Cell surface membrane protein changes during the differentiation of cultured human promyelocytic leukemia HL-60 cells

The human promyelocytic leukemia cell line HL-60 was induced to differentiate in vitro by treatment with dimethyl sulfoxide or retinoic acid. Morphological maturation was accompanied by a total loss of transferrin binding and a 7-fold increase in the percentage of cells reducing nitro blue tetrazolium. Cell surface membrane proteins and glycoproteins were labeled with 125I by the lactoperoxidase-H2O2 or 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodo-Gen) methods and analyzed by two-dimensional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A minimum of 12 cell surface proteins were unchanged, 3 proteins (Mr 95,000, 87,000, and 77,000) were lost, and up to 7 new proteins (Mr 270,000, 240,000, 150,000, 135,000, 58,000, 56,000, and 50,000) appeared during HL-60 cell differentiation. The kinetics of disappearance of one major labeled cell surface protein (Mr 95,000) within two days during treatment with retinoic acid correlated with the loss of cellular transferrin binding. This protein was identified as the transferrin receptor by affinity absorption of extracts of 125I-surface protein-labeled cells to transferrin-Sepharose beads. The affinity-purified component had molecular weights of 190,000 and 95,000 under nonreducing and reducing conditions, respectively, confirming its dimeric structure. Two-dimensional electrophoresis of cell surface membrane-labeled proteins of normal human granulocytes confirmed the absence of the transferrin receptor and identified cell surface proteins with molecular weight and pI values corresponding to three of the new cell surface proteins which appeared during HL-60 maturation. The most intensely labeled of these had a molecular weight of about 55,000, and was confirmed as being identical to the corresponding Mr 58,000 HL-60 cell surface membrane protein by one-dimensional peptide-mapping analysis. This prominent new Mr 55,000 to 58,000 protein increased continuously throughout retinoic acid-induced maturation and was identified as a major terminal myeloid differentiation cell surface membrane protein.