THE DEVELOPMENT OF THE EFFICIENT CELLULAR AND BIOCHEMICAL ASSAY SYSTEM OF APOPTOSIS REGULATING CHEMICALS
Yasushi Yamaguchi, Jun Maeda, Toshiaki Nakano, J Nishikawa, Kenichi Mikitani · 2007
Programmed cell death is genetic and biochemical process caused by specific chemicals and stress stimulation like UV-irrad iation. Using Hela cell line, we have developed efficient assay system for determining apoptotic (and anti-apoptotic) properties of a number of chemical substances. Cell death inducing activities are assessed by measuring cell viability (WST-8) and cytotoxicity (LDH) using 96-well plates. Further characterization of cell death inducing chemicals was performed by one dish-based Annexin-V and DNA ladder formation assay. Additionally, activation of the cell death executing caspase-3 was detected by western blot analysis using the protein extracted from the same cell population as Annexin-V and DNA ladder formation assay. As for the initial test chemicals, we used staurosporine, TRAIL+IFNγ, tunicamycin, and brefeldin-A. External apoptotic stimuli (staurosporine) and death receptor-mediated inducer (TRAIL+IFNγ) showed rapid apoptosis induction 4-8 hr after treatment in Annexin-V, DNA ladder formation, and caspase-3 assay. In contrast, ER stress inducers (tunicamycin, brefeldin-A) showed rather slow and modest apoptosis induction activities in all the three assays. Cell nuclear and mitochondrial morphological investigation further su pports the distinct properties of these acute and modest apoptosis inducers. Taken together, the apoptosis assay system established here would be strong tools to evaluate a number of chemicals including dioxins.