1. ¹Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan, USA
2. ²Department of Pathology, Medical College of Ohio, Toledo, OH, USA

Correspondence: Dr DS Shewach, Department of Pharmacology, 4713 Upjohn Center, University of Michigan Medical Center, 1310 East Catherine, Ann Arbor, MI 48109-0504, USA

Received 14 October 2004; Accepted 3 January 2005; Published online 24 March 2005.

Abstract

The role of gap junctional intercellular communication (GJIC) in bystander killing with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) was evaluated in U251 cells expressing a dominant-negative connexin 43 cDNA (DN14), and in HeLa cells, reportedly devoid of connexin protein. These cell lines both exhibited 0% GJIC when assayed by Lucifer Yellow fluorescent dye microinjection. Bystander cytotoxicity was still apparent in 50:50 cocultures of DN14 and HSV-TK-expressing U251 cells, but not in 50:50 cocultures of HeLa cells. However, the sensitivity of HeLa HSV-TK-expressing cells to GCV decreased nearly 100-fold (IC₉₀=109 M) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC₉₀=1.2 M). A more sensitive flow cytometry technique to measure GJIC over 24 h revealed that the DN14 and HeLa cells exhibited detectable levels of communication (29 and 23%, respectively). Transfer of phosphorylated GCV to HeLa bystander cells occurred within 4 h after drug addition, and GCV triphosphate (GCVTP) accumulated to 21384 pmol/10⁶ cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (86733 pmol/10⁶ cells) compared to 100% cultures of HSV-TK-expressing cells (1773188 pmol/10⁶ cells). The half-life of GCVTP in the HSV-TK-expressing cells was approximately four times that measured in the bystander cells (12.3 and 3.1 h, respectively). These data suggest that the lack of bystander cytotoxicity in HeLa cocultures is due to low transfer of phosphorylated GCV and a rapid half-life of GCVTP in the bystander cells. Thus, GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate either bystander cell killing or sparing of HSV-TK-positive cells, depending upon the cell specific drug metabolism.