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Cytotoxicity and Accumulation of Ganciclovir Triphosphate in Bystander Cells Cocultured with Herpes Simplex Virus Type 1 Thymidine Kinase-expressing Human Glioblastoma Cells¹

Departments of Pharmacology [L. Z. R., P. D. B., P. J. M., D. S. S.] and Internal Medicine [M. K.], University of Michigan Medical Center, Ann Arbor, Michigan 48109

The ability of herpes simplex virus type 1 thymidine kinase (HSV-TK)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-TK-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs through the transfer of phosphorylated GCV, there is little direct proof that bystander cells can accumulate GCV nucleotides. We have studied the ability of U251 human glioblastoma cells expressing HSV-TK (U251tk cells) to induce cytotoxicity in neighboring U251 bystander cells that lack the viral kinase (U251ßgal cells) and evaluated whether this bystander cell killing is mediated by GCV nucleotides. The cytotoxicity studies demonstrated that the ratio of HSV-TK-expressing cells:bystander cells was important in determining the sensitivity of both cell types to GCV. U251tk cells cocultured with an equal number of U251ßgal cells (a 50:50 ratio) exhibited a sensitivity to GCV similar to that observed in the absence of bystander cells, with >99.8% cell kill at 1 µM GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 µM GCV decreased the survival of U251tk cells by only 54%. Strong bystander cell killing was observed at both ratios. In a 50:50 coculture of U251tk and U251ßgal cells, the survival of bystander cells was decreased by >99.5% with 3 µM GCV, whereas 30 µM GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251ßgal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. A U251 bystander cell line was developed from the parental cell line by transfection with the cDNA coding for green fluorescent protein (U251gfp cells), which permitted the separation of U251gfp cells from nonfluorescing U251tk cells by flow cytometry with cell sorting. With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. The results demonstrated that significant levels of the triphosphate of GCV (GCVTP) accumulated in bystander cells within 4 h of coculture, and this accumulation was dependent upon the percentage of HSV-TK-expressing cells as well as the concentration of GCV and the length of incubation. The proportion of GCVTP in bystander cells was consistently 50–80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. However, the actual amount of GCVTP was as much as 8-fold lower in both the U251tk and U251ßgal cells cocultured at a ratio of 10:90 compared to those cocultured at a ratio of 50:50, which is consistent with the lesser effect on cell survival. When U251tk and U251gfp cells were cultured with 1-ß-D-arabinofuranosylthymine (araT), the 5'-triphosphate of araT accumulated in the bystander cells, demonstrating that the transfer of phosphorylated compounds between these cell types is not restricted to GCV nucleotides. However, the proportion of araT-5'-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population.

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