Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.     

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.     

HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells

Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wild-type ER. To isolate clonal lines of HeLa cells stably expressing epitope-tagged ER, we used a construction encoding a single bicistronic mRNA, in which FLAG-epitope-tagged human ER alpha (fER) was translated from a 5'-translation initiation site and fused to the neomycin resistance gene, which was translated from an internal ribosome entry site. One stable HeLa-ER-positive cell line (HeLa-ER1) produces 1,300,000 molecules of fER/cell (approximately 20-fold more ER than MCF-7 cells). The HeLa fER is biologically active in vivo, as judged by rapid death of the cells in the presence of either 17 beta-estradiol or trans-hydroxytamoxifen and the ability of the cell line to activate a transfected estrogen response element (ERE)-containing reporter gene. The FLAG-tagged ER was purified to near homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified fER exhibited a distribution constant (KD) for 17 beta-estradiol of 0.45 nM. Purified HeLa fER and HeLa fER in crude nuclear extracts exhibit similar KD values for the ERE (0.8 nM and 1 nM, respectively), which are approximately 10 times lower than the KD of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both crude and purified HeLa fER to the ERE (KD of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17 beta-estradiol-dependent transactivation. At high levels of transfected HMG-1 expression plasmid, transactivation by ER became partially ligand-independent, and transactivation by trans-hydroxytamoxifen was increased by more than 25-fold. These data describe a system in which ER, stably expressed in HeLa cells and easily purified, exhibits extremely high affinity for the ERE, and suggest that intracellular levels of HMG-1 may be limiting for ER action.     

Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells

Ligand-gated ion channels are structurally complex transmembrane proteins that all neurons must synthesize for rapid chemical synaptic transmission. The most abundant nicotinic acetylcholine receptor serving as a ligand-gated ion channel in the nervous system is a species that contains α7 subunits, binds α-bungarotoxin, and has a high relative permeability to calcium. The ability of neurons to make such receptors was compared with that of nonneuronal cells stably transfected with an α7 cDNA to determine whether neuron-specific machinery is likely to aid in their assembly or stabilization. Transfected cells expressed α7 protein and assembled it into a species that was indistinguishable in size and pharmacology from native receptors, but much of the α7 protein they synthesized was rapidly degraded without becoming receptor. Neurons were not only more efficient than the best transfectants at assembling the receptors but also produced a subpopulation of receptors on the cell surface that was relatively stable and resistant to solubilization. This subpopulation, which was absent from transfected cells, may be tethered to cytoskeletal elements in the neurons. The results support the contention that neurons contain components that facilitate the production and stabilization of ligand-gated ion channels. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 968–982, 1997     

Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots.

The major heat-shock protein, hsp-70, is synthesized by cells from a wide variety of organisms in response to heat shock or other stresses. It is assumed that hsp-70 may have an important thermal protective function. To test this hypothesis directly, we have transfected rat fibroblast cells with appropriate expression plasmids containing a cloned human hsp-70 gene. Stable transfectants expressing the human hsp-70 gene product were identified by Western blot analysis. During the course of selecting successful transfectants, we found that when standard methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining were employed with the monoclonal antibody raised against the human hsp-70 antigen, we were unable to differentiate the human hsp-70 from the heat-inducible rat hsp-70. This was because the monoclonal antibody cross-reacts with the human and rat proteins, which have the same mobility in SDS-PAGE, and it is difficult to determine which protein is expressed. To improve the resolution of the Western blot technique, we performed additional immunoblot analysis of cellular proteins separated by slab gel isoelectrofocusing. Our study shows that the isoelectrofocusing technique, when combined with antibodies against hsp-70, gave a better resolution for the separation of exogenous human hsp-70 and the endogenous rat hsp-70 than the commonly used SDS-PAGE Western blot analysis. It provides a rapid and specific method to identify positive colonies that express the human hsp-70 gene in transfected rodent cells.     

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese‐hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human β‐globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 5′ or 3′ site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 5′ site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 3′ site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.     

