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)

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.
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PMID:BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors. 145 57

Cultured epithelial grafts have proven to be life-saving in the treatment of large skin losses. It has become apparent that one of the main difficulties of this technology is the overall poor take of the grafts as a consequence of severely damaged dermal beds. Skin substitutes providing both cultured keratinocytes, as an epidermal layer, and a dermal analogous offer a more suitable material for skin repair. Ex vivo transfer of stroma regeneration-promoting genes to keratinocytes appears to be an attractive strategy for improving the therapeutic action of these grafts. The use of epidermal-specific promoters as expression drivers of exogenous genes results in both high expression levels and stratum specificity, as shown in transgenic mice studies. Most current gene transfer protocols to primary keratinocytes involve transduction of epidermal cells with retroviral vectors. However, transfer of gene constructs harboring these long DNA fragment promoters cannot be achieved through viral transduction. In this paper, we describe a protocol consisting of lipid-mediated transfection, G418 selection and an enhanced green fluorescence protein (EGFP)-based enrichment step for obtaining high levels of transgene-expressing primary keratinocytes. Using this protocol, the cDNA for vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen driven by the 5.2 kb bovine keratin K5 promoter, was stably transfected into pig primary keratinocytes. Genetically modified keratinocytes, expanded on live fibroblast-containing fibrin gels and transplanted to nude mice as a composite material, elicited a strong angiogenic response in the host stroma as determined by fresh tissue examination and CD31 immunostaining. Since the formation of a well-vascularized wound bed is a crucial step for permanent wound closure, the use of an 'angiogenic' composite material may improve wound bed preparation and coverage with cultured keratinocyte grafts.
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PMID:Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter. 1051 23

Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase (NTR) gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% 02 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.
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PMID:Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy. 1192 90

A replication-deficient recombinant retrovirus containing the cDNA coding for human vascular endothelial growth factor (VEGF) was generated, and then infected rabbit primary skin fibroblasts. After selection with G418, the transduced colonies have the ability of producing VEGF. The integration and expression of VEGF in transduced cells were confirmed by Southern blot, PCR, Northern blot and RT-PCR assay. The VEGF secreted by transduced cells has strong bioactivity when assayed by endothelial proliferation and Miles vascular permeability assay. Thus, this study pave the way for future study of biological and physiological effect of VEGF in vivo.
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PMID:[Expression of VEGF on rabbit skin fibroblasts using retroviral vector]. 1254 73

The goal of this study was to test the efficiency and possible functional effects of a Friend Leukemia derived retrovirus vector (FOCH29-NeoR) on cultured human keratinocytes, obtained from skin biopsy samples. The keratinocytes were grown and infected with filtered Friend vector supernatant. After one or two doses of infection, one duplicate of the culture was submitted to selection with G418; the other one was utilized for DNA extraction and PCR modification detection. Transduction efficiency was 46.66 percent and 47.22 percent for one and two doses of infection respectively (range 100 to 15 %). Colony Forming Efficiency (CFE) assays were done with Rodhamine-B staining in nonselected modified cultures and negative controls. There was no difference in CFE (% CFE= 10.74+/-6.53 negative control vs % CFE= 9.22+/-5.45 with one dose, and % CFE= 10.03+/-5.74 with two doses of infection). Nevertheless, the cell-cycle analysis done by Propidium Iodade (PI) incorporation and colchicine-arrest assays in nonselected transduced and nontransduced cells show that transduced keratinocytes have a longer time to enter G2. As far as we know, this is the first report of retroviral transduction-induced changes in the cell cycle done on human keratinocytes. This observation is very important because retroviral vectors of genes, such as platelet derived growth factor (PDGF) or vascular endothelial growth factor (VEGF), are expected to facilitate the implementation of these modified cultures for tissue grafting and skin substitute development and potentiate the effectiveness of the grafts.
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PMID:Human skin keratinocytes modified by a Friend-derived retroviral vector: a functional approach. 1615 Feb 10

