Apigenin inhibits growth and motility but increases gap junctional coupling intensity in rat prostate carcinoma (MAT-LyLu) cell populations

Apigenin (4',5,7,-trihydroxyflavone) is a flavonoid abundant in the common fruits, herbs and vegetables constituting the bulk of the human diet. This study was aimed at quantifying the effects of apigenin on the basic cellular traits determining cancer development, i.e. cell proliferation, gap junctional coupling, and motility, using the Dunning rat prostate MAT-LyLu cell model. We demonstrated that apigenin considerably inhibits MAT-LyLu cell proliferation and significantly enhances the intensity of connexin43-mediated gap junctional coupling. This effect correlates with an increased abundance of Cx43-positive plaques at the cell-to-cell borders seen in apigenin-treated variants. Moreover, we observed an inhibitory effect of apigenin on the motility of MAT-LyLu cells. The basic parameters characterising MAT-LyLu cell motility, especially the rate of cell displacement, considerably decreased upon apigenin administration. This in vitro data indicates that apigenin may affect cancer development in general, and prostate carcinogenesis in particular, via its influence on cellular activities decisive for both cancer promotion and progression, including cell proliferation, gap junctional coupling and cell motility and invasiveness.     

LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca(2+) ([Ca(2+)](i)). Although agonist or ionomycin exposure can raise bulk [Ca(2+)](i) to levels higher than those caused by capacitative Ca(2+) influx, the LAMP assay revealed that only Ca(2+) influx through the plasma membrane store-operated Ca(2+) channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.     

In vitro cell culture methods for investigating Campylobacter invasion mechanisms

Studying the mechanisms of Campylobacter pathogenesis is complicated by the lack of simple animal models that mimic the disease seen in humans. In vitro cell culture methods provide a useful alternative to investigate the interactions between Campylobacter and the host epithelium that occur during infection. In the genomics era there is an increasing use of in vitro cell culture techniques to interrogate the potential role of different genes in pathogenesis. The aim of this review was to discuss the suitability and limitations of the various experimental approaches that might be adopted. We review current knowledge concerning the influence of cell-specific as well as bacterial factors required for Campylobacter invasion such as flagella and secreted proteins. The involvement and effects of phase variation on the results of invasion studies in cell culture emphasise the need to verify observed strain variations. We present the use of a mathematical Invasion Success Model to analyse Campylobacter invasion and show that it can be used to derive three strain dependent characteristics Imax, k, and I0. Even by combining data from independent experiments the Invasion Success Model can be used to statistically compare Campylobacter strains for their invasion of epithelial cells. Recommendations are given for the adoption of standard assay parameters and analytical methods such as the Invasion Success Model in order to facilitate comparison of data generated in different laboratories.     

In situ gellable oxidized citrus pectin for localized delivery of anticancer drugs and prevention of homotypic cancer cell aggregation

The aim of this study was to develop in situ gellable hydrogels composed of periodate oxidized citrus pectin (OP) for localized anticancer drug delivery and evaluate the potential of OP to inhibit cancer metastasis. Doxorubicin (Dox) was coupled to OP by imine bonds. Adipic dihydrazide (ADH) was used for cross-linking of the Dox-OP conjugates. The Dox-OP conjugate solution gelled within 2 min after addition of ADH. The release rate of Dox from the hydrogels was controllable by an additive amount of ADH. The released Dox retained anticancer activity. OP was shown to have a potency to prevent homotypic cancer cell aggregation compared to unmodified citrus pectin, strongly suggesting that OP released from hydrogels in vivo will inhibit cancer metastasis. These results indicate that OP hydrogels have the potential to prevent progression of primary cancer by the released Dox and generation of metastatic cancer by the released OP.     

Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0–1000 V/cm for a selected duration in the range 10–1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.     

Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide,vasoactive intestinal peptide (VIP) and a VIP-antagonist

Cholecystokinin-octapeptide (CCK-OP) induces a time- and dose-dependent decrease of gap Junctional conductance in isolated pairs of pancreatic acinar cells. In double whole-cell experiments, the time course could be described by the latency and the half-life time (t _1/2 ) of cell-to-cell uncoupling. The latency shows a biphasic dependence on [CCK-OP] with a minimum of about 50 sec at 10^–9 m CCK-OP. In the presence of vasoactive intestinal peptide (VIP), the biphasic relationship is shifted to lower CCK-OP concentrations. The increase of latency at high concentrations of CCK-OP (> 10⁰⁹ m) was blocked by addition of a VIP-antagonist. t _1/2 decreases monophasically with increasing [CCKOP]. Addition of GTPS to the pipette solution suppresses the [CCK-OP] dependence of the latency and potentiates the uncoupling phase. The kinetic data are discussed in terms of CCK binding to receptors of high and low affinity. Evidence is presented that secretion and cell-to-cell coupling are not related by an all-ornone process, but that for physiological CCK-OP concentrations, gap junctional uncoupling follows secretion.The authors would like to thank Dipl. Biol. F. Mendez for his support in software development for analysis of gap junctional conductance. The work was supported by the Graduiertenkolleg Biochemische Pharmakologie, the Herrmann und Lilly Schilling Stiftung and the Sonderforschungsbereich 156 of the Deutsche Forschungsgemeinschaft.     

In situ localized amplification and contact replication of many individual DNA molecules

We describe a method to clone and amplify DNA by performing the polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the linear DNA molecules so that the amplification products remain localized near their respective templates. At the end of the reaction, a number of PCR colonies, or 'polonies', have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. If an Acrydite modification is included at the 5' end of one of the primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology. Other applications are also discussed.     

