Glioma genetic profiling: The role of DNA repair and telomerase

Brain cancers inflict a disproportional burden of mortality upon sufferers. Due to the difficulties of diagnosis and treatment, survival is low with most patients succumbing to the disease within a year of diagnosis. This has been the imperative factor to underpin some of the molecular mechanisms regulating gliomas and to aid with the development of novel disease biomarkers specifically targeting immortalisation (telomerase). 1[mmortalisation not only requires telomerase but also an imbalance/inactivation of DNA repair functions such as 06 methylguanine-DNAmethyltransferase (MGMI) which is a major obstacle with regards to glioma chemotherapy. The primary focus of this study was to evaluate whether there was an association betweenh TERT and its many subunits: TEPI (human telomerase-associated protein 1), TNKS ( tankyrase), DCKI (dyskerin) and PARPI (poly (ADP-ribose) polymerase1 ) in human glioma compared to normal brain tissues and cell lines. Evidence suggests that hTERT was the only gene that was transcribed in all the glioma cell lines and tissues, while absent in the normal. Thus hTERT may represent a simple but reliable biological marker for distinguishing glioma tissues from normal. The hTERT gene is subjected to its own highly coordinated regulation in normal and cancer cells however, whether hTERT is regulated via methylation/demethylation in glioma is unknown. 5azadC treatment reduced hTERT expression, which resulted in the downregulation of telomerase protein and its activity. However, an inverse correlation between hTERT and MGMT expression was observed after treatment. Evidence shows that hTERT inactivation enhances sensitivity towards some chemotherapeutic agents, thus a combination of 5azadC with various chemotherapeutic agents proved to be more effective than chemotherapy administered on its own. Although 5azadC treatment activates MGMT and subsequently hyposensitises glioma cells toward alkylating agents, specifically TMZ (temozolomide), it may compensate by offering telomerase sensitised cells, thus providing an alternative avenue of therapy for the 50% of the glioma patients chosen for this study that have an unmethylated MGMT promoter. However, further clinical developments concerning this approach will be required. 5azadC is known for its toxicity and its effects are diverse, thus it is not favoured as a therapeutic agent. To address these issues three DNMTI (methylation gene) siRNAs were used, downregulating DNMTI directly and hTERT indirectly (possibly via the promoter methylation inhibition mechanism). Combining siRNA and chemotherapeutic agents (TMZ or taxol) enhanced the effects of both the drugs, thus offering an alternative method of treatment using lower concentrations of the drug and hence, reducing side effects, and improving the life expectancy of glioma patients. All glioma tissues used in this study, irrespective of grade or invasiveness, transcribed hTERT at approximately similar copy numbers however, only some of these tissues translated this to protein. This could be due to hTERT mRNA splicing or the involvement of other regulating factors. Several studies have reported that protein translation decreases with age, this would explain why only two of the older patient samples had detectable telomerase activity when they all expressed hTERT mRNA. However, additional studies using a larger cohort of glioma tissues would be needed to further understand the precise mechanism for the discrepancy in hTERT translation in the older glioma patients. Four novel sets of investigations were documented in this thesis resulting in publications: a) 77VKS expression was evaluated in glioma tissues for the first time, b) the effects of the demethylating agent 5-aza-2'-deoxycytidine (5azadC) on the expression of hTERT and MGMTin glioma cells was evaluated, C) combining DNMTI siRNA and chemotherapeutic agents (TMZ or taxol) enhancedth e efficacy of the chemotherapeutiacgents and, finally d) although, h TERT transcription was found in all glioma tissues used in this study irrespective of grade or invasiveness (at similar copy numbers), a discrepancy in translation was documented. Some of the finding from this study may well become the starting point for integrating translational research in to future clinical trial designs for cancer, specifically for glioma patients