High expression levels of the EEN gene in leukemic cells with AML1-ETO. (A) RT-PCR analyses of EEN and CAF1 mRNA in leukemic cell lines. Myeloid lineages: Kasumi-1, HL60, U937, and NB4; lymphoid lineages: Jurkat, Namalwa, and Raji. (B) RT-PCR (top 6 rows) and Western blot analyses (bottom 4 rows) of AML1-ETO, EEN, Sp1, and CAF1 in the Ponasterone A (PA)–inducible, AML1-ETO–expressing U937 cell line. Cells were treated with 5 μM Ponasterone A to induce AML1-ETO expression. A 2-fold increase of EEN expression was observed 48 hours after PA treatment, in contrast to Sp1, which showed no obvious change in expression level. (C) RT-PCR analysis (bottom) and quantitative real-time RT-PCR analysis (top) of EEN in leukemic patient samples with or without AML1-ETO. Each leukemia subtype included 8 cases. The EEN-expression level in M2 t(8;21)+ was significantly higher than the other 3 subtypes without t(8;21) (P < .01), whereas no difference was detected between patients negative for t(8;21) (P > .1). Each column represents the mean ± SD in 8 cases. AML indicates acute myeloid leukemia; APL, acute promyelocytic leukemia; CML, chronic myeloid leukemia.