TY - JOUR
T1 - Use of live imaging analysis for evaluation of cytotoxic chemicals that induce apoptotic cell death
AU - Koike-Kuroda, Yoshiko
AU - Kakeyama, Masaki
AU - Fujimaki, Hidekazu
AU - Tsukahara, Shinji
N1 - Funding Information:
This work was supported in part by the Environment Technology Development Fund from the Ministry of the Environment Japan ( KS-02 ) to S. Tsukahara and by a Health and Labour Sciences Research Grant to M. Kakeyama. We thank Dr. M. Miura (University of Tokyo, Tokyo, Japan) for providing the pcDNA-SCAT3 vector.
PY - 2010/10
Y1 - 2010/10
N2 - 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 300min after exposure to sodium arsenite (NaAsO2: 0, 1, 5, or 10μM) 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 NaAsO2. The mean ECFP/Venus emission ratio between 200 and 300min after exposure to NaAsO2 at a dose of 5 or 10μM, but not at 1μM, was significantly higher than that in the control group. We showed by other methods that NaAsO2 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 NaAsO2 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.
AB - 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 300min after exposure to sodium arsenite (NaAsO2: 0, 1, 5, or 10μM) 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 NaAsO2. The mean ECFP/Venus emission ratio between 200 and 300min after exposure to NaAsO2 at a dose of 5 or 10μM, but not at 1μM, was significantly higher than that in the control group. We showed by other methods that NaAsO2 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 NaAsO2 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.
KW - Apoptosis
KW - Caspase-3
KW - Live imaging
KW - Sodium arsenite
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U2 - 10.1016/j.tiv.2010.07.022
DO - 10.1016/j.tiv.2010.07.022
M3 - Article
C2 - 20682336
AN - SCOPUS:77957018427
VL - 24
SP - 2012
EP - 2020
JO - Toxicology in Vitro
JF - Toxicology in Vitro
SN - 0887-2333
IS - 7
ER -