The expression of ganglioside GD3, which plays crucial roles in normal brain development, decreases in adults but is upregulated in neoplastic cells, where it regulates tumor invasion and survival. Normally a buildup of GD3 induces apoptosis, but this does not occur in gliomas due to formation of 9-O-acetyl GD3 by the addition of an acetyl group to the terminal sialic acid of GD3; this renders GD3 unable to induce apoptosis. Using human biopsy-derived glioblastoma cell cultures, we have carried out a series of molecular manipulations targeting GD3 acetylation pathways. Using immunocytochemistry, flow cytometry, western blotting, and transwell assays, we have shown the existence of a critical ratio between GD3 and 9-O-acetyl GD3, which promotes tumor survival. Thus, we have demonstrated for the first time in primary glioblastoma that cleaving the acetyl group restores GD3, resulting in a reduction in tumor cell viability while normal astrocytes remain unaffected. Additionally, we have shown that glioblastoma viability is reduced due to the induction of mitochondrially mediated apoptosis and that this occurs after mitochondrial membrane depolarization. Three methods of cleaving the acetyl group using hemagglutinin esterase were investigated, and we have shown that the baculovirus vector transduces glioma cells as well as normal astroctyes with a relatively high efficacy. A recombinant baculovirus containing hemagglutinin esterase could be developed for the clinic as an adjuvant therapy for glioma.

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INTRODUCTION: Recent evidence suggests that glioblastoma is driven by a subset of tumor initiating (TI) cells characterized by their capacity to form tumors in xenograft models and self-renew in vitro. These TI cells share many properties of neural stem/progenitor cells, including the expression of certain cell surface markers. With serial passage, many cells lose their capacity to TI. The transition between TI-proficient and -deficient states remains poorly understood. METHODS AND RESULTS: There are two theoretic models for the maintenance of TI states. In the "elite" model, TI activity is restricted to a predetermined subpopulation of cells. The alternative "stochastic" model suggests that any tumor cell has a finite chance of acquiring TI capacity through random fluctuations in cell physiology. To address this issue, we examined the TI capacity of distinct subclones isolated from a distinct glioblastoma line as well as the TI capacity of single cells derived from each distinct clone. We found that only a subset of subclones from a single glioblastoma line displayed capacity for TI, suggestive of the elite model. However, single cells derived from any single subclone exhibited a wide range of TI capacity, suggesting a stochastic component to this process. Transcriptome profiling of the subclones of differing TI revealed a gene signature associated with TI capacity. Analysis of this signature showed enrichment for genes regulated by c-Myc. Indeed, clones with increased TI capacity tend to harbor increased c-Myc expression. Additionally, over-expression of c-myc increased the TI capacity of glioblastoma cells in xenograft models and led to the formation of intracranial tumor in an Ink4a/ARF null transgenic murine model. CONCLUSION: Our results are most consistent with a threshold model in which TI states in glioblastomas are driven by expression levels of critical factors such as c-Myc.