NUCLEOSIDE TRANSPORTERS, BCL-2 AND APOPTOSIS IN CLL CELLS EXPOSED TO NUCLEOSIDE ANALOGS IN-VITRO

The purine nucleoside analogues fludarabine (F1) and chlorodeoxyadenos ine (2-CdA) are considered to be cell cycle specific agents which requ ire DNA synthesis for cytotoxicity. However, their efficacy in the tre atment of CLL, an indolent lymphoid malignancy suggests additional mec hanisms of action. Like cytosine arabinoside (AraC), F1 and 2-CdA gain access to the cell via a specific nucleoside transporter (NST) protei n. To investigate the mode of action of these drugs in CLL, we used a fluorescent ligand for the NST (5-(SAENTA- x 8)-fluorescein) and 3-col our flow cytometry to determine NST expression on CD5(+)/CD19(+) B-cel ls from the peripheral blood (PB) of patients with CLL. NST levels on these cells was found to be not significantly different from normal co ntrol lymphocytes (mean = 485 +/- 425) vs. (mean = 553 +/- 178). Expos ure to varying concentrations (0, 3 mu M and 30 mu M) of F1 and 2-CdA, however, resulted in an upregulation of NST (mean = 1552 +/- 775 with 30 mu M FL; mean = 3392 +/- 2197 with 30 mu M 2-CdA) after 48 h. ''La rge'' lymphoid cells (not present in normal PB) were found to express significantly more NST (mean = 2540 +/- 2861) and have a higher prolif erative capacity than ''small'' cells (mean = 357 +/- 517 NST/cell). I ncubation of CLL cells with F1 (n = 6) and 2-CdA (n = 8) in vitro over 48 h also resulted in an increase in the proportion of cells in S-pha se (0 mu M = 0.2 +/- 0.1; 30 mu M FL = 2.4 +/- 2.0; 30 mu M 2-CdA = 3. 3 +/- 1.3) and a significant increase in morphologically identifiable apoptosis. Apoptosis was confirmed by flow cytometric DNA analysis (0 mu M = 13 +/- 8%; 30 mu M FL = 40 +/- 20%; 30 mu M 2-CdA = 48 +/- 11%) . In situ hybridization using a biotinylated cDNA bcl-2 probe demonstr ated that bcl-2 mRNA expression was markedly decreased in treated cell s after 24 h. These studies have demonstrated that: (1) NST expression on CLL lymphocytes is low; (2) in vitro exposure to the analogues inc reases both the level of NST expression and the % cells in S-phase; (3 ) exposure to the analogues downregulates bcl-2 expression and increas es apoptosis.