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At least 19 recordsLinked to original sources
PubMed2000

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

Blotting, Northern↗
PubMed1999

Ligand binding and activation of rat nicotinic alpha4beta2 receptors stably expressed in HEK293 cells.

HEK293 cells were stably transfected with rat neuronal nicotinic alpha4 and beta2 subunits. Binding of tritiated cytisine and nicotine to cell homogenates revealed the presence of a single class of high-affinity sites (dissociation constants 0.1 nM and 0.4 nM, respectively). Activation of nicotinic receptors was studied using whole-cell patch clamp methods, and acetylcholine, nicotine, dimethylphenylpiperazinium, and cytisine all produced a conductance increase. Responses desensitized to prolonged applications, at both positive and negative membrane potentials. The conductance was strongly rectifying, and outward currents were essentially absent. Responses were maximal at about 2 mM external calcium ion concentration and were reduced by about one-half at either nominally 0 or 10 mM external calcium. Di-hydro-beta-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (Ki 20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocked responses to acetylcholine (IC50, 1.3 microM), but had no effect on cytisine binding at a concentration of 30 microM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of alpha4 and beta2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited.

Acetylcholine↗
PubMed1997

Functional expression of odorant receptors of the zebrafish Danio rerio and of the nematode C. elegans in HEK293 cells.

Odorant receptors of zebrafish and C elegans were functionally expressed in vertebrate kidney cells (HEK293) using the eucaryotic expression vector pSMyc. Receptor-encoding cDNA cloned into this vector was expressed as a fusion protein with the N-terminal membrane import sequence of the guinea-pig serotonin receptor followed by a myc tag. Immunocytochemical evidence indicates that this strategy directs a protein with the predicted immunoreactivity and approximate molecular weight to the plasma membrane. Fish food extract (TetraMin) evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for three fish odorant receptors and converted to stable cell lines. The effect of the extract was concentration dependent and limited to the fraction of the extract < 5 kDa. Pretreating the transfected cells with the PLC inhibitor U73122 reduced the odor-evoked signal. Fish food extract also evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for single fish odorant receptors. Diacetyl evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids encoding the cDNA of ODR10, an odorant receptor of C. elegans suggested in other work to be specific for diacetyl. These results strongly imply that odorant receptors can be functionally expressed in HEK293 cells using this novel expression protocol.

Animals↗
PubMed1998

Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.

Caffeine↗
PubMed2000

The influence of an endogenous beta3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells.

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant alpha1 and gamma2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of alpha1 and gamma2 subunits and suggest the existence of an endogenous beta3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma2 subunit, WSS-1 cell receptors also contain beta2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta3 mRNA. We examined the contribution of the beta3 subunit in the function of receptors formed by expression of alpha1 and gamma2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with alpha1 and gamma2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha1 and gamma2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha1 and gamma2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain alpha1, beta3 and gamma2S subunits.

Animals↗
PubMed1998

[Pathways of signal transduction of intracellular Ca2+ increase mediated by three subtypes of alpha 1-adrenoceptor in HEK293 cells].

In HEK293 cell lines in which alpha 1a-, alpha 1b- or alpha 1d-adrenoceptor subtypes were stably expressed, fluorescence intensities due to traces of Ca2+ were investigated using fura-2/AM with the aim of exploring signal transduction pathways of intracellular Ca2+ increase mediated by these receptor subtypes. Pertussis toxin had no effect on [Ca2+]i increase mediated by all three subtypes of alpha 1-adrenoceptors. However, U-73122 and PMA could inhibited the [Ca2+]i increase induced by NE, which was not affected by Foskolin and Rp-cAMPs. Pretreatment with calphostin C abolished the [Ca2+]i response to PMA. Quercetin and tyrphostin inhibited maximal [Ca2+]i increase but had no effect on the plateau phase in all the three transfected cells. In the HEK293 cells, the phosphatide-Ca2+ signalling system mediated by the 3 subtypes of alpha 1-AR was brought into play by activation of phospholiphase C via coupling with PTX-insensitive G proteins. PKC system inhibited both mobilization of internal Ca2+ stores and the Ca2+ influx across the plasma membrane. Tyrosine kinase, but not cAMP system, might participate in such Ca2+ signalling.

Animals↗
PubMed2004

Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells.

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K+ current (IKr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K+ channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K+ channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC50) of the antimalarials was: halofantrine (0.04 microM)<chloroquine (2.5 microM)<mefloquine (2.6 microM)<desbutyl-lumefantrine (5.5 microM)<lumefantrine (8.1 microM). Lumefantrine and desbutyl-lumefantrine showed a slower inhibition of IKr than the other tested antimalarials. In conclusion, lumefantrine and desbutyl-lumefantrine inhibited significantly the hERG tail current with a higher IC50-value than mefloquine, chloroquine and halofantrine. This, together with the calculated cardiac safety indices, suggests that lumefantrine and desbutyl-lumefantrine have a weaker proarrhythmic potential than their comparator compounds.

