Superior cytotoxicity with ganciclovir compared with acyclovir and 1-beta-D-arabinofuranosylthymine in herpes simplex virus-thymidine kinase-expressing cells: a novel paradigm for cell killing.

Enzyme-prodrug therapy using ganciclovir and herpes simplex virus-thymidine kinase (HSV-TK) has demonstrated excellent antitumor activity in many different types of malignant cells. Previously, we noted that ganciclovir was substantially more cytotoxic than other HSV-TK substrates. Therefore, we embarked on a study to determine the basis for the superior cytotoxicity of ganciclovir. In U251tk human glioblastoma cells that stably express HSV-TK, ganciclovir elicited a >4 log cell kill instead of the < or =1.5 log cell kill mediated by two other HSV-TK substrates, 1-beta-D-arabinofuranosylthymine (araT) and acyclovir. Study of the metabolism of these drugs demonstrated that acyclovir was poorly phosphorylated to its active triphosphate with DNA incorporation below the limit of detection, which may explain the < 1 log cell kill in these cells. Lower levels of ganciclovir triphosphate accumulated compared with araT triphosphate (araTTP) under conditions that induced < or =1 log cell kill (67 versus 1235 pmol/10(7) cells, respectively), and the half-life for the triphosphate of ganciclovir was shorter than that of araT (terminal half-lives of 15 and 41 h, respectively). Incorporation of ganciclovir monophosphate into DNA was less than that of araT monophosphate, and both analogues were retained in DNA for > or =48 h. Thus, the superior cytotoxicity of ganciclovir was not due to enhanced metabolism to active forms. Highly cytotoxic concentrations of ganciclovir produced only weak inhibition of DNA synthesis. This allowed cells to proceed through S and G2-M phases during and after drug exposure, resulting in a doubling of cell number by 48 h after drug washout. As they attempted to progress through the cell cycle a second time, ganciclovir-treated cells accumulated in early S-phase and remained there until cell death, suggesting that ganciclovir incorporation in the DNA template was important for cytotoxicity. In contrast, strong inhibition of DNA synthesis by araTTP prevented cells from traversing the cell cycle for at least 12 h after drug washout, when the active metabolite was largely degraded araT-treated cells were unable to divide for at least 72 h after drug exposure, at which point the surviving cells displayed a normal cell cycle distribution pattern. Based on the results presented here, we propose a novel paradigm in which the ability of ganciclovir to incorporate into DNA without inhibiting progression through S-phase, combined with high cytotoxicity for incorporated ganciclovir monophosphate, produces multilog cytotoxicity.

Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells.

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 (U251beta gal 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 U251beta gal 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 microm GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 microM 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 U251beta gal cells, the survival of bystander cells was decreased by >99.5% with 3 microM GCV, whereas 30 microM GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251beta gal 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 U251beta gal 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-beta-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.

Improved method to prepare RNA-free DNA from mammalian cells.

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.