Development of a Bicistronic Vector Driven by the Human Polypeptide Chain Elongation Factor 1α Promoter for Creation of Stable Mammalian Cell Lines That Express Very High Levels of Recombinant Proteins

Hobbs, Steve; Jitrapakdee, Sarawut; Wallace, John C.

doi:10.1006/bbrc.1998.9646

Cited by 244 publications

(168 citation statements)

References 23 publications

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“…To test this possibility, we have constructed an NIH3T3-derived cell line constitutively overexpressing AzI. For this purpose, mouse AzI cDNA was cloned into the pEFIRES-p bicistronic vector (Hobbs et al, 1998), the resulting construct was transfected into NIH3T3 cells, and stable transformants were selected for their ability to grow in the presence of puromycin. NIH3T3 cells stably transfected with an empty pEFIRES-p vector served as a control (NIH3T3-E).…”

Section: Resultsmentioning

confidence: 99%

“…Mouse wild-type AzI, AzI/MTM mutant (described previously) (Bercovich and Kahana, 2004) and ODC cDNA were cloned between the XhoI and NotI sites of the bicistronic vector pEFIRES-p (Hobbs et al, 1998). The resulting constructs and the empty vector (pEFIRES-E) were transfected into NIH3T3 mouse fibroblasts using jetPEI (Poly Transfection).…”

Section: Expression Plasmids Construction Cell Lines and Transfectionmentioning

confidence: 99%

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Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

et al. 2006

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Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.

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Section: Resultsmentioning

confidence: 99%

Section: Expression Plasmids Construction Cell Lines and Transfectionmentioning

confidence: 99%

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

et al. 2006

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“…TLR4 and CD14 were cloned into pcDNA3 and MD-2 was subcloned into pEFIRES (25). TLR4 and MD2 chimeras were constructed by overlap extension PCR.…”

Section: Constructsmentioning

confidence: 99%

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Walsh

Gangloff

Monie

et al. 2008

The Journal of Immunology

115

137

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LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure ( L ipopolysaccharide molecules are complex glycolipids that form the outer layer of the outer membrane of Gramnegative bacteria (1). The lipid A domain of LPS is responsible for cellular activation and consists of a disaccharide to which various substituents, including acyl chains of variable length and number, are attached (2). Escherichia coli lipid A is usually hexa-acylated whereas a tetra-acylated lipid A, lipid IVa, is also produced by E. coli as an intermediate in the lipid A biosynthetic pathway (2). Lipid IVa was originally identified as an inhibitor at the human LPS receptor and was considered a candidate to be developed for clinical use as an endotoxin antagonist. Lipid A signals to host cells through a transmembrane complex consisting of the lipid-binding protein MD-2 and the type 1 receptor TLR4 (1, 3-5). MD-2 is probably the key player in lipid A recognition whereas TLR4, unlike other TLRs, is thought not to participate directly in lipid A binding (5).LPS and lipid A are believed to be recognized by MD-2 following transfer from CD14, which does not participate in the signaling complex (6). Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7, 8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13). Mutagenesis studies identified amino acids 79 -83, 121-124, 125-129 (12), K128, and K132 of human MD-2 as being important in lipid A binding (13), but in the crystal structure of the inactive MD-2-lipid IVa complex, only residues I46, L78, I80, and F121-I124 contact the ligand. The other residues...

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“…The pEF1-iFGFR1-IRES-Neo construct was made by excising the iFGFR1 gene from pMMP-iFGFR1 (Welm et al, 2002) with NcoI and NotI and ligating into similarly digested pEF1-Luc-IRES-Neo, which was originally made by excising the XhoIXbaI fragment of the firefly luciferase gene from pGL3-Basic (Promega, Madison, WI, USA) and ligating into similarly digested pEF1-IRES-N (Hobbs et al, 1998). To generate HA-FGFR1, mouse prostate cells derived from the TRAMP model were harvested using Trizol (Invitrogen, Carlsbad, CA, USA) to isolate total RNA.…”

Section: Constructs and Stable Cell Linesmentioning

confidence: 99%

Conditional activation of FGFR1 in the prostate epithelium induces angiogenesis with concomitant differential regulation of Ang-1 and Ang-2

et al. 2007

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The expression of fibroblast growth factor receptor (FGFR)-1 correlates with angiogenesis and is associated with prostate cancer (CaP) progression. To more precisely define the molecular mechanisms whereby FGFR1 causes angiogenesis in the prostate we exploited a transgenic mouse model, JOCK-1, in which activation of a conditional FGFR1 allele in the prostate epithelium caused rapid angiogenesis and progressive hyperplasia. By labeling the vasculature in vivo and applying a novel method to measure the vasculature in three dimensions, we were able to observe a significant increase in vascular volume 1 week after FGFR1 activation. Although vessel volume and branching both continued to increase throughout a 6-week period of FGFR1 activation, importantly, we discovered that continued activation of FGFR1 was not required to maintain the new vasculature. Exploring the molecular mediators of the angiogenic phenotype, we observed consistent upregulation of HIF-1a, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2), whereas expression of Ang-1 was lost. Further analysis revealed that loss of Ang-1 expression occurred in the basal epithelium, whereas the increase in Ang-2 expression occurred in the luminal epithelium. Reporter assays confirmed that the Ang-2 promoter was regulated by FGFR1 signaling and a small molecule inhibitor of FGFR activity, PD173074, could abrogate this response. These findings establish a method to follow spontaneous angiogenesis in a conditional autochthonous system, implicate the angiopoietins as downstream effectors of FGFR1 activation in vivo, and suggest that therapies targeting FGFR1 could be used to inhibit neovascularization during initiation and progression of CaP.

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Development of a Bicistronic Vector Driven by the Human Polypeptide Chain Elongation Factor 1α Promoter for Creation of Stable Mammalian Cell Lines That Express Very High Levels of Recombinant Proteins

Cited by 244 publications

References 23 publications

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Conditional activation of FGFR1 in the prostate epithelium induces angiogenesis with concomitant differential regulation of Ang-1 and Ang-2

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