Mutation of the binding site for PI3K reduces factor-independent growth of MIHC-D816V.
(A) Factor-independent colony formation of MIHC-D816V/Y721F is reduced in comparison with MIHC-D816V. Parental MIHCs and MIHCs expressing wild-type c-Kit, Y721F c-Kit, D816V/Y721F c-Kit, or D816V c-Kit were tested for the ability to form colonies in methylcellulose culture in the presence (■) and absence (▪) of GM-CSF. Colonies comprising more than 50 cells were scored after 1 week. Data shown are means ± SEM derived from 2 independent experiments, each carried out in triplicate. (B) Factor-independent growth of MIHC-D816V/Y721F is reduced in liquid culture compared with MIHC-D816V. MIHCs infected with the indicated c-Kit mutant seeded at a density of 5 × 104/mL in media without exogenous growth factors on day 0 were assessed for the total number of cells on days 2, 4, and 6 by CellTitre Assay. Mean ± SEM is shown from a representative experiment of 3. Because of overgrowth, several points on day 6 are not shown in panels B and C. ♦ indicates parent; ●, wild-type Kit; ▴, Y721F Kit; ○, D816V/Y721F Kit; ■, D816V Kit. (C) GM-CSF–induced growth is not altered in MIHCs expressing c-Kit mutants. Proliferation assays were performed as described in panel B, except in the presence of murine GM-CSF.
