Identification of a novel nuclear protein synthesized in growth-arrested human hepatoblastoma HepG2 cells.

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth-inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth-arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein.     

Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2)

Summary The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line.     

The role of dileucine in the expression and function of human organic anion transporter 1 (hOAT1)

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. In the current study, we investigated the role of dileucine (L6L7) at the amino terminus of hOAT1 in the expression and function of the transporter. We substituted L6L7 with alanine (A) simultaneously. The resulting mutant transporter L6A/L7A showed no transport activity due to its complete loss of expression at the cell surface. Such loss of surface expression of L6A/L7A was consistent with a complete loss of an 80 kDa mature form and a dramatic decrease in a 60 kDa immature form of the mutant transporter in the total cell lysates. Treatment of L6A/L7A-expressing cells with proteasomal inhibitor resulted in a significant increase in the immature form of hOAT1, but not its mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters, suggesting that the mutant transporter was degraded through proteasomal pathway. The accumulation of mutant transporter in the endoplasmic reticulum (ER) was confirmed by coimmunolocalization of L6L7 with calnexin, an ER marker. Furthermore, treatment of L6A/L7A-expressing cells with sodium 4-phenylbutyrate (4PBA) and glycerol, two chemical chaperones, could not promote the exit of the immature form of the mutant transporter from the ER. Our data suggest that L6L7 are critical for the stability and ER export of hOAT1.     

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function remains unclear. A complete definition of the physiological activities of this oncofetal protein has been severely limited, until now, by the lack of a purification procedure appropriate to obtain pure AFP in appreciable amount. The present report describes a purification procedure extremely rapid and simple and takes advantage of the well-known fact that AFP contains copper. We have developed a single-step purification procedure by immobilized copper-chelate affinity chromatography using the culture medium from human hepatoblastoma cell line HepG2 grown in the absence of serum. This method yields AFP at high purity and high yield. Purified AFP amino acid sequence, molecular mass, carbohydrate structure, and copper content were found to be in line with previous studies. Moreover, we found that the purified AFP has superoxide dismutase activity with efficiency similar to that of the native Cu, Zn SODs at physiological pH. This result may provide further support to the idea that AFP is a bifunctional protein, acting in cellular defence against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells

Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.     

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon

Summary The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog -methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 m phlorizin, 1mm phloretin, and 25mm d-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na⁺-dependent, phlorizin-and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na⁺-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na⁺-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na⁺-dependent transport pathway(s) ford-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.     

Activation of Na(+), K(+), Cl(-)-cotransport mediates intracellular Ca(2+) increase and apoptosis induced by Pinacidil in HepG2 human hepatoblastoma cells

The role of Na(+), K(+), Cl(-)-cotransport (NKCC) in apoptosis of HepG2 human hepatoblastoma cells was investigated. Pinacidil (Pin), an activator of ATP-sensitive K(+) (K(ATP)) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 cells. Pin increased intracellular K(+) concentration ([K(+)](i)). Bumetanide and furosemide, NKCC inhibitors, significantly inhibited the Pin-induced increased [K(+)](i) and apoptosis, whereas K(ATP) inhibitors (glibenclamide and tolbutamide) had no effects. The Pin-induced [K(+)](i) increase was significantly prevented by reducing extracellular Cl(-) concentration, and Pin also increased intracellular Na(+) concentration ([Na(+)](i)), further indicating that these effects of Pin may be due to NKCC activation. In addition, Pin induced a rapid and sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which was completely prevented by the NKCC inhibitors. Treatment with EGTA or BAPTA/AM markedly inhibited the Pin-induced apoptosis. Inhibitors of Na(+), Ca(2+)-exchanger, bepridil, and benzamil significantly prevented both [Ca(2+)](i) increase and apoptosis induced by Pin. Taken together, these results suggest that Pin increases [Na(+)](i) through NKCC activation, which leads to stimulation of reverse-mode of Na(+), Ca(2+) exchanger, resulting in [Ca(2+)](i) increase, and in turn, apoptosis. These results further suggest that NKCC may be a good target for induction of apoptosis in human hepatoma cells.     

In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris

The use of Pichia pastoris for protein production was simplified by creation of fusion proteins containing green fluorescent protein (GFP) and the product of interest. Human interleukin-2 (hIL-2) was used as a model product: GFP enabled clear identification of fusion protein expression and, more importantly, the quantification of hIL-2. Although GFP fusions for bioprocess monitoring have been demonstrated in other hosts, this is its first use in P. pastoris.     

