PHORBOL ESTER-INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
基本信息
- 批准号:3186192
- 负责人:
- 金额:$ 15.68万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1987
- 资助国家:美国
- 起止时间:1987-05-01 至 1995-06-30
- 项目状态:已结题
- 来源:
- 关键词:SDS polyacrylamide gel electrophoresis antisense nucleic acid autoradiography cell differentiation enzyme substrate gene expression genetic regulation genetic transcription human tissue laboratory rabbit leukemia mutant neoplastic cell nucleic acid sequence phorbols protein kinase C protein sequence radiotracer tissue /cell culture western blottings
项目摘要
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.
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已产生
有希望的结果。 佛波酯是一组诱导剂
人类白血病细胞的体外分化能力比药物更有效
目前正在评估其临床用途。 然而,促有丝分裂作用
在其他组织中的存在妨碍了它们在“临床环境中的使用。佛波醇
酯类 (TPA) 通过激活蛋白质成员发挥作用
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 诱发的事件。 因此,研究有必要直接
评估各个亚型在介导中的参与
TPA 的影响。
使用可分化的人单母细胞 U937 白血病细胞系
暴露于 TPA 后进入巨噬细胞样细胞,我们有: (1)
证明了以前未表征的 PKC 同工型的存在;
(2)观察到U-937细胞中含有PKC zeta,并且不存在
响应 TPA 进行易位或下调; (3)发现TPA
治疗功能性诱导部分下调 PKC a 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 的情况下组成型激活确定
各个亚型对基因产生不同质的影响
表达。 该数据构成了本次修订的基础
提交的提案。
当前提案的目标是通过以下方式确定机制:
TPA 刺激 U937 分化。 该计划的主要推力是
提议是确定各个亚型在介导 TPA 中的作用
刺激分化和其他 TPA 诱导的反应。 根据
为了实现这一目标,我们将:(1)直接检查各个亚型的作用
通过评估 PKC 突变体组成型激活的能力
缺乏 TPA 来引发 U937 分化和其他 TPA 诱导的分化
回应; (2)分析各个异构体对TPA刺激的作用
通过用反义 DNA 选择性地消除同种型来进行分化; (3)
确定激活 PKC-zeta 的因素,即细胞反应
通过激活该亚型引起并检查第二个亚型的作用
Cl 结构域中富含半胱氨酸的重复序列赋予 TPA 响应性
PKC; (4)分析TPA引起的改变的机制
PKC 中具有底物特异性; (5) 识别并表征新的
识别 U937 细胞中的 PKC 活性; (6) 确定效果
改变内源性 PKC 亚型表达对 TPA 反应性的影响
U937细胞; (7) 分析 DNA 区域 5' 对 β 的作用
和 γ 亚型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 构建体。 经过
确定各个异构体在介导 TPA 诱导的过程中的作用
分化和其他佛波酯刺激反应、信息
从这些研究中获得的结果可能具有直接的临床意义
白血病的治疗。 这些结果将集中于发展
激动剂选择性地激活所涉及的亚型并可以形成
设计可用作选择性形式的 PKC 突变体的基础
某些白血病的基因治疗。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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DOUGLAS K. WAYS其他文献
DOUGLAS K. WAYS的其他文献
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{{ truncateString('DOUGLAS K. WAYS', 18)}}的其他基金
PHORBOL ESTER-INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3186193 - 财政年份:1994
- 资助金额:
$ 15.68万 - 项目类别:
CHARACTERIZATION OF PROTEIN KINASE C GENE RESPONSE ELEME
蛋白激酶 C 基因反应元件的表征
- 批准号:
3023319 - 财政年份:1991
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3457923 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER-INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3186194 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3457925 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3447011 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3457922 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3457926 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
PHORBOL ESTER INDUCED DIFFERENTIATION OF LEUKEMIC CELLS
佛波酯诱导白血病细胞分化
- 批准号:
3457924 - 财政年份:1987
- 资助金额:
$ 15.68万 - 项目类别:
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