Synthesis and spectroscopy of near infrared fluorescent dyes for investigating dichromic fluorescence

We developed a series of near infrared (NIR) cyanine dyes to study dichromic fluorescence phenomenon, which provides new protocols for in vivo optical imaging. Preliminary spectroscopic studies show that dichromic fluorescence correlates with structural symmetry. This feature suggests the potential use of dichromic fluorescent molecules to study biological processes that can alter the structural symmetry of the molecular probes.     

Retinoic acid modulates gap junctional permeability: a comparative study of dye spreading and ionic coupling in cultured cells

All-trans retinoic acid (RA), which was recently identified as a morphogen, affects gap junctional permeability in a dose- and time-dependent manner. In five different established mammalian cell lines (FL, BRL, BICR/M1Rk, HEL37, BT5C1) 100 mumol/liter RA reduced Lucifer yellow spreading within 30 min to 20-50% of the control. Ionic coupling, however, remained almost unaffected under the same conditions. Freeze-fractured membranes of untreated and RA-treated cells were similar with regard to frequency and sizes of gap junction plaques. With concentrations of less than 10 mumol/liter RA the dye spreading increased significantly in the human amniotic cell line FL, pointing to a possible modulatory effect of RA on junctional communication.     

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.     

In vivo single-cell electroporation for transfer of DNA and macromolecules

Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body-rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.     

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

In vivo quantification of cell coupling in plants with different phloem-loading strategies

Uptake of photoassimilates into the leaf phloem is the key step in carbon partitioning and phloem transport. Symplasmic and apoplasmic loading strategies have been defined in different plant taxa based on the abundance of plasmodesmata between mesophyll and phloem. For apoplasmic loading to occur, an absence of plasmodesmata is a sufficient but not a necessary criterion, as passage of molecules through plasmodesmata might well be blocked or restricted. Here, we present a noninvasive, whole-plant approach to test symplasmic coupling and quantify the intercellular flux of small molecules using photoactivation microscopy. Quantification of coupling between all cells along the prephloem pathways of the apoplasmic loader Vicia faba and Nicotiana tabacum showed, to our knowledge for the first time in vivo, that small solutes like sucrose can diffuse through plasmodesmata up to the phloem sieve element companion cell complex (SECCC). As expected, the SECCC was found to be symplasmically isolated for small solutes. In contrast, the prephloem pathway of the symplasmic loader Cucurbita maxima was found to be well coupled with the SECCC. Phloem loading in gymnosperms is not well understood, due to a profoundly different leaf anatomy and a scarcity of molecular data compared with angiosperms. A cell-coupling analysis for Pinus sylvestris showed high symplasmic coupling along the entire prephloem pathway, comprising at least seven cell border interfaces between mesophyll and sieve elements. Cell coupling together with measurements of leaf sap osmolality indicate a passive symplasmic loading type. Similarities and differences of this loading type with that of angiosperm trees are discussed.     

In situ observation of spherical DNA assembly in water and the controlled release of bound dyes

Three strands of 30-mer oligodeoxyribonucleotides (ODNs) were designed to form three-way junctions that possess self-complementary sticky ends. The morphology of self-assembled ODNs in water was observed in situ by confocal laser scanning fluorescence microscopy. The three-way junctions self-assembled into spherical assemblies, in accordance with transmission and scanning electron microscopy. The size of nucleospheres was in the range of several tens of nanometers to micrometers, which varied depending on the concentration of ODNs and added salts. Fluorescence images of spherical ODN assemblies suggested that the nucleospheres possess multiwalled structures. The fluorescence of sodium 1-anilinonaphthalene-8-sulfonate in the presence of nucleospheres revealed that the interior of nucleospheres possesses polarity corresponding to that between methanol and ethanol. A dye-inclusion experiment showed that cationic and even anionic dyes were adsorbed to the interior of the nucleospheres. The dye-included nucleospheres released dyes by thermal dissociation or digestion of the constituent ODNs.     

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.     

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

Vanadium compounds were found to promote the induction of morphological transformation of hamster embryo cells. Exposure of the cells to Na−O-vanadate, vanadin (V) oxide or vanadin (IV) oxide sulfate following pre-exposure to a low concentration of benzo[a]pyrene, potentiated the induction of transformed colonies similar to 12-O-tetradecanoylphorbol-13-acetate. Unlike this phorbol ester, vanadium compounds did not inhibit intercellular communication, or activate protein kinase C. Nor did vanadate influence the reoccurrence of communication after removal of a communication blocking phorbol ester. On the other hand, vanadate showed strong synergism with the phorbol ester on induction of transformed morphology in the phorbol ester sensitive cell line BPNi. This suggests that vanadium and tumor promoting phorbol esters mediate their effect on the induction of morphological transformation of hamster embryo cells through different mechanisms.     

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK)

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.     

Preparation,characterization, and structure of half gap junctional layers split with urea and EGTA

Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60–90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca²⁺ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.The antibodies used to analyze the protein composition in our samples were the generous gift of Dr. David Paul. We thank Dr. Camillo Peracchia for sending us a preprint of his chapter on molecular mechanisms of connexon gating and docking which helped guide our thinking about interconnexon forces and John Badger for information on divalent cation sites in protein structure. We thank Amani Thomas-Yusuf for technical assistance. The photographic expertise of Marie Craig is gratefully acknowledged. This work was funded by National Institutes of Health GM43217 to G.E.S. and GM18974 to D.A.G.