The phenotypic expression of leukemia appears to be related to a disorganization in the normal processes of cellular commitment and differentiation within the hematopoietic system. Myeloid leukemic cells have been induced to undergo """"""""normal"""""""" differentiation in vitro and in vivo. The resulting cells are no longer malignant in vivo and no longer multiply in vitro. The long-term objective of our studies is elucidation of the molecular mechanisms underlying the operation and regulation of normal and leukemic terminal myeloid differentiation with particular emphasis on the differentiation processes underlying granulogenesis. Our experimental approach has centered on the enzyme myeloperoxidase (MPO), an abundant, functionally important, granule-specific protein of neutrophilic polymorphonuclear leukocytes (PMN). MPO synthesis and incorporation into primary or azurophilic granules occurs exclusively during the promyelocyte stage of cytodifferentiation. We are studying these processes using the human promyelocytic leukemia cell line HL-60, which may be induced to differentiate in vitro to more mature PMN using agents such as dimethylsulfoxide. We have recently shown that PMN contains three forms of MPO that exhibit differences in enzymatic activity and subunit structure. There appears to be a differential distribution of these forms in different azurophilic granule subpopulations. Moreover, these different granule subpopulations appear to be under separate secretory control. We have developed a Percoll density gradient centrifugation system for the isolation of 13 granule subpopulations from normal human PMN. Biochemical and ultrastructural characterization of these subpopulations revealed striking qualitative and quantitative differences. We have isolated the mRNA for MPO from HL-60 cells and are studying translation and processing in vitro. We have developed a new cationic detergent-PAGE system for the determination of molecular weights of biologically active proteins including MPO. Current work is aimed at using these procedures to correlate the biosynthesis and packaging of MPO in leukemic cells at the cellular and molecular levels. We feel the information to be developed by this project has clear implications for the future development of new strategies for the clinical management of myeloid leukemia. (M)
Showing the most recent 10 out of 28 publications
