PHORBOL ESTER-INDUCED DIFFERENTIATION OF LEUKEMIC CELLS

肿瘤是一种以增殖异常为特征的疾病。 和差异化。使用疗法的初步临床试验 旨在刺激白血病细胞分化的基因已经产生 结果令人振奋。佛波酯是一组能够诱导 人白血病细胞的体外分化比药物更有效 目前正在进行临床使用评估。然而，有丝分裂效应 在其他组织中排除了它们在临床环境中的使用。佛波醇 酯(TPA)通过激活蛋白质的成员来发挥作用 激酶C(PKC)基因家族。因为七个PKC之间的差异 异构体，假设个别异构体可以选择性地 调节TPA诱导的事件。因此，有必要直接进行研究 评估个体异构体在调节 TPA的影响。 人单核细胞样白血病细胞系U937的体外诱导分化 在暴露于TPA后变成巨噬细胞样细胞，我们有：(1) 证明存在一种以前没有特征的PKC亚型； (2)观察到U-937细胞中含有PKC Zeta 转位或下调对TPA的反应；(3)发现TPA 治疗从功能上诱导部分下调PKC a活性 通过改变其底物特异性和(4)利用制备的PKC突变体 通过重组聚合酶链式反应技术 在没有TPA的情况下被宪法激活的决定 不同亚型对基因的影响存在质的差异 表情。这些数据构成了对以前版本的修订的基础 提交的建议书。 目前提案的目标是确定机制(S)由 其中TPA刺激U937分化。该计划的主旨是 建议确定个体异构体在调节TPA中的作用 刺激分化和其他TPA诱导的反应。根据 为了达到这个目标，我们将：(1)直接研究个体亚型的作用 通过评估PKC突变体在体内结构性激活的能力 TPA缺失诱导U937分化及其他TPA诱导 (2)分析个体亚型在TPA刺激中的作用 反义DNA选择性耗尽异构体诱导分化； 确定激活PKC-Zeta的因子，细胞反应 通过激活这种异构体，并研究第二种异构体的作用 氯结构域中富含半胱氨酸的重复序列赋予TPA对 PKC；(4)分析TPA诱导改变的机制 在PKC中有底物特异性；(5)鉴定和鉴定一种新的 在U937细胞中识别的PKC活性；(6)确定 内源性PKC亚型表达对TPA反应性的影响 U937细胞；以及(7)分析DNA区域5‘对β基因的作用 和伽马异构体调节这些基因的转录活性 在U937细胞中使用包含这些区域的CAT构建物。通过 确定各亚型在介导TPA诱导中的作用 分化和佛波酯等刺激反应、信息 从这些研究中获得的信息可能会对 白血病的治疗。这些结果将集中于 激动剂选择性地激活所涉及的异构体，并可以形成 设计可作为选择性形式的PKC突变体的基础 针对某些白血病的基因疗法。

Neoplasia is a disorder characterized by abnormalities in proliferation and differentiation. Preliminary clinical trials using therapies designed to stimulate leukemic cell differentiation have yielded promising results. Phorbol esters are a group of agents which induce in vitro differentiation of human leukemic cells more potently than agents currently being evaluated for clinical use. However, mitogenic effects in other tissues preclude their use in a "clinical setting. Phorbol esters (TPA) exert their effects by activating members of the protein kinase C (PKC) gene family. Because of differences among the seven PKC isoforms, it is hypothesized that individual isoforms may selectively mediate TPA-induced events. Thus, studies are warranted to directly assess the involvement of the individual isoforms in mediating the effects of TPA. Using the human monoblastoid U937 leukemic cell line which differentiates into a macrophage-like cell after exposure to TPA, we have: (1) demonstrated the existence of a previously uncharacterized PKC isoform; (2) observed that PKC zeta is contained in the U-937 cell and does not translocate or down regulate in response to TPA; (3) found that TPA treatment functionally induces a partial down regulation PKC a activity by altering its substrate specificity and (4) using PKC mutants prepared by recombinant polymerase chain reaction techniques that are constituitively activated in the absence of TPA determined that individual isoforms elicit qualitatively different effects on gene expression. This data form the basis for this revision of the previously submitted proposal. The goal of the current proposal is to determine the mechanism(s) by which TPA stimulates U937 differentiation. The main thrust of the proposal is to determine the role of individual isoforms in mediating TPA stimulated differentiation and other TPA-induced responses. Pursuant to this goal, we will: (1) directly examine the role of individual isoforms by assessing the ability of PKC mutants constituitively activated in the absence to TPA to elicit U937 differentiation and other TPA-induced responses; (2) analyze the role of individual isoforms on TPA-stimulated differentiation by selectively depleting isoforms with antisense DNA; (3) determine the factors activating PKC-zeta, the cellular responses elicited by activation of this isoform and examine the role of the second cysteine-rich repeat in the Cl domain in conferring TPA-responsiveness to PKC; (4) analyze the mechanisms responsible for TPA-induced alterations in PKC a substrate specificity; (5) identify and characterize a newly recognized PKC activity in the U937 cell; (6) determine the effects of altering endogenous PKC isoform expression on TPA-responsiveness in the U937 cell; and (7) to analyze the role of the DNA region 5' to the beta and gamma isoforms in regulating transcriptional activity of these genes in the U937 cell using CAT constructs containing these regions. By determining the role of individual isoforms in mediating TPA-induced differentiation and other phorbol ester stimulated responses, information obtained from these studies could have direct clinical implications in the treatment of leukemias. These results would focus the development of agonists selectively activating the involved isoform and could form the basis for designing PKC mutants which could be used as a selective form of gene therapy for certain leukemias.