Modulation of cIAP-1 by novel antitubulin agents when combined with bryostatin 1 results in increased apoptosis in the human early pre-B acute lymphoblastic leukemia cell line Reh

Previous studies have shown that bryostatin I induces a decrease in the exp ression of the antiapoptotic protooncogene Bcl-2 in the human acute lymphob lastic leukemia (ALL) cell line Reh. This down-regulation has been shown to reduce drug resistance of the Reh cells to anti-tubulin polymerization age nts. In the present study we investigated the effect of bryostatin I alone and in combination with novel anti-tubulin agents (dolastatin 10 and aurist atin PE) and the chemotherapeutic vincristine on the inhibitor of apoptosis protein cIAP-1. Cells were cultured with bryostatin 1 (I nM), dolastatin 1 0 (0.1 ng/ml), auristatin PE (0.1 ng/ml), or vincristine (0.5 ng/ml) alone or the combination of these anti-tubulins with bryostatin 1. Western blots were conducted to assess the effects of the above agents on cIAP-1 protein level. Flow-cytometric analysis [7-amino-actinomycin D (7AAD)] was conducte d to assess apoptosis as well as staining for morphology using tetrachrome stain. Our results show that cIAP-1 is induced in a time-dependent fashion after bryostatin I exposure up to 72 h. However, upon treatment of cells wi th a combination of bryostatin 1 and dolastatin 10 or auristatin PE, the in duction of cIAP-1 was abolished, leading to a significant increase in apopt osis. The initial 24- and 48-h reduction in cIAP-1 protein level recorded i n the bryostatin 1 and vincristine combination recovered to control levels by 72 h. We believe that this phenomenon is responsible for the reduced apo ptosis recorded in this combination. Results of this study should prove use ful in guiding the clinical application of these novel agents in the treatm ent of ALL. (C) 1999 Academic Press.