RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines

Narendrula, Rashmi; Mispel-Beyer, Kyle; Guo, Baoqing; Parissenti, Amadeo M.; Pritzker, Laura B.; Pritzker, Ken; Masilamani, Twinkle; Wang, Xiaohui; Lannér, Carita

To assess whether docetaxel induces apoptosis and whether this is concurrent with the induction of RNA disruption, A2780 cells were treated with 0.2 μM docetaxel for varying times up to 72 h. Cells were stained with annexin V-fluorescein isothiocyanate (annexin V-FITC) and propidium iodide (PI) and analyzed by flow cytometry (Fig. 6a) [53] and Wakeman et al. [28] . Location of the probes in the 28S rRNA sequence is shown above the diagram using arrows and the location of the cleavage sites and resulting bands are shown below the diagram increase in the number of cells stained with annexin V-FITC only, which persisted at 48 and 72 h, indicating that the cells were in early apoptosis. No increase in PI staining was observed up to 72 h, suggesting that cells had retained plasma membrane integrity and had not undergone necrosis. Next, the effect of docetaxel treatment on cell cycle progression was investigated using PI staining of fixed cells following 8, 24, 48 and 72 h of docetaxel exposure (Fig. 6b) . A sub G 1 peak, often associated with apoptotic bodies, was evident in the docetaxel treated cells after 24 h, and by 72 h, almost all the PI signal was in the sub G 1 peak. This shows that extended docetaxel treatment generated cell fragments with less than a diploid amount of DNA content, representing apoptotic bodies or micronuclei. Finally, to see if DNA laddering (a late apoptosis biomarker) also occurred, A2780 and Jurkat cells were treated with or without docetaxel (A2780 cells) or etoposide (Jurkat cells) for similar lengths of time. Genomic DNA was prepared from the cells and resolved by agarose gel electrophoresis (Fig. 6c) . Interestingly, docetaxel-treated A2780 cells did not show any evidence of DNA fragmentation while the DNA from etoposidetreated Jurkat cells was clearly degraded. To confirm that A2780 cells treated with docetaxel were no longer viable despite the lack of DNA degradation, a recovery assay was performed to determine if docetaxel-treated A2780 cells were capable of resuming growth when transferred into drug-free cell culture medium. Following exposure to 0.005 or 0.2 μM docetaxel for up to 72 h, cells were replated in fresh, drug-free medium and cultured for up to 96 additional hours (Fig. 7) . Cells treated with 0.2 μM docetaxel never recovered (regardless of incubation time), while those treated with 0.005 μM docetaxel could recover after 24 and 48 h of docetaxel exposure but not after 72 h docetaxel exposure. When one relates these observations to the extent of RNA disruption induced in A2780 cells treated with 0.005 or 0.2 μM docetaxel over time (Fig. 1d ), it appears that cells can only tolerate a specific level of RNA disruption (RDI =~0.5), above which cells become nonviable.