cytochrome-c-t and lucifer-yellow

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

Programmed cell death (apoptosis) occurs both during normal development and as a result of various pathological conditions. An in vitro system was used to explore the transmission of death signals from apoptotic cells to cells with which they were coupled via gap junctions. Confluent cultures of baby hamster kidney (BHK) cells, stably transfected with the gap-junctional protein connexin32, were scrape loaded with cytochrome C (cyC), a mitochondria-derived apoptotic agent, to introduce the protein into cells injured by the cut. The cultures were subsequently analyzed for the presence of activated caspases, the distribution of TUNEL staining, and the binding of annexin V. Although cyC is too large to traverse the gap junctional channel, each of the assays revealed that apoptosis had spread from dying cells at the margin of the scrape to otherwise healthy neighboring cells to which they were coupled. This "bystander effect" was significantly reduced in the presence of agents that block gap junctional intercellular communication.

Topics: Animals; Apoptosis; Carbenoxolone; Caspases; Cell Line; Connexins; Cricetinae; Cytochromes c; Dextrans; Fluorescent Dyes; Gap Junction beta-1 Protein; Gap Junctions; In Situ Nick-End Labeling; Isoquinolines; Octanols; Rhodamines; Transfection