IL-3 does not alter cellular sensitivity to STI-571 and fails to activate β-chain or Jak-2 tyrosine phosphorylation in bcr-abl–expressing cells.

(A) MBA cells were incubated with STI-571 in normal growth media at the dose level indicated or were pretreated and maintained in IL-3 (30 ng/mL) before STI-571 incubation. After 24 hours, cells were incubated with MTT reagent to estimate changes in cell growth and survival (Figure 1). Each point represents the average ± SEM of 4 determinations. In apoptosis studies, IL-3 did not alter STI-571–mediated caspase 3 activation in MBA cells (results not shown). Similar results were obtained in K562 cells. (B) Mo7e, MBA, K562, or KBM5 cells were cultured in media without exogenous cytokines for 8 hours and subsequently incubated with IL-3 (30 ng/mL) for 5 or 30 minutes before harvesting. β-Chain was immunoprecipitated from 150 μg protein lysate and immunoblotted for phosphotyrosine (top) or β-chain (bottom). (C) Mo7e (left) or MBA (right) cells were grown in the absence of cytokine (as described above) before the addition of IL-3 for 5 or 30 minutes. Jak-2 activation was determined in equal protein cell lysates (30 μg) by immunoblotting with phosphospecific Jak-2 (p-Jak-2), and relative Jak-2 protein levels were determined in stripped blots with anti–Jak-2. Similar results were obtained in immunoprecipitation/immunoblotting analysis of Jak-2 tyrosine phosphorylation (data not shown). Migration of the 130-kd Jak-2 protein is shown.