PHOSPHOLIPID-CA2+ AND PHOSPHOPROTEINS IN LEUKEMIC CELLS

这项研究的具体目标涵盖四个主要领域 新发现的磷脂/钙离子刺激的蛋白质磷酸化 人类白血病细胞中的系统。(1)\磷脂敏感 钙依赖蛋白激酶将从 白血病患者的细胞及其特性和机制 将研究磷脂和钙离子的激活作用。免疫细胞化学 正常中性粒细胞、患者白血病细胞和HL60中该酶的表达 和培养中的K562细胞将使用多克隆抗体进行研究 这种酶已经在这个实验室里研发出来了。(2)内源底物 这种酶的蛋白质将被搜索、鉴定和 特色化的。应注意衬底的变化。 以便提出它们的病理生理学相关性。单克隆 针对白血病细胞的底物的抗体将是 以及它们抑制某些白细胞功能的能力，如 以及它们对白血病的诊断和治疗作用，将是 探索过了。刺激内源性蛋白质磷酸化的研究 还将进行钙调蛋白/钙离子、cAMP和cGMP，以便 细胞介质之间在蛋白质水平上的相互作用 人类白细胞的生物学和病理学中的磷酸化可以 学习。(3)酶的变化(活性水平和亚细胞 分布)和底物将被研究在HL-60刺激到 通过二甲基亚砜和佛波酯进行末端区分。 (4)烷基溶血磷脂类似物的构效关系 将对抑制酶系统进行检测，并将结果 与他们的临床疗效(如果知道)和他们的能力进行比较 影响培养中白血病细胞的生长。(B)

The specific aims of this research cover four major areas of the newly-identified phospholipid/Ca2+-stimulated protein phosphorylation system in human leukemic cells. (1)\Phospholipid-sensitive Ca2+-dependent protein kinase will be purified to homogeneity from leukemic cells from patients, and its properties and mechanism of activation by phospholipid and Ca2+ will be studied. Immunocytochemistry of the enzyme in normal neutrophils, leukemic cells from patients, and HL60 and K562 cells in culture will be studied using polyclonal antibodies to the enzyme already developed in this lab. (2)\Endogenous substrate proteins for the enzyme will be searched for, identified, and characterized. Attention will be paid to the variation in the substrates so that their pathophysiologic relevance can be suggested. Monoclonal antibodies to the substrates "specific" to leukemic cells will be developed; and their ability to inhibit certain leukocyte functions, as well as their diagnostic and therapeutic usefulness in leukemias, will be explored. Endogenous protein phosphorylation stimulated by calmodulin/Ca2+, cAMP, and cGMP also will be carried out so that the interplays among the cellular mediators at the level of protein phosphorylation in the biology and pathology of human leukocytes can be studied. (3)\Alterations in the enzyme (activity level and subcellular distribution) and substrates will be investigated in HL-60 stimulated to terminally differentiate by dimethylsulfoxide and phorbol ester. (4)\Structure-activity relationship of alkyl-lysophospholipid analogs to inhibit the enzyme system will be examined, and the results will be compared with their clinical efficacy (if known) and their ability to affect leukemic cell growth in culture. (B)