Increase of intracellular dGTP and dCK phosphorylation at Ser-74 correlates with forodesine-induced cell death in CLL cells. (A) Cell viability (analyzed by annexin V binding) after forodesine 2 μM and dGuo 10 μM treatment for 48 hours was plotted against the intracellular dGTP fold increase observed after 18 hours of treatment as described in “Methods” in 26 CLL cases. (B) Cells from 4 patients with CLL were treated with forodesine and dGuo, fludarabine, or the combination of fludarabine and forodesine for 18 hours. Intracellular dGTP levels were quantified as described in “Methods.” (C) Primary CLL cells were incubated with forodesine 2 μM and dGuo (10 and 20 μM), fludarabine (7.5 μM), or bendamustine (25 μM) in the presence or absence of deoxycytidine (5 and 10 μM), and cell viability was analyzed at 24 hours. One representative CLL case of 5 cases analyzed is shown. (D) Cells from 4 representative patients with CLL were treated with forodesine 2 μM and dGuo (10 and 20 μM) or bendamustine (25 μM) for 10 (CLL no. 31) and 16 (CLL nos. 6, 36, and 42) hours. Protein extracts were subjected to Western blot with the antiphospho-Ser-74 dCK antibody and with the goat anti-dCK antibody as a control for dCK expression. α-Tubulin was used as loading control, and the ratio phospho dCK/dCK is shown. Four representative cases are shown. (E) Densitometric protein quantification of phospho dCK/dCK ratio was performed in 6 CLL cases treated with forodesine (2 μM) and dGuo (10 and 20 μM) and plotted against cell viability (analyzed by annexin V binding) after 48 hours of forodesine and dGuo treatment. Correlation coefficient and P value are shown.