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。 使用治疗的初步临床试验 旨在刺激白血病细胞分化的药物 有希望的结果。 佛波酯是一组药物， 人白血病细胞的体外分化比药物更有效 目前正在评估临床使用。 然而，促有丝分裂作用 排除了它们在“临床环境”中的使用。佛波 酯（TPA）通过激活蛋白质的成员发挥作用 激酶C（PKC）基因家族。 由于七个PKC之间的差异， 同种型，假设单个同种型可以选择性地 介导TPA诱导的事件。 因此，研究有必要直接 评估个体同种型参与介导 TPA的影响。 使用分化为人单核细胞样U937白血病细胞系， 进入巨噬细胞样细胞后暴露于TPA，我们有：（1） 证明了以前未表征的PKC亚型的存在; (2)观察到PKC zeta包含在U-937细胞中，并且不 （3）发现TPA可使细胞内的蛋白质易位或下调， 治疗在功能上诱导部分下调PKC α活性 通过改变其底物特异性和（4）使用制备的PKC突变体 通过重组聚合酶链反应技术， 在没有TPA的情况下激活的组成型抗体确定， 不同的同种型对基因表达产生不同的影响， 表情 这些数据构成了本次修订的基础， 提交的提案。 本提案的目标是通过以下方式确定机制： TPA刺激U937分化。 的主旨 一项建议是确定个体亚型在介导TPA中的作用 刺激的分化和其它TPA诱导的反应。 根据 为了实现这一目标，我们将：（1）直接研究单个异构体的作用 通过评估PKC突变体的能力， 缺乏TPA以引发U937分化和其他TPA诱导的分化 反应;（2）分析个体亚型对TPA刺激的 通过用反义DNA选择性地消耗同种型来分化;（3） 确定激活PKC-zeta的因素， 引起的激活这种亚型，并检查第二个的作用， C1结构域中富含半胱氨酸的重复序列在赋予TPA-应答性中的作用 （4）分析TPA诱导的细胞凋亡的机制 在PKC中底物特异性;（5）鉴定和表征新的 在U937细胞中识别的PKC活性;（6）确定 改变内源性PKC亚型表达对TPA反应性的影响， U937细胞;和（7）分析DNA区域5'到β-葡聚糖酶的作用。 和γ亚型在调节这些基因的转录活性中的作用 在U937细胞中使用含有这些区域的CAT构建体。 通过 确定单个同种型在介导TPA诱导的细胞凋亡中的作用， 分化和其他佛波酯刺激的反应，信息 从这些研究中获得的信息可能具有直接的临床意义， 白血病的治疗 这些结果将集中在发展 激动剂选择性激活相关亚型，并可形成 设计可用作选择性形式的PKC突变体的基础 基因疗法的应用。

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.