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c-myc and c-myb Oncoproteins during Induced Maturation of Myeloid and Erythroid Human Leukemic Cell Lines

LRF Preleukaemia Unit, Department of Haematology, University of Wales College of Medicine, Cardiff, CF4 4XN [P. P., M. A. B., J. F., T. G. H., A. J.], and Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, Dominion House, Bartholomew Close, London, EC1A 7BE [R. W.], United Kingdom

c-myc and c-myb mRNAs have been found to be tightly regulated during hemopoietic differentiation. We have studied nuclear c-myc and c-myb oncoproteins through the cell cycle, during macrophage, granulocyte, erythroid, and megakaryocytic differentiation of KG1, HL60, and HEL cells. p62^c-myc and p75^c-myb content of propidium iodide-stained nuclei was quantitated by flow cytometry using fluoresceinated antibodies CT14-G4 and MB4.3, respectively. In uninduced cells p62^c-myc content is highest in HL60, followed by HEL, then KG1, while p75^c-myb is highest in HEL, followed by HL60 and KG1. All lines showed a <2-fold increment in both oncoproteins over the cell cycle. Macrophage induction of KG1 and HL60 resulted in early increase in both oncoproteins, followed by a decline to less than starting values by 48 h, concurrent with a reduction of S phase cells and the appearance of adherent -naphthyl acetate esterase-positive cells. p62^c-myc changes were more pronounced in HL60 and p75^c-myb changes in KG1. Different patterns of oncoprotein expression were found when different inducing agents were used for granulocyte differentiation of HL60. Under all conditions, however, both oncoproteins declined to basal levels before granulocyte maturity. Hemin-induced erythroid differentiation of HEL to hemoglobin-containing cells resulted in biphasic p62^c-myc and p75^c-myb kinetics. In contrast, dimethyl sulfoxide-induced megakaryocytic differentiation of HEL was accompanied by an early and steady decline in both oncoproteins. Despite considerable reduction in oncoprotein levels, HEL cells were still actively cycling at 120 h.

It appears that c-myc and c-myb proteins decline with differentiation, well before proliferation ceases in some lineages. The kinetics of the decline differ between the two oncogenes and vary with the lineage induced and the nature of the inducing agent used. The cell cycle distribution of the oncoproteins does not change during maturation. These data suggest disparate roles for c-myc versus c-myb during hemopoietic differentiation and the existence of multiple signal transduction pathways for down-regulation of these genes.

¹ To whom requests for reprints should be addressed.

Received 2/ 8/89. Revised 6/26/89. Revised 9/11/89. Accepted 9/19/89.