PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS

白血病细胞的特征是成熟停滞在 造血发育阶段， 扩散得到维持。 体外培养的人白血病细胞 可以被刺激发育成非增殖细胞。 因此，在本发明中， 白血病可以部分地被看作是一种 分化和增殖。 其中最有效的 刺激白血病细胞分化的试剂， 120十四烷酰基佛波醇13-乙酸酯（TPA），佛波醇酯。 通过 取代甘油二酯，TPA激活CA 2 +- 磷脂依赖性激酶，蛋白激酶C。 的能力 模拟TPA刺激的细胞的二酰基甘油衍生物 反应表明蛋白激酶C激活可能介导 TPA所产生的部分细胞效应。 然而，在这方面， 二酰基甘油衍生物不能刺激分化 人类白血病细胞系U937 在我们的实验室里， 蛋白激酶C，TPA刺激的激酶活性已经被 鉴定 蛋白激酶C和TPA刺激的激酶 活性可以通过底物的差异来区分 的特异性 二酰基甘油衍生物不能 刺激分化或激活这种激酶表明， TPA刺激的激酶的激活可能是重要的 介导TPA刺激分化。 这个提议的目的是描述非蛋白激酶 C，TPA刺激的激酶，并确定是否激活这种激酶， 激酶是介导信号刺激的关键 白血病细胞的分化。 使用柱 层析，这种激酶将被表征和比较 其他非蛋白激酶C磷酸转移酶活性 由TPA刺激。 使用32 P标记的完整细胞和两个 三维电泳分析，内源性底物 将定义由该激酶磷酸化的蛋白质。 的影响 诱导U937分化的其它试剂（淋巴因子、二丁酰 cAMP等）将评估对该激酶活性的影响。 更好地理解控制机制 人类白血病细胞的分化将有助于发展 专门设计用于刺激内分泌的 白血病细胞的体内分化。 此外，这种疗法 提供一种替代性治疗，理论上， 避免了许多目前药物固有的毒副作用， 使用了康复疗法。 拟议的研究还可以 提供深入了解调解许多不同的机制 佛波醇酯在其他生物系统中发挥的作用。

The leukemic cell is characterized by maturational arrest at a stage of hematopoietic development where the ability to proliferate is maintained. Human leukenic cells cultured in vitro can be stimulated to develop into a non-proliferating cell. Thus, the leukemias can be viewed, in part, as a disorder of differentiation as well as proliferation. One of the most potent agents stimulating differentiation of leukemic cells is 120tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester. By substituting for diacylglycerol, TPA activates the CA2+- phospholipiddependent kinase, protein kinase C. The ability of diacyglycerol derivatives to mimic TPA stimulated cellular responses indicates that protein kinase C activation may mediate a portion of the cellular effects exerted by TPA. However, diacylglycerol derivatives are unable to stimulate differentiation of the human leukemic cell line U937. In our laboratory, a non- protein kinase C, TPA stimulated kinase activity has been identified. Protein kinase C and the TPA stimulated kinase activity can be distinguished by differences in substrate specificity. The inability of diacylglycerol derivatives to stimulate differentiation or to activate this kinase suggests that activation of the TPA stimulated kinase may be important in mediating TPA stimulated differentiation. The goal of this proposal is to characterize the nonprotein kinase C, TPA stimulated kinase and to determine if activation of this kinase is crucial in mediating the signal stimulating differentiation of the leukemic cell. Using column chromatography, this kinase will be characterized and compared to other non-protein kinase C phosphotransferase activities stimulated by TPA. Using 32P-labeled intact cells and two dimensional electrophoretic analysis, endogenous substrates phosphorylated by this kinase will be defined. The effects of other agents inducing U937 differentiation (lymphokines, dibutryl cAMP, etc.) will be evaluated for effects on this kinase activity. A better understanding of the mechanism controlling differentiation of human leukemic cells would aid in developing therapeutic modalities specifically designed to stimulate the in vivo differentiation of leukemic cells. Such a therapy, in addition to providing an alternative form of treatment, would theoretically avoid many of the toxic side effects inherent to the currently used antineoplastic therapy. The proposed research could also provide insight into the mechanisms mediating the many diverse effects exerted by phorbol esters in other biological systems.