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Title: Tools development for the investigation of p53 in neuroblastoma
Author: Nelson, Rachel Helena
ISNI:       0000 0004 2704 8936
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2010
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The tumour suppressor p53 is a transcription factor which is mutated in over 50% of adult human cancers. However, it is rarely mutated in primary neuroblastoma, nor in a number of other childhood cancers. Neuroblastomais a cancer derived from precursor cells of the peripheral sympathetic nervous system, apparently occurring due to a lack of differentiation of neuronalcrest cells. It is the most commonsolid extra-cranial tumour in childhood. Due to the complexity of the activation and regulation of p53, the presence of a wild type genomic sequenceis not indicative of a fully functioning pathway. Mutations upstream of the p53 protein can result in functional inactivation of the protein, as well as mutations downstream resulting in a lack of p53 effects, including cell cycle arrest and apoptosis. Many chemotherapy treatments function by activating p53 via causing DNA damage. This project studied the effect of chemotherapy treatment on the functional status of p53 in a selection of neuroblastomacelllines. Wescreened neuroblastomacell lines for TP53 gene status and identified a genomic mutation at the end of intron 6 and the start of exon 7 of the SK-N-AScell line. This mutation resulted in loss of the wild type p53 protein and expression of a C-terminal truncated protein. The truncated form of the protein was transcriptionally inactive, displaying little or no increase in protein levels post-DNA damage, or in the mRNA of p53 target genes; mdm2, pumaorbax. Neuroblastoma cell lines SH-SY-SY, SHEP and SK-N-SH were determined to be TP53 wild type. Upstream mutations appeared unlikely, as an increase in p53 protein was observed post-DNA damage, together with an associated increase in downstream target gene mRNA.Effects downstream of target gene activation were notfully investigated, so it was not formally confirmed whethercell cycle arrest or apoptosis occurred. The existence of several p53 isoforms adds to the significant complexity of the p53 system. We studied a set of p53 isoforms that had already been partially characterised. The data was consistent with the hypothesis that these p53 isoforms functioned in a similar way to isoforms of the p53 family members, p63 and p73. Those isoforms have been shown to control preferential downstream target gene activation and can act as dominant negative inhibitors. Alterations in the expression profile of these isoforms might have tumourigenic effects. The RNA expression levels of the p53 isoforms were analysed in the neuroblastoma cell lines. Fluorescently conjugated p53 isoform expression vectors were also produced for characterisation oftheir cellular localisation. Due to the negative feedback loop that exists between p53 and its E3 ubiquitin ligase, MDM2,out-of-phase protein oscillations have been proposed and demonstrated to exist in this system. Theoscillations are thought to aid robust control of cell cycle arrest or apoptosis. We observed p53:MDM2oscillations at the single cell level using p53 and Mdm2 fluorescently conjugated vectors. To improve upon the previous p53 expression system,a fluorescently conjugated p53 bacterial artificial chromosome was producedthat allowed expression of normal p53 as a fluorescent fusion protein under the control of its normal promoter. This vector was found to be able to restore p53 nuclear expression to the p53 mutantcell lines, SK-N-AS and p53 oscillations were observed. Novel tools were produced in this project to investigate aspects of the p53 system. They represent a future resource to further increase our knowledge of how p53 is regulated and functionally inactivated in cancer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral