Induction of apoptosis and differentiation by fludarabine in human leukemia cells (U937): interactions with the macrocyclic lactone bryostatin 1

We have examined interactions between the purine nucleoside analog fludarab ine (9-beta-arabinofuranosyl-2-fluoroadenine) and the macrocyclic lactone b ryostatin 1 in the human monocytic leukemic cell line U937. Fludarabine exe rted dose-dependent effects on U937 cell viability and growth which were as sociated with both induction of apoptosis, as well as cellular maturation. Incubation of cells with bryostatin 1 (10 nM; 24 h) after, but not before a 6-h exposure to 10 mu M fludarabine resulted in a modest but significant i ncrease in apoptosis, and was associated with greater than a 1 log reductio n in clonogenicity. Subsequent exposure to bryostatin 1 also increased the percentage of fludarabine-treated cells displaying differentiation-related features (eg plastic adherence, CD11b positivity) compared to cells exposed to fludarabine alone. Bryostatin 1 did not increase the retention of the a ctive fludarabine metabolite, Fara-ATP, nor did it increase H-3-F-ara-A inc orporation into DNA. Despite its capacity to trigger cellular maturation, f ludarabine exposure (either with or without bryostatin 1) failed to induce the cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/CIP1) and p27(KIP1) . Nevertheless, dysregulation of p21 (resulting from stable transfection of cells with a p21(WAF1/CIP1) antisense construct) reduced fludarabine-media ted differentiation, while inducing a corresponding increase in apoptosis. Enforced expression of Bcl-2 partially protected cells from fludarabine-rel ated apoptosis, an effect that was overcome, in part, by subsequent exposur e of cells to bryostatin 1. Interestingly, Bcl-2-overexpressing cells were as or In some cases, more susceptible to differentiation induction by fluda rabine (+/- bryostatin 1) than their empty vector-containing counterparts. Collectively, these results indicate that the antiproliferative effects of fludarabine toward U937 leukemic cells involve both induction of apoptosis and cellular maturation, and that each of these processes may be enhanced b y bryostatin 1.