Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.
Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.
Zhang
Zhihong
Z
Huazhong University of Science and Technology, Wuhan National Laboratory for Optoelectronics, Britton Chance Center for Biomedical Photonics, Wuhan 430074, China.
Lin
Juqiang
J
Chu
Jun
J
Ma
Yan
Y
Zeng
Shaoqun
S
Luo
Qingming
Q
D024201:Bystander Effect; D003528:Carcinoma, Adenoid Cystic / Q000378:metabolism* / Q000821:virology*; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015774:Ganciclovir / Q000378:metabolism*; D006801:Humans; D013937:Thymidine Kinase / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; C099241:thymidine kinase, Canid herpesvirus 1; D013937:Thymidine Kinase; D053148:Caspase 3; D015774:Ganciclovir