lucifer-yellow and Glioma

Some cell lines grown for only two cell doublings as multicell spheroids develop a form of resistance to killing by ionizing radiation that has been called the "contact" effect. While our previous results have implicated a role for higher order chromatin structure in the contact effect, another possible explanation is the presence of intercellular gap junctions that might facilitate communication between cells grown as spheroids and thereby enhance the ability of cells to resist or recover from radiation damage. To examine the role of gap junctions in the contact effect, rat glioma C6 and mouse EMT6 cell lines were transfected with a gene encoding the gap junctional protein connexin43. While C6 glioma cells are deficient in gap junctional communication, cells from spheroids were nonetheless more resistant than monolayers to killing by ionizing radiation, and the contact effect was present to a similar extent in the three transfected clones. For mouse EMT6 cells, radiosensitivity was similar whether cells were grown as monolayers or spheroids. Transfection of EMT6 cells with connexin43 increased gap junctional communication but did not promote development of a contact effect. Tumor volume doubling time in SCID mice increased significantly for one transfected clone; however, doubling time in vitro was also increased relative to the EMT6 parent. We conclude that extensive gap junctional communication is not a requirement for the increased radiation resistance observed when some cell lines are grown as spheroids.

Topics: Animals; Carcinoma; Cell Communication; Connexin 43; Cytoplasm; Fluorescent Dyes; Gap Junctions; Glioma; Isoquinolines; Mammary Neoplasms, Experimental; Mice; Mice, SCID; Radiation Tolerance; Rats; Spheroids, Cellular; Transfection; Tumor Cells, Cultured

Glial cells in the brain are known to provide structural and functional supports to neurons. To sustain such a supportive role, they have developed cell-to-cell communicating gap junctional channels. The authors studied the effect of dbcAMP on gap junctional channels mediated communication in C6 cells, a rat glioma cell line. Quantitative assessment of coupled cells under microscope after microinjection of a fluorescent dye was taken as a measure of junctional permeability. An enhanced coupling between cells was observed following dbcAMP treatment and this elevated coupling was found to be dependent on the duration of exposure of cells to dbcAMP. The studies have focused on a subtle shift in the spatial organization of the functional channels to the processes of dbcAMP induced differentiated cells from the cell cytoplasms and membranes of dbcAMP untreated cells. Immunofluorescence study with affinity purified antibody against gap junction further confirmed the spatial distribution of gap junctional protein(s) in the processes and also showed an increase in the density of the protein at the intercellular spaces in dbcAMP induced differentiated C6 glioma cells.

Topics: Animals; Bucladesine; Cell Communication; Cell Differentiation; Cell Division; Fluorescent Antibody Technique; Fluorescent Dyes; Gap Junctions; Glioma; Isoquinolines; Microinjections; Rats; Tumor Cells, Cultured

Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.

Topics: Animals; Carbenoxolone; Cell Communication; Connexin 43; Electric Conductivity; Fluorescent Dyes; Gap Junctions; Glioma; Glycyrrhetinic Acid; Isoquinolines; Rats; Succinates; Tumor Cells, Cultured

A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin-stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components.

Topics: Animals; Cell Line; Cholera Toxin; Chromatography, Thin Layer; Cyclic AMP; Fluorescent Dyes; G(M1) Ganglioside; Glioma; Isoquinolines; Kinetics; Rats; Receptors, Cell Surface; Receptors, Immunologic