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

Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine kinase

“…7 When exposed to 1 to 50 m (ie 20-to 100-fold the respective IC 50 ) of GCV in the medium, NG4TL4/TK-C3 sarcoma, 1MEA 7R.1/TK-C1 hepatoma and H6/TK-C10 hepatoma showed similar response of p53 elevation, cell cycle arrest and finally signs of apoptosis, although with slight variations in the kinetics and extent of these responses ( Figure 1). Signs of apoptosis, which included electrophoretic laddering of nucleosomal DNA, an increase in in situ DNA-end labeling of nuclei, cells with sub-G1 DNA contents in cytofluorometric analysis, and specific binding of cell surface annexin V, appeared to be more marked with B16F10/TK-C19 cells and 1MEA 7R.1/TK-C1 cells than NG4TL4/TK-C3 sarcoma and H6/TK-C10 hepatoma cells (data not shown).…”

“…Previously no alteration of Bax protein level was detected in B16F10/TK-C19 melanoma cells during GCV-induced apoptosis, while Bcl-2 content was below the detectable level by the immunoblotting method used, making it impossible to determine whether Bcl-2 played a role in the HSVtk/GCV-induced apoptosis. 7 Figure 2, the cellular levels of FasL and Fas proteins in HSVtk-transduced cells were increased in 30 h to 60 h after GCV treatment. The increases were more obvious in B16F10/TK-C19 melanoma and H6/TK-C10 hepatoma, which had relatively low basal levels of FasL and Fas, than in 1MEA 7R.1/TK-C1 hepatoma and NG4TL4/TK-C3 sarcoma with relatively high basal levels of Fas and FasL.…”

“…An additional important feature of this strategy is the 'bystander' effect, namely the spread of the cytocidal effect of GCV to neighboring cells that do not receive the suicide gene. 6 Studies with colorectal carcinoma, glioma and melanoma cells 7 have concluded that the GCV killing of HSVtk-transduced tumor cells, as well as the 'bystander' effect is manifested by apoptosis, such as fragmentation of chromatin DNA revealed by the electrophoretic 'laddering pattern', 8 although another study using the same parameter on rat hepatoma cells reached the opposite conclusion. 9 In our previous study, kinetic analysis with additional indicators showed that apoptosis could indeed be induced by the HSVtk/GCV strategy but was slow in occurrence and preceded sequentially by p53 elevation and cell cycle S-phase retardation as well as G2 arrest, which might vary in different tumors.…”

“…9 In our previous study, kinetic analysis with additional indicators showed that apoptosis could indeed be induced by the HSVtk/GCV strategy but was slow in occurrence and preceded sequentially by p53 elevation and cell cycle S-phase retardation as well as G2 arrest, which might vary in different tumors. 7 Transduction with the bcl-2 gene was reported to confer resistance to the HSVtk/GCV induction of apoptosis and the bystander effect. 10 However, we did not detect alterations of Bcl-2 or Bax protein levels during induction of apoptosis in HSVtk-transduced murine B16F10 melanoma cells treated with GCV.…”

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

“…The GCV-TP metabolite has been implicated in the initiation of the apoptosis pathway, though the exact mechanism whereby the cell becomes apoptotic is still unknown. Briefly, GCV-TP is incorporated in the elongating DNA chain resulting in S-phase cell-cycle arrest, and/or GCV-TP competitively inhibits the cellular DNA polymerases, resulting in inhibition of DNA synthesis, and ultimately apoptosis [9][10][11][12][13][14][15].…”