Fig. 4

The induction of CLL cell apoptosis by metal-containing nucleosides is ROS dependent. Flow cytometry with representative dot plots of 3 CLL samples. (a) Increasing ROS levels (detected by CM-H2DCFDA probe) following exposure to metal-containing nucleosides (MCNA; example here Huni132) were noticeable at 4-6 h and were associated with the induction of early apoptosis (Annexin-V). Apoptosis induced by bendamustine was not paralleled by increases of cellular ROS. * For bendamustine data are shown at 24 h instead of 12 h due to different cell-death kinetics, however, at no time-point of bendamustine exposure a ROS increase was noted. (b) The increasing ROS levels (CM-H2DCFDA, solid grey histograms represent Dmso controls) at 6 h that precede the induction of cell death by the MCNA (x: AnnexinV, y: 7AAD; max. at 24 h), but not by bendamustine, are preferentially observed in CLL tumor cells as compared to healthy donor peripheral blood mononuclear cells (PBMC). Pre-incubation (1 h) with several ROS scavengers, here Tiron (10 mM), partially abrogated the MCNA mediated ROS increase and reduced the extent of overall apoptosis (AnnexinV+ fraction). MF: mean fold changes in mean fluorescence intensity. Lower-left quadrant %-ages indicate AnnexinV/7AAD double-negative viable cells