Characterization of Na+ transport in normal human fibroblasts and neoplastic H.Ep.2 cells and the role of inhibitin

Summary Na⁺ transport was characterized in normal human fibroblasts and neoplastic H.Ep. 2 cells in order to investigate the role of the endogenous peptidic factor inhibitin that is secreted by a variety of neoplastic cells (including H.Ep. 2) and inhibits Na⁺/Na⁺ exchange in human erythrocytes. Although active (Na⁺, K⁺-ATPase mediated) Na⁺ fluxes were similar in the two cell types, H.Ep. 2 cells maintained higher intracellular Na[su+] concentration (26mm) compared to fibroblasts (12mm). An analysis of passive Na⁺ fluxes showed a difference in the handling of Na⁺ via ouabain and bumetanide-insensitive transport between the two cell types: H.Ep. 2 cells achieved net Na⁺ influx via an amiloride-sensitive pathway that was only demonstrated in fibroblasts when 10% fetal calf serum (FCS) was present. Kinetic studies were undertaken to investigate the interaction between Na⁺ flux via Na⁺/H⁺ and Na⁺/Na⁺ exchanges. for this purpose, an outwardly directed Na⁺ gradient was created by loading the cells with Na⁺ (Na _i >100mm) to activate the reverse functioning of Na⁺/H⁺ exchange (i.e., Na _out ⁺ H _in ⁺ ). The rates of ouabain-and bumetanide-insensitive Na⁺ efflux were measured over a range of extracellular Na⁺ concentrations (Na _o ⁺ 14–140mm). In the presence of 10% FCS, the two cell types showed different responses: in fibroblasts the Na⁺ efflux rate showed an inverse correlation with extracellular Na⁺ concentration, while H.Ep. 2 cells significantly increased their rate of Na⁺ efflux as extracellular Na⁺ concentration increased. So although the thermodynamic force would direct net Na⁺ efflux when Na _i ⁺ >Na _o ⁺ , H.Ep.2 cells were under kinetic control to perform Na⁺/Na⁺ exchange.When exogenous inhibitin was tested on fibroblasts, the steady-state intracellular Na⁺ concentration increased from 14 to 19mm (p<0.01). In Na⁺-loaded fibroblasts, serum-stimulated Na⁺ efflux was partially inhibitin sensitive and the maximal inhibitory effect was seen when extracellular Na⁺ concentration was 14mm and presumably the Na⁺/H⁺ exchanger operating in the reverse mode. This study demonstrated that, in contrast to fibroblasts, H.Ep.2 cells have a modified Na⁺/H⁺ exchange system whereby it acts in the Na _in ⁺ H _out ⁺ mode without exogenous growth factor activation and resists functioning in the reversed mode. It is proposed that inhibitin, is the endogenous modifier of this transport system in H.Ep.2 cells with the result that H.Ep.2 cells maintain a higher concentration of intracellular Na⁺ compared to fibroblasts.     

Quantitative monitoring for secreted production of human interleukin-2 in stable insect Drosophila S2 cells using a green fluorescent protein fusion partner

Human interleukin-2 (hIL-2) production in Escherichia coli and insect cell/baculovirus expression systems can be inefficient. Here we investigated secreted production of hIL-2 fused with green fluorescent protein (GFP) as a versatile fusion partner in optimized stably transfected insect Drosophila melanogaster S2 cells. This nonlytic S2 insect cell expression system employs a plasmid vector and allows for secretion of functional human proteins. We report that, following stable transfection and induction, S2 cells secreted hIL-2 as a fusion protein (approximately 2.3 microg/mL yield), with a secretion efficiency of approximately 90%. Regression analysis indicated a single linear relationship existed between GFP fluorescence and hIL-2 mass in both whole cell and secreted medium samples, indicating that in vivo monitoring and quantification of target foreign protein expression and even secretion is possible using this system. The simple comparative measurement of GFP fluorescence also allowed monitoring of secretion efficiency during periods of high GFP/hIL-2 expression.     

Stable high-level expression of truncated human papillomavirus type 16 L1 protein in Drosophila Schneider-2 cells

To improve the existing human papillomavirus type 16 (HPV16) virus-like particle (VLP) preparation, the Drosophila inducible/secreted expression system, a highly efficient, economical method, was used to produce HPV16 VLPs. Drosophila Schneider-2 cells were cotransfected with pMT/BiP/V5-His expression vector containing the target gene encoding HPV16L1 protein without nucleus localization sequence and the selection vector pCoHygro plasmids at the ratio of 4:1. The stabled hygromycin-resistant cell line was obtained 1 month later, and the protein expression was induced by copper sulfate. The molecular mass of expressed HPV16L1 protein was 66 kDa, as revealed by SDS-PAGE, and confirmed by Western blot analysis. The yield of HPV16L1 protein was 0.554 mg per 1×10⁷ cells. The characteristics of HPV16L1 protein were further analyzed by mouse erythrocyte hemagglutination assay, hemagglutination inhibition assay, and transmission electron microscopy. Results showed that the truncated protein was as biologically active as natural HPVL1 protein, inducing murine erythrocyte agglutination and VLP formation. These findings indicate that the Drosophila inducible/secreted expression system is promising as a convenient and economical method for the preparation of HPV16VLP vaccine.     

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.     

Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.     

Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx.

The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.     

Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis

AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.     

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.