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)

Primary fetal human adrenocortical cells of definitive zone origin were transfected by electroporation with pSV3neo, a plasmid coding for SV40 T antigen and neo, which confers resistance to the antibiotic G418. The clones obtained proliferated for 30 to 40 population doublings after isolation when grown under standard medium conditions, and then entered 'crisis'. When early-passage clones were incubated with cyclic AMP (1:1 N6-monobutyryl and 8-bromo analogues), cell rounding was observed, as in primary cultures of human adrenocortical cells. As previously shown in bovine adrenocortical cells, rounding was inhibited with a monoclonal antibody against urokinase plasminogen activator but not with a monoclonal antibody against tissue plasminogen activator. The regulation of the steroidogenic pathway in clones was investigated. The effects of cyclic AMP and activation of protein kinase C were examined in cells maintained in defined medium or in the presence of serum. 17 alpha-Hydroxylase was strongly induced by cyclic AMP, as evidenced by Northern blotting and by the conversion of progesterone or 25-hydroxy-[1,2-3H]cholesterol, this induction being blocked by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA). Cholesterol side-chain cleavage enzyme was strongly induced by cyclic AMP, and clones also showed low activities of 21-hydroxylase and 11 beta-hydroxylase. Under all circumstances levels of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as assessed by Northern blotting or by conversion of 25-hydroxycholesterol, were very low. 3 beta-HSD was not induced by cyclic AMP or TPA alone, but was induced by the combination of the two agents. The regulation of 17 alpha-hydroxylase and 3 beta-HSD resembles that previously described in primary cultures of human fetal adrenocortical cells. Thus, transfection with SV40 T antigen resulted in the production of clones which preserve the unique characteristics of the human adrenal cortex.
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PMID:Expression of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase in fetal human adrenocortical cells transfected with SV40 T antigen. 132 52

Cultured bovine adrenocortical cells reach replicative senescence after 100-120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17+ to CYP17-, where '+' and '-' refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17 alpha-hydroxylase). We used luciferase reporter constructs to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing -2544 to +29 and -488 to +29 of the 5' region of CYP17 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the CYP17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the -488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the -2544 and -488 constructs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. 133 23

We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.
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PMID:Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors. 137 12

To test the putative role of c-fos in F9 differentiation, we have attempted to inhibit c-fos expression in these cells using an SV40-based expression vector (pSVneo-sof) that programs expression of c-fos antisense (sof) sequences as a 3' extension of a neo mRNA transcript. Of six G418-resistant clones isolated in transfection experiments, five expressed neo-sof transcripts. Two clones synthesized polyadenylated mRNA of the expected size (3.8 kb), two were smaller than expected, and one was larger. Two clones that expressed reduced levels of c-fos protein were inhibited in the induction of laminin, type IV collagen, and proteoglycan-19 RNA transcripts measured after 4 days of differentiation induction with RA and dibutyryl cyclic AMP. Also inhibited was the induction of the differentiation markers, TROMA-1 and TROMA-3. Antisense-expressing cells were not inhibited in the differentiation pathway to visceral endoderm since the alpha-fetoprotein gene was activated normally. We conclude that c-fos antisense expression inhibits some aspects of differentiation in F9 cells.
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PMID:Expression of c-fos antisense RNA inhibits the differentiation of F9 cells to parietal endoderm. 245 27

In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.
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PMID:Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: transfection with SV40 as a means for dissociating effects of senescence on growth and on differentiated function gene expression. 253 19

Expression of the biologically active beta-subunit of mouse nerve growth factor (beta-NGF) was conferred onto cultured AtT-20 mouse pituitary cells via a replication-defective retroviral vector. The retroviral LTR promoter was used for expression of a cDNA for beta-NGF corresponding to the shorter mRNA species produced by most tissues that receive sympathetic innervation. The vector included the TU5 gene conferring resistance to the neomycin analogue G418 under the control of an SV40 early promoter. AtT-20 cells, which produce essentially no endogenous beta-NGF, were infected and then cloned under G418 selection. Clones were evaluated for release into the medium of biologically active beta-NGF using a bioassay for neurite extension from PC-12 cells. The biological activity was equivalent to 1 to 10 ng of beta-NGF per mg cell protein over 24 hours. Immune precipitation and SDS/polyacrylamide gel electrophoresis of labelled proteins in the medium showed that the major form of immunoreactive beta-NGF secreted from cells comigrated with authentic mature beta-NGF, apparent Mr 13,000. Release of this beta-NGF from cells was stimulated by addition of 1 mM-8-bromocyclic AMP or 10 nM-corticotropin releasing factor, suggesting that at least some of the processed factor is stored in secretory vesicles. These studies, together with those on other cultured cells, which produce beta-NGF and lack secretory granules, e.g. L cells, suggest that the beta-NGF precursor synthesized from the shorter mRNA species can be processed and secreted through either the regulated or constitutive route. This retroviral vector provides a potential means of conferring beta-NGF expression onto a number of different cell types in culture and in vivo.
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PMID:Retrovirus-mediated gene transfer of beta-nerve growth factor into mouse pituitary line AtT-20. 283 91

The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO4, the cells expressed the receptor at a level of 250 pmol/mg of membrane protein. The glucagon receptor was functionally coupled to increases in cyclic AMP in S2 cells. Protein immunoblotting with anti-peptide antibodies revealed the expressed receptor to have an apparent molecular mass of 48 kDa, consistent with low levels of glycosylation in this insect cell system. Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Anisotropy data suggest that the mobility of the fluorescein is restricted when the ligand is bound to the receptor. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. These data would be consistent with a conformational change upon the binding of agonist to the receptor. The combination of [fluorescein-Trp25]glucagon with the S2 cell expression system should be useful for analyzing glucagon receptor structure and function.
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PMID:Interaction of [fluorescein-Trp25]glucagon with the human glucagon receptor expressed in Drosophila Schneider 2 cells. 759 63

Binding activity and biologic effect of a novel alpha-melanocyte-stimulating hormone analogue were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. The human MC1 and MC5 receptor genes were cloned into the expression vector pcDNA3. 1/ myc-his(-) B. The vectors were transferred to HEK-293 cells by the calcium phosphate method. Stable receptor populations were generated using G418 selection (900 microg x mL(-1)) for subsequent bioassay analysis. K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were obtained in competition with [125I]-NDP-MSH for binding studies. The cyclic AMP level was tested by using [3H]-cyclic AMP kit. It is showed that K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were (0.159 +/- 0.040), (35.430 +/- 6.743), (19.293 +/- 2.780) and (2.230 +/- 0.670) nmol L(-1), respectively. Its EC50 values for MC1, MC3, MC4, and MC5 receptors were (0.45 +/- 0.07), (7.80 +/- 0.65), (2.55 +/- 0.23) and (0.33 +/- 0.09) nmol L(-1), respectively. In these tests, the novel alpha-MSH analogue is a MC1R and MC5R selective agonist.
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PMID:[Detection of binding activity and biologic effect of a novel alpha-melanocyte-stimulating hormone analogue]. 1752 Aug 25