Treatment of a de novo fludarabine resistant-CLL xenograft model with bryostatin 1 followed by fludarabine

WSU-CLL is a de novo fludarabine resistant cell line established from a pat ient with advanced chronic lymphocytic leukemia (CLL) refractory to chemoth erapy including fludarabine (Flud). Our previous studies indicate that bryo statin 1 (Bryo 1) induces differentiation of WSU-CLL and increases the rati o of dCK/5'-NT activity and Bax/Bcl-2. This study tests the hypothesis that Bryo 1-differentiated cells are more susceptible to Flud than the parent W SU-CLL cells. Flud, given sequentially after Bryo 1, in vitro and in vivo a nimal studies resulted in significantly higher rates of growth inhibition a nd improved animal survival. Flud at 100 to 600 nM exhibited a dose-depende nt growth inhibitory effect on the WSU-CLL cell line. The sequential exposu re to Bryo 1 (10 nM for 72 h) followed by Flud (100 nM) resulted in signifi cantly higher rates of growth inhibition than either the reverse addition o f these two agents or each agent alone, but was not significantly different than the concurrent addition of Bryo 1 + Flud. Using 7-amino-actinomycin D staining and flow cytometry, apoptosis was seen in 40.8% of cells treated with Bryo 1 (10 nM, 72 h) followed by Flud, compared with Flud (100 nM, 72 h) followed by Bryo 1 (18.1%). To demonstrate that Bryo 1 enhancement of Fl ud efficacy was not restricted to in vitro culture, we used the WSU-CLL xen ograft model in mice with severe combined immune deficiency (SCID). Bryo 1 + Flud at the maximum tolerated doses (75 mu g/kg i.p. and 200 mg/kg i.v., respectively) were administered to mice in different combinations. The surv ival in days, the tumor growth inhibition ratio (T/C), the tumor growth del ay (T-C) in days, log(10), kill, as well as mean turner weight (mtw) of mic e treated with Bryo 1 followed by Flud, were significantly better than cont rol and other groups. T/C%, T-C, log(10), kill and mtw were as follows: Bry o 1 (36.8%, 10 days, 0.8, 375 mg); Flud (100%, 0.0 day, 0.0, 1130 mg); Bryo 1 + Flud (14.3%, 12 days, 0.95, 288 mg); Bryo 1 followed by Flud (4.6%, 17 days, 1.35, 35 mg); Flud followed by Bryo (40.3%, 10 days, 0.80, 175 mg). We conclude that: i) Bryo 1 sensitizes WSU-CLL cells to Flud and enhances a poptosis;) the sequential treatment with Bryo 1 followed by Flud resulted i n higher anti-tumor activity compared with either agent alone, in combinati on, or the reverse addition of these agents and iii) these results are comp arable to those of Bryo 1 followed by 2-CdA suggesting common pathway(s) of interaction between Bryo 1 and purine analogues.