QUANTITATIVE STUDIES ON GRANULOCYTE DIFFERENTIATION

白血病的表型表达似乎与 细胞承诺的正常过程中的紊乱， 造血系统内的分化。 髓性白血病细胞 已经在体外和体内被诱导经历“正常”分化， vivo. 由此产生的细胞在体内不再是恶性的， 在试管中繁殖。 我们研究的长期目标是阐明 的运作和调节的分子机制， 正常和白血病终末髓系分化， 强调颗粒形成的分化过程。 我们 实验方法集中在酶髓过氧化物酶（MPO）， 丰富的，功能重要的，颗粒特异性蛋白质的嗜酸性粒细胞 多形核白细胞（PMN）。 MPO合成和掺入 初级或嗜天青颗粒只发生在早幼粒细胞 细胞分化阶段。 我们正在研究这些过程， 人早幼粒细胞白血病细胞系HL-60，其可被诱导 在体外分化为更成熟的PMN，使用试剂， 二甲亚砜 我们最近发现PMN包含三种形式的 MPO表现出酶活性和亚基结构的差异。 这些形式似乎在不同的地方有不同的分布。 嗜天青颗粒亚群。 此外，这些不同的颗粒 亚群似乎处于单独的分泌控制下。 我们有 开发了Percoll密度梯度离心系统， 从正常人中性粒细胞中分离出13个颗粒亚群。 生化 这些亚群的超微结构特征显示， 质和量的巨大差异。 我们已经分离出 从HL-60细胞中提取MPO的mRNA，并正在研究翻译和加工 体外 我们研制了一种新的阳离子洗涤剂-聚丙烯酰胺凝胶电泳系统， 生物活性蛋白质分子量的测定 包括MPO。 目前的工作旨在利用这些程序， 相关的MPO的生物合成和包装在白血病细胞中， 细胞和分子水平。 我们认为， 该项目对未来新的发展具有明显的影响， 骨髓性白血病的临床治疗策略。 （百万）

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)