Potentiation of 2-Chlorodeoxyadenosine Activity by Bryostatin 1 in the Resistant Chronic Lymphocytic Leukemia Cell Line (WSU-CLL): Association with Increased Ratios of dCK/5'-NT and Bax/Bcl-2

The activities of 2-chlorodeoxyadenosine (2-CdA) metabolizing enzymes, deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase (5'-NT) were measured in control and bryostatin 1 treated CLL cells using an EBV-negative WSU-CLL cell line. This cell line was established from a patient with CLL resistant to fludarabine. The results revealed a significant increase in dCK activity in bryostatin 1 treated cells at 48 and 72 h compared with the control. 5'-NT activity decreased significantly at 48 h. The ratio of dCK to 5'-NT activity was significantly increased in bryostatin 1 treated WSU-CLL cells after 48 h. WSU-CLL cells treated with bryostatin 1 exhibited an increase in the percentage of apoptotic and dead cells from control levels of 16% to 40%. This percentage was further increased to 67% following the addition of 11.2 microM 2-CdA to WSU-CLL cells pretreated with bryostatin 1. Results from Western blot analysis indicate that WSU-CLL cells express high levels of Bcl-2, Bcl-xL and c-myc, and a low level of Bax. p53 in untreated WSU-CLL cells is undetectable. WSU-CLL cells treated with bryostatin 1 showed a significant increase in the ratio of Bax to Bcl-2. To demonstrate that the bryostatin 1 mediated enhancement of 2-CdA efficacy was not restricted to in vitro cell culture, we have studied the tumor growth delay of WSU-CLL xenografts treated with placebo, bryostatin 1, 2-CdA, and bryostatin 1 followed by 2-CdA. SCID mice given bryostatin 1 at 75 microg x kg(-1) x d(-1) for 5 days followed by 30 mg x kg(-1) x d(-1) 2-CdA for 5 days in two cycles, had significantly improved tumor growth delay (P = 0.05). We conclude that bryostatin 1 is not only capable of inducing apoptosis by itself, but also sensitizes de novo resistant WSU-CLL cells to the chemo-therapeutic effects of 2-CdA. The bryostatin 1-induced increased ratio of dCK/5'-NT activity and an increased ratio of Bax/Bcl-2 are at least two mechanisms through which this natural compound is able to potentiate the anti-tumor activity of 2-CdA in otherwise resistant CLL cells.