MECHANISMS OF ERYTHRODIFFERENTIATION OF LEUKEMIC CELLS

白血病细胞红细胞分化的机制

• 批准号: 2144485
• 负责人: Dorothy C Moore
• 金额: $ 10.88万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1998-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144485
• 关键词: 1,25 dihydroxycholecalciferol; DNA replication; apoptosis; autoradiography; cell differentiation; clone cells; cycloheximide; cytosine arabinoside; electroporation; enzyme activity; enzyme induction /repression; erythroid stem cell; erythroleukemia; erythropoietin; gene expression; genetic markers; genetic regulation; growth factor receptors; hemoglobin; hemoprotein biosynthesis; human tissue; isozymes; messenger RNA; nuclear runoff assay; phenotype; protein kinase C; protooncogene; puromycin; receptor expression; stainings

An understanding of the controls and mechanisms of hematopoietic differentiation will contribute to the cure of human diseases. It is proposed to study the in vitro induction of the erythroid phenotype in human leukemia K562 cells, the inhibition by 1,25(OH)2 vitamin D3 of such differentiation, and its potentiation by inhibitors of protein synthesis. The mechanisms of inhibition of erythrodifferentiation by 1,25(OH)2D3 to be investigated will include the prevention of down-regulation of c-myc proto-oncogene, modulation of receptor (R) density for growth factors e.g. erythropoietin (Epo) R, the reduction of the window of sensitivity in the cell cycle to induction of differentiation, and changes in the level and activity of protein kinase C (PKC). For most experiments there will be four standard groups: untreated K562 cells, 1,25(OH)2D3 treated cells, Ara-C treated cells, and cells pretreated with 1,25(OH)2D3 followed by Ara-C. Lineage restricted phenotypic and molecular markers of differentiation will be compared between the test groups after the various manipulations described below are performed. K562 cells will be transfected by electroporation with DNA constructs of sense and antisense c-myc and subsequently exposed to various agents. In addition, mRNA stabilization will be examined as a cause of prevention of c-myc down-regulation. Messenger RNA levels, nuclear transcriptional rates, protein levels and enzyme activity of the predominant PKC isozymes will be examined. Cell cycle studies will utilize benzidine staining and autoradiography to simultaneously assess the state of differentiation and the presence or absence of DNA synthesis during initiation of the induction. The potentiation of Ara-C induced hemoglobinization of K562 cells by inhibitors of protein synthesis will be investigated by determining if, 1) the increased proportion of detectably hemoglobinized cells is due to an increased level of Hb accumulation or is the result of recruitment of cells into erythroid program, 2) observed differences between cycloheximide (CHX) and puromycin (PM) as potentiators of differentiation are due to different mechanisms of action of these drugs, or due to different degrees of inhibition of protein synthesis, and 3) CHX reduces apoptosis in this system. The acquisition of this new knowledge will increase the understanding of the control of hematopoiesis and should lead to the ability to complement the cytotoxic drug therapy of human leukemias.

对造血调控及其机制的认识 分化将有助于人类疾病的治愈。它是 建议进行红系表型体外诱导的研究。 1，25(OH)2维生素D3对人白血病K562细胞的抑制作用 分化，以及蛋白质合成抑制剂对其增强作用。 1，25(OH)2D3抑制红系分化的机制 将被调查包括预防c-myc下调 原癌基因与生长因子受体密度的调节 例如，促红细胞生成素(EPO)R，减少敏感窗口 在细胞周期中诱导分化，并在细胞周期中改变 蛋白激酶C(PKC)水平和活性。对于那里的大多数实验来说 将设四个标准组：未处理的K562细胞、1，25(OH)2D3处理组 细胞、Ara-C处理的细胞和1，25(OH)2D3预处理的细胞 然后是Ara-C。谱系限制性表型和分子标记 之后，将在试验组之间进行差异性比较 执行下面描述的各种操作。K562细胞将被 正、反义DNA构建体电穿孔转染法 C-myc，随后暴露于各种制剂。此外，mRNA. 稳定性将被视为预防c-myc的一个原因。 下调监管。信使核糖核酸水平，核转录速率， 主要的PKC同工酶的蛋白质水平和酶活性将 接受检查。细胞周期研究将利用联苯胺染色和 放射自显影同时评估分化状态和 DNA合成的存在或不存在 归纳法。阿糖胞苷对K562细胞血红蛋白化的增强作用 通过蛋白质合成抑制剂对细胞进行研究 确定是否，1)可检测到的血红素化比例增加 细胞是由于Hb蓄积水平增加或是 细胞在红系程序中的招募，2)观察到的差异 放线菌酮(CHX)与嘌呤霉素(PM)作为促进剂的比较 分化是由于这些药物的作用机制不同， 或由于蛋白质合成受到不同程度的抑制，以及3) ChX可减少该系统中的细胞凋亡。收购这一新的 知识将增加对造血控制的理解 并应导致对细胞毒药物治疗的补充能力 人类白血病。