BMI1 expression in CML as assessed by Q-RT/PCR. (A) BMI1 expression in CD34⁺ immunomagnetically selected hematopoietic progenitors from CML patients at diagnosis in chronic phase (CP) compared with patients in more advanced disease stage (acceleration phase and blast crisis). The definition of CP and advanced phases (accelerated phase and blast crisis) was based on previously established criteria^2,11,12 : CP, less than 10% blasts; accelerated phase 10% to 30% blasts or less than 10% blasts with clonal evolution; and blast crisis, more than 30% blasts. Bone marrow (BM)-derived CD34⁺ cells from healthy donors and G-CSF-mobilized CD34⁺ stem cells (PBSCs) from non-CML donors were used as controls. As previously described, G-CSF-mobilized CD34⁺ PBSCs express high levels of BMI1 compared with nonstimulated normal cells.¹⁰  (B) BMI1 expression in total unfractionated PBMCs from CML patients at diagnosis in CP compared with patients in more advanced disease stage (accelerated phase and blast crisis). Total PBMCs from healthy donors were used as controls. (C) E2F1 expression in total unfractionated PBMCs from CML patients at diagnosis in CP compared with patients in more advanced disease stage (accelerated phase and blast crisis). Total PBMCs from healthy donors were used as controls. Horizontal bars denote the medians. Values of genes represent the Q-RT/PCR expression as a ratio of the gene of interest to the GAPDH control gene. For establishment of the Q-RT/PCR assay, the Jurkat and HeLa cell lines were used as positive controls for BMI1 and E2F1 expression, respectively, with a standard curve being generated for the amplification of logarithmic dilutions (10⁻¹ to 10⁻⁵) of their cDNAs. An average of the duplicates of each data point was taken and plotted against the cycle threshold (Ct). The technical variability between duplicate samples in our RT/PCR assays has been established for a number of different genes as less than 1.3-fold at the 95% level of confidence (data not shown).