The long-term objective of this project is to better understand the mechanisms underlying the expression and regulation of terminal differentiation of normal and leukemic human neutrophilic granulocytes. Acute myeloid leukemia (AML) is associated with a disorganization in the expression of this differentiative program, and is characterized clinically by an expanding population of relatively immature, functionally incompetent cells that often exhibit abnormal granulogenesis. Seventy percent of AML patients die of infections. Our experimental approach focuses on the enzyme myeloper-oxidase (MPO) which plays a major role in the microbicidal system of mature, functional neutrophils. We are studying the differentiation-stage synthesis, post-translational processing and packaging of different forms of MPO into a class of specialized lysomsomes known as azurophilic granules (AG). AG exhibit considerable heterogeneity that appears to reflect both maturational and functional differences.
Our specific aims are directed at further defining AG heterogeneity by correlating biochemical properties of isolated granules with ultrastructural morphology and cytochemical and immunocytochemical staining of intact cells and granules. Using these criteria, we have so far identified four types of AG, each of which can be classed as immature of mature. Such granule typing, together with automated size-frequency granule histograms, will be used to classify the granules from patients with myeloid leukemia. We plan to examine the possible peroxisomal nature of certain azurophilic microgranules and whether MPO and catalase are co-localized as in the specific granules of eosinophils. We will study the relationship between the processing of MPO, the addition of heme, the acquisition of enzymatic activity and the assembly of MPO into different dimeric forms using pulse-chase procedures, automated, high resolution liquid chromatography, immunoprecipitation, SDS- PAGE/fluorography and scintillation counting methods to separate, identify and quantitate specific molecular species. These studies will be coordinated with efforts to identify and characterize the intracellular compartments involved in the transport and packaging of MPO into different AG using a combination of Percoll density gradient and immunoselective separation techniques. We will correlate these molecular and cellular events at the biochemical and ultrastructural levels and as a function of granule maturation. This project will provide new information concerning the basic mechanisms of neutrophil differentiation that should be potentially useful in the diagnosis and classification of myeloid leukemia and in the design and/or selection of different therapeutic strategies for the clinical management of leukemic patients.
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