This study was purposed to investigate the inhibitory effect of short hairpin RNA (shRNA) on expression of vascular endothelial growth factor (VEGF) receptor flt-1 gene in leukemia cells line K562, and to explore the influence of shRNA invasive ability on leukemia cells and its mechanism. The recombinant eukaryotic expression plasmid containing flt-1 shRNA gene was transfected into K562 cells by lipofectamine mediation and positive clones were screened by G418. shRNA gene in K562 cells was confirmed by PCR. RT-PCR and Western blot were employed to detect the expression of flt-1 mRNA and protein in leukemia cells. The invasive ability of K562 cells was studied by Boyden chamber invasion assay before and after flt-1 shRNA transfection. MMP-2 and MMP-9 mRNA expressions were detected by RT-PCR after transfection of the recombinant plasmid C1/U6/FltS2 into K562 cells through liposome. The results showed that the recombinant eukaryotic expression plasmid had been transfected into the human leukemia cell line K562 and positive clones had been screened by G418 for 2 weeks. PCR detection revealed the stable expression of the shRNA gene in K562 cells. Flt-1 gene and protein expressions were inhibited by plasmid-expressed shRNA after transfection of recombinant vetors C1/U6/FltS into K562 cells. The inhibitory efficiency of two different shRNA sequences targeting Flt-1 gene were 46.1% and 65.4% respectively. The expression of MMP-2 and MMP-9 mRNA decreased, and the mean invasion rate in C1/U6/fltS2-transfected K562 cells was lower than that in nontransfected cells. It is concluded that shRNA eukaryotic expression vector specific to VEGF receptor flt-1 gene can high efficiently be transfected into leukemia cell line K562, effectively inhibits the expression of flt-1 gene, weakens the in vitro invasive ability of leukemia cells and the expression levels of MMP-2 and MMP-9 mRNA, which suggests that the VEGF involves in the migration of leukemia cells by regulating the MMP-2 and MMP-9 through joints with the receptor.
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PMID:[Inhibition effect of short hairpin RNA on VEGF receptor flt-1 gene expression in leukemia cell line K562]. 2003 Sep 17

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.
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PMID:[Anti-sense nucleic acid of CyclinD1 induces apoptosis of lung adenocarcinoma cancer cell A549]. 2168 45

Biglycan is an important component of the extracellular matrix, which belongs to the small leucine-rich proteoglycan family. Recent studies have shown that biglycan expression is elevated in many tumor tissues and implies poor prognosis, such as colon cancer. However, the molecular mechanism of biglycan in colon cancer has not been investigated. The present study aimed to investigate the effects of biglycan on vascular endothelial growth factor (VEGF) expression in colon cancer cells and on tumor angiogenesis in vivo. Biglycan overexpression vectors were constructed, and the stable biglycan overexpression in human colon cancer cell lines (HCT116 cells) was established by G418 screening. The stable cell clones were subsequently used to initiate tumor xenografts in nude mice. Our results showed that biglycan overexpression notably up-regulated the levels of VEGF in colon cancer cells, which was further confirmed by immunohistochemistry analysis in the xenograft colon tumors. Moreover, high levels of biglycan promoted angiogenesis and colon tumor growth, as evidenced by the increased cell viability, colon tumor size, and weight, as well as the CD34 expression. Additionally, we found that the extracellular signal-regulated kinase (ERK) signaling pathway was activated by biglycan in colon cancer cells. The ERK inhibitor PD98059 dramatically reversed the increased expression of VEGF induced by biglycan. Taken together, our results indicated that biglycan up-regulated VEGF expression in colon cancer cells and promoted tumor angiogenesis. Biglycan-mediated VEGF regulation may correlate with the activation of the ERK signaling pathway. Therefore, biglycan may be a promising target for anti-angiogenic therapy for cancer.
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PMID:Biglycan up-regulated vascular endothelial growth factor (VEGF) expression and promoted angiogenesis in colon cancer. 2537 Oct 74