Samantha C. Higgins

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.

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

Abstract Background Glioblastoma (GBM) is the most common primary brain tumor in adults, with a dismal prognosis. Treatment is hampered by GBMs 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.

Drug Repurposing to Circumvent Chemotherapy Resistance in Brain Tumours

The incidence of primary brain tumours in the UK is steadily rising; prognosis for this devastating disease is dismal and not significantly improving. The relative failure for effective therapy may be attributed to heterogeneity with over 120 histological entities. Additionally, the biology is incredibly complex with numerous pathways and cascades working simultaneously or in harmonisation. Moreover, we find that as the tumour evolves, it commandeers alternative pathways for its survival. At its most aggressive form, glioblastoma produces guerrilla cells that trek into the normal brain parenchyma where they are protected by the intact blood-brain barrier (B-BB). The majority of drugs cannot cross the intact B-BB; the dosage required to cross the damaged area around the tumour would be toxic systemically. Effective therapy, therefore, has a number of hurdles against chemotherapy resistance. In this chapter we look at the tumour biology and conventional therapeutic resistance; we then look at the potential of using drugs originally used to treat other diseases. Indeed, repurposed drugs are drawing increasingly more interest, especially as after approximately ten years of development; the new drug failure rate of 25,000:1 costs the industry an astonishing fortune of around

The in vitro effects of tricyclic drugs and dexamethasone on cellular respiration of malignant glioma

2.6 billion per year. Here, we refer to UK regulations and doctor trepidation for prescribing outside of the drug’s licence—‘off-label’—and give examples of some of the repurposed agents which have gained attention within the laboratory and within our current legislation require additional clinical trials for marketing authorisation and prescription without fear of potential litigation.