Characterization of somatostatin receptor subtype 2 expression in stably transfected A-427 human cancer cells

Although radiolabeled somatostatin analogs have become highly prevalent in the diagnosis and treatment of somatostatin receptor subtype (sst)-positive tumors, there are relatively few options with respect to sst-positive tumor cell lines and animal models. It would be highly beneficial, particularly for therapeutic purposes, to have several clones of one human sst2-positive cell line that express a range of sst2 concentrations for evaluating the dose response and intracellular processing of radiolabeled somatostatin analogs. The human non-small cell lung cancer line A-427 was stably transfected with a hemagglutinin-tagged human sst2. Expression of the receptor was evaluated in vitro using flow cytometry, saturation binding analysis, internalization assays, and quantitative polymerase chain reaction. The receptor expression was also validated in an in vivo mouse model in biodistribution and micro-positron emission tomography (microPET) studies using the somatostatin analog octreotide (OC), which was linked to the (64)Cu chelator 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or (64)Cu-TETA-OC. Stable clones were isolated, and four clones (2, 4, 5, and 7) were chosen for further examination. In vitro assays showed that clone 4 had no expression of sst2, whereas the others had various levels in the order of 7 > 2 > 5. Biodistribution studies with (64)Cu-TETA-OC showed the same rank order, with tumor uptake of the clones ranging from 0.8 to 6.5% injected dose/g. These studies showed that there was a strong correlation among the in vitro assays and between the in vitro assays and the biodistribution. MicroPET confirmed significant uptake of (64)Cu-TETA-OC in clone 7 and background uptake in clone 4. These studies show that clones of a human cell line can be produced expressing various levels of sst2 that should be useful for the future evaluation of radiolabeled somatostatin analogs.     

Embryonic stem cells stably transfected with mRAR beta 2-lacZ exhibit specific expression in chimeric embryos.

Using the embryonic stem (ES) cell/chimera approach, we have studied the activity of the mouse retinoic acid receptor beta 2 (mRAR beta 2) promoter during ES cell differentiation and during embryonic development. Stable ES clones were isolated after introduction of a 1.8 kb mRAR beta 2-lacZ expression cassette. LacZ expression in these stable clones was specifically induced by retinoic acid (RA) in a similar fashion as the endogenous RAR beta 2 gene. Following introduction of three different ES clones into blastocysts, an integration-independent mRAR beta 2-lacZ expression pattern was obtained in chimeric embryos similar to that described by in situ hybridization and transgenic studies. Moreover, mRAR beta 2-lacZ expression was also detected at some additional sites not described before, e.g. body wall, ureter, mesonephric duct and optic stalk. Maternal RA administration at 8.5 days of pregnancy extended lacZ expression to more anterior and posterior regions. Transgenic mice were generated from germ-line transmission of the transfected ES cells; expression pattern and changes in expression upon RA induction in these transgenic embryos were identical to those in chimeric embryos. We conclude that by using the ES/chimera approach, the proximal 1.8 kb of the mRAR beta 2 promoter produces a reliable and reproducible expression pattern of the reporter gene, and that the ES cell/chimera approach is invaluable for the study of gene expression and regulation.     

Syndecan-2 expression enhances adhesion and proliferation of stably transfected Swiss 3T3 cells

Syndecans (heparan sulfate proteoglycans) participate in cell-cell and cell-matrix adhesion and are co- and low-affinity receptors for growth factors and enzymes, respectively. We examined the influence of stable syndecan-2 expression in Swiss 3T3 cells on cell-adhesion and proliferation. Higher syndecan-2 expression changed cell morphology and increased spreading and adhesion in these cells and proliferation induced by FCS and FGF-2. This emphasizes the role of syndecan-2 in the integration of signals from soluble and insoluble factors.     

Characterization of stably transfected fusion protein GFP-estrogen receptor-alpha in MCF-7 human breast cancer cells

Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.     

Urea transport in MDCK cells that are stably transfected with UT-A1

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 ·cm². Transepithelial [¹⁴C]urea fluxes were measured at 37°C in a HCO₃^–/CO₂ buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [¹⁴C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells     

Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic

The human Golgi enzyme CMP-NeuAc:Gal(β1–4)GlcNAc-R α2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous α2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human α2,6-sialyltransferase is capable of sialylating, apart from Gal(β1–4)GlcNAc-R, also GalNAc(β1–4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell α2,3-sialyltransferase. This revised version was published online in July 2006 with corrections to the Cover Date.     

