Effect of doublecortin on self-renewal and differentiation in brain tumor stem cells.

Abstract

Analysis of microarray probe data from glioma patient samples, in conjunction with patient Kaplan-Meier survival plots, indicates that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, time-lapse microscopic video recording, and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSC) in human primary glioma (YU-PG, HF66) cells from surgically removed human glioma specimens and U87 cells in vitro and in vivo. Time-lapse microscopic video recording revealed that double transfection of YU-PG, HF66, and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, that is, endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10 nM) treatment of DCX(+) neurabin II(+) BTSC from YU-PG, HF66, and U87 cells induced terminal differentiation into neuron-like cells. dUTP nick end labeling data indicated that JNK1 activation also induced apoptosis only in double transfected BTSC with DCX and neurabin II, but not in single transfected BTSC from YU-PG, HF66, and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66, and U87 BTSC. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data show that DCX synthesis induces apoptosis in BTSC through a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway.