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 mu M or higher, HSVtk-transduced B16F10 melanoma cell clones became susceptible to GCV with IC50 of 0.1 to 0.3 mu M. By means of various parameters including characteristic morphological changes, in situ DNA 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 on HSVtk-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 Bar or Cdc2. These results suggest that apoptosis occurred as a result of gamciclovir-induced cell cycle arrests rather than direct chemical effect on HSVtk-transduced B16F10 melanoma cells. (C) 1998 Academic Press.
