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Title: The pathogenesis and regulation of cell death in glioblastoma : experimental studies using glioma spheroid cultures
Author: Bell, Helen Susan
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Understanding the pathways by which endogenous cell death occurs in tumours may be of considerable value when identifying potential therapies. In glioblastoma, two main types of cell death are observed, apoptosis and necrosis. Although the regulatory mechanisms leading to necrotic and apoptotic morphologies are thought to vary widely, their close association in glioblastoma suggests they may share some regulatory function. High levels of Fas (APO-1), HIF-la and PARP found within perinecrotic tissue in vivo suggests regulatory factors do exist. The presence of these regulatory factors, sub-lethal stress and a high apoptotic index suggests the p53 pathway may be involved in the cell death response around these areas. In order to investigate cell death regulatory factors in vitro, particularly those relating to the p53 pathway, the glioma spheroid system was utilised. Glioma spheroids are known reproduce some of the regional heterogeneity found in vivo and they form areas of necrosis as the spheroids become larger and the central core of cells becomes metabolically compromised. Using spheroids derived from four glioma cell lines, the aims of this project were (i) to fully characterise the glioma spheroid system for the four glioma cell lines, U87, U373, MOG-GCCM and A172 and to establish that the system adequately reflects many of the features found in glioblastoma cell populations in vivo; (ii) to define the exact time point in spheroid growth when central cell death occurs and to identify the modes of cell death present (iii) to establish which p53-related proteins are associated with areas of cell death within stressed glioblastoma cell populations and to determine any correlations between this distribution and the genetic status of the cell lines; and finally, (iv) to ascertain whether modulation of the P53 status of the cells has an effect on the development of cell death and the expression of p53-related proteins within glioma spheroid cultures. Regions of proliferation, death and differentiation were first assessed using a variety of immunohistochemical and microscopical techniques. Spheroid growth and apoptotic index were also quantitatively recorded. Onset of central cell death was seen to occur over week 3 and these spheroids were examined using electron microscopy to establish the primary mode of cell death (apoptosis or necrosis). HIF-la expression was used as a marker to determine the metabolic status of spheroid central regions at this time point. The P53 genotype of the cell lines was then determined and the reactivity of p53, Bax, p21 and MDM2 in monolayer cultures of the 4 lines was assessed following exposure to hypoxia and free radical stress. Perinecrotic expression of p53, Bax, p21 and MDM2 was recorded in both week 3 spheroid cultures and in glioblastoma biopsy material. The cell lines were transfected with wild-type and dominant negative P53 transcripts to investigate the effect of increasing and decreasing levels of endogenous p53 on cell death susceptibility within 3-dimensional spheroid cultures. Cross-sections of large glioma spheroids appeared highly representative of GBM tissue, modeling the span from blood vessels to nutritionally compromised, necrotic tissue distinct from the vasculature. The primary cell death morphology observed was necrosis, followed by increases in perinecrotic apoptotic index. Increases in HIF-la expression coincided with the onset of necrosis. HIF-la, p53 and p21 accumulation were associated with U87 and A172 monolayer cultures (p53 wild-type), but not U373 and MOG-G-CCM monolayer cultures (.P53 mutant), following exposure to oxidative stress. No increases in p53, Bax and p21 expression were found within perinecrotic tissue in spheroid cultures derived from any of the cell lines. A 60kDa MDM2 isoform was found to be upregulated within perinecrotic tissue in both spheroids and in 80% of biopsy cases irrespective of P53 status. The addition of a wildtype P53 transcript to U87 cells did not effect cell death susceptibility within U87 spheroids. U373 cells transfected with wild-type P53 died 3 days post transfection. Cell death susceptibility was not altered in either U87 and U373 spheroids following transfection with dominant negative P53. In conclusion, in large 3-dimensional glioma spheroid cultures, necrosis is the primary cell death event, followed by increases in perinecrotic apoptotic index. Although increases in p53-related expression are observed in P53 wild-type lines in response to oxidative stresses, p53, Bax and p21 accumulation do not appear to be important for the regulation of cell death in spheroid cultures, irrespective of the P53 status of the cell lines. Although the TGdgene appears to be essentially redundant within perinecrotic tissue, high levels of 60kDa MDM2 may associate with other cell death regulatory factors thus significantly influencing cell death susceptibility in both P53 wild-type and mutant cell populations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available