Use of live imaging analysis for evaluation of cytotoxic chemicals that induce apoptotic cell death.
We carried out live imaging of PC12 cells expressing SCAT3, a caspase-3 cleavage peptide sequence linking two fluorescent proteins, ECFP and Venus, which function respectively as the donor and acceptor for FRET. Live imaging of SCAT3-expressing cells was performed from 60 to 300 min after exposure to sodium arsenite (NaAsO(2): 0, 1, 5, or 10 μM) was initiated. We then measured the emission ratio of ECFP to Venus to monitor the activity of caspase-3 and found that the ratio was temporally and dose-dependently increased by NaAsO(2). The mean ECFP/Venus emission ratio between 200 and 300 min after exposure to NaAsO(2) at a dose of 5 or 10 μM, but not at 1 μM, was significantly higher than that in the control group. We showed by other methods that NaAsO(2) significantly increased the amount and activity of mature caspase-3 and the amount of nucleosomes generated from DNA fragmentation, and decreased cell viability. However, methods other than live imaging required a longer time and higher doses of NaAsO(2) than did live imaging to detect significant effects. This result suggests that live imaging using SCAT3 is a useful method for the screening of chemical toxicities and for improving the efficiency of toxicity evaluation.
Koike-Kuroda
Yoshiko
Y
Department of Regulation Biology, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo 255, Sakura-ku, Saitama City, Saitama 338-8570, Japan.
Kakeyama
Masaki
M
Fujimaki
Hidekazu
H
Tsukahara
Shinji
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018053:Arsenites / Q000008:administration & dosage / Q000633:toxicity*; D053148:Caspase 3 / Q000187:drug effects* / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009707:Nucleosomes / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D017670:Sodium Compounds / Q000008:administration & dosage / Q000633:toxicity*; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods
D018053:Arsenites; D009707:Nucleosomes; D017670:Sodium Compounds; C017947:sodium arsenite; D053148:Caspase 3