Fig. 5

Higher levels of ROS and an increased sensitivity towards metal-containing nucleosides are associated with high TCL1 oncogene expression. (a) Left: overall cellular ROS levels (CM-H2DCFDA flow cytometry) positively correlate with the percentage of TCL1-expressing tumor cells in 20 freshly isolated CLL (Spearman’s rho = 0.66, P = 0.0016). Adjustments by DoHH2 control cells in each individual measurement to address inter-experimental variations are outlined in Materials and Methods. Right-top: basal cellular ROS levels in splenic B-cells from 7 months old TCL1-transgenic mice (Eμ-TCL1 tg) are higher than in those from age-matched animals of identical C3HxC57BL/6 (C3B6) wild-type (WT) background (representative histogram with mean fluorescence intensities (MFI) from measurements on 3 different mice). Right-bottom: Introduction of TCL1 into JCM3 CLL-like cells (stable transfectants JVM3-TCL1) leads to higher ROS levels (MFI; representative example of 3 independent measurements). (b) Top: representative Western blot of 3 experiments of 24 h of metal-containing nucleoside treatment of JVM3 cells. A stronger PARP cleavage is observed in the TCL1-expressing sub-line. The opposite is noted for the ROS-independent alkylator bendamustine. Numbers represent densitometric ratios to Dmso controls of cleaved/non-cleaved PARP normalized to ß-Actin loading. Bottom-left: the same pattern of differential induction of PARP cleavage across the substances is seen in leukemic Eμ-TCL1-tg mice (24 h, 1 representative of 3). Bottom-right: immunoblot showing PARP activation in a selected pair of 6 CLL patient samples demonstrating a stronger PARP cleavage in TCL1-high cases than observed for tumors expressing this oncogene at hardly detectable levels (Huni132 and Me-N69, both at 10 μM)