HSV-tk/GCV system, which is the virus-directed enzyme/prodrug therapy of herpes simplex virus (HSV) thymidine
kinase (tk) gene / the anti-viral reagent ganciclovir (GCV), is one of the promising approaches in the rapidly growing
area of gene therapy. As gene therapy of cancer such as suicide gene therapy has entered the clinic, another therapy
effect which is called 'bystander effect' was reported. Bystander effect can lead to killing of non-transduced tumor cells
in the immediate vicinity of GCV-treated HSV-TK-positive cells. Now the magnitude of 'bystander effect' is an
essential factor for this anti-tumor approach in vivo. However, the mechanism which HSV-tk/ACV brings "bystander
effect" is poorly understood. In this study, we monitor the activation of caspase-3 in HSV-tk/GCV system by a FRET
probe CD3, a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent
protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property.
Through application of CD3 we have visualized the activation of caspase-3 in tk gene positive human adenoid cystic
carcinoma (ACC-M) cells but not in bystander effect of HSV-tk/GCV system induced by GCV. This finding provides
needed information for understanding the mechanisms by which suicide gene approaches actually kill cancer cells, and
may prove to be helpful for the clinical treatment of cancers.