Junctional communication of embryonic cells after induction

Cell couplings before and after neural induction in embryos of Cynops orientalis were studied by means of single cell injection of Lucifer Yellow.Differences both in incidence and the extent of cell couplings were demonstrated.Results of cell couplings were correlated with electron microscopic observations of freeze-etching replicas.     

Intercellular communication in cultured human vascular smooth muscle cells

Intercellular communication through gap junction channelsplays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,capacitance vessel). Northern and Western blots documented the presenceof connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA incultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority ofrecords, in both vessels, single-channel activity was dominated by amain-state conductance of 120 pS, with subconducting events comprisingless than 10% of the amplitude histograms. However, some recordsshowed "atypical" unitary events that had a conductance similar toCx40 (~140-160 pS), but gating behavior like that of Cx43. Assuch, it is conceivable that the presence and coexpression of Cx40 andCx43 in IMA and SV myocytes may result in heteromeric channelformation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

     

Junctional communication is induced in migrating capillary endothelial cells

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.     

Laser scanning analysis of cell-cell communication in cultured human prostate tumor cells

OBJECTIVE: To investigate gap-junctional intercellular communication (GJIC) in LNCaP and DU145 human prostate cancer cells. STUDY DESIGN: Normal rat liver F344 (WB1) cells were used as positive controls. Functional GJIC was inspected using either the scrape-loading/dye transfer (SL/DT) method or fluorescence recovery after photobleaching (FRAP) analysis. In the former, GJIC activity was expressed as a measure of the extent of diffusion of Lucifer Yellow after cell monolayers were scraped using a surgical blade and exposed to dye for a few minutes at room temperature. In the latter, cells were incubated for 15 minutes at 37 degrees C with 5,6-carboxyfluorescein diacetate dye and the dye transfer visualized by photobleaching individual cells with a 488-nm laser and monitoring the recovery of fluorescence using a laser cytometer. RESULTS: The preliminary results obtained indicate that neither LNCaP nor DU145 cells have functional GJIC, while, as expected, WB1 cells show unimpaired GJIC activity. Equivalent results were consistently obtained using either SL/DT or the FRAP approach. However, using FRAP analysis, DU145 cells only showed weak recovery of fluorescence after a total observation interval of 15 minutes. CONCLUSION: The present data, though preliminary, suggest that disruption of GJIC may play a role in development of malignancy in the human prostate.     

Purification of mitotic spindles from cultured human cells

In eukaryotes, both chromosome segregation and the determination of the cell division cleavage plane depend on the mitotic spindle apparatus. Spindle malfunctioning can lead to chromosome mis-segregation and cytokinesis defects and hence result in aneuploidy. Thus, the understanding of the structure and function of mitotic spindles is of interest not only from the perspective of basic science, but has implications also for human health and disease. Until recently, this complex microtubule-based structure was studied mainly by cell biological techniques in mammalian cells, by biochemical assays in Xenopus egg extracts, and by genetic approaches in genetically tractable organisms such as yeast, flies, and nematodes. With the rapid development of mass spectrometry and its increasing application to biological problems, it has become possible to subject highly complex structures, such as the mitotic spindle apparatus, to proteomics approaches. Such studies require the isolation of the mitotic spindle, or its substructures, in sufficient amounts and free of excessive contaminants. A number of methods for the isolation of mitotic spindles from mammalian tissue culture cells have been developed in the past. We have compared these methods and found that protocols based on the stabilization of microtubules by taxol were most efficient and reproducible. Here, we describe the further optimization of a taxol-based method, originally developed by Zieve and Solomon [Cell 28 (1982) 233-242], and its application to the isolation of human mitotic spindles at a scale suitable for mass spectrometric analysis [G. Sauer, R. Korner, A. Hanisch, A. Ries, E.A. Nigg, H.H.W. Sillje, Mol. Cell. Proteomics 4 (2005) 35-43].     

Histochemical identification of cultured cells from human endometrium

Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, gamma-glutamyltranspeptidase, peroxidase, and beta-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelia in sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Characterization of cells isolated and cultured from human bone

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.     

Identification of cultured progenitor cells from human marrow stroma

The marrow stromal cells (MSC) are essential for regulation of bone remodeling and hematopoiesis. It is of prime importance to isolate MSC and to expand the proliferating cells ex vivo. In this study, we analyzed cultured MSC for various cellular parameters, including cell morphology, cell cycle, and expression of cell surface antigens by flow cytometry. MSC were divided based on cell size to small (S-cells) and large (L-cells) and were visualized by light and electron microscope. The S-cells were proliferating cells correlated with G0/G1 phase of cell cycle, and expressed cFOS. The expression of surface markers CD-34, -44, -51, -61, -62E, -62P, -62L was quantified using flow cytometry. CD-44 was ubiquitously expressed by S and L cells, CD-51 and -61 were expressed by 30%-38% of S-cells. CD-34 and -62 expressed 20% positive of the analyzed cells that were of the proliferating progenitors (S-cells). This study enables the identification of subpopulations from MSC with special attention paid to the proliferating cells from ex vivo cultures of marrow stroma.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.     

Effect of serum on cells cultured from human mammary tumors

Summary Clusters of cells derived from biopsy specimens of human mammary ductal carcinomas form two morphologically distinct epithelial colonies in culture, designated as E and E′. The proportion of E′ cell clusters that attached and formed colonies ranged from 0.3 to 13.0% with different tumors. Attachment was independent of tumor grade. Microscopic observations revealed that the survival of E′ cell colonies was limited to approximately 10 days with rapid cell degeneration commencing about 7 days. A comparison of sera showed that colony formation by cells from malignant tumors during the 1st week of culture was maximum in the presence of fetal bovine serum. Human serum alone was 70 to 100% less effective in promoting E′ colonies. The most significant finding was that human serum from normal donors inhibited E′ colony development in the presence of FBS. Although human serum was less effective than FBS in promoting colony formation by clusters of E cells, an inhibition was not observed. Inhibitory activity could not be attributed to either antagonistic hormones or the source of human serum. THese results demonstrate that normal human serum contains a factor(s) that exhibits an inhibitory activity specific for human epithelial cells (E′) derived from malignant tumors. Supported by NCI Contract CB-33898 and a Fellowship from the Imperial Cancer Research Fund, London, England.     

Androstenedione metabolism in epithelial cells derived from early-lactation human milk

Epithelial cells derived from duct epithelium were cultured from early lactation human milk in medium supplemented with 15% fetal calf serum, insulin (0.3 u/ml), cortisol 21-sodium succinate (6 micrograms/ml) and amikacin (50 micrograms/ml). The capacity of these cells to metabolize androstenedione to estrone, estradiol and C19 metabolites was studied during continuous culture. After extraction of the medium, the products were subjected to phenolic partition and separated by thin-layer and paper chromatography, followed by recrystallization to constant specific activity. The study demonstrated a progressive increase in the formation of estrone and testosterone over the first 24 h in culture, while estradiol formation showed an initial 2-4 h lag, then increased slowly. The C19 compounds identified were androsterone, 5 alpha-androstanedione, epiandrosterone, dihydrotestosterone and etiocholanolone. 5 alpha-Androstanedione and androsterone were the major 5 alpha-reduced metabolites. Since these cells are derived from normal duct epithelium, their metabolic characteristics may be more representative of normal breast tissue than those of tissue removed from patients with pathological breast disorders.     

Immunogenicity of human B cell antigens solubilized from cultured human lymphoid cells.

Antigens solubilized from culured human lymphoid cells WI-L2 and RPMI 1788 were partially purified by ultracentrifugation on a KBr gradient. These antigens injected into rabbits produced xenoantibodies which after absorption with melanoma cells became specific to B cell antigens. Three such xenoantisera were submitted to the Second Histocompatibility Workshop of the Americas and reacted much like alloantisera to B cell antigens against a large panel of B peripheral lymphocytes and cells from patients with chronic lymphocytic leukemia. Xenoantisera to B cell antigens inhibited the mixed lymphocyte reaction, but did not affect the mitogenic activity of phytohemagglutinin or the functional properties of C3 receptors, monkey red blood cell receptors, or T cell receptors.