MECHANISMS OF ERYTHRODIFFERENTIATION OF LEUKEMIC CELLS

对造血调控和机制的理解 分化将有助于人类疾病的治疗。 是 建议研究体外诱导红细胞表型， 人白血病K562细胞，1，25（OH）2维生素D3对这种细胞的抑制作用， 分化，以及通过蛋白质合成抑制剂的增强作用。 1，25（OH）_2D_3抑制红细胞分化的机制 研究将包括预防c-myc下调 原癌基因，生长因子受体密度的调节 例如促红细胞生成素（Epo）R，敏感性窗口的减少 在细胞周期中诱导分化， 蛋白激酶C（PKC）水平和活性。 对于那里的大多数实验来说 将有四个标准组：未处理的K562细胞，1，25（OH）2D 3处理的 细胞、Ara-C处理的细胞和用1，25（OH）2D 3预处理的细胞 其次是阿糖胞苷 谱系限制性表型和分子标记 将比较试验组之间的差异， 执行下面描述的各种操作。 K562细胞 通过电穿孔用有义和反义DNA构建体转染 c-myc并随后暴露于各种试剂。 此外，mRNA 稳定性将作为预防c-myc的原因进行检查 下调。 信使RNA水平，核转录率， 蛋白质水平和主要PKC同工酶的酶活性将 接受检查。 细胞周期研究将利用联苯胺染色， 放射自显影以同时评估分化状态， DNA合成的存在或不存在，在启动过程中， 诱导 阿糖胞苷对K562细胞血红蛋白化的增强作用 蛋白质合成抑制剂对细胞的影响将通过以下方式进行研究： 确定是否，1）可检测的血红蛋白化的增加的比例， 细胞是由于Hb积累水平的增加，或者是由于 将细胞募集到红细胞程序中，2）观察到的差异 放线菌酮（CHX）和嘌呤霉素（PM）作为增效剂之间的 分化是由于这些药物的不同作用机制， 或由于蛋白质合成的不同程度的抑制，以及3） CHX减少该系统中的细胞凋亡。 收购这一新 知识将增加对造血控制的理解 并且应该导致补充细胞毒性药物治疗的能力 人类白血病

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.