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 microM or higher, HSVtk-transduced B16F10 melanoma cell clones became susceptible to GCV with IC50 of 0.1 to 0.3 microM. 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 Bax or Cdc2. These results suggest that apoptosis occurred as a result of ganciclovir-induced cell cycle arrests rather than direct chemical effect on HSVtk-transduced B16F10 melanoma cells.
Wei
S J
SJ
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Chao
Y
Y
Hung
Y M
YM
Lin
W C
WC
Yang
D M
DM
Shih
Y L
YL
Ch'ang
L Y
LY
Whang-Peng
J
J
Yang
W K
WK
D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000187:drug effects; D017690:Cell Transplantation; D019926:Cyclin B / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D016195:G2 Phase / Q000187:drug effects*; D015774:Ganciclovir / Q000008:administration & dosage* / Q000627:therapeutic use; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D007700:Kinetics; D008546:Melanoma, Experimental / Q000737:chemistry / Q000473:pathology / Q000628:therapy*; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D010718:Phosphatidylserines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016196:S Phase / Q000187:drug effects*; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000998:Antiviral Agents; D018797:Cell Cycle Proteins; D019926:Cyclin B; D008563:Membrane Lipids; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D013937:Thymidine Kinase; D015774:Ganciclovir