lucifer-yellow and Cell-Transformation--Neoplastic

Phorbol ester tumor promoters bind to specific cellular receptors (probably protein kinase C) and modulate membrane structure and function and gene expression of target cells. Using cell culture systems, we are studying the interaction of phorbol esters with the cellular membrane and subsequent modulation of gene expression. Our recent results can be summarized as follows: 1) specific binding of phorbol esters to mammalian cells can be inhibited by a human placental factor, which we have partially purified and characterized. 2) Phorbol ester tumor promoters reversibly inhibit intercellular communication, as measured by electrical coupling and dye transfer between cultured cells, suggesting that they inhibit both ionic and molecular transfer between cells. 3) In vitro transformation of Balb/c 3T3 cells results in blockage of intercellular communication between normal and transformed cells, indicating that blocked intercellular communication may play a role in cell transformation. 4) 12-O-Tetradecanoylphorbol-13-acetate (TPA) can continuously inhibit differentiation and globin gene expression in Friend erythroleukemia cells, without affecting their growth rate, for about 3 years. Both globin gene expression and terminal differentiation of Friend cells occur again upon removal of TPA from culture medium during such long-term culture.

Topics: Animals; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Communication; Cell Differentiation; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Female; Gene Expression Regulation; Humans; Isoquinolines; Leukemia, Erythroblastic, Acute; Mice; Phorbol Esters; Phorbols; Pregnancy; Protein Kinase C; Protein Kinases; Receptors, Drug; Receptors, Immunologic

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap-junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myc/ras all displayed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src- and neu-transformed cells, but not in the ras- and myc/ras-transformed cells. Nuclei isolated from WB-neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB-neo control cells, supporting the idea that the nuclear-localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC-deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane-localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane-associated Cx43 immunostaining. These data suggest that altered localization of the gap-junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells.

Topics: Animals; Blotting, Western; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Connexin 43; Epithelial Cells; Epithelium; Gap Junctions; Gene Expression; Isoquinolines; Liver; Mice; Microinjections; Microscopy, Phase-Contrast; Mutation; Oncogenes; Phosphorylation; Rats; Rats, Inbred F344; Staining and Labeling; Transduction, Genetic; Transfection

One of the effects of neoplastic transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). The investigation of junctional permeability is usually conducted through the microinjection of the fluorescent dye, Lucifer yellow, followed by observation of its migration into neighboring cells. This is a time-consuming approach, requiring expensive equipment. To overcome these problems, a novel technique was devised which takes advantage of the ability of short electric pulses to create transient "pores" on the cell membrane through which Lucifer yellow can enter, simultaneously and into large numbers of cells, with minimal disturbance to cellular metabolism. Cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse was applied in the presence of Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area was microscopically observed under fluorescence illumination. Using this technique, we investigated the relationship between expression of the middle tumor antigen of polyoma virus (mT) and GJIC in two representative cell systems with different responses to mT. The results show that low mT expression levels, although unable to transform rat F111 cells fully, are able to interrupt GJIC. Although parts of this mechanism might be mediated through protein kinase C (PKC), mT appears to have additional functions. PKC, however, had the opposite effect upon junctional permeability in a clone of mouse NIH-3T3 fibroblasts; intercellular communication in these cells appears to require PKC activity.

Topics: 3T3 Cells; Animals; Antigens, Polyomavirus Transforming; Cell Communication; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Electroporation; Fibroblasts; Fluorescent Dyes; Gap Junctions; Gene Expression Regulation, Neoplastic; Indium; Isoquinolines; Mice; Protein Kinase C; Tin Compounds

The ability of 18 different hydrocarbons to induce and promote morphological transformation and to inhibit intercellular communication in primary Syrian hamster embryo cells in culture have been studied. The compounds were: the n-alkanes octane, nonane, decane, undecane, dodecane and tridecane; the iso-alkanes 2-methylheptane, 2-methyloctane and 2-methylnonane; the naphthenes 1,2-dimethylcyclohexane, 1,2,4-trimethylcyclohexane and tert-butylcyclohexane; the aromates 1,2-dimethylbenzene, 1,2,4-trimetylbenzene and tert-butylbenzene; and the alkenes 1-octene, 1-nonene and 1-decene. None of the hydrocarbons induced morphological transformation of Syrian hamster embryo cells. When the hydrocarbons were incubated together with benzo(a)pyrene, enhancement of the transformation frequency was observed for the naphthene 1,2-dimethylcyclohexane and the iso-alkanes 2-methylheptane and 2-methyloctane. None of the n-alkanes, alkenes or aromates enhanced the transformation frequency induced by benzo(a)pyrene. The alkane tridecane and the iso-alkanes 2-methyloctane and 2-methylnonane reduced intercellular communication in the primary Syrian hamster embryo cells.

Topics: Animals; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Colony-Forming Units Assay; Cricetinae; Embryo, Mammalian; Fluorescent Dyes; Hydrocarbons; Intercellular Junctions; Isoquinolines; Mesocricetus

Disruption of intercellular communication (IC) by tumor promoters has been implicated as one of the major events in the promotion process. We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on IC in relation to colony formation (CF) in a coculture system of mouse epidermal JB6 cells, including unpromotable, promotable, and transformed clones. CF was evaluated in cocultures where cells were overlaid onto irradiated mat cells. IC was evaluated by the dye transfer assay in cocultures where overlaid cells were labeled with fluorescent beads. Enhancement of CF by TPA was observed in combinations where promotable clones were used as overlays. However, suppression of IC by TPA was observed in all clones of overlaid cells (day 1) and did not correlate satisfactorily to subsequent CF. Growth-arrested cells retained their capability to communicate with mat cells, while IC between colony-forming cells and mat cells was disrupted during CF (day 5), implying that selective communication is an event secondary to CF. It is suggested that in our experimental model, short-term suppression of IC by TPA may not be sufficient to explain subsequent colony formation and that other factors should be considered.

Topics: Animals; Cell Aggregation; Cell Communication; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Epidermis; Fluorescent Dyes; Isoquinolines; Kinetics; Mice; Tetradecanoylphorbol Acetate

Diminished intercellular communication has been associated with heightened sensitivity of cultured cells to morphological transformation and enhancement of transformation by tumor promoters. Microinjection of Lucifer yellow dye was employed to evaluate intercellular communication between transformable C3H/10T1/2 murine fibroblasts under a variety of culture conditions. Intercellular communication assayed by dye transfer from injected cells to surrounding cells in contact occurred in logarithmically growing cultures, declined to very low levels as confluence was attained, and then resumed upon the formation of mature confluent monolayers. Dye-transfer networks of 50 or more cells resulted from injection of single monolayer cells. Freeze-fracture electron microscopy confirmed the presence of gap junction structures in confluent cultures. Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidine and/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit intercellular communication between C3H/10T1/2 cells during 6-week transformation experiments. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate produced a transient inhibition of dye-coupling upon first introduction to cultures and prolonged the period of diminished dye-coupling at the attainment of confluence, but did not inhibit subsequent interactions between monolayer cells. A simple relationship thus could not be established between levels of dye-coupling within monolayers and focus formation events. Curiously, although the cells of foci in early phases of development did not exhibit dye-transfer capacity, dye-coupling was observed in mass cultures of most transformed cell lines cloned from foci. Co-cultivation of communication-competent transformed cells with nontransformed cells to produce reconstructed foci generally resulted in a cessation of dye-transfer by transformed cells. An often reversible loss of communication competence thus accompanies the growth of transformed C3H/10T1/2 cells as foci and may constitute an adaptive response which facilitates focus growth in the presence of intercellular communication between monolayer cells.

Topics: Animals; Carcinogens; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Isoquinolines; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Polychlorinated Dibenzodioxins; Tetradecanoylphorbol Acetate

Cultures of C3H/10T1/2 mouse embryo cells were treated in accordance with several treatment regimens that induced the focal growth of morphologically transformed cells. Intercellular communication between focus cells, and between focus and monolayer cells, was examined in late stages of transformation experiments by microinjection of Lucifer yellow dye into cells and observation of dye transfer to surrounding cells. Transformed foci produced by treatment with 3-methylcholanthrene, by initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or by initiation with MNNG and promotion with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. These included focus types thought to possess tumorigenic potential (Types II and III) and those believed to lack such potential (Type I). Cells within all focus types exhibited only limited communication with each other or with surrounding monolayer cells. In contrast, microinjection of monolayer cells typically resulted in dye transfer to an average of approximately 50 other monolayer cells. The presence of the tumor promoters TPA or TCDD did not alter intercellular communication between monolayer cells. These studies demonstrate that alterations in intercellular communication are evident during the growth of transformed foci. These changes are relatively independent of both the treatment regimen used to produce foci and the presumed oncogenic potential of different focus types.

Topics: Animals; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Isoquinolines; Methylcholanthrene; Mice; Polychlorinated Dibenzodioxins; Tetradecanoylphorbol Acetate

Topics: Animals; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Isoquinolines; Mice; Mice, Inbred BALB C; Phorbol Esters; Protein Kinase C

In order to study the possible role of intercellular communication in the process of in vitro cell transformation, the communicating capacity of BALB/c 3T3 cells transformed by 20-methylcholanthrene was investigated using a dye transfer method. Morphologically transformed foci, detectable 4 to 5 weeks after treatment with 20-methylcholanthrene (1.0 microgram/ml), are clearly distinguishable under the phase-contrast microscope from surrounding nontransformed monolayer cells; therefore, a tracer dye can be injected into individual cells, and gap-junctional communication between and among transformed and nontransformed can be studied directly. When fluorescent Lucifer Yellow CH was microinjected into a cell within the transformed focus, the dye was transferred to other transformed cells but not to cells in the adjacent nontransformed area, although they were in physical contact. Similarly, dye injected into a nontransformed cell was transferred to neighboring nontransformed cells but not to cells in an adjacent transformed focus. These results indicate that when BALB/c 3T3 cells are transformed by 20-methylcholanthrene they can no longer communicate with surrounding normal cells, although they maintain the ability to communicate with other transformed cells in the focus. These results suggest that loss of the ability to communicate with surrounding nontransformed cells may be one of the important determinants of induction and expression of the final malignant transformation.

Topics: Animals; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Fluorescent Dyes; Isoquinolines; Methylcholanthrene; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence