Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We constructed a retroviral vector, pLhIL-9RSN, containing CDNA encoding the human interleukin-9 receptor (IL-9R) along with a neomycin phosphotransferase gene (Neo). In order to study the biological effects of the IL-9R, high titer (1-5 x 10(5) CFU/ml) viral supernatant, generated from the packaging cell lines, ecotropic GPE86 and amphotropic PA317, was used to transduce the IL-9R gene into sorted populations of CD34++ CD33-cells from human cord blood which are highly enriched for erythroid progenitor cells (BFU-E). Colony formation by BFU-E transduced with the IL-9R gene and grown without selection in G418 and in the presence of erythropoietin (Epo) and interleukin (IL)-9 was significantly increased up to three-fold and the size of the erythroid colonies was significantly increased 50-100% compared to colony formation by mock virus transduced cells. Moreover, colony formation by IL-9R-transduced cells was more sensitive to stimulation with lower doses of IL-9 and Epo. Individual colonies formed with or without selection in G418 were evaluated. Proviral integration and mRNA expression were respectively assessed by polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR analysis and were apparent in 93% and 84% of the G418-resistant colonies and 52% and 48% of the colonies grown in the absence of G418. Our study demonstrates that a functional human IL-9R gene can be efficiently transduced into human cord blood hematopoietic progenitors using retroviral vectors with increased cytokine-dependent erythroid colony formation.
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PMID:Transduction of human interleukin-9 receptor gene into human cord blood erythroid progenitors increases the number of erythropoietin-dependent erythroid colonies. 897 79

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.
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PMID:The 30/35 kDa chymotryptic fragment of fibronectin enhances retroviral-mediated gene transfer in purified chronic myelogenous leukemia bone marrow progenitors. 900 33

IFN-gamma has a direct antitumor effect on many tumor cell lines mediated through the IFN-gammaR. One effect of IFN-gamma is to induce the nuclear transcription factor IFN regulatory factor-1 (IRF-1), which may function as a tumor suppressor. In this study, mouse IRF-1 cDNA under a high constitutive expression promoter was transfected into the highly aggressive, nonimmunogenic MCA 101 murine sarcoma. Clones were obtained by G418 selection and screened for IRF-1 mRNA expression by reverse transcriptase-PCR (RT-PCR). High expression clones had high levels of two MHC class I proteins (H-2Kb and H-2Db) on the cell surface that correlated with increased levels of class I mRNA by RT-PCR. Furthermore, these clones also had increased levels of MHC class II protein (I-Ab), which correlated with increased levels of one subunit of class II mRNA by RT-PCR. IRF-1-expressing clones had markedly diminished cell growth in vitro and decreased anchorage-independent growth in a soft agar assay. These clones also demonstrated markedly prolonged tumor latency and slowed growth in syngeneic C57BL/6 mice. IRF-1 gene-transfected cells had shortened tumor latency and formed faster growing tumors in gamma-irradiated immunodeficient mice compared with results in immunocompetent mice. Mice immunized with IRF-1-transfected cells were protected against subsequent challenge with IRF-1 transfected cells and also demonstrated greater tumor latency and slower tumor growth against subsequent challenge with untransfected cells compared with mice immunized with empty vector-transfected cells. These studies demonstrate a tumor suppressor effect of IRF-1, which acts in vivo through both partial reversion of the malignant phenotype and enhanced immune recognition and may play a role in the antitumor effects of IFN-gamma.
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PMID:IFN regulatory factor-1 gene transfer into an aggressive, nonimmunogenic sarcoma suppresses the malignant phenotype and enhances immunogenicity in syngeneic mice. 901 71

Overexpression of p185erbB2/neu has been detected in many adenocarcinomas, including prostatic cancer. In this study, a nontumorigenic cell line isolated from the rat prostatic epithelium (NbE) transfected with the activated oncogene p185neu-T was used to investigate the role of this oncogene in tumor progression. When clones overexpressing p185neu-T were injected orthotopically (1.5 to 2 x 10(6) cells) into the dorsal-lateral prostates of nude mice, prostatic tumors were detected in all mice injected and metastasis to the skeletal muscle in the rib area in 60-80% of the mice injected. Tumor and metastasis origin was confirmed by reselection with G418 and reverse transcriptase-polymerase chain reaction. Control cell lines produced no prostatic tumors or metastases. Incubation at low density (12500 cells/2 cm2) in serum-free medium revealed that clones overexpressing p185neu-T had a higher rate of [3H]thymidine incorporation than did control clones on 3, 5, and 7 d after plating (P < or = 0.0001) and constitutively overexpressed the 2.6-kb ornithine decarboxylase transcript. Additionally, clones overexpressing p185neu-T demonstrated an increased expression of epidermal growth factor receptor and p180erbB4, as judged by RNA blot analysis. Together these data support the hypothesis that overexpression of p185neu-T fosters tumor progression by several pathways, including induction of the metastatic cascade, increased proliferative capabilities, and increased expression of other members of the erbB2 gene family.
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PMID:Metastasis induced by overexpression of p185neu-T after orthotopic injection into a prostatic epithelial cell line (NbE). 925 83

Progressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter; i.e., basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter-specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c-NOS), and the growth-associated protein GAP-43, three genes known to be expressed in central cholinergic neurons. A "nondestructive" method of screening cultured cells for their expression of c-NOS was established using depolarization with medium containing 50 mM potassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E-115 (c-NOS-positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature-sensitive SV-40 transforming activity and neomycin resistance. Cell colonies surviving in G418-containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33 degrees C, differentiated by switching to a low-serum medium and growth at 39 degrees C, and screened for depolarization-induced accumulation of nitrite in the medium. The subset of putative c-NOS-positive clones (about 4%) were then screened for their expression of mRNAs using RT/PCR in combination with CE/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cells cloned from embryonic rat brainstem.
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PMID:Use of capillary electrophoresis with laser-induced fluorescence detection to assess messenger ribonucleic acid molecules amplified by the polymerase chain reaction: applications in the cloning of cells. 937 66

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
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PMID:Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage. 952 44

Gastrin (G-17) is a trophic hormone with a high affinity for the cholecystokinin-B receptor (CCK-BR); the mechanisms linking receptor binding and activation of downstream events to cell growth are not known, and these studies have been hampered by the lack of a cell model. We have established a pancreatic carcinoid cell line, BON, which produces a number of gut hormones; however, these cells lack native CCK-BR. The purpose of our study was to develop a model cell line containing the CCK-BR and to characterize the cellular mechanisms involved in gastrin regulation of human cell growth. BON cells were transfected with an expression plasmid containing the human CCK-BR, and stable clones were selected using G418. Functional CCK-BR was confirmed by reverse transcriptase-polymerase chain reaction, 125I-gastrin binding, and mobilization of intracellular calcium ([Ca2+]i) in response to G-17. Stable transfectants were treated with G-17 (+/-) the CCK-BR antagonist, L365,260 (L-60); growth was assessed using a Coulter counter. G-17 stimulated the growth of the stable clones, whereas the selective CCK-BR antagonist, L-60, abolished this G-17-mediated trophic effect. We have shown that G-17, acting through the CCK-BR, mobilizes [Ca2+]i as a second messenger and stimulates cell growth. Our unique BON cell line, stably transfected with the human CCK-BR, provides a novel paradigm to further delineate signaling mechanisms in gastrin regulation of human cell growth.
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PMID:A functional in vitro model to examine signaling mechanisms in gastrin-mediated human cell growth. 983 32

We investigated a strategy for gene therapy, intracellular expression of anti-HIV-1 Rev single-chain variable fragments (SFvs), in promonocytic (U1) and T (ACH-2) cell lines latently infected with HIV-1. The cellular and molecular mechanisms leading to activation of latent integrated HIV-1 provirus in U1 and ACH-2 cells have been well delineated. These cells produce HIV-1 in response to stimulation with certain cytokines. U1 and ACH-2 cells were transduced with a murine retroviral shuttle vector that expresses anti-Rev SFv (pLXSN-D8SFv-Rev) or with a control murine leukemia virus (MLV) vector (pLXSN). Tumor necrosis factor alpha (TFNalpha)-, interleukin 6 (IL-6)-, and phorbol myristate acid (PMA)-induced HIV-1 expression, as determined by reverse transcriptase (RT) assay, was significantly inhibited in cells transduced with pLXSN-D8SFv-Rev, compared with cells transduced with pLXSN. In addition, pLXSN-D8SFv-Rev-transduced cells, when incubated with monokine-enriched supernatants of human peripheral blood monocyte cultures, produced significantly less HIV-1 than did cells transduced with pLXSN. This resistance to cytokine-induced HIV-1 expression was demonstrated in SFv-transduced U1 and ACH-2 cells maintained in G418-free medium for 2 months. These data suggest that feasibility of utilizing various anti-HIV-1 SFvs to block activation of HIV-1 infection in vivo.
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PMID:Inhibition of HIV type 1 replication in chronically infected monocytes and lymphocytes by retrovirus-mediated gene transfer of anti-Rev single-chain variable fragments. 984 Feb 90

