S- and G2-phase Cell Cycle Arrests and Apoptosis Induced by Ganciclovir in Murine Melanoma Cells Transduced with Herpes Simplex Virus Thymidine Kinase

Mechanism of cell killing by transfer of Herpes simplex virus type-1 thymidine kinase (HSVtk) and subsequent ganciclovir (GCV) treatment was examined in B16F10 murine melanoma model. While parental B16F10 melanoma cells were resistant to GCV at 100 μM or higher,HSVtk-transduced B16F10 melanoma cell clones became susceptible to GCV with IC50of 0.1 to 0.3 μM. By means of various parameters including characteristic morphological changes,in situDNA end-labeling, DNA ladder pattern, flow cytometric detection of sub-G1 DNA content, and annexin V binding of inverted cell surface phosphatidylserine, apoptosis was shown to be associated with the cell killing of ganciclovir onHSVtk-transduced melanoma B16F10 cells. Kinetic analysis showed that the signs of apoptosis were observed not until 60 h of continued GCV treatment and preceded first by a rise in p53 protein level in 12 h and then by S-phase/G2-phase cell cycle arrest associated with corresponding increases in the level of cyclin B1 protein but no apparent change in protein level of Bax or Cdc2. These results suggest that apoptosis occurred as a result of ganciclovir-induced cell cycle arrests rather than direct chemical effect onHSVtk-transduced B16F10 melanoma cells.