TPA induced expression and function of human connexin 26 by post-translational mechanisms in stably transfected neuroblastoma cells

Connexin 26 (Cx26) has been proposed to be a tumor suppressor gene and its expression may modulate development, cell growth and differentiation in various tissues, including the brain. 12-O-tetradecanoylphorbol-13-acetate (TPA) may serve as either tumor promoter (in mammary gland amd skin) or as a differentiating agent (in neuroblastoma and leukemic cells) and may also modulate expression, function and phosphorylation of gap junctions. In this study, to determine the effects of TPA on Cx26 expression and its function in neuroblastoma, we transfected N2A mouse neuroblastoma cells (which are gap junction deficient) with the coding region of human Cx26 gene (which lacks TPA response elements) and examined the changes of expression and function of Cx26 following 10 nM TPA treatment. Individual clones of transfectants stably expressed distinct levels of exogenous Cx26 as judged by Northern and Western blots, immunocytochemistry and electrophysiological recordings. Cx26 channels displayed unitary conductances of about 140-155 pS. Increase of Cx26 expression following TPA treatment was markedly observed using immunocytochemistry and Western blots of membrane fractions although it was not detected in Northern or Western blots of whole cells. This increase in Cx26 expression in the plasma membrane was accompanied by an increase of function as evidenced in measurements of junctional conductance. These results suggest that induction of exogenous Cx26 in neuroblastoma cells by TPA treatment is controlled by post-translational mechanisms.     

Distance effect of matrix attachment regions on transgene expression in stably transfected Chinese hamster ovary cells

The β-globin matrix attachment regions (MARs) were inserted into the 5′-site of the eukaryotic expression vector cassette and DNA fragments 350 and 750 bp in length were inserted into the site to generate expression vectors with varying distances between the expression cassette and MAR. The vectors containing MARs increased chloramphenicol acetyltransferase (CAT) expression levels compared to the negative control vector lacking the MAR; the highest expression increase was 3.8-fold. A greater MAR-transgene distance (750 bp) correlated with a greater increase in transgene expression when compared to the control vector that lacked separation between the MAR and transgene. CAT gene copy numbers were higher in cells transformed with the vector possessing a smaller MAR-transgene distance (350 bp) than in cells belonging to the other three groups. However, MAR-induced transgene expression levels did not exhibit a direct relationship with gene copy number.     

High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.     

High-dose gamma-irradiation enhance expression of transgene under control of immediate-early CMV promoter in stably transfected tumor cells

Gamma-irradiation is a usual method to inactivate whole-cellular anticancer vaccines consisting viable tumor cells. To evaluate the effect of gamma-irradiation to transgene expression in tumor cells we constructed several stably transfected clones of human and mouse cell lines expressing transgenic GM-CSF or GFP under control of IE-CMV promoter. Irradiation of those cells with different doses (ranged from 20 to 100 Gr) of gamma-radiation caused loss of proliferation capacity with survival of the cells population clearly depended on irradiation dose. Cell-cycle staining reveals accumulation of the cells with G2/M DNA content and almost loss of cells in S-phase. Substantial proportion of irradiated cells shows beta-galactosidase activity and morphological changes associated with cell senescence. An irradiated cell shows no changes in the level of mitochondrial dehydrogenase activity regardless irradiation dose exposed. Irradiated cells retain their ability to express transgene. Moreover, amount of the secreted GM-CSF as well as MFI in GFP-expressing cells significantly increases after gamma-irradiation up to 10 fold for cells exposed with 100 Gr. Enhancing of the transgene expression in both human and mouse cells positively correlates with total dose of gamma-irradiation gained by the cells and demonstrates gradual nature. Overall, our results supports using of 100 Gr of gamma-irradiation as the optimal dose for whole-cell anticancer vaccine inactivation.     

Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression

Cingulin is a component of the cytoplasmic domain of vertebrate tight junctions (TJ). Mutation or down-regulation of cingulin in cultured cells results in changes in gene expression. Some of these changes are dependent on RhoA, whose activity is regulated by GEF-H1, which is inactivated by binding to cingulin at junctions. To gain further insights on the function of cingulin through dominant-negative effects, we cloned and sequenced canine cingulin, and developed stable MDCK cell lines where either full-length cingulin, or head or rod+tail domains were inducibly overexpressed. Surprisingly, analysis of these clones by immunoblotting, microarray, immunofluorescence, measurement of transepithelial resistance, and cell density showed that the overexpression of either full-length cingulin or its domains does not significantly affect TJ protein levels, gene expression, RhoA activity, cell density, doubling time, and the organization and function of TJ. These results suggest that compensatory mechanisms prevent dominant-negative effects in this model system, and that modulation of cellular functions by cingulin occurs within physiological protein levels.