Constitutive activation of STAT5 mediates growth factor independence and increased in vitro and in vivo.

(A) 5 × 10⁵ Ba/F3 tet-on cells, inducibly expressing either p210^Bcr/Abl or wild-type STAT5 (STAT5-WT) or the constitutive active mutant STAT5-1*6 were cultured in RPMI 1640 + 10% FCS with or without doxycycline, as indicated. The number of cells was counted by trypan blue exclusion at the indicated time points. (B) Ton.B.210 and Ton.B.1*6 cells were cultured for 30 hours in RPMI 1640 + 10% FCS with or without doxycycline. 1 × 10⁶ cells were harvested and used for an annexin-V–PI staining procedure, and analyzed as described in “Material and methods.” Results are represented as the percentage of apoptotic cells. (C) 5 × 10⁶ Ton.B.STAT5 or Ton.B.1*6 cells were intraveneously injected into nude mice. For each cell line, the mice were split into 2 groups (5 mice in each group), receiving regular water or water supplemented with 500 μg/mL doxycycline. Mice were killed after 6 weeks, blood smears were prepared and stained with Wright-Giemsa stain, and spleen and lymph nodes were sectioned and smears stained with hematoxylin and eosin. Here are shown blood smears and a lymph node section from Ton.B.1*6 injected mice, treated (pictures 2 and 4) or not (pictures 1 and 3) with doxycycline. Magnification: 60 ×.