LasBD suppresses b3a2-mRNA and p210^BCR/ABL expression and restores normal function. MO7e^p210 or 32D^p210 cells were transduced with LasBD and cultured in IMDM+ 0.25 μmol/L MTX + rHuIL-3 or rMuIL-3 (Bulk) for 14 days. In addition, 1 × 10³ transduced cells were cultured in wells of a 48-well plate in IMDM+ 0.25 μmol/L MTX + IL-3 (clones 32D^p210 A and TH and MO7e^p210 A and E). (A) Molecular analysis of antisense and BCR/ABL expression: MTX-selected LasBD-32D^p210 and LasBD-M07e^p210 cells and untransduced cells were analyzed by semiquantitative RT-PCR to detect AS mRNA, BCR/ABL mRNA, and β-actin mRNA and by Western blot to detect p210^BCR/ABL and p145^ABL protein, as described in the Materials and Methods. Expression of AS-mRNA and BCR/ABL mRNA as well as p210^BCR/ABL protein for untransduced cells (indicated with a minus sign), bulk-selected (indicated as Bulk) and clonal cell populations (for 32D^p210 cells, Th and A; for M07e^p210 cells, E and A) are shown. For Western blot analysis, the bands representing the p145^ABL protein and the p210^BCR/ABL protein are indicated with an arrow. Equal loading of the gels is shown by the presence of the p145^ABL protein. (B) Effect of LasBD on growth in the presence and absence of IL-3. (Upper panels) Untransduced 32D^p210 and MO7e^p210 cells and transduced, bulk-selected, or cloned 32D^p210 and MO7e^p210 cells cultured for 6 days in IL-3–containing medium. On days 1, 3, 5, and 6, the number of viable cells was determined by enumerating cells stained with trypan blue in a hemocytometer. Total viable cell expansion is depicted. Bulk-selected LasBD-32D^p210 and LasBD-MO7e^p210 cells and clones 32D^p210-Th and MO7e^p210-A expanded significantly less than untransduced 32D^p210 and MO7e^p210 cells or clones that continued to express BCR/ABL mRNA and protein (32D^p210-A and MO7e^p210-E cells). (Lower panels) Untransduced 32D^p210 and MO7e^p210 cells and transduced, bulk-selected, or cloned 32D^p210 and MO7e^p210 cells cultured for 6 days without IL-3. On days 1, 3, 5, and 6, the number of viable cells was determined by enumerating cells stained with trypan blue in a hemocytometer. The percentage of viable cells is depicted. All bulk-selected LasBD-32D^p210 cells and LasBD-MO7e^p210 cells and clones 32D^p210 -Th and MO7e^p210-A died by day 6, whereas a significant proportion of untransduced 32D^p210 and MO7e^p210 cells or clones that continued to express BCR/ABL mRNA and protein (32D^p210-A and MO7e^p210-E cells) survived even in the absence of IL-3. (C) Untransduced 32D^p210 cells and transduced, bulk-selected, or cloned 32D^p210 cells cultured for 6 days without IL-3 (BCL-2, BAX, and p53) or with IL-3 (C-MYC). After 3 days, cells were recovered and protein extracts were subjected to Western analysis for expression of p145^ABL, C-MYC, BCL-2, BAX, and p53. Decreased cell expansion of bulk-selected LasBD-32D^p210 cells and clone LasBD-32D^p210-Th that express only low levels of p210^BCR/ABL in the presence of IL-3 was associated with decreased expression of C-MYC (upper panel). Cell death of bulk-selected LasBD-32D^p210 cells and clone LasBD-32D^p210-Th that express only low levels of p210^BCR/ABL was associated with increased levels of p53 and BAX and decreased levels of BCL-2 (lower panel).