Evi-1 is up-regulated in myeloid progenitor cells immortalized by MLL oncoproteins. (A) Murine c-Kit⁺ BM progenitor cells were retrovirally transduced with pMXs-neo-MLL-ENL, pMXs-neo-MLL-AF9, pMXs-neo-E2A-HLF, or pMYs-HoxA9-ires-Meis1. The expression level of Evi-1 in immortalized cells from the third to fourth round of serial replating in semisolid medium was quantified relative to BM MNCs with real-time polymerase chain reaction. Data are shown as mean ± SD. *P < .05. (B) Murine c-Kit⁺ BM progenitor cells were retrovirally transduced with leukemia oncogenes. Four types of myeloid leukemia genes cloned into MIG were retrovirally transduced into c-Kit⁺ BM progenitor cells. Forty-eight hours after initiation of retroviral transduction, GFP-positive cells were isolated and the expression level of Evi-1 was quantified relative to BM MNCs. Data are shown as mean ± SD. *P < .05. (C) Immortalization of c-Kit⁺ BM progenitor cells by MLL-ENL-ER is dependent on the presence of 4-OHT. Graph indicates the number of colonies, with SD, generated from 10⁴ pMXs-neo-MLL-ENL-ER–transduced BM cells in the presence (□) or absence (■) of 1μM 4-OHT at each round after retroviral transduction. The number of G418-resistant colonies obtained by transduction of MLL-ENL-ER into 10⁴ BM cells in the presence of 4-OHT is shown in the first round (). (D) Expression level of HoxA9 or Evi-1 in MLL-ENL-ER–transformed cells cultured with or without 1μM 4-OHT for 72 hours. The averages of the relative expression ratio of 4-OHT⁻ cells (■) to 4-OHT⁺ cells (□) are shown with SD. *P < .05.