Growth inhibition of transformed fibroblasts and primary bone marrow transduced with NPM-ALK by PI 3-kinase inhibitors.
(A) Rat-1 soft agar assay of NPM-ALK–transfected cells. Rat-1 cells were retrovirally transduced with either empty vector or NPM-ALK as described in the “Materials and methods” section. After 2 days, cells were selected for growth in soft agar medium. Colonies were scored after 12 days. Numbers of colonies represent means ± SD from triplicate experiments. (B) Primary murine bone marrow cells infected with NPM-ALK (upper 2 panels) or control-vector (lower 2 panels) were plated in methyl-cellulose without growth factors (upper 2 panels) or in the presence of 10 pg/mL IL-3 (lower 2 panels). Ten micromolars Ly290004 was added once were indicated. Representative pictures obtained after 10 days of culture from 2 independent experiments are shown. Absolute numbers of colonies are listed in the table. Data represent mean ± SD of 2 independent experiments. (C) Effect of PI3-kinase inhibitor wortmannin on growth of NPM-ALK–infected murine bone marrow (BM) cells in liquid culture. The 1 × 105 BM cells retrovirally transduced with empty vector (∅) or NPM-ALK were plated in BM-medium with or without 100 pg/mL mIL-3 as indicated. Wortmannin (200 nmol/L) was added daily to half of the wells (▪); ■, no wortmannin. The bars represent the percentage of cells present in the supernatant after 6 days of culture and the number of cells infected with control vector and supplemented with IL-3 was arbitrarily set as 100%. Data represent means ± SD of at least 2 independent experiments.
