Objective: To investigate the mechanism of variability of herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir (GCV)-mediated suicide effect obtained in three different cell lines and the ways in which this system-mediated cell killing occurs.
Methods: Recombinant retroviral vector expressing HSV-TK was transduced into three cell lines known with different growth rate (PC-2, PC-7 and LLC-PK1). The doubling time of the transduced and parental cells was calculated. MTT method was used to detect the concentration of GCV at which cell growth was inhibited by 50% (IC(50) value). Cell cycle was analyzed by flow cytometry. Necrosis or apoptosis of cultured cells and/or xenografts was observed under light and electron microscope, and by in situ apoptosis detection.
Results: The doubling time of the three parental cell lines was (31.2 +/- 0.1) h, (48.3 +/- 0.1) h, and (53.9 +/- 0.1) h, separately. The IC(50) values of their HSV-TK-transduced cell lines to GCV were (0.73 +/- 0.12) micromol/L, (0.93 +/- 0.16) micromol/L and (1.22 +/- 0.06) micromol/L, respectively and the IC(50) value was correlated with the cell doubling time. Flow cytometry revealed S arrest. The majority of cells under treatment of GCV displayed swelling and collapse, but very few cells showed apoptosis. Large areas of necrosis were observed in the xenografts.
Conclusions: The cells with high growth rate are more susceptible to HSV-TK/GCV-mediated killing effect. Necrosis is the main way in which cells of the three HSV-TK-transduced cells lines die.