Molecular mechanism of apoptosis induced by ricin in HeLa cells

AIM: To study the morphological changes and molecular mechanism of HeLa cel l apoptosis induced by ricin. METHODS: HeLa cells were coincubated with ric in 0.05 mu mol.L-1 for 1, 2, 3, 6, 12, 18, and 24 h, then scanning electron microscopy (SEM), transmission electron microscopy (TEM), Western blot, ce ll cycle, cell cytotoxicity, and cell viability were assayed. RESULTS: The typical apoptosis was induced by ricin 0.05 mu mol.L-1 and necrotic cells i ncreased after being cultured with ricin 0.05 mu mol.L-1 for more than 12 h . The apoptotic cells mainly showed cytoplasmic membrane blebbing, chromati n condensation and fragmentation, and crescentic nuclear and membrane bound apoptotic bodies formation. No detectable levels of p53, Bar, Bcl-2 and th e subunit p20 of interleukin-1 beta-converting enzyme (ICE) were found by W estern blot, but the active subunit pi? of 32-kDa putative cysteine proteas e (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 ric in 0.05 mu mol.L-1 and reached the peak at 6 h of treatment. There was no s ignificant difference of ICE activity between the ricin treated cells and c ontrol cells. The percentage of G(2)/M cells increased from 13.9% +/- 0.5% to 33.2% +/- 0.5% after 24 h of ricin 0.05 mu mol.L-1 treatment. CONCLUSION : CPP32 but not ICE was involved in the ricin-induced apoptosis in HeLa cel ls. Ricin 0.05 mu mol.L-1 had no effect on the G(0)/Gr(1) phase of cell cyc le, but induced C-2/M arrest.