Characterization of glioma initiating cells and its role in radiation response

Lim, Yi Chieh (2012). Characterization of glioma initiating cells and its role in radiation response PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Lim, Yi Chieh
Thesis Title Characterization of glioma initiating cells and its role in radiation response
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Martin Lavin
Tara Roberts
David Walker
Total pages 237
Total colour pages 47
Total black and white pages 190
Language eng
Subjects 111208 Radiation Therapy
060111 Signal Transduction
111201 Cancer Cell Biology
Formatted abstract Glioma is the most frequent primary brain tumour and accounts for approximately 80% of all diagnosed cases. Amongst the different grades of glioma set by the World Health Organization, Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most malignant brain tumour. What differentiates GBM from other forms of brain cancer is its rapid progression and lethality, resulting in a relatively short survival time of 12 to 15 months. Surgical excision followed by radiotherapy increases survival, however patients still fall victim to the recurrent brain tumour. This dismal outcome highlights the need for new approaches to treat GBM. A new avenue has been unveiled with the discovery of cancer stem cells (CSCs) in solid tumours. CSCs ability to self-renew and differentiate may explain the recurrence of disease. Molecular studies have also demonstrated that these cells are highly resistant even to the most effective form of therapeutic treatment, ionizing radiation (IR). Unfortunately, no mechanistic pathways have been implicated in their enhanced DNA repair capability.

Using glioma initiating cells (GICs) as a CSC model, this study first demonstrates features of stem cell as characterized by their non-tumourgenic counterpart - neural progenitor cell (NPC). Depending on the mitogen concentration in vitro, GICs are able to self-renewal or differentiate into various lineages. Most importantly, when engrafted into animal models, tumours with GBM-like phenotypes are formed.

In terms of radiotherapy responses, the role of DNA double strand break (DSB) repair pathways during cell-cycle progression was explored. Initial analysis showed that the DNA damage response (DDR) machinery in GICs was ineffective at activating G1/S cell-cycle arrest thereby limiting the use of the non-homologous end joining repair (NHEJ) pathway. Mainly because of an attenuated Chk2 phosphorylation after IR-induced damage, GICs circumvented checkpoint arrest and progressed directly into S-phase. Complementary data from BrdU pulse-labeling and cell-cycle analysis confirmed continuous DNA synthesis after IR treatment, hence supporting the hypothesis that GICs transited rapidly into S-phase. NPCs on the other hand, were capable of activating the DDR machinery to initiate G1/S cell-cycle arrest followed by the more rapid NHEJ repair. DSB repair, as determined by the disappearance of γH2AX foci confirmed slow repair kinetics of GICs. With the examination of RAD51 and BRCA1 foci, GICs showed inclined homologous recombination (HR). Additional data from the analysis of an integrated pDR-GFP excised by I-Sce I endonuclease to generate a single DSB, demonstrated GICs have several fold higher in HR repair than NPC.

By targeting the ataxia-telangiectasia mutated (ATM) protein activity with the use of a specific kinase inhibitor, downstream HR repair was inactivated. Delays in resolving γH2AX foci were noted also in GICs. As NHEJ is the dominant pathway for repair in NPCs, they were able to compensate from IR-induced DNA damage and maintain better survival. HR-dependent GICs had limited radioresistance following ATM inhibitor treatment with survival profile being similar to the NPCs. Complementing this finding, DNA-PKcs inhibitor which impedes specifically NHEJ and not HR was also used to treat GIC and NPC. As expected, inhibition of the NHEJ pathway increased apoptosis in NPC but not in GIC.

Apart from DDR initiating downstream DNA DSB repair, several GIC lines in our study also showed induction of Interleukin-6 (IL-6) expression after IR. Presence of IL-6 alone increases the rate of proliferation of GICs. This includes better survival following IR, albeit to different degrees. On the other hand, IL-6 acts differently on NPC where increases in neither proliferation nor radioresistance were observed. As DDR is central to IL-6 activation, inhibition of the ATM kinase activity prevented the cytokine from conferring improved survival on GIC. It is clear from this investigation, non-DNA repair pathways are also involved in the radioresistant properties of GIC, and through ATM inhibition, both DNA DSB repair and proliferation can be impeded. This study supports targeting the central DDR machinery, particularly ATM as a potential therapeutic approach in prolonging patients’ survival and reduces the rate of tumour recurrence.
Keyword DNA damage response
Glioma initiating cells
Neural progenitor cells

 
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Created: Tue, 30 Oct 2012, 09:19:58 EST by Ernie Lim on behalf of Scholarly Publishing and Digitisation Service