MECHANISMS OF ERYTHRODIFFERENTIATION OF LEUKEMIC CELLS

白血病细胞红细胞分化的机制

• 批准号: 2144484
• 负责人: Dorothy C Moore
• 金额: $ 10.65万
• 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 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144484
• 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.

了解造血的控制和机制 分化将有助于治愈人类疾病。 这是 提议研究红斑表型的体外诱导 人白血病K562细胞，抑制1,25（OH）2维生素D3 分化及其通过蛋白质合成抑制剂增强。 1,25（OH）2d3抑制红细胞增生的机制 被调查将包括预防C-MYC的下调 原始癌基因，受体密度调节生长因子的密度 例如促红细胞生成素（EPO）R，敏感性窗口的降低 在细胞周期中，诱导分化和变化 蛋白激酶C（PKC）的水平和活性。 对于大多数实验 将是四个标准组：未处理的K562细胞，1,25（OH）2d3处理 细胞，ARA-C处理的细胞和用1,25（OH）2d3预处理的细胞 其次是Ara-C。 谱系限制表型和分子标记 将比较测试组之间的分化 下面描述的各种操作将进行。 K562细胞将是 通过电穿孔使用有义务和反义的DNA构建体转染 C-Myc，随后暴露于各种代理。 另外，mRNA 稳定将被检查为预防C-MYC的原因 下调。 信使RNA水平，核转录率， 主要PKC同工酶的蛋白质水平和酶活性将 被检查。 细胞周期研究将利用苯甲胺染色和 放射自显影，以同时评估分化状态和 在开始期间的存在或不存在DNA合成 就职。 ARA-C诱导的K562血红蛋白的增强 细胞通过蛋白质合成的抑制剂将通过 确定，1）是否增加了可检测到的血红蛋白的比例 细胞是由于HB积累水平升高，或者是由于 将细胞募集到红细胞计划中，2）观察到差异 在环己酰亚胺（CHX）和呼毒素（PM）之间 分化是由于这些药物的作用机理的不同， 或由于蛋白质合成的抑制程度不同，3） CHX减少了该系统中的凋亡。 收购这个新的 知识将增加对造血控制的控制 并应导致补充细胞毒性药物治疗的能力 人类白血病。