lucifer-yellow and Carcinoma--Squamous-Cell

Down-regulation of Cx43 expression had been shown to occur in nasopharyngeal carcinoma cells. The present study was undertaken to estimate if methylation of the promoter region in Cx43 gene was responsible for the repression of Cx43 expression in the CNE-1 nasopharyngeal carcinoma cells. Calcein transfer and lucifer yellow transfer were detected to evaluate gap junction intercellular communication (GJIC) in CNE-1 cells. It was found that the control CNE-1 cells showed no fluorescent dye transfer. After treatment with DNA methyltransferase inhibitor 5-aza-CdR, fluorescent dye transfer between cells became obvious. RT-PCR and Western blot were performed to determine the expression of Cx43 gene. The control CNE-1 cells showed a low expression level of Cx43, whereas 5-aza-CdR-treated CNE-1 cells showed an enhanced level of Cx43 expression. Methylation-sensitive restriction enzyme and PCR analysis showed that the methylation of the Cx43 gene promoter region occurred in CNE-1 cells. In addition, treatment with 5-aza-CdR inhibited the growth (including anchorage-independent growth) of CNE-1 cells, and resulted in an accumulation of cells in G0/G1 phase. These results indicate the promoter methylation as an important role in inactivation of Cx43 in CNE-1 cells.

Topics: Azacitidine; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Connexin 43; Decitabine; DNA Methylation; DNA Modification Methylases; Down-Regulation; Fluoresceins; Gap Junctions; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Nasopharyngeal Neoplasms; Promoter Regions, Genetic

Gap junction intercellular channels are required for metabolic cooperation between cells and regulate normal tissue homeostasis by means of the transfer of small molecules between contacting cells. Not surprisingly, the gap junction phenotype is frequently lost during carcinogenesis in human tissues (including those of the upper aerodigestive tract), freeing individual cancer cells from the growth control signals of normal surrounding tissues and less aggressive adjacent cancer cells. We hypothesized that gap junctional intercellular communication (GJIC) could mediate a bystander effect (apoptotic cell death) in squamous cell carcinoma of the head and neck (SCCHN) cells adjacent to individually targeted SCCHN cells.. Single-cell microinjection of cytochrome c was used to induce apoptosis in target SCCHN cells with endogenous GJIC activity and in an SCCHN cell line with exogenously introduced GJIC activity. Apoptosis was followed in target and surrounding bystander cells through light and time course microscopic characterization. All of the preceding experiments were carried out in the absence and presence of 18-beta-glycerretinic acid, a pharmacologic inhibitor of GJIC.. When cytochrome c was introduced into SCCHN cells with endogenous GJIC activity through single-cell microinjection, bystander effects (apoptosis of nontarget cells) were observed. When GJIC activity was blocked with the specific pharmacologic inhibitor of gap junctions, 18-beta-glycerretinic acid, a bystander effect was never seen in GJIC active SCCHN cell lines.. Gap junction intercellular channels can mediate a bystander effect in SCCHN. Inconsistencies in our data will be discussed in the context of recent advances in this field, as well as our future research directions.

Topics: Apoptosis; Bystander Effect; Carcinoma, Squamous Cell; Cell Line; Connexin 43; Cytochromes c; Fluorescent Dyes; Gap Junctions; Glycyrrhetinic Acid; Head and Neck Neoplasms; Humans; Isoquinolines; Microinjections; Microscopy; Transfection; Video Recording