Action Potentials↗
PubMed1999

Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current.

OBJECTIVE: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. METHODS: The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. RESULTS: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to -40 mV and the steady-state inactivation curve had a midpoint of -47.4 +/- 0.3 mV and a slope of 5.9 +/- 0.2 mV. Rat ventricular Ito current was activated at potentials positive to -20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of -29.1 +/- 0.7 mV and 4.5 +/- 0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2 +/- 4.1 msec and 23. +/- 1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54 +/- 0.13 mM and 0.14 +/- 0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogeneous. CONCLUSION: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogeneous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current.

4-Aminopyridine↗
PubMed2003

Low-level arsenite induced gene expression in HEK293 cells.

Chronic, low-level exposure to arsenic frequently results in skin, lung, bladder, and kidney cancer. Since arsenic is primarily excreted via the kidney, this study focused on this target tissue. Gene array was used as a sensitive low-level monitor of the impact of arsenic on this target tissue. Arsenite [As(III)] was chosen as the chemical species of arsenic since As(III) species are touted as the cellular toxic form of arsenic. Human embryonic kidney cell line HEK293 cells were incubated with 1, 10, and 25 microM arsenite [As(III)] for 6 or 24 h. Total RNA from treated and control cells was isolated, reverse transcribed, and labeled with Cy3 or Cy5, and hybridized to a human cDNA microarray. Hybridizations were performed four times using independent total RNA preparations to ensure reproducibility. Raw data from 10 and 25 microM treated cells exposed for 6 h was normalized within, and between, hybridizations followed by identification of genes affected by arsenite exposure based on practical significance (2-fold change up or down) and reproducibility (affected in four of six measurements). In these studies, 20 genes (HMOX1, MT1E, or FOSL1, etc.) were up-regulated, and 19 genes (MYC, JAK1, or CENPE, etc.) were down-regulated. Genes identified at 10 and 25 microM arsenic exposure were then examined after 1 microM treatment for 6 or 24 h. Expression of affected genes showed a dose-dependent (1-25 microM) trend that was apparently not time-dependent (6 vs. 24 h). The affected genes indicate that even this realistic, low-level arsenite exposure was recognized by the HEK293 cells (e.g. metallothionein genes) and produced an oxidative stress (e.g. heme oxygenase gene). These affected genes were characterized as stress response genes, proto-oncogene, signaling molecules, transcription factors, chemokine receptors, proteolytic enzymes, ESTs, and unknown genes. These findings imply that arsenite induces complex cellular injury and the cellular adaptation to As(III) is associated with alterations in the expression of many genes.

Arsenites↗
PubMed1998

Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells.

The extracellular calcium (Ca2+o)-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.

Biotinylation↗
PubMed2005

Sphingosine-1-phosphate induces a PDGFR-dependent cell detachment via inhibiting beta1 integrin in HEK293 cells.

Several different types of interactions between sphingosine-1-phosphate (S1P) receptors and platelet-derived growth factor receptor (PDGFR) have been revealed recently. In this work, we used HEK293 cells to further investigate the potential crosstalk. Interestingly, we observed that S1P specifically induced a PDGFR-dependent cell detachment in HEK293 cells, which could be inhibited by AG1296, a specific inhibitor for PDGFR. EGFR on the other hand, did not have any effect on cell detachment. The detachment was extracellular matrix (ECM) protein specific, suggesting the involvement of specific integrin molecules. When beta(1) integrin was engaged into an active state, S1P-induced cell detachment was blocked, suggesting that S1P induced an inside-out inhibitory effect on beta(1) integrin. G(i) protein and ERK activation were required for the cell detachment induced by S1P, suggesting an endogenous receptor for S1P is likely to be involved.

Blotting, Western↗
PubMed2026

Multi-omics reveal molecular changes during suspension adaptation of HEK293 cells.

Human embryonic kidney 293 (HEK293) cells have been successfully adapted from adherent to suspension culture and widely applied in both scientific research and the pharmaceutical industry. Although some studies investigated the variances between established adherent and suspension HEK293 cells of different strains, specific alterations in the cells during this consecutive process of suspension adaptation and possible factors driving this process have not been well described. Here, we adapted adherent HEK293 to suspension with desirable cell growth and high productivity for recombinant adenoviral vectors, and cells at several stages throughout the process were characterized. Slower cell growth, lower glucose uptake, increased lactate production, and weaker cell-surface adhesion were observed in suspension cells compared to their adherent counterparts. We further performed transcriptomics, proteomics, and metabolomics analysis to identify key cellular switches. A total of 2476 differentially expressed genes were found, including 1218 upregulated and 1258 downregulated genes in suspension cells. A similar and correlated pattern was observed in the proteomic study, and 702 differentially expressed metabolites were identified by untargeted metabolomics. In light of enrichment analysis, we summarized that HEK293 adherent cells survived and adapted to suspension culture via structural remodeling, metabolic shift and stress resistance. Our results provide a molecular enlightenment for suspension adaptation and potential directions for rational modification of HEK293 cell lines for future use. KEY POINTS: &#x2022; Suspension adaptation reduced adhesion and reshaped the HEK293 cytoskeleton. &#x2022; Multi-omics revealed metabolic rewiring and enhanced stress resistance. &#x2022; An optimized suspension line outperformed an internal HEK293 suspension reference.

Humans↗
PubMed2003

Dihydropyridine Ca2+ channel antagonists and agonists block Kv4.2, Kv4.3 and Kv1.4 K+ channels expressed in HEK293 cells.

(1) We have determined the molecular basis of nicardipine-induced block of cardiac transient outward K(+) currents (I(to)). Inhibition of I(to) was studied using cloned voltage-dependent K(+) channels (Kv) channels, rat Kv4.3L, Kv4.2, and Kv1.4, expressed in human embryonic kidney cell line 293 (HEK293) cells. (2) Application of the dihydropyridine Ca(2+) channel antagonist, nicardipine, accelerated the inactivation rate and reduced the peak amplitude of Kv4.3L currents in a concentration-dependent manner (IC(50): 0.42 micro M). The dihydropyridine (DHP) Ca(2+) channel agonist, Bay K 8644, also blocked this K(+) current (IC(50): 1.74 micro M). (3) Nicardipine (1 micro M) slightly, but significantly, shifted the voltage dependence of activation and steady-state inactivation to more negative potentials, and also slowed markedly the recovery from inactivation of Kv4.3L currents. (4) Coexpression of K(+) channel-interacting protein 2 (KChIP2) significantly slowed the inactivation of Kv4.3L currents as expected. However, the features of DHP-induced block of K(+) current were not substantially altered. (5) Nicardipine exhibited similar block of Kv1.4 and Kv4.2 channels stably expressed in HEK293 cells; IC(50)'s were 0.80 and 0.62 micro M, respectively. (6) Thus, at submicromolar concentrations, DHP Ca(2+) antagonist and agonist inhibit Kv4.3L and have similar inhibiting effects on other components of cardiac I(to), Kv4.2 and Kv1.4.

Calcium Channel Agonists↗
PubMed2006

Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP.

Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation.

Amino Acid Sequence↗
PubMed1998

Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells.

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.

Cell Line↗
PubMed2005

Effect of single wall carbon nanotubes on human HEK293 cells.

The influence of single-walled carbon nanotubes (SWCNTs) on human HEK293 cells is investigated with the aim of exploring SWCNTs biocompatibility. Results showed that SWCNTs can inhibit HEK293 cell proliferation, decrease cell adhesive ability in a dose- and time-dependent manner. HEK293 cells exhibit active responses to SWCNTs such as secretion of some 20-30 kd proteins to wrap SWCNTs, aggregation of cells attached by SWCNTs and formation of nodular structures. Cell cycle analysis showed that 25 microg/ml SWCNTs in medium induced G1 arrest and cell apoptosis in HEK293 cells. Biochip analysis showed that SWCNTs can induce up-regulation expression of cell cycle-associated genes such as p16, bax, p57, hrk, cdc42 and cdc37, down-regulation expression of cell cycle genes such as cdk2, cdk4, cdk6 and cyclin D3, and down-regulation expression of signal transduction-associated genes such as mad2, jak1, ttk, pcdha9 and erk. Western blot analysis showed that SWCNTs can induce down-regulation expression of adhesion-associated proteins such as laminin, fibronectin, cadherin, FAK and collagen IV. These results suggest that down-regulation of G1-associated cdks and cyclins and upregulation of apoptosis-associated genes may contribute to SWCNTs induced G1 phase arrest and cell apoptosis. In conclusion, SWCNTs can inhibit HEK293 cells growth by inducing cell apoptosis and decreasing cellular adhesion ability.

Antibodies, Monoclonal↗
PubMed2003

Substance P evokes cation currents through TRP channels in HEK293 cells.

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 microM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 +/- 120.0 s, mean +/- SE, n = 20), had a peak density of 11.3 +/- 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 +/- 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC.

Calcium Channels↗