Characterisation of lens epithelium derived growth factor isoforms in chronic lymphocytic leukemia: Studies and significance

Blocksidge, Jemma
Characterisation of lens epithelium derived growth factor isoforms in chronic lymphocytic leukemia: Studies and significance. Doctor of Philosophy thesis, University of Liverpool.

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Over expression of lens epithelium derived growth factor (LEDGF) is described in a number of different tumours. In addition, self-antibodies against this protein are detectable in patients with lymphomas and chronic lymphocytic leukemia (CLL). Alternative splicing of LEDGF results in a number of different isoforms, and thus the initial aim of this thesis was to determine the variability in LEDGF isoform expression in a cohort of CLL cases. Utilising RT-PCR, it is shown that CLL cells variably express four different LEDGF isoforms. The study was then extended to quantitate the expression levels of these four isoforms in individual CLL cases, and to compare them to normal B cells. RT-qPCR analysis showed that the longest of these isoforms, p75, is significantly over expressed, whilst the shortest form, p52bΔE6 is significantly under expressed in CLL, compared to normal B cells. All four isoforms of LEDGF are generally over expressed in CD38 positive CLL cases compared to those that are deficient for surface expression of this molecule. Alternative splicing occurs as a result of the inclusion or exclusion of different exons within the mRNA. This process is governed by the splicing machinery, of which Splicing Factor 3B, subunit 1 (SF3B1) is a critical component. Recently, it has been shown that mutations within SF3B1 are recurrent in CLL. Such mutations impact on the splicing of a significant array of genes within the cell. One of the genes predicted to be affected is LEDGF. Therefore, the mutational status of SF3B1 was examined in the same cohort of CLL cases and correlated with the expression of the LEDGF isoforms. As well as the commonly reported mutations in SF3B1, a novel nonsense mutation was identified. Although not statistically significant, cases with unmutated SF3B1 have higher levels of all isoforms of LEDGF and a possible explanation for this is discussed. That two of the major isoforms of LEDGF (p75 and p52) have differing functions within the cell is well documented and can be explained by virtue of their participation in distinct protein complexes. In order to identify components of the complexes generated by the four LEDGF isoforms identified in this study, stable cell lines exogenously expressing these isoforms were generated. Each of the isoforms was ‘double-tagged’, and a purification protocol optimised that would allow successful characterisation of the complex associated with each isoform. The results provide useful insights and provoke thought for the role of alternative splicing in the altered phenotype of cancer cells and its contribution as an additional epigenetic mechanism in normal and malignant gene regulation.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2014-05 (completed)
Subjects: ?? RE ??
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 05 Aug 2014 11:51
Last Modified: 16 Dec 2022 04:42
DOI: 10.17638/00018297