Connexin-independent ganciclovir-mediated killing conferred on bystander effect-resistant cell lines by a herpes simplex virus-thymidine kinase-expressing colon cell line

A novel gap junction-independent mechanism for ganciclovir-mediated bystand er effect killing by a herpes simplex virus thymidine kinase (HSV-TK)-expre ssing SW620 human colon tumor cell line has been characterized. The mechani sm of the HSV-TM/GCV bystander effect for many tumor cell lines has been de monstrated to be due to connexin gap junction transfer of phosphorylated ga nciclovir (GCV) metabolites; however, there may be as yet uncharacterized c onnexin-independent mechanisms for the effect. To address this, the bystand er effect was further evaluated in a panel of cell lines mixed with homolog ous HSV-TK-expressing cell lines, a SW620.TK cell line, or a high connexin4 3-expressing PA-317.TK cell line. Of the 10 cell lines tested, 4 were found to be resistant to bystander effect killing by their homologous HSV-TK-exp ressing cell lines and the PA-317.TK cells, but all of the cell lines were sensitive to GCV killing when mixed with the SW620.TK cells. The SW620.TK c ells were then further evaluated for any indication of extracellular GCV me tabolite efflux. Culture medium from SW620.TK cells labeled with [H-3]GCV w as evaluated for the presence of GCV nucleotides by ion-exchange column sep aration and HPLC analysis. The presence of GCV mono-, di-, and triphosphate metabolites in the medium was detected. Inclusion in the medium of inhibit ors of extracellular phosphatases and edo-ATPases increased the proportion of GCV metabolites recovered. These results indicate that phosphorylated GC V metabolites can be effluxed from SW620.TK cells and that some type of cel lular uptake mechanism independent of gap junctions exists for nucleotide e ntry into neighboring cells.