Identification and functional prediction of mitochondrial complex III and IV mutations associated with glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults, with a dismal prognosis. Treatment is hampered by GBM's unique biology, including differential cell response to therapy. Although several mitochondrial abnormalities have been identified, how mitochondrial DNA (mtDNA) mutations contribute to GBM biology and therapeutic response remains poorly described. We sought to determine the spectrum of functional complex III and IV mtDNA mutations in GBM. METHODS: The complete mitochondrial genomes of 10 GBM cell lines were obtained using next-generation sequencing and combined with another set obtained from 32 GBM tissues. Three-dimensional structural mapping and analysis of all the nonsynonymous mutations identified in complex III and IV proteins was then performed to investigate functional importance. RESULTS: Over 200 mutations were identified in the mtDNAs, including a significant proportion with very low mutational loads. Twenty-five were nonsynonymous mutations in complex III and IV, 9 of which were predicted to be functional and affect mitochondrial respiratory chain activity. Most of the functional candidates were GBM specific and not found in the general population, and 2 were present in the germ-line. Patient-specific maps reveal that 43% of tumors carry at least one functional candidate. CONCLUSIONS: We reveal that the spectrum of GBM-associated mtDNA mutations is wider than previously thought, as well as novel structural-functional links between specific mtDNA mutations, abnormal mitochondria, and the biology of GBM. These results could provide tangible new prognostic indicators as well as targets with which to guide the development of patient-specific mitochondrially mediated chemotherapeutic approaches.

Dual targeting NG2 and GD3A using Mab-Zap immunotoxin results in reduced glioma cell viability in vitro.

BACKGROUND: Effective treatments for glioblastoma multiforme (GBM) are lacking due, in part, to cellular heterogeneity. Consequently, single-target therapeutic strategies are unlikely to succeed. Simultaneous targeting of different neoplastic cell populations within the same tumour may, therefore, prove of value. Neuron-glia 2 (NG2), a transmembrane chondroitin sulphate proteoglycan, present on developing glial cells, and GD3(A), a ganglioside expressed on developing migratory glia, are re-expressed in GBM. MATERIALS AND METHODS: The aims of this study were to conduct 'proof of concept' experiments in human GBM cell lines to show that proliferative high NG2-expressing cells and high GD3(A) -expressing migratory cells could be effectively ablated using a Mab-Zap saporin immunotoxin system. RESULTS: The combinatorial ablation of both NG2 and GD3(A)-expressing cells resulted in significant reduction in GBM cell viability compared to single epitope targeting and controls (p<0.0001); non-neoplastic astrocytes were not affected. CONCLUSION: Multiple targeting of GBM sub-populations may, therefore, help inform novel therapeutic approaches.

Expression of the chondroitin sulphate proteoglycan, NG2, in paediatric brain tumors.

BACKGROUND/AIM: While neuron-glia 2 (NG2) is well-characterized in the developing brain and in adult high-grade gliomas, little is known about NG2 expression in paediatric brain tumors. Here, NG2 expression was examined in a range of paediatric brain tumors. MATERIALS AND METHODS: A retrospective immunohistopathological analysis of 57 paediatric brain tumor biopsies of various tumor types was carried out. Paediatric cell lines, including two medulloblastomas and one dysembryoplastic neuroepithelial tumor, in addition to one adult high-grade glioma, were also assessed for NG2 expression. RESULTS: NG2-positive staining was seen in all dysembryoplastic neuroepithelial tumors (DNETs) examined; however, only two of the fourteen medulloblastomas examined were NG2-positive. Compared to adult glioma, there was a lack of NG2 staining in the vasculature of paediatric brain tumors. CONCLUSION: NG2 expression in paediatric brain tumors differs depending upon type and, unlike adult glioma, includes expression on lower-grade tumors.

Human, mouse or rat? Species authentication of glioma-derived cell cultures.

Cell culture and the use of cell lines are often fundamental requirements in basic scientific research. It is of the utmost importance for researchers to ensure that the cell lines in use have a well defined origin and are routinely re-analysed to highlight possible areas of contamination. In this preliminary study species specific primers were designed to easily distinguish between human, mouse and rat DNA with standard agarose gel electrophoresis. Inter-species contamination is often the most common form of contamination experienced, with the most common of cell lines in use being of human, mouse and rat derivation. A PCR-based assay was therefore developed to ensure an accurate, quick and cost effective means of determining any cell line contamination which could be easily executed on a routine basis. Furthermore, this simple PCR is able to identify the species in the inter-species mixture of DNA and therefore provides a valuable tool for the authentication of human cell lines.