Biochemical pharmacology and resistance to 2-chloro-2 '-arabinonuoro-2 '-deoxyadenosine, a novel analogue of cladribine in human leukemic cells

The objective of the present study was to investigate the biochemical pharm acology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)-a fluorinat ed analogue of cladribine: [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] wi th improved acid and metabolic stability-in human leukemic cell lines and i n mononuclear cells isolated from patients with chronic lymphocytic leukemi a (CLL) and acute myelocytic leukemia (AML). We have also made and characte rized two cell lines that are not sensitive to the growth inhibitory and cy totoxic effects of CAFdA. Incubation of cells isolated from the blood of CL L and AML patients with various concentrations of CdA or of CAFdA accumulat ed CdA and CAFdA nucleotides in a dose-dependent manner. A significantly hi gher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA. in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphospha tes in both CLL (n = 14; P = 0.001) and AMT, (n = 4; P = 0.04) cells, Reten tion of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5 '-triphosphate (CdATP) in cells from leukemic patients, The relative effica cy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (d CK), the key enzyme in the activation of nucleoside analogues, was very hig h and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The K-m for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia c ell line HL60, Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell li nes was directly correlated to the decreased activity of the nucleoside pho sphorylating enzyme, dCK, Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observa tions demonstrated that dCK phosphorylates CAFdA more efficiently than CdA, Furthermore, CAFdATP is apparently more stable than CdATP and the mechanis ms of resistance to CAFdA are similar to those leading to CdA resistance. T hese results encourage studies on the clinical effect of CAFdA in lymphopro liferative diseases.