Using retroviral supernatants derived from the amphotropic murine packaging cell line PA317 and the amphotropic canine packaging cell line (DA), cord blood and mobilized peripheral blood CD34+ cells were transduced with the vector LN (neomycin resistance) and the vector L-TR/TAT neo (neomycin resistance in conjunction with a double-hammerhead ribozyme conferring anti-HIV activity). Different multiplicities of infection (MOI) were applied in the setup according to vector titrations on NIH-3T3 cells. PA317-based supernatants were tested at MOI of 10 and 30. Purified concentrated DA-derived vector preparations were tested at MOI of 10, 30, 100, and 300. Immediately after transduction, CD34+ cells were plated into colony assays in the presence and absence of G418 to evaluate the amount of gene transfer and potential toxic effects of the vectors on colony growth. The remaining cells were subjected to G418 selection in liquid culture for 12 days and subsequently challenged with HIV-1JR-FL to test for efficacy of the anti-HIV gene in macrophages derived from transduced CD34+ cells. Transduction by the PA317-packaged vectors was maximal at the lowest MOI used and did not increase with increasing MOI. In contrast, transduction by the DA-packaged vectors could be progressively increased using increased MOI. The net transduction efficiency per unit of reverse transcriptase activity in the DA vector preparations was 8.7-fold higher than in the PA317 vector supernatants. HIV-1 challenge of the cells transduced by the ribozyme vector derived from the PA317 packaging cells resulted in a 1.5 log inhibition of p24 output compared with the control cells containing neomycin resistance only. A 2.5 log inhibition of p24 output could be observed in the cell population transduced with DA-packaged vector supernatants. Compared with retroviral supernatants from PA317 packaging cell lines, DA packaging line-derived vector preparations demonstrated higher transduction efficiency into CD34+ cells, particularly at higher MOI, and increased efficacy of the transferred anti-HIV gene when challenged with HIV-1JR-FL. The increase in transduction efficiency may be due to a higher ratio of intact vs. defective vector particles in the DA-derived vector preparations.
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PMID:Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line. 992 9

To determine whether overexpression of the human MnSOD transgene protected 32D cl 3 hematopoietic progenitor cells from ionizing irradiation, 32D cl 3 cells were co-electroporated with the pRK5 plasmid containing the human MnSOD transgene and SV2-neo plasmid with G418-resistant colonies selected. Two clones (1F2 and 2C6) were identified to overexpress the human MnSOD transgene by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and increased biochemical activity. Measurement of irradiation-induced damage was determined in cells removed from G418 for 1 week before irradiation. Irradiation survival curves, apoptosis tunnel assay, and Comet assay was performed. Cell cycle distribution was determined for each line at 0, 1, 3, 6, 24, and 48 hr after 500 cGy by fixing the cells in 70% ethanol, staining with propidium iodide, and analysis by flow cytometer. Biochemical MnSOD activity in U/mg protein was 2.6 for 32D cl 3 and significantly elevated to 8.4 and 6.6 (P < 0.001) U/mg protein for subclones 1F2 and 2C6, respectively. Irradiation survival curves demonstrated an increased shoulder on the irradiation survival curve for 1F2 and 2C6 cells with an n of 4.95 +/- 0.48 (P = 0.042) and 4.95 +/- 0.13 (P = 0.011), compared with 2.77 +/- 0.20 for 32D cl 3. A higher percent of 32D cl 3 cells demonstrated apoptosis at 24 and 48 hr after 1,000 cGy irradiation, compared with 1F2 and 2C6 cells (at 24 hr, 29.37% +/- 2.01% of 32D cl 3 cells were apoptotic compared with 5.21 +/- 2.61 (P = 0.018) and 5.27 +/- 2.58 (P = 0.004) for 1F2 and 2C6, respectively). Significantly more DNA strand breaks were detected by Comet assay in 32D cl 3 cells (Comet length at 600 cGy of 103.4 +/- 50.3 units, compared with 69.7 +/- 36.3 (P < 0.001) and 48.9 +/- 27.5 (P < 0.001) for 1F2 and 2C6, respectively). In contrast, irradiation-induced cell cycle arrest was similar between the cell lines with a G2/M phase arrest at 6 hr and a G1/S phase arrest at 24 and 48 hr after irradiation. While overexpression of MnSOD increases the shoulder on the irradiation survival curve of 32D cl 3 cells, decreases irradiation-induced apoptosis, and DNA strand breaks by Comet assay, irradiation-induced alterations in cell cycle distribution were not significantly altered. These 32D cl 3 subclonal lines overexpressing MnSOD provide a potentially valuable system with which to study the mechanism of irradiation-induced cell cycle arrest separate from irradiation-induced apoptosis.
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PMID:Overexpression of the human manganese superoxide dismutase (MnSOD) transgene in subclones of murine hematopoietic progenitor cell line 32D cl 3 decreases irradiation-induced apoptosis but does not alter G2/M or G1/S phase cell cycle arrest. 1064 56


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