ROLE OF G-KINASE IN DIFFERENTIATION--USE OF HL-60 MUTANT
ROLE OF G-KINASE IN DIFFERENTIATION--USE OF HL-60 MUTANT
批准号:
3080020
负责人:
RENATE B PILZ
金额:
$8.32万
依托单位国家:
美国
项目类别:
财政年份:
1990
资助国家:
美国
项目状态:
已结题
起止时间:
1990-08-16 至 1995-07-31
关键词:
autoradiography cell differentiation complementary DNA cyclic AMP cyclic GMP enzyme substrate gel electrophoresis high performance liquid chromatography molecular cloning mutant myeloid stem cell phosphoproteins phosphorylation protein biosynthesis protein kinase protein structure function tissue /cell culture transfection
中文摘要
骨髓增生异常综合征和急性骨髓增生异常综合征患者的异常细胞
骨髓性白血病不能正常分化。更好的
了解调节髓系细胞的生化事件
分化可能会导致更具体的治疗这些
紊乱人早幼粒细胞系HL-60提供了机会,
在体外研究骨髓细胞分化,
粒细胞对几种诱导剂的反应,包括二甲基亚砜
(DMSO),以及cAMP和cGMP升高剂。由于cAMP和cGMP发挥
它们的作用几乎完全通过它们各自的蛋白激酶,
HL-60细胞的分化似乎可以通过
关键调节蛋白的磷酸化。私家侦探最近发现
对分化有抗性的稳定突变HL-60亚系
cGMP浓度升高的影响,但在重新分化正常,
对其他诱导剂包括DMSO和8-Br-cAMP也有反应。初步
这些突变体的特征表明,
cGMP依赖性蛋白激酶底物(或底物)。三大
该提案的目标是:(i)鉴定磷酸化蛋白质,
野生型HL-60细胞在cGMP诱导的分化过程中,
在突变HL-60细胞中磷酸化;(ii)纯化和部分纯化
对这些磷蛋白中的一种或几种进行测序;以及(iii)分离一种
一种磷蛋白的cDNA克隆。这些蛋白质将被鉴定
通过2-D PAGE/放射自显影,并将使用
放射性HPLC和PAGE/放射自显影的组合。的部分氨基酸
蛋白质的序列将提供关于它们的生理学特性的信息。
功能,并将允许合成变性寡核苷酸,
肽特异性抗体。cDNA克隆和肽特异性抗体
将允许研究磷蛋白合成的调节,
HL-60细胞的分化和突变体中的分子缺陷
HL-60细胞。克隆的基因将被转染到突变细胞中,
确定它是否会纠正他们的缺陷,磷蛋白将被
显微注射到野生型HL-60细胞中,以确定其是否足以
以诱导分化。这些研究将为PI提供培训
在蛋白质生物化学和分子生物学中的应用,
在骨髓增生异常综合征和急性
骨髓性白血病
英文摘要
The abnormal cells from patients with myelodysplastic syndromes and acute
myelogenous leukemias fail to differentiate normally. A better
understanding of the biochemical events regulating myeloid cell
differentiation could potentially lead to more specific therapies of these
disorders. The human promyelocytic cell line HL-60 provides the opportunity
to study myeloid cell differentiation in vitro as it differentiates into
granulocytes in response to several inducers, including dimethylsulfoxide
(DMSO), and cAMP- and cGMP-elevating agents. Since cAMP and cGMP exert
their effects almost exclusively through their respective protein kinases,
it appears that differentiation of HL-60 cells can be induced by
phosphorylation of key regulatory protein(s). The PI has recently isolated
stable mutant HL-60 sublines which are resistant to the differentiating
effects of elevated cGMP concentrations but differentiate normally in re-
sponse to other inducing agents including DMSO and 8-Br-cAMP. Preliminary
characterization of these mutants indicates a defect in phosphorylation of
a cGMP-dependent protein kinase substrate (or substrates). The three major
goals of this proposal are: (i) to identify the proteins phosphorylated in
wild type HL-60 cells during cGMP-induced differentiation that are not
phosphorylated in the mutant HL-60 cells; (ii) to purify and partially
sequence one or several of these phosphoproteins; and (iii) to isolate a
cDNA clone of one of the phosphoproteins. The proteins will be identified
by 2-D PAGE/autoradiography and selected ones will be purified using a
combination of radio-HPLC and PAGE/autoradiography. The partial amino acid
sequence of the proteins will provide information about their physiological
function and will allow synthesis of degenerative oligonucleotides and
peptide specific antibodies. A cDNA clone and peptide-specific antibodies
will allow study of the regulation of the phosphoprotein's synthesis during
differentiation of HL-60 cells and of the molecular defect in the mutant
HL-60 cells. The cloned gene will be transfected into the mutant cells to
determine if it will correct their defect and the phosphoprotein will be
microinjected into wild type HL-60 cells to determine if it is sufficient
to induce differentiation. These studies will provide the PI with training
in protein biochemistry and molecular biology and lay the foundation for
future studies in patients with myelodysplastic syndromes and acute
myelogenous leukemias.
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