Identification of CNDAC and its metabolites in cells and DNA. (A) ML-1 cells were incubated with 0.3μM [³H]CNDAC for 18 hours and washed into fresh RPMI medium, and DNA was extracted and enzymatically hydrolyzed. Components of DNA hydrolysates were separated by reversed-phase HPLC, and radioactivity was quantitated. (Top panel) An authentic standard of CNDAC, CNddC, and CNDC; the ordinate unit represents ultraviolet absorption at 270 nm (AU). (Bottom panel) An HPLC analysis of DNA hydrolysis products degraded with DNase I, phosphodiesterase, and alkaline phosphatase; the ordinate represents radioactivity (CPM). (B) Number of CNddC molecules per cell, calculated as described in “Quantitation of CNDAC and its metabolites in cells and DNA.” (C) Cytotoxicity of CNDAC in ML-1 cells (left) and purified primary samples from AML patients (right) determined by clonogenic assay. Exponentially growing ML-1 cells were exposed to a range of concentrations of CNDAC for 18 hours, washed into fresh medium, and allowed to form colonies. The survival curve is mean ± SD values from triplicates. AML primary samples (patients 11-17) were incubated with CNDAC at indicated concentrations for 5 to 6 days without washing out of drug. The curves are mean values from duplicates or triplicates. (D) HPLC analysis of intracellular accumulation of CNDAC-TP in AML primary samples exposed to CNDAC at indicated concentrations for 2 hours (left, patients 1, 2, 4, 6, 7, 8, 9, and 10) or 5μM CNDAC for indicated times (middle, patients 3-10), and elimination of CNDAC-TP from AML primary cells over 10 hours after a 4-hour incubation with 10μM CNDAC (right, patients 6, 7, and 10).