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Title: Role of FUS (TLS) in differentiation in acute myeloid leukaemia
Author: Walsby, Elisabeth Jane
ISNI:       0000 0004 2750 6562
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2004
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Leukaemia is the result of molecular abnormalities which lead to a block in differentiation that is the defining characteristic of myeloid leukaemia cells. Work to identify the genes that are associated with this defective differentiation led to the identification of the involvement of the FUS gene. FUS (TLS) is a housekeeping gene that is capable of binding DNA and RNA. FUS has roles in coupling mRNA transcription and processing and in the repair of double stranded DNA breaks. Previous work within this department has shown that FUS is down-regulated in human leukaemia cell lines in response to induction of differentiation with ATRA and that FUS is up-regulated in acute myeloid leukaemia (AML) patients. The aim of this study was to identify whether this is a causative or correlative relationship and also to identify target genes associated with FUS dysregulation. Transduction of human (HL- 60, NB4, NB4R2) and murine (32D, 32D AML-ETO, 32D B2A2) leukaemia cell lines with retroviral constructs expressing FUS or antisense FUS resulted in some over-expression or down-regulation of FUS in these cells. The effect of FUS modulation in the transduced cell lines was assessed through cell growth and viability. No effect on the growth and viability of transduced cells was observed as a result of FUS modulation. To determine whether FUS had a role in differentiation, the transduced cell lines were induced to differentiate using ATRA and G-CSF. Differentiation was assessed by measurement of cell growth, viability and the expression of cell surface markers by flow cytometry. The result of expression of FUS antisense in the ATRA sensitive NB4 and 32D cell lines was to generate resistance to differentiation induction using ATRA. Conversely in the ATRA resistant NB4R2 and 32D B2A2 cells, expression of FUS antisense reinstated the ability to differentiate in these cells. In response to treatment with G-CSF, the 32D cells expressing FUS antisense developed a resistance to differentiation while the previously G-CSF resistant 32D B2A2 cells became capable of differentiation when FUS antisense was expressed in these cells. Following the demonstration of an altered phenotype in response to treatment with different differentiation inducers in cells containing FUS antisense constructs, genes acting as target genes of FUS were identified using the Affymetrix gene expression system. The effect of FUS over-expression and down- regulation were studied in all the murine 32D derived cell lines and in the human NB4 cell line. In addition to this, gene expression changes resulting from FUS modulation in the NB4 cells during treatment with ATRA over 96 hours was investigated in this manner. Expression levels of genes associated with FUS dysregulation were verified using quantitative RT-PCR. Genes identified as having altered expression as a result of the expression of the FUS antisense construct included transcription factors, genes involved in apoptosis and differentiation and genes that have previously been shown to interact with, or have homology to, FUS itself. Further analysis of these candidate genes suggested that they were not likely to have a dominant effect in the altered phenotype seen in the transduced cells but were more likely to play a participatory role in the effects observed. This study has concluded that FUS may have a role in haematopoietic differentiation induced by both ATRA and G-CSF but this role appears to be context dependent making it important to study the effects of its modulation in primary AML blasts. The mechanism through which FUS affects the ability of the cells to differentiate remains unresolved.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available