ETO AND AML1 PHOSPHOPROTEINS ARE EXPRESSED IN CD34(- IMPLICATIONS FORT(8-21) LEUKEMOGENESIS AND MONITORING RESIDUAL DISEASE() HEMATOPOIETIC PROGENITORS )

To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ETO protein in normal hematopo iesis and in leukemia, we raised rabbit antisera to a bacterially expr essed polypeptide containing amino acid residues 1 to 220 of ETO and t o synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross- reactivity with other members of the PEBP2 alpha family. With affinity -purified reagents, we observed immunofluorescent staining for both AM L1 and ETO in the nucleus of HEL, K562, and Kasumi-1 leukemic cell lin es, the last from a t(8;21) AML. Biochemical analysis confirmed specif icity of the antibodies and the nuclear localization of the antigens, the latter being exclusive for AML1 and primary for ETO. Immunoprecipi tations of metabolically labeled P-32-proteins from Kasumi-1 cells sho w that AML1 and ETO are phosphorylated on serine and threonine. Invest igations with normal bone marrow reveal AML1 and ETO are coexpressed i n megakaryocytes and that each is expressed in a portion of the simila r to 10-mu m-diameter cells residing there. Using a CD34(+) enriched p opulation mobilized to peripheral blood, we found AML1 and, unexpected ly, ETO present in these cells. Because of this, we conclude that the expression of ETO in hematopoietic cells is not by itself leukemogenic . Also, because ETO would not be exclusively expressed as part of chim eric AML1/ETO in leukemic patients, its presence cannot be used to mon itor t(8;21) AML residual disease. (C) 1996 by The American Society of Hematology.