MOLECULAR INHIBITION OF BCR-ABL TYROSINE KINASE BY BCR SEQUENCES
BCR 序列对 BCR-ABL 酪氨酸激酶的分子抑制
基本信息
- 批准号:6102549
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1998
- 资助国家:美国
- 起止时间:1998-09-30 至 1999-01-31
- 项目状态:已结题
- 来源:
- 关键词:SCID mouse cell cycle cell line chimeric proteins chronic myelogenous leukemia enzyme inhibitors gene therapy genetic transduction hematopoietic stem cells human tissue immunoprecipitation intermolecular interaction liposomes mutant neoplasm /cancer therapy oncoproteins phosphorylation protein kinase protein sequence protein structure function protein tyrosine kinase transfection transfection /expression vector
项目摘要
The Bcr-Abl oncoprotein is the primary causative factor in Philadelphia
chromosome (Ph) associated leukemias. The activated tyrosine kinase of the
Bcr-Abl oncoprotein is the primary driving force behind its oncogenic
activity. We have shown that a deleted form of Bcr [Bcr(64-413)],
encompassing the Abl SH2 binding domains of Bcr, reduced the
phosphotyrosine content of c-Abl and Bcr-Abl within cells, and inhibited
Bcr-A autophosphorylation activity in vitro. Similarly, a Bcr peptide
phosphorylated on Ser 354 blocked the c-Abl and Bcr-Abl kinases in vitro
whereas the unphosphorylated peptide was not inhibitory. Bcr(64-413) was
also resistant to tyrosine phosphorylation by either activated c-Abl or
Bcr-Abl. Importantly, Bcr(64-413) interfered with the growth of BCR-Abl
expressing cell liners. Our findings indicate that the Abl SH2 binding
domain of Bcr in the phospho-serine form inhibits the Bcr-Abl oncoprotein,
but that tyrosine phosphorylation of this domain of Bcr reverse its
inhibitory effects of Bcr-Abl. The results of these findings form the
basis of a new strategy to treat chronic phase CML patients. The specific
aims are: 1) Determine the optimum form of Bcr(64-413) will be tested in
human hemopoietic cells expressing Bcr-Abl for their ability to reverse
growth/apoptosis effects induced by Bcr-Abl. These constructs will be
compared to vector only, BCR(1-413), and S354A BCR(64-413) transfected
cells. These studies will be done using transfection and positive
selection with the tetracycline repressor promoter system. 2) Construct a
BCR(64-413) recombinant, replication-defective adenovirus for insertion of
the BCR inhibitor sequence into Bcr-Abl positive and negative cell lines.
3) Test the effectiveness of a recombinant defective Bcr adenovirus in
SCID/NOD SCID mouse systems programmed with cell lines derived from CML
patients. We plan to test the effects of Bcr(64-413) on patient cell lines
growing in a SCID mouse model. We plan to infect K562 cells and KBM-5
cells with Ad5 CMV BCR(64-413) to determine its effects on the growth
properties of these patient cell lines in the appropriate SCID model
compared to an adenovirus preparation encoding a unrelated gene (anti-
sense C-CAM). Recent findings indicate that Ad5 CMV BCR(64-413) induced
cell death in 75% in K562 cells infected for 2 days. The cell killing
effects was specific to BCR sequences because Ad5 CMV anti-sense C CAM did
not affect cell growth or induce cell death. 4) Test the effects of
recombinant, replication defective BCR(64-413) adenovirus infection in
human bone marrow preparations from CML chronic phase patients, Ph-
positive ALL, and normal bone marrow. These effects will be compared to
negative control adenoviruses. Cell death (50%) has been induced in a low
density marrow preparation from an untreated CML patient. We anticipate
that Bcr(64-413) will inhibit growth of Ph-positive colonies. 5)
Investigate the inhibitory effects of synthetic peptides derived from
Bcr(64-413) for their utility as inhibitors of Bcr-Abl expressing cell
lines and marrow samples. Positive results from either virus-mediated
introduction of Bcr(64-413) or soluble peptides from this region of Bcr
will be useful for inclusion in therapeutic protocols for treatment of
chronic phase CML patients.
