Mechanism for the induction of cell death in ONS-76 medulloblastoma cells by Zhangfei/CREB-ZF

Cells from medulloblastoma lines do not contain detectable amounts of the basic leucine-zipper protein Zhangfei. However, we have previously shown that expression of this protein in cells of the ONS-76 and UW228 medulloblastoma lines causes the cells to stop growing and develop processes that resemble neurites. Our objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. We infected ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control protein LacZ and then compared the following parameters in Zhangfei and LacZ-expressing cells: (a) markers of apoptosis, autophagy and macropinocytosis, (b) transcripts for genes involved in neurogenesis and apoptosis, (c) phosphorylation of peptide targets of selected cellular protein kinases, and (d) activation of transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. Within our analysis, patterns of gene expression and phosphorylation-mediated signal transduction activity in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active. In addition, we found that the transcription factor Brn3a as well as factors implicated in differentiation were also active in Zhangfei-expressing cells. We tested the hypothesis that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then engages NGF in an autocrine manner triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells-a process that eventually brings about cell death. We showed that: (a) Zhangfei could enhance transcription from the isolated Brn3a promoter, (b) ONS-76 cells produced NGF and (c) antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells.