (DNA binding mutant form). We examined growth rate, checked the neutrophil markers of Gr-1 and Mac-1, and counted differentiated cells in the transduced 32Dcl3 cells. The transduced #1111 cells were injected into five mice and survival times were analyzed. RESULTS: The mean number of green fluorescent protein (GFP) (+) 32Dcl3 cells transduced with C/EBP was 244, 045 at day 2, 582, 938 at day 4, and 873, 963 at day 6; mean expression of Gr-1 was 31.3% and Mac-1 32.6%; mean count of immature form was 41.0%, intermediate form 48.3%, and mature form 10.7%. In case of C/EBP epsilon (R211A) transduced 32Dcl3 cells, the respective figures were 707, 226, 1, 106, 736, and 2, 133, 819; 0.1% and 0.1%; and 91.7%, 4.3%, and 4.0%. The mean survival time of #1111 cells transduced with C/EBP epsilon was 26.0 days in placebo group and 34.0 days in 4-hydroxytamoxifen (4HT; C/EBP epsilon group); in case of C/EBP epsilon(R211A) transduced #1111 cells, the respective figures were 36.4 and 37.4 days. CONCLUSIONS: The growth of 32Dcl3 and #1111 cells was inhibited by C/EBP epsilon, but not by C/EBP epsilon(R211A). Also C/EBP epsilon was involved in the differentiation of 32Dcl3 cells, but C/EBP epsilon(R211A) was not. The DNA binding domain of C/EBP epsilon is a very important site for differentiating and inhibiting early myeloid cells.
Animals ; Carrier Proteins ; DNA* ; Mice ; Myeloid Cells ; Neutrophils ; Survival Rate ; Transcription Factors ; Zidovudine

BACKGROUND AND OBJECTIVES: Compared to other target cells examined for gene therapy, vascular smooth muscle cells (VSMCs) have the unique advantages including proximity to blood stream and relative abundance in vasculature. With an ultimate goal of developing VSMC-based therapies for cardiovascular disorders, we explored the utility of VSMC as a target cell for ex vivo gene therapy using a set of retroviral vectors. MATERIALS AND METHODS: Cultured VSMCs were transduced with replication-defective recombinant retroviruses harboring LacZ, nlsLacZ, mVEGF, mGM-CSF or bacterial CAT reporter. The VSMCs were examined for G418-selection, transduction efficiency, the level of transgene expression, and longevity of gene expression. ResultsVSMCs were readily transduced with different kinds of retroviral vectors. The bacterial neo r gene-transduced VSMCs were successfully selected with G418. The G418-selected VSMCs could express the transduced genes at a level comparable to NIH3T3. The level of transgene expression did not appear to be affected by the increasing number of passages. CONCLUSION: The results demonstrate an efficient transduction of VSMCs by retroviral vectors in vitro and an sustained expression of retrovirally transduced genes in VSMCs. VSMCs could be one of the ideal target cells for ex vivo cardiovascular gene therapy employing retroviral vector.
Animals ; Cats ; Gene Expression ; Genetic Therapy* ; Longevity ; Muscle, Smooth, Vascular* ; Retroviridae ; Rivers ; Transgenes ; Zidovudine

PURPOSE: We tried to assess the optimal conditions to improve low transduction efficiency and their effect on target cells. METHODS: Cultured NIH 3T3 cells were incubated with retroviral vectors bearing an enhanced green fluorescent protein (eGFP) gene. We varied the ratio of viral vectors to target cells (1:1-1:8) and the number of transfections (x1, x2), and compared transduction efficiencies. Also, the effects of polybrene on transduction efficiency and viability of target cells were assessed. Transduction of the eGFP gene was evaluated by observing NIH 3T3 cells under a fluorescence microscope and efficiencies were measured by the percentage of eGFP positive cells using FACscan. RESULTS: As the ratio of retroviral vectors to target cells increased, transduction efficiency was greatly improved, from 7% (1:1) to 38% (1:4). However, transduction efficiency did not increase any more when the ratio increased from 1:4 to 1:8. Cells transfected twice showed higher transduction efficiencies than cells transfected once, at a ratio of 1:8. The eGFP gene transduced to NIH 3T3 cells sustained its expression during repeated passages. However, after the third passage (day 9), the percentage of eGFP positive cells began to decline. The degree of this decline in eGFP expression was lower in cells transfected twice than in cells transfected once (P<0.05). The addition of polybrene did not have any toxic effect on NIH 3T3 cells and greatly increased transduction efficiency (P=0.007). In addition to vector component, transduction efficiency was very sensitive to culture confluence. Cells cultured and transfected in 24-well plate showed higher transduction efficiency, although cells cultured in 6- well plate proliferated more (P=0.024). CONCLUSION: Our data could be used as a basis for retrovirus-based gene therapy. Further study will follow using human cells as target cells.
Fluorescence ; Genetic Therapy ; Hexadimethrine Bromide ; Humans ; NIH 3T3 Cells* ; Retroviridae ; Transfection ; Zidovudine

Background: WDR1 is thought to be correlating with polymerization and depolymerization of actin protein. Though WDR1 was found to be within nucleus, in which actin could not be present by previous studies, the exact distribution pattern of WDR1 protein under various circumstances was not elucidated up to the present time. In this regard, we tried to see a change in the distribution of WDR1 protein within artificially induced senescent PC 12 pheochromocytoma cells for the first time. Methods: PC12 pheochromocytoma cells (ATCC CRL-1721) were grown in the culture media including 1 micrometer 3'-Azido-3'-deoxythymidine (AZT, Sigma-Aldrich, USA). The senescence of the cells was confirmed by senescence detection kit (Calbiochem, San Diego, CA). Immunocytochemical study by using WDR1 antibody was also performed in the cells treated with AZT during 0, 75 and 153 days. Results: WDR1 protein was mainly observed within the cytoplasm of the cells not treated with AZT. However, the distribution of the same protein was changed into the nucleus after 153 day-AZT treatment. Conclusion: The distribution of WDR1 protein was changed into nucleus in the artificially senescent PC12 cells.
Actins ; Aging ; Animals ; Culture Media ; Cytoplasm ; PC12 Cells* ; Pheochromocytoma ; Polymerization ; Polymers ; Zidovudine