293E Cells Research Articles

Corl1, a Novel Neuronal Lineage-specific Transcriptional Corepressor for the Homeodomain Transcription Factor Lbx1

During development, neuronal identity is determined by a combination of numerous transcription factors. However, the mechanisms of synergistic action of these factors in transcriptional regulation and subsequent cell fate specification are largely unknown. In this study, we identified a novel gene, Corl1, encoding a nuclear protein with homology to the Ski oncoprotein. Corl1 was highly selectively expressed in the central nervous system (CNS). In the embryonic CNS, Corl1 was expressed in a certain subset of postmitotic neurons generated posterior to the midbrain-hindbrain border. In the developing spinal cord, Corl1 was selectively expressed in the dorsal horn interneurons where a homeodomain transcription factor, Lbx1, is required for proper specification. Corl1 was localized in a nuclear dot-like structure and interacted with general transcriptional corepressors. In addition, Corl1 showed transcriptional repression activity in the GAL4-fusion system, indicating its involvement in the regulation of transcriptional repression. Furthermore, Corl1 interacted with Lbx1 and cooperatively repressed transcription, suggesting that it acts as a transcriptional corepressor for Lbx1 in regulating cell fate determination in the dorsal spinal cord. Corl1 corepressor activity did not depend on Gro/TLE activity, and Gro/TLE also functioned as a corepressor for Lbx1. Thus, Lbx1 can select two independent partners, Corl1 and Gro/TLE, as corepressors. Identification of a novel transcriptional corepressor with neuronal subtype-restricted expression might provide insights into the mechanisms of cell fate determination in neurons.

135. A New Ligand-Pseudoreceptor System for the Generation of Adenoviral Vectors with Altered Tropism

Top of pageAbstract One strategy for the development of Ad vectors (AdV) for the delivery of transgenes in specific tissues relies both on the ablation of native tropism and the introduction of new tropism for target cells. Ablation of AdVs intereaction with their natural receptors renders them unable to grow in current cell lines. Consequently such ablated AdVs require new packaging cells for their generation. Here we describe the use of a couple of high affinity complementary peptides E-coil and K-coil, to create a new cell entry pathway for the native tropism-ablated AdV. E-coil and K-coil are artificial peptides containing from 3 to 5 repeats of 7 amino acids. The strength of interaction between the artificial coil peptides increases with the number of repeats. An E-coil (5 repeats)-containing pseudo-receptor was first expressed into 293 cells (293E). Then, we have cloned in the fiber knob 3, 4 and 5 repeats of K-coil. Insertion of K-coil with 3 or 4 repeats had no deleterious effect on fiber trimerization whereas insertion of 5 repeats dramatically reduces it. Therefore K-coil of 4 repeats (K4) was inserted into the fiber of a CAR-ablated virus (AdFK4m/GFP), since K4 can keep high affinity to 5 repeats of E-coil, without disturbing fiber trimerization. The transduction level of the tropism modified-virus AdFK4m/GFP was 20 times higher in 293E stable cell line then in 293 cells without the pseudo-receptor. Moreover, the soluble peptide K-coil inhibited the transduction of AdF4Km/GFP into 293E cells, whereas soluble fiber had no effect. This indicates that the transduction of the 293E packaging cell line by K-coil-incorporated AdV was taken place via E-coil and K-coil interaction. Such a system is therefore suitable for the production of re-targeted AdV.

Membrane-bound form of fractalkine induces IFN-gamma production by NK cells.

Natural killer (NK) cells participate in both innate and adaptive immunity, in part by their prompt secretion of cytokines including IFN-gamma, a pro-inflammatory cytokine with an important role in Th1 polarization. To assess the involvement of fractalkine in inflammatory processes, we examined the effect of fractalkine on IFN-gamma production by NK cells. Although soluble chemokines, including MCP-1 and RANTES as well as fractalkine, had a negligible effect on IFN-gamma production, immobilized fractalkine markedly induced IFN-gamma production by NK cells in a dose-dependent manner. Pretreatment of NK cells with the phosphatidylinositol 3-kinase (PI 3-K) inhibitor, wortmannin, completely inhibited the production of IFN-gamma induced by fractalkine, and pretreatment with the protein tyrosine kinase inhibitor, herbimycin A, partially suppressed the response, suggesting that augmentation of IFN-gamma production in response to fractalkine treatment of NK cells involves signaling through PI 3-K and protein tyrosine kinases. Furthermore, co-culture of NK cells with fractalkine-transfected 293E cells markedly enhanced IFN-gamma production by NK cells compared with co-culture with control 293E cells. These findings may indicate a paracrine feedback loop system in which endothelial cells may be activated to produce more fractalkine, and also suggest a role for fractalkine expressed on endothelial cells in Th1 polarization through the stimulation of IFN-gamma production by NK cells.

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.

A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein-Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.

