Figure 1.

TK/GCV-induced apoptosis involves activation of caspases and is attenuated by caspase inhibitors. (A) Activation of caspases by TK/GCV. HSV-TK cells (1 × 10⁷) containing (TK+) SH-EP pHyTK11 cells and SH-EP pLSXN cells that lack TK (TK−) were treated with 10 μM of GCV for the times indicated. Protein was extracted and separated by SDS/PAGE. Caspase-8, caspase-3, and PARP were detected by immunoblot. Caspase-8 was seen as a double-band corresponding to the isoforms caspase-8/a and 8/b (14). p43 and p41 are cleavage intermediates of caspase-8 leading to the active subunit p18. The cleavage products of CPP32 and PARP are p17 and p85, respectively. Cleavage products were seen only in TK+ but not in TK− cells. (B) TK/GCV-mediated cell death is attenuated by the caspase inhibitor zVAD-fmk. Cells were treated with 10 μM of GCV alone or in the presence of 80 μM of zVAD-fmk. Apoptosis, as determined by flow cytometry (Left) and counting of trypan blue-positive cells (Right), was measured after 72 h. Results are means of triplicates, and similar results were obtained in three separate experiments.