Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.714197
Title: Deacetylation of GD3A as a potential therapeutic strategy for paediatric medulloblastoma
Author: Mather, Rebecca Louise
Awarding Body: University of Portsmouth
Current Institution: University of Portsmouth
Date of Award: 2016
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Abstract:
Medulloblastoma survivors frequently suffer from low quality of life as a result of aggressive treatment. New treatments are urgently needed to reduce down-stream sequelae. GD3, an oncofoetal ganglioside, has roles in embryonic brain development and is commonly modified to 9-O-acetyl GD3 (GD3A). GD3A plays a role in protecting cells from pro-apoptotic GD3 during brain development, after which GD3 accumulates above a threshold to collapse the mitochondrial membrane potentials of supernumary progenitor cells. GD3 and GD3A then become minor ganglioside species of the postpartum brain. In medulloblastoma however, GD3 and GD3A are re-expressed as shown here by the medulloblastoma cell lines RES256, UW402 and CHLA-01-Med where expression of GD3 is seen in 56.7%, 61.3% and 45.1% of the cell populations respectively. GD3A is expressed by 84.5%, 74.4% and 79.4% of the populations respectively. By bioinformatic analysis we also show that the human endogenous deacetylation enzyme of GD3A, Sialate-O-acetylesterase (SIAE), is significantly down-regulated in medulloblastoma patient tissue (p<0.001). This enzyme is thought to restore GD3 levels by cleaving the acetyl groups from GD3A. In order to evaluate this pathway as a potential therapeutic target we used an inducible SIAE overexpression approach in RES256 cells, the highest intracellular GD3A expresser. This transfection resulted in two SIAE wild-type expressing clones. Upon over-expression of SIAE (in clone 2) we show a significant increase in GD3 expression (p<0.05), and in preliminary experiments a significant increase in depolarisation of the mitochondrial membrane potential was seen in SIAE expressing clone 1 (p<0.05). These changes were not seen on induction of expression of a catalytic mutant SIAE-S127A or vector controls. We also demonstrate that wild-type SIAE expressing clones do not secrete the enzyme, however the catalytic mutant SIAE-S127A protein is secreted. We hypothesise that possible cleavage of caspase 1 could be responsible for this secretion. The expression of SIAE and SIAE-S127Ain cell lysates were confirmed by Western blot and demonstrated a possible cleavage product. We hypothesise that this possible cleavage may result in different abilities for these clones to secrete the enzyme. Furthermore, we show that induction of SIAE overexpression in combination with etoposide treatment resulted in a significant reduction inIC50 in SIAE overexpressing clones after 72 hours of treatment (p<0.001; clone 2). Changes in IC50 for empty vector controls and SIAE-S127A were not significant.
Supervisor: Pilkington, Geoffrey John ; Fillmore, Helen Louise Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.714197  DOI: Not available
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