Doublecortin (DCX) is one of the three genes found from Affymetrix gene chip analysis related to glioma patient survival. Two other genes (e.g., osteonectin and semaphorin 3B) are well characterized as antioncogenic and tumor suppressor genes. However, there is no report about the involvement of DCX in cancer. Here, we show that gene transfer technology into DCX-deficient glioblastoma cell lines, such as A172, U87, U251N, RG2, and 9L, with DCX cDNA significantly suppressed growth of these glioma cells. U87 cells with ectopic expression of DCX exhibit a marked suppression of the transformed phenotype as growth arrested in the G 2 phase of the cell cycle progression, small colony formation in soft agar, and no tumor formation in nude rats. This transformed phenotype can be restored by knocking down DCX expression with DCX small interfering RNA. DCX was highly phosphorylated in glioma cells. Phosphorylation in the glioma cells was greater than in noncancer cells such as mouse NIH 3T3 and human embryonic kidney 293T cells. Coimmunoprecipitation of the phosphorylated DCX and spinophilin/neurabin II from DCX-synthesizing glioma cells indicated their interaction. This interaction would lead to a block of anchorage-independent growth as neurabin II is a synergistic inhibitor of anchorage-independent growth with p14ARF (ARF). Interaction between phosphorylated DCX and neurabin II may induce the association of the protein phosphatase 1 catalytic subunit (PP1) with neurabin II and inactivate PP1 and block mitosis during G 2 and M phases of the cell cycle progression. Thus, DCX seems to be a tumor suppressor of glioma. (Cancer Res 2006; 66(24): 11726-35)

Background: Thymosin ␤4 (T␤4) promotes differentiation of oligoprogenitor cells (OPCs) to oligodendrocytes in animal models of neurological injury. Results: T␤4 increased expression of microRNA-146a and suppressed expression of TLR (Toll-like) proinflammatory cytokines. Conclusion: T␤4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a to promote OPC differentiation. Signficance: Learning how T␤4 promotes oligodendrogenesis supports its development for clinical studies.

Doublecortin (DCX) is a microtubule (MT) binding protein that induces growth arrest at the G2-M phase of cell cycle in glioma and suppresses tumor xenograft in immunocompromised hosts. DCX expression was found in neuronal cells, but lacking in glioma cells. We tested the hypothesis that DCX inhibits glioma U87 cell mitosis and invasion. Our data showed that DCX synthesizing U87 cells underwent mitotic MT spindle catastrophe in a neurabin II dependent pathway. Synthesis of both DCX and neurabin II were required to induce apoptosis in U87 and human embryonic kidney 293T cells. In DCX expressing U87 cells, association of phosphorylated DCX with protein phosphatase-1 (PP1) in the cytosol disrupted the interaction between kinesin-13 and PP1 in the nucleus and yielded spontaneously active kinesin-13. The activated kinesin-13 caused mitotic MT catastrophe in spindle checkpoint. Phosphorylated-DCX induced depolymerization of actin filaments in U87 cells, down-regulated matrix metalloproteinases-2 and -9, and inhibited glioma U87 cell invasion in a neurabin II dependent pathway. Thus, localization of the DCX-neurabin II-PP1 complex in the cytosol of U87 tumor cells inhibited PP1 phosphatase activities leading to antiglioma effects via (1) mitotic MT spindle catastrophe that blocks mitosis and (2) depolymerization of actin that inhibits glioma cell invasion.

Thymosin beta 4 (Tβ4), a G-actin sequestering peptide, increases oligodendrogenesis and improves functional outcome in models of neurological injury. The molecular mechanisms of Tβ4 mediated oligodendrogenesis are unclear. The p38 mitogen-activated protein kinase (p38MAPK) regulates oligodendrocyte (OL) differentiation and myelin gene expression in other models. Therefore, we investigated p38MAPK signaling pathways. We used primary rat neural progenitor cells (NPCs) and a mouse oligodendrocyte progenitor cell (OPC) line (N20.1 cells) to investigate the molecular mechanisms of Tβ4-enhanced oligodendrogenesis. NPCs were isolated from rat subventricular zone (SVZ) of the lateral ventricles (n=12). Primary NPCs and N20.1 cells were grown in the presence of 0, 25 and 50 ng/ml of Tβ4 (RegeneRx Biopharmaceuticals Inc, Rockville, MD) for 14 days. Quantitative real-time PCR and Western blot data showed significant induction of both expression and phosphorylation of p38MAPK with simultaneous inhibition of phosphorylation of extracellular signal regulated kinase (ERK1), c-Jun N-terminal kinase 1 (JNK1), leading to reduction of phosphorylation of c-Jun, a potent negative regulator of transcription of myelin genes. These effects were reversed with transfection of Tβ4siRNA. Our data indicate that Tβ4 treatment induces OL differentiation by inducing p38MAPK with parallel inactivation of ERK1 and JNK1, thus preventing the accumulation of phosphorylated c-Jun.

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