Involvement Of Gap Junctional Intercellular Communication Research Articles

Inhibition of Balb/c 3T3 cell transformation by synthetic acyclic retinoid NIK-333; possible involvement of enhanced gap junctional intercellular communication

Background: In an attempt to develop effective and non-cytotoxic cancer chemopreventive derivatives of retinoids, a novel acyclic retinoid has previously been synthesized. This acyclic retinoid, NIK-333, had been reported to suppress recurrence of primary hepatocellular carcinomas. We explored the molecular mechanisms by which NIK-333 exerts anti-proliferative effects. Methods: We employed Balb/c 3T3 cells, since they can be used as a quantitative transformation assay and since we can study a possible involvement of gap-junctional intercellular communication (GJIC) in their growth control. We included all- trans-retinoic acid (ATRA) for comparison. Results: We first confirmed that these cells express the retinoid receptors, RARα, γ and RXRα, suggesting that they respond to NIK-333 and ATRA. When NIK-333 or ATRA was added to Balb/c 3T3 cells during the induction of cell transformation by a standard (3-methylcholanthrene (MCA) alone) or two-stage (low dose of MCA plus 12- O-tetradecanoylphorbol-13-acetate (TPA)) protocol, there was a significant decrease in the number of transformed foci. The extent of inhibition of transformation by NIK-333 was similar to that exerted by ATRA. Employing the microinjection dye-transfer assay, we found that both retinoids increase GJIC level when measured 24 h after treatment. The extent of GJIC increase by NIK-333 was similar to that of ATRA. These retinoids also increased the amount of connexin 43 (Cx43) on the plasma membrane as revealed by immunostaining. Conclusion: These data indicate that NIK-333 suppresses chemical carcinogenesis in vitro and support the hypothesis that enhancement of GJIC is involved in this process.

Cell proximity is a prerequisite for the proliferative response of bystander cells co-cultured with cells irradiated with gamma-rays.

In a recent study, we showed that unirradiated cells, when they are in the presence of cells irradiated with gamma-rays, are characterized by enhanced cell growth (Cytometry 2003;54A:1-7). However, the mechanisms and factors involved in the proliferative response of bystander cells are largely unknown. The aim of the current work was to investigate the possible role of spatial proximity of cells, including gap junctional intercellular communication (GJIC), in transmitting proliferation signals from cells irradiated with gamma-rays to unirradiated cells (bystander cells). Confluent monolayers of rat liver epithelial cells (WB-F344) were irradiated with 137Cs gamma-rays at doses ranging between 0.5 and 10 Gy. The impact of cell proximity on the proliferative response of bystander cells was determined by co-culturing different densities of irradiated and unirradiated cells (ratio 1:1) followed 24 h later by two-scheme flow cytometric analysis of their proliferation. To determine whether soluble extracellular factors play a role in this response, irradiated cells were plated together with unirradiated cells at the same ratio (1:1), but they were not allowed to directly contact unirradiated cells by using porous low-protein binding polyethylene terephthalate membranes. After 24 h, the numbers of unirradiated cells in the co-culture with irradiated cells were compared with the numbers of unirradiated cells in the control (unirradiated cells co-cultured with unirradiated cells). To investigate the possible involvement of GJIC in mediating the proliferative response in the bystander cells, the proliferation status of unirradiated GJIC-incompetent WB-aB1 cells was compared with that of unirradiated WB-F344 cells that were GJIC competent. In all of the protocols, the two populations of co-cultured cells were distinguished by labeling one population with fluorescent cell tracers, such as membrane-resident or CFDA SE (carboxy) fluorescein diacetate, succinimidyl ester that metabolizes intracellularly. Unirradiated cells that were co-cultured with irradiated cells, but were not allowed to directly contact them, did not show any changes in the proliferation rate compared with that of unirradiated cells in the control. Unirradiated cells that were co-cultured as a mixture with irradiated cells showed enhanced and statistically significant proliferation, particularly when they were plated together more densely. The proliferation rate of bystander WB-aB1 cells was apparently higher than that of bystander WB-F344 cells; however, the difference was statistically insignificant. In the present experimental model, the spatial proximity of cells is a crucial element for transmitting growth stimulation signals from irradiated cells to neighboring unirradiated cells. Direct cell-to-cell contact appears to be required for transmitting these signals. Neither functional GJIC nor soluble extracellular factors released by irradiated cells into the culture medium appear to play significant roles in this process under the present experimental conditions.

Involvement of gap junctional communication in myogenesis

Cell-to-cell communication plays important roles in development and in tissue morphogenesis. Gap junctional intercellular communication (GJIC) has been implicated in embryonic development of various tissues and provides a pathway to exchange ions, secondary messengers, and metabolites through the intercellular gap junction channels. Although GJIC is absent in adult skeletal muscles, the formation of skeletal muscles involves a sequence of complex events including cell-cell interaction processes where myogenic cells closely adhere to each other. Much experimental evidence has shown that myogenic precursors and developing muscle fibers can directly communicate through junctional channels. This review summarizes current knowledge on the GJIC and developmental events involved in the formation of skeletal muscle fibers and describes recent progress in the investigation of the role of GJIC in myogenesis: evidence of gap junctions in somitic and myotomal tissue as well as in developing muscle fibers in situ, GJIC between perfusion myoblasts in culture, and involvement of GJIC in cytodifferentiation of skeletal muscle cells and in myoblast fusion. A model of intercellular signaling is proposed where GJIC participates to coordinate a multicellular population of interacting myogenic precursors to allow commitment to the skeletal muscle fate.

Close relationship between modulation of serum-induced stimulation of DNA synthesis and changes in gap-junctional intercellular communication in quiescent 3T3-L1 cells caused by cyclic AMP and the tumor-promoting phorbol ester TPA

Involvement of gap-junctional intercellular communication in the stimulation of growth was investigated in quiescent 3T3-L1 cells. When the cells in monolayer were growth-arrested by culture in a low concentration of calf serum, addition of dibutyryl cyclic AMP enhanced dye-coupling and suppressed the enhancement of DNA synthesis, induced by calf serum, in quiescent cells. 12- O-Tetradecanoylphorbol-13-acetate (TPA) suppressed dye-coupling in quiescent cells and enhanced DNA synthesis in both quiescent and serumtreated cells. When about 5000 cells were cultured in contact to form a colony, growth arrest of the cells was observed in the central region of such colonies rather than in the peripheral region, but addition of calf serum induced DNA synthesis in the cells in both the peripheral and central regions of the colonies. Addition of TPA enhanced serum-induced DNA synthesis in the cells in the central region of colonies rather than in the peripheral region. These results suggest that the ability of quiescent cells to escape from growth arrest is inversely correlated to the extent of gap-junctional intercellular communication.