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)

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.
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PMID:Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells. 248 39

Expression of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) was obtained in human cells from the gB-1 gene cloned in the episomal replicating vector pBK-1, which contains the origin of replication and early region of the human papovavirus BK. Selective systems for the TK+ phenotype in TK-143B cells and for resistance to G418 in adenovirus 5-transformed 293 cells were used to obtain stable transformants that produced gB-1. While gB-1 expression in 143B cells required induction by HSV-1 early proteins, constitutive gB1 production was observed in 293 cells, where endogenous trans-acting factors probably replace the need for early viral products in the activation of the cloned gB-1 gene. The amount of recombinant gB-1 was comparable to that produced during HSV-1 lytic infection in human cells, due to amplification of the inserted gene in the replicating episomal vector. Expression of gB-1 was induced by cadmium and zinc when the promoter of the mouse metallothionein-I gene was placed upstream of gB1 structural sequences. The inducible system where the gB-1 gene is under the control of its own promoter could be employed to clarify the role of early viral products in induction of gB-1 synthesis. Constitutive expression of gB-1 in human cells could provide useful material for diagnostic purposes and for the preparation of a subunit vaccine against HSV infections.
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PMID:Constitutive expression in human cells of herpes simplex virus type 1 glycoprotein B gene cloned in an episomal eukaryotic vector. 284 17

We have studied the ability of plasmids encoding a normal human myc protein to stimulate growth of primary rat embryo fibroblasts. We measured growth stimulation by the number of G418-resistant colonies obtained after co-transfection with plasmid pSV2neo and by the percentage of these colonies that grew in long-term culture (immortalization). Using a normal human myc gene, we detected a weak growth stimulation at the colony formation stage and a low frequency of immortalization. Replacement of the myc promoter by a heterologous promoter (mouse metallothionein I promoter) and deletion of the first non-coding exon led to a more efficient growth stimulation by both criteria. Thus, disregulation of c-myc is essential for an altered pattern of growth. Using zinc, a metallothionein inducer, we observed a slight increase in the growth rate of some transfectants, which can be measured by thymidine incorporation. However, the relative inefficiency of immortalization we observed suggests that either a high level of myc expression or participation of other genes is required for establishment in culture. Under our experimental conditions, we could not detect a transforming activity for the human myc gene and none of our myc-containing cell lines was tumorigenic in nude mice.
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PMID:Growth stimulation of rat primary embryo fibroblasts by the human myc gene. 374 60

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.
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PMID:p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3. 758 25

The feasibility of somatic cell gene therapy as a method of insulin delivery has been studied in mice. Murine pituitary AtT20 cells were transfected with a human preproinsulin DNA in a plasmid containing a metallothionein promoter and a gene conferring resistance to the antibiotic G418. The AtT20MtIns-1.4 clone of cells was selected because of its higher insulin-releasing activity compared with other clones. After culturing for 24 h in Dulbecco's medium containing 10 mM glucose, the AtT20MtIns-1.4 cells released human insulin at about 5 ng/10(6) cells per 24 h. Insulin release was not significantly altered by raised concentrations of glucose, potassium or calcium, but insulin release was increased by 20 mM arginine, 5 mM isomethylbutylxanthine and 90 microM zinc. AtT20MtIns-1.4 cells (2 x 10(6)) were implanted intraperitoneally into non-diabetic athymic nude (nu/nu) mice, and the mice were made diabetic by injection of streptozotocin after 7 days. Release of human insulin in vivo was assessed using a specific plasma human C-peptide assay. Human C-peptide concentrations were maintained at about 0.1 pmol/ml throughout the 29 days of the study. The development of streptozotocin-induced hyperglycaemia was delayed in recipients of the cells releasing human insulin, compared with a control group receiving an implant of non-transfected cells. At autopsy the implanted AtT20MtIns-1.4 cells in each recipient had formed a tumour-like aggregation, with an outer region of insulin-containing cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Insulin delivery by somatic cell gene therapy. 814 41

Using a differential hybridization technique, we have identified a mouse cellular gene, high mobility group protein HMG-I(Y), whose expression is up-regulated by the E6 protein of human papillomavirus (HPV) type 16. This gene was overexpressed in E6-expressing mouse 10T1/2 cells, but not in G418-resistant 10T1/2 cells. The expression of the HMG-I(Y) gene was up-regulated by the transient expression of E6 from a zinc-inducible human metallothionein-IIA gene promoter. Expression was found to be more efficient at a confluent cell density than at a subconfluent cell density. The up-regulation of HMG-I(Y) gene expression by E6, in particular at a confluent cell density, may be part of an altered genetic program in host cells infected with HPV-16.
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PMID:Human papillomavirus type 16 E6 protein up-regulates the expression of the high mobility group protein HMG-I(Y) gene in mouse 10T1/2 cells. 880 79

Calmodulin (CaM) is involved in cellular processes that are vital to cell proliferation and viability. Elevated CaM content is seen in transformed cells. Anti-CaM compounds alone are cytotoxic to tumor cells and are synergistic with certain cancer chemotherapeutic agents. However, all known CaM antagonists are nonselective, complicating interpretation of these studies. To more rigorously analyze the relationship between CaM protein expression and the behavior of cancer cells, tumor-derived cell lines were engineered such that CaM concentration could be manipulated by overexpressing CaM RNA. A full-length rat CaM I cDNA was inserted into the mammalian expression vector pMTCB6+ so that either CaM mRNA (sense) or antisense RNA was expressed under the control of an inducible metallothionein promoter. Constructs were introduced into C6 glioblastoma cells by liposome-mediated transfection and colonies were selected in G418. Significantly fewer clones were recovered from transfections with antisense vectors compared to CaM sense RNA or control (empty) vector alone. This difference was attributed to the cytotoxic effects of antisense CaM RNA as opposed to differences in transfection efficiencies. CaM expression was analyzed at the RNA level by Northern blotting and CaM protein concentrations were quantitated by immunofluorescence. Clones were identified in which CaM protein could be increased or decreased following exposure to zinc ions. Changes in CaM mRNA preceded changes in CaM protein by several hours. Overexpression of CaM had no significant effects on the growth of C6 cells. However, reductions in CaM lead to decreased growth rates of C6 cells and lowered cell viability.
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PMID:The effects of altered cellular calmodulin expression on the growth and viability of C6 glioblastoma cells. 911 56

Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by LipofectamineTM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by TranswellTM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.
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PMID:[Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells]. 2887 49