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Molecular mechanism of apoptosis induced by ricin in HeLa cells
1Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China.
Aim:
To study the morphological changes and molecular mechanism of HeLa cell apoptosis induced by ricin.
Methods:
HeLa cells were coincubated with ricin 0.05 mumol.L-1 for 1, 2, 3, 6, 12, 18, and 24 h, then scanning electron microscopy (SEM), transmission electron microscopy (TEM), Western blot cell cycle, cell cytotoxicity, and cell viability were assayed.
Results:
The typical apoptosis was induced by ricin 0.05 mumol.L-1 and necrotic cells increased after being cultured with ricin 0.05 mumol.L-1 for more than 12 h. The apoptotic cells mainly showed cytoplasmic membrane blebbing, chromatin condensation and fragmentation, and crescentic nuclear and membrane bound apoptotic bodies formation. No detectable levels of p53, Bax, Bcl-2 and the subunit p20 of interleukin-1 beta-converting enzyme (ICE) were found by Western blot, but the active subunit p17 of 32-kDa putative cysteine protease (CPP32) was detected at 3, 6, and 9 h after ricin treatment. The activity of CPP32 in HeLa cells increased 4 to 5 folds after being treated with ricin 0.05 mumol.L-1 and reached the peak at 6 h of treatment. There was no significant difference of ICE activity between the ricin treated cells and control cells. The percentage of G2/M cells increased from 13.9% +/- 0.5% to 33.2% +/- 0.5% after 24 h of ricin 0.05 mumol.L-1 treatment.
Conclusion:
CPP32 but not ICE was involved in the ricin-induced apoptosis in HeLa cells. Ricin 0.05 mumol.L-1 had no effect on the G0/G1 phase of cell cycle, but induced G2/M arrest.