Single-cell microinjection of cytochrome c can result in gap junction-mediated apoptotic cell death of bystander cells in head and neck cancer.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Frank
Douglas K
DK
Department of Otolaryngology-Head and Neck Surgery and the Head and Neck Cancer Molecular Biology Research Laboratory, The New York Eye and Ear Infirmary, New York, New York 10003, USA. dfrank@bethisraelny.org
Szymkowiak
Bozena
B
Josifovska-Chopra
Olgica
O
Nakashima
Torahiko
T
Kinnally
Kathleen W
KW
D017209:Apoptosis / Q000187:drug effects*; D024201:Bystander Effect / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics; D045304:Cytochromes c / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D006258:Head and Neck Neoplasms / Q000473:pathology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008845:Microinjections; D008853:Microscopy; D014162:Transfection; D014741:Video Recording
D018031:Connexin 43; D005456:Fluorescent Dyes; D007546:Isoquinolines; D045304:Cytochromes c; C017475:lucifer yellow; D006034:Glycyrrhetinic Acid