Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.     

Differentiation-dependent induction of CYP1A1 in cultured rat small intestinal epithelial cells,colonocytes, and human colon carcinoma cells: basement membrane-mediated apoptosis

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells.     

AH receptor antagonist inhibits constitutive CYP1A1 and CYP1B1 expression in rat BP8 cells

The BP8 variant of the 5L rat hepatoma cell line is completely devoid of aryl hydrocarbon receptor (AHR) and is a useful model to examine AHR function. Previous studies showed that BP8 cells, when transfected with mouse AHR, exhibit induction of a plasmid-based reporter even in the absence of exogenous ligands. We transfected BP8 cells with full-length human AHR and found that presence of the AHR alone was sufficient to induce substantial CYP1A1 and CYP1B1 mRNA without any exogenous AHR ligand. An AHR antagonist, 3,4-dimethoxyflavone, inhibited CYP1A1 and CYP1B1 expression in a dose-dependent manner. When we transfected BP8 cells with a mutated human AHR that is defective in ligand binding, expression of CYP1A1 and CYP1B1 was diminished but not abolished. Inhibition by the AHR antagonist along with the diminished response to the mutated AHR indicates that BP8 cells contain some agent that acts as an agonist ligand for the AHR.     

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intracellular accumulation of mercury enhances P450 CYP1A1 expression and Cl- currents in cultured shark rectal gland cells

The effects of acute and subchronic exposure to mercury on the Cl- current (ICl) were investigated in cultured shark rectal gland (SRG) cells. The effects of intracellular accumulation of mercury on cytochrome P450 (P450) were also assessed. Bath perfusion of a cocktail solution containing forskolin, 1-isobutyl-3-methylxanthine, and 8-bromoadenosine monophosphate enhanced ICl. Addition of 10 microM HgCl2 significantly inhibited the cAMP-activated ICl (p < 0.05, n = 11). Intracellular dialysis with ATP gamma S did not prevent the inhibitory effect of mercury on ICl. In contrast, incubation of SRG cells with 10 microM HgCl2 for 48 hrs markedly increased ICl (p < 0.01, n = 12). Dephosphorylation of the channel by intracellular dialysis with phosphatase I and II abolished the mercury-incubated increase in ICl. The P450-mediated metabolite of arachidonic acid, 11,12-epoxyeicosatrienoic acid (11,12-EET), significantly increased ICl. However, application of 11,12-dihydroxyeicosatrienoic acid (11,12-DHT) did not alter ICl. Mercury incubation for 48 hrs did not alter the protein expression of Cl- channels, but caused an induction of CYP1A1 in cultured SRG cells. In addition, co-incubation of SRG cells with mercury and the P450 inhibitor clotrimazole prevented the mercury-incubated increase in ICl. Our results demonstrate that acute and subchronic application of mercury has opposing effects on ICl in cultured SRG cells. The acute effect of mercury on ICl may result from mercury blockade of Cl- channels. The subchronic effect of mercury on ICl may be due to an induction of P450 CYP1A1 and its mediated metabolites, but not due to an over-expression of Cl- channels.     

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.     

Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression

Non-transformed, rat intestinal epithelial cells (IEC-6), and human intestinal colonic carcinoma cells (CACO-2) have both been used to study processes of epithelial cell differentiation. However, only CACO-2 cells have been described as spontaneously expressing phenotypic changes of differentiation in culture. We report here that when IEC-6 cells are grown in post-confluent culture, they develop structural changes similar to those seen in cells induced to differentiate by culture on Englebreth-Holm-Swarm (EHS) extracellular matrix proteins. Correlated with this morphological change is loss of nuclear localization of c-myc protein and development of cell surface alkaline phosphatase (ALP) enzymatic activity. Messenger RNAs for liver and intestinal isoforms of ALP were expressed in both pre- and post-confluent cells. Inhibition of ALP activity in post-confluent cells by levamisole indicated the expressed ALP activity to be of the liver isoform. We suggest the expression of ALP activity, which occurs concomitantly with morphological alterations in post-confluent IEC-6 cells, represents increased expression and localization to the cell surface of the liver isoform of ALP. Cultured IEC-6 cells may provide a non-transformed, in vitro alternative to CACO-2 cells for study of epithelial cell differentiation.     

Prekeratin expression of simple epithelia in the cells of long-term cultured epithelial lines of the rat liver

Prekeratin of simple epithelia with m.m. 55 kD (PK55) was found in all the studied tumorigenic and non-tumorigenic liver epithelial cell lines of the IAR series by means of indirect immunofluorescent methods in combination with corresponding monoclonal antibodies. The most prominent expression was observed in some tumorigenic cell lines. Expression of PK55 was reversible--the cells lost prekeratin in low density cultures. It has been found that the synthesis of prekeratins with m.m. 49 kD (PK49) and 40 kD (PK40) began on reaching higher cell densities than those needed for PK55 synthesis in IAR6-7 line. The PK40 appeared in cells spread on the substratum, while the PK49 was observed in upper poorly spread cells of ridges in multilayered dense cultures. Thus, the synthesis of prekeratins is not constitutive at least for some types of epithelial cells. Specific cell-to-cell interactions are presumably needed for each particular prekeratin synthesis induction.     

Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.     

Lipopolysaccharide induces functional ICAM-1 expression in rat alveolar epithelial cells in vitro

Lipopolysaccharide (LPS)-induced lung inflammation is known to increase pulmonary intercellular adhesion molecule-1 (ICAM-1) expression. In the present study, L2 cells, a cell line of alveolar epithelial cells, were stimulated with LPS, and ICAM-1 expression was studied. ICAM-1 protein on L2 cells peaked at 6 (38% increase; P < 0.01) and 10 (48% increase; P < 0.001) h after stimulation with Escherichia coli and Pseudomonas aeruginosa LPS, respectively. ICAM-1 mRNA expression was markedly increased, with a peak at 2-4 (E. coli) and 4-6 (P. aeruginosa) h. Adherence assays of neutrophils to LPS-stimulated L2 cells showed a threefold increase in adherence (P < 0.001). Pretreatment of the neutrophils with anti-lymphocyte function-associated antigen-1 and anti-Mac-1 antibodies reduced adherence by 54% (P < 0.001). Analysis of immunofluorescence staining for ICAM-1 showed an exclusive apical expression of ICAM-1. These results indicate that LPS upregulates functional active ICAM-1 on the apical part of the membrane in rat pneumocytes.     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.