A Reporter Gene Assay for High-Throughput Screening of G-Protein-Coupled Receptors Stably or Transiently Expressed in HEK293 EBNA Cells Grown in Suspension Culture

We describe in detail a robust, sensitive, and versatile functional assay for G-protein-coupled receptors (GPCRs) expressed in human embryonic kidney (HEK) 293-EBNA (Epstein–Barr virus nuclear antigen) (designated 293E) cells. The ability to grow these cells in suspension, in conjunction with the use of the secreted form of the human placental alkaline phosphatase (SEAP) as the reporter enzyme transcriptionally regulated by 5-cyclic AMP (cAMP) response elements (CREs) (Chen et al., Anal. Biochem. 226, 349–354 (1995)), makes this CRE-SEAP assay potentially attractive for high-throughput screening (HTS). A 293E clonal cell line, stably transfected with the CRE-SEAP plasmid, was initially characterized with compounds known to activate intracellular signal transduction pathways similar to those activated by GPCRs. Forskolin and cAMP analogues were potent at inducing SEAP expression but calcium ionophores (A23187 and ionomycin) were without effect. The forskolin response was also potentiated by the protein kinase C activator phorbol myristate acetate as well as the phosphodiesterase inhibitor isobutylmethylxanthine. Previously established cell lines expressing the Gαs-coupled DP or the Gαq-coupled-EP1 prostanoid receptors were stably transfected with the reporter gene construct and clones were selected based on their ability to secrete SEAP upon agonist challenge. Pharmacological characterization of the DP and EP1 receptors displayed a similar rank order of potency for several known prostanoids and related compounds to that previously reported using classical binding assays or other functional assays. The CRE-SEAP assay was also used to characterize the EP1 receptor antagonists SC-51322, SC-51089, and AH6809. In summary, we have established a reporter gene assay for GPCRs that couple to both Gαs and Gαq and is amenable to HTS of both agonists and antagonists.

Combinatorial gene expression using multiple episomal vectors.

Episomal vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combinations of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2)alpha, were stably expressed from separate episomal vectors in 293-EBNA (293E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of functional receptors and G protein. Cell lines expressing genes from multiple autonomously replicating vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5months. Co-expression of supplementary G(i2)alpha with receptor amplifies the magnitude of signal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfection is the strategy of choice for generating stable cell lines expressing multiple genes for the study of signal-transduction pathways or the evaluation of receptor ligands.

Small molecule antagonists of the bradykinin B1 receptor

Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arg10-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax=20,000 receptors/cell, KD=0.5±0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax=8,000 receptors/cell, KD=0.5±0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6±1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (KBapp=0.4±0.2 nM) and calcium mobilization (KBapp=17±2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are >1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.

Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK

Background: The rsk1 gene encodes the 90 kDa ribosomal S6 kinase 1 (RSK1) protein, which contains two kinase domains. RSK1, which is involved in regulating cell survival and proliferation, lies at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases. ERK activation and subsequent phosphorylation of the RSK1 carboxy-terminal catalytic loop stimulates phosphotransferase activity in the RSK1 amino-terminal kinase domain. When activated, RSK1 phosphorylates both nuclear and cytoplasmic substrates through this amino-terminal catalytic domain. It is thought that stimulation of the ERK/MAP kinase pathway is sufficient for RSK1 activation, but how ERK phosphorylation activates the RSK1 amino-terminal kinase domain is not known.Results: The individual isolated RSK1 kinase domains were found to be under regulatory control. In vitro kinase assays established that ERK phosphorylates RSK1 within the carboxy-terminal kinase domain, and the phosphoinositide-dependent kinase 1 (PDK1) phosphorylates RSK1 within the amino-terminal kinase domain. In transiently transfected HEK 293E cells, PDK1 alone stimulated phosphotransferase activity of an isolated RSK1 amino-terminal kinase domain. Nevertheless, activation of full-length RSK1 in the absence of serum required activation by both PDK1 and ERK.Conclusions: RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.

Ca 2+-dependent interaction of N-copine, a member of the two C2 domain protein family, with OS-9, the product of a gene frequently amplified in osteosarcoma

N-copine is a novel two C2 domain protein that shows Ca 2+-dependent phospholipid binding and membrane association. By using yeast two-hybrid assays, we identified OS-9 as a protein capable of interacting with N-copine. We further revealed that the second C2 domain of N-copine bound with the carboxy-terminal region of OS-9. Their interaction in vivo was also confirmed by co-immunoprecipitation from 293E cells co-expressing transfected N-copine and OS-9. In vitro binding assays showed that this interaction was Ca 2+-dependent. By Northern blot analysis, N-copine and OS-9 were co-expressed in the same regions of human brain. These results reveal that OS-9 is a potential target of N-copine.

Rapid Generation of Stable Cell Lines Expressing Corticotropin-Releasing Hormone Receptor for Drug Discovery

Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein α subunit (Gαs) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.