The goal of this proposal is to elucidate the mechanism by which the protein kinase C (PC-C) activator, bryostatin 1, as well as other agents acting through the PK-C signal transduction pathway, enhance the ability of 1-Beta-D-arabinofuranosylcytosine (ara-C) to induce apoptosis (programmed cell death; PCD) in myeloid leukemia cells, resulting in synergistic antileukemic effects for the combination. This approach is based on the hypothesis that basal activity of PK-C protects myeloid leukemia cells from PCD, and that down-regulation of the enzyme, or of one or more of its isoforms, sensitizes cells to drug-induced apoptosis. Initially, the dose and schedule-dependent effects of PK-C activators, both physiologic (e.g., phospholipase C, diacyglycerol) and non- physiologic (e.g., bryostatin 1, PDBu, mezerein), as well as inhibitors (e.g., H07, staurosporine, calphostin, chelerythrine, gossypol, hypericin), will be fully characterized with respect to ara-C-induced endonucleolytic DNA cleavage, apoptotic morphology, and inhibition of clonogenicity in HL-60 cells and other leukemia cell lines (e.g., U937). The total activity and subcellular distribution (nuclear, cytoplasmic, and membrane) of PK-C will be monitored in parallel, and correlations sought between specific perturbations and potentiation (or antagonism) of ara-C-induced apoptosis. Northern analysis, immunoblotting techniques, and isoform assays will be employed to determine whether alterations in the activity (e.g., down-regulation) of individual PK-C isoforms (e.g., alpha, Beta, or gamma) are specifically related to augmentation of ara-C actions. Perturbations in PK-C activity associated with potentiation of apoptosis will be assessed with respect to the expression of oncogenes (e.g., c-jun, bcl-2, c-myc) implicated in this process; these associations may subsequently be evaluated more definitely through the use of antisense blockade strategies and dominant-negative transfectants. Attempts will be made to determine whether agents acting through signal transduction pathways also potentiate ara-C-induced apoptosis in primary AML cell cultures, and whether this strategy selectively spares normal primitive human hematopoietic progenitors exhibiting stem-cell characteristics (e.g., HPP-CFC). It is anticipated that these studies will provide a rational basis for employing bryostatin 1 and other agents acting through second messenger pathways to improve the antileukemic efficacy and selectivity of ara-C and possibly other nucleoside analogs.
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