Bcr-Abl癌蛋白是费城的主要致病因素
染色体(Ph)相关白血病。激活的酪氨酸激酶,
Bcr-Abl癌蛋白是其致癌的主要驱动力,
活动我们已经证明了Bcr [Bcr(64-413)]的删除形式,
包括Bcr的Abl SH 2结合结构域,降低了Bcr的表达。
细胞内c-Abl和Bcr-Abl的磷酸酪氨酸含量,并抑制
体外Bcr-A自身磷酸化活性。类似地,Bcr肽
Ser 354的磷酸化在体外阻断c-Abl和Bcr-Abl激酶
而未磷酸化的肽没有抑制性。Bcr(64-413)为
也抵抗由活化的c-Abl或
重要的是,Bcr(64-413)干扰BCR-Abl的生长
表达细胞系。我们的研究结果表明,Abl SH 2结合
磷酸丝氨酸形式的Bcr结构域抑制Bcr-Abl癌蛋白,
但是Bcr这个结构域的酪氨酸磷酸化逆转了其
Bcr-Abl的抑制作用。这些发现的结果形成了
治疗慢性期CML患者的新策略的基础。具体
目的是:1)确定Bcr(64-413)的最佳形式,
表达Bcr-Abl的人造血细胞逆转
Bcr-Abl诱导的生长/凋亡作用。这些构建体将被
与仅载体、BCR(1-413)和S354 A BCR(64-413)转染相比,
细胞这些研究将使用转染和阳性对照进行。
用四环素阻遏物启动子系统进行选择。2)构建
BCR(64-413)重组复制缺陷型腺病毒,用于插入
将BCR抑制剂序列导入Bcr-Abl阳性和阴性细胞系。
3)检测重组缺陷型Bcr腺病毒在
用源自CML的细胞系编程的SCID/NOD SCID小鼠系统
患者我们计划测试Bcr(64-413)对患者细胞系的影响
在SCID小鼠模型中生长。我们计划感染K562细胞,
用Ad 5 CMV BCR(64-413)感染细胞,以确定其对生长的影响
这些患者细胞系在适当的SCID模型中的特性
与编码不相关基因的腺病毒制剂(抗-
有义C-CAM)。最近的研究结果表明,Ad 5 CMV BCR(64-413)诱导了
感染2天的K562细胞死亡率为75%。的细胞杀伤
作用是特异性的BCR序列,因为Ad 5 CMV反义CCAM
不影响细胞生长或诱导细胞死亡。4)测试的影响
重组复制缺陷型BCR(64-413)腺病毒感染
CML慢性期患者的人骨髓制备物,Ph-
ALL阳性骨髓正常这些影响将与
阴性对照腺病毒。细胞死亡(50%)已被诱导在一个低的
来自未经治疗的CML患者的高密度骨髓制备物。我们预计
Bcr(64-413)可抑制Ph阳性菌落的生长。第五章)
研究来源于以下的合成肽的抑制作用:
Bcr(64-413)作为Bcr-Abl表达细胞抑制剂的用途
和骨髓样本病毒介导的
引入Bcr(64-413)或来自Bcr该区域的可溶性肽
将可用于包括在治疗方案中,
慢性期CML患者。
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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RALPH BERNARD ARLINGHAUS其他文献
RALPH BERNARD ARLINGHAUS的其他文献
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{{ truncateString('RALPH BERNARD ARLINGHAUS', 18)}}的其他基金
Identification of New Targets and Novel Treatment Strategies for Chronic Mye
确定慢性髓性白血病的新靶点和新治疗策略
- 批准号:
8000085 - 财政年份:2010
- 资助金额:
-- - 项目类别:
Jak2 involvement in Bcr-Abl oncogenic transformation
Jak2参与Bcr-Abl致癌转化
- 批准号:
6611511 - 财政年份:2003
- 资助金额:
-- - 项目类别:
Jak2 involvement in Bcr-Abl oncogenic transformation
Jak2参与Bcr-Abl致癌转化
- 批准号:
6749553 - 财政年份:2003
- 资助金额:
-- - 项目类别:
Jak2 involvement in Bcr-Abl oncogenic transformation
Jak2参与Bcr-Abl致癌转化
- 批准号:
6891574 - 财政年份:2003
- 资助金额:
-- - 项目类别:
MOLECULAR INHIBITION OF BCR-ABL TYROSINE KINASE BY BCR SEQUENCES
BCR 序列对 BCR-ABL 酪氨酸激酶的分子抑制
- 批准号:
6332466 - 财政年份:2000
- 资助金额:
-- - 项目类别:
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