Kinetics of hallmark biochemical changes in paclitaxel-induced apoptosis.

Apoptosis is associated with cascades of biochemical changes, including caspase activation, cleavage of poly-ADP-ribose polymerase (PARP), and fragmentation of genomic DNA. Knowledge of the kinetics of these changes in drug-induced apoptosis is important for designing pharmacodynamic studies. We have shown that the slow manifestation of apoptosis contributes to the delayed pharmacological effects of paclitaxel (Cancer Res. 58:2141-2148, 1998). The present study examined the timing of the biochemical changes in paclitaxel-induced apoptosis in human prostate PC3 cancer cells. After treatment with 20 nM paclitaxel, the fraction of cells that detached from the culture flask increased with time to reach 68% at the end of the 96-hour experiment. In contrast, the control samples showed <1% detachment. The attached and detached paclitaxel-treated cells showed different biochemical properties. The detached cells exhibited the full spectrum of apoptotic changes, whereas the attached cells only showed activation of caspase-3-like proteases but not PARP cleavage, DNA fragmentation, nor release of DNA fragments to the cytoplasm. Activation of caspases in the attached cells was several-fold lower and occurred at a later time (ie, 24 vs 12 hours) compared to the detached cells. In the detached cells, caspase activation was first detected at 12 hours and peaked at 36 hours, whereas PARP cleavage was first detected at 24 hours and was completed prior to 72 hours. In contrast, the extent of internucleosomal DNA fragmentation and the release of DNA-histone complex to the cytoplasm (both were first detected at 24 hours) were cumulative over time up to the last time point of 96 hours. In summary, in paclitaxel-induced apoptosis, caspase activation was followed with a 12-hour lag time by PARP cleavage, internucleosomal DNA fragmentation, and release of DNA-histone complex to the cytoplasm. There was no detectable lag time between PARP cleavage and DNA fragmentation. The observation that only the detached cells but not the attached cells showed the full spectrum of apoptotic changes suggests that detachment is either a part of the initiation/execution phases of apoptosis and/or is required for their completion.