The goal of this renewal application is to elucidate further the basis by which agents that down-regulate (bryostatin 1) or inhibit (UCN-01, safingol) protein kinase C (PKC) promote leukemic cell apoptosis by ara-C and other nucleoside analogs. Evidence generated during the preceding period of support suggests that interruption of the PKC signal transduction pathway may promote cell death by three distinct mechanisms: (a) dysregulation of cell cycle-related events, particularly induction of cyclin-dependent kinase inhibitors (CDKIs); (b) redirection of signals away from cytoprotective survival (e.g., MAPK/ERK) toward stress-related (e.g., SAPK/JNK) pathways; and (c) phosphorylation of the Bcl-2 protein, promoting mitochondrial permeability transition and circumvention of the block to capase activation. To examine these possibilities, (1) Human leukemic cells (HL-60, U937) stably overexpressing Bcl-2, Bcl-xL, and phosphorylation loop deletant mutants will be employed to determine whether phosphorylation of anti-apoptotic proteins is responsible for potentiation of ara-C mediated apoptosis by PKC inhibition/down-regulation, (2) Analogously, HL-60 and U937 cells expressing antisense p21WAF1+/-p27KIP1 will be used to characterize the effect of CDKI dysregulation on ara-C mediated apoptosis and its potentiation by PKC inhibitors/down-regulators, (3) The effects of enforced expression of p53 will be examined with respect to leukemic cell differentiation and modulation of ara-C induced cell death by bryostatin 1/PKC inhibitors, (4) Direct evidence for the participation of stress and survival pathways in cell death decisions will be obtained through the use of SEK1/JNK1 dominant-negative and ER-inducible Raf/MAPK mutants, (5) Findings will be extended to include another clinically important nucleoside analog, 2,2(1)-difluorodeoxcytidine (gemcitabine) and (6) Finally, the ability of pharmacologically relevant concentrations of bryostatin 1, UCN-01, and safingol to potentiate ara-C- (and gemcitabine)-mediated apoptosis in primary leukemic myeloblasts ex vivo will be explored. Information derived from these studies will lay the foundation for a novel approach to leukemia therapy aimed at enhancing the activity of effective antileukemic drugs via combination with agents that interrupt the PKC signal transduction pathway.
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