MECHANISMS OF ERYTHRODIFFERENTIATION OF LEUKEMIC CELLS

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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.