ROLE OF G-KINASE IN DIFFERENTIATION--USE OF HL-60 MUTANT

骨髓增生异常综合征和急性白血病患者的异常细胞 髓系白血病不能正常分化。更好的 对调节髓系细胞的生化事件的认识 差异化可能会导致对这些疾病的更具体的治疗 精神错乱。人类早幼粒细胞系HL-60提供了这样的机会 在体外研究髓系细胞分化为 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提高剂。由于cAMP和cGMP发挥作用 它们的作用几乎完全是通过各自的蛋白激酶实现的， 提示HL-60细胞可被诱导分化。 关键调节蛋白的磷酸化(S)。私家侦探最近分离了 耐分化的稳定突变型HL-60亚系 CGMP浓度升高但分化正常的Re 应答其他诱导剂，包括DMSO和8-溴-cAMP。初步 对这些突变体的鉴定表明其磷酸化存在缺陷。 依赖于cGMP的蛋白激酶底物(或底物)。三大 这项建议的目标是：(I)鉴定磷酸化蛋白 野生型HL-60细胞在cGMP诱导分化过程中 突变的HL-60细胞中的磷酸化；(Ii)纯化和部分 对其中一种或几种磷酸蛋白进行测序；以及(Iii)分离一种 其中一种磷蛋白的基因克隆。这些蛋白质将被鉴定出来 通过2-D PAGE/放射自显影，所选的将使用 放射-高效液相色谱和PAGE/放射自显影的结合。部分氨基酸 蛋白质的序列将提供关于它们的生理信息 功能，并将允许合成简并的寡核苷酸和 多肽特异性抗体。一个cDNA克隆及其多肽特异性抗体 将允许研究磷蛋白合成的调节在 HL-60细胞分化及突变型分子缺陷的研究 HL-60细胞。克隆的基因将被导入突变细胞以 确定它是否能纠正他们的缺陷，磷蛋白将被 显微注射入野生型HL-60细胞以确定是否足够 诱导分化。这些研究将为PI提供培训 并在蛋白质生物化学和分子生物学方面奠定了基础 骨髓增生异常综合征和急性骨髓增生异常综合征患者的未来研究 骨髓性白血病。

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.