lucifer-yellow and Lung-Neoplasms

Studies using normal or neoplastically transformed established mouse lung epithelial cell lines revealed a reduction in gap junctional, intercellular communication (GJIC) with transformation. To determine the stage in tumor development at which GJIC is interrupted, we used the well-established model of lung tumors induced in strain A/J mice by urethane. In this system, tumor development follows a well-characterized pattern; hyperplasias, adenomas, and carcinomas are manifested at approximately 8, 16, and 40 weeks after urethane treatment, respectively. GJIC levels were examined using a novel technique where cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse that opens transient pores on the plasma membrane is applied in the presence of the fluorescent dye, Lucifer yellow, causing dye penetration into cells growing on the conductive part of the slide. Migration of the dye through gap junctions to nonelectroporated cells growing on the nonconductive area is then microscopically observed under fluorescence illumination. Unexpectedly, primary cells cultured from urethane-induced tumors, even late stage carcinomas, possessed extensive GJIC immediately upon isolation. Upon passage for several months however, these cells lost GJIC. These results suggest that the molecular changes that lead to the formation of the tumor in vivo are not sufficient to interrupt gap junctions. Propagation of tumor cells in culture induces additional alterations that can lead to gap junction closure.

Topics: Animals; Electroporation; Fluorescent Dyes; Gap Junctions; Isoquinolines; Lung Neoplasms; Mice; Signal Transduction

In addition to its antioxidant activity, beta-carotene (BC) is known to enhance gap junction intercellular communication (GJIC) by up-regulation of connexin 43 (Cx43), an action that may be important in its control of tumor growth. Surprisingly, two clinical trials on supplemental BC suggest that BC may increase lung cancer incidence in smokers. Recently, an animal study indicated that a very high dose of BC (50 mg/kg b.w./day for 5 days) decreases GJIC in rat liver, while a lower dose (5 mg/kg b.w./day) increases GJIC. It is unclear how high-doses of BC inhibit GJIC. In this study, we tested whether oxidized BC (OBC, obtained by heating BC at 60 degrees C in open air for 1 h) may inhibit GJIC. We incubated a human lung cancer cell line (A549) with OBC or BC at 2-10 microM for 5 days. Cell viability (by Trypan-blue assay), GJIC (by scrape-loading dye transfer) and Cx43 expression (by western blotting and immunocytochemical localization) were measured to investigate the effects of OBC and BC on GJIC and the possible mechanisms. The results show that OBC at concentrations lower than 10 microM did not significantly affect cell viability. However, OBC at 5 muM inhibited GJIC, whereas BC at 5 microM markedly increased GJIC. The loss of GJIC in A549 induced by OBC accompanied the aberrant localization and phosphorylation of connexin43 (Cx43). These changes in the expression of Cx43 induced by OBC were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter. Thus, our results suggest that in vivo inhibition of GJIC by a high dose of BC on GJIC is, at least in part, attributable to the effect of OBC.

Topics: Adenocarcinoma; Antioxidants; beta Carotene; Blotting, Western; Cell Communication; Cell Line, Tumor; Connexin 43; Fluorescent Dyes; Gap Junctions; Humans; Immunohistochemistry; Indicators and Reagents; Isoquinolines; Lung Neoplasms; Oxidation-Reduction; Phosphorylation

Topics: Animals; Carcinoma; Cell Communication; Electric Conductivity; Electroporation; Endothelium, Vascular; Fibroblasts; Fluorescent Dyes; Gap Junctions; Glass; Humans; Isoquinolines; Lung Neoplasms; Mice; Mice, Inbred A; Tin Compounds; Tumor Cells, Cultured; Umbilical Veins

Animal tumor models have demonstrated a close correlation between gap junctional, intercellular communication (GJIC) and tumor metastasis. To examine GJIC levels in human lung carcinoma cells, a novel technique was developed: cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse which opens transient pores on the plasma membrane was applied in the presence of the fluorescent dye, Lucifer yellow, causing the dye's penetration into the cells growing on the conductive part of the slide. The migration of the dye through gap junctions to the non-electroporated cells growing on the non-conductive area was then observed microscopically under fluorescence illumination. The results show that this is a rapid, precise and highly reproducible assay for GJIC assessment in lines established from lung carcinomas or freshly explanted lung tumor cells. Out of 17 established lines only two had extensive junctional communication, while out of 16 fresh tumor specimens none displayed GJIC. On the other hand, fibroblasts isolated from the same tumors had extensive junctional permeability. The examination of GJIC in a large number of samples could establish a correlation between GJIC and metastasis which might have prognostic value.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Communication; Electroporation; Fibroblasts; Fluorescent Dyes; Gap Junctions; Humans; Isoquinolines; Lung Neoplasms; Tumor Cells, Cultured

Topics: Carcinoma; Cell Communication; Electrophysiology; Fluorescent Dyes; Humans; Intercellular Junctions; Isoquinolines; Lung Neoplasms; Tumor Cells, Cultured