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.

Mutations of Ras and activation of the Ras pathway are amongst the most common abnormalities detected in human cancer (-20%), and in myeloid neoplasia. In addition, excessive production of reactive oxygen species (ROS) is a common feature of human malignancy and is often triggered by activation of Ras oncogenes. ROS act as second messengers and can influence a variety of cellular process including growth factor responses and cell survival. This study examined the contribution of ROS production to the phenotype of mutationally-activated Ras in normal human CD34+ haematopoietic progenitor cells. For the first time, this study demonstrated that Ras strongly upregulated the production of both superoxide and hydrogen peroxide (H₂O₂) in these cells, through the stimulation of NOX oxidase activity, without affecting the expression of endogenous antioxidants or the production of mitochondrial ROS. Ras also promoted both the survival and the growth factor independent proliferation of CD34+ cells. Using oxidase inhibitors and antioxidants, it was found that excessive ROS production by these cells did not contribute to their enhanced survival rather, this study presents the first data demonstrating that ROS promoted their growth factor-independent proliferation. While Ras-induced ROS production specifically activated the p38MAPK oxidative stress response, this failed to induce expression of the cell cycle inhibitor pl6INK4A instead, ROS promoted the expression of cyclin Dl and D3. Expression of activated Ras in human haematopoietic progenitors drives hyperphosphorylation of PKC family members, which mediates several phenotypes of mutant Ras in haematopoietic cells including dysregulated development. This study demonstrated that endogenous H₂O₂ production contributes to hyperphosphorylation of PKC in this model, and that exogenous H₂O₂ can drive phosphorylation of PKC in a similar manner. Finally, this study presents preliminary data obtained by kinomic PepChip analysis suggesting that endogenous ROS production driven by mutant Ras can influence the kinase activity of these cells, consistent with the hypothesis that ROS may promote protein phosphorylation via phosphatase inhibition. In summary, this study presents novel data showing endogenous ROS production makes a significant contribution to the phenotype of human haematopoietic progenitor cells expressing mutant Ras and suggests that targeting ROS may be a valid approach in acute myeloid leukaemia therapy.

This study concerns a specific molecular genetic mutation, the mixed lineage leukaemia partial tandem duplication ( MLL PTD). MLL is a transcriptional regulator that known to be instrumental in both normal haematopoiesis and in leukaemogenesis. This study aimed to evaluate the prognostic importance of this abnormality through investigation of its influence on clinical outcome, its utility as a marker of minimal residual disease (MRD) and the downstream effects of its expression. A qualitative PCR assay was established and determined the frequency of MLL PTD to be 5.2% in <italic>de novo</italic> AML. MLL PTD was found to be a useful independent marker being associated with a greater risk of relapse and a reduction of relapse free survival and overall survival. A quantitative PCR (QPCR) assay was developed that reliably distinguished AML-related MLL PTDs from the low level of background MLL PTDs occurring normally. Despite a high rate of cytogenetic clonal evolution MLL PTD expression remained stabled between diagnosis and relapse, making it a suitable marker for MRD. Results also suggested the QPCR assay could determine onset of remission and prediction of relapse. Gene expression profiling was used to identify a gene signature unique to the MLL PTD and was found to be distinct from that associated with MLL translocation. Analysis of the gene signature also identified three candidate chemotherapy compounds predicted to antagonise the effects of the MLL PTD. The results of this study could prove instrumental in improving the treatment of MLL PTD patients.

In the next part of this study I employed microarray analysis to compare CD38+ and CD38" cells from the same patient purified by high speed cell sorting. CD38+ CLL cells demonstrated a unique gene expression profile when compared to their CD38" counter-parts with 61 genes found to be consistently statistically different. Six genes were investigated further, VEGF, IL-lp, CXCL2, CCR3, ZAP-70 and Akt. Using flow cytometry and Western blotting techniques it was confirmed that these proteins were expressed at significantly higher levels in CD38+ sub-populations when compared to the CD38" sub-populations. CD38+ and CD38" cells from the same patient showed similar drug sensitivity to fludarabine in the majority of samples tested. However, samples from five patients who had received the most prior treatment showed greater resistance to drug therapy in their CD38+ CLL cells. The apoptotic proteins Bcl-2, Bax and Mcl-1 were up-regulated in the CD38+ cells. The combination of CD38 expression data with other prognostic factors such as Vh gene mutational status and ZAP-70 expression will be beneficial in determining the prognosis of the patients and the course of treatment. Also determining the role of CD38 within CLL cells could lead to new therapeutic targets.

In this study, I applied a range of techniques in an attempt to enhance our knowledge of the role that CD38 plays in the pathogenesis of CLL. Investigation of a number of techniques to genetically modify the CLL cells led to the development of a lentiviral transduction system that was able to induce a marked increase in the ectopic expression of CD38 on the CLL cell surface. Subsequent molecular analysis identified changes in gene expression which may enhance disease progression. The pro-angiogenic growth factor VEGF and the DNA mismatch repair protein Msh6 were both identified as candidates for further investigation. This work also highlighted the challenges and limitations involved in using a lentiviral knock-in system and led to the design of experiments utilising CD31-expressing co-cultures to stimulate CD38 on the CLL cell surface. The CD31-expressing co-culture system induced survival within the CLL sample compared to cells incubated with the control, non-transfected co-culture. Increased proliferation was illustrated through the incorporation of BrdU and induction of the cell cycle protein Ki-67. Multi-colour flow cytometry was employed to observe the expression of surface and intracellular molecules which may be involved in CLL cell activation and signalling. Changes in the phenotype of the CLL cells were consistently observed which support the notion that these cells can be activated in vitro and can thereby enhance B-cell receptor signalling. Specifically, CD 19, CD38 and the aberrantly expressed tyrosine kinase Zap-70 were all induced following incubation with CD31-expressing co-culture. This is the first time that a lentiviral transduction system has been developed which efficiently expresses CD38 in a CLL cell population with little cell death. The work carried out in this project also highlights the importance of using co-culture to stimulate CD38 on the surface of the CLL cells in vitro. The novel findings within this project have given insight into some of the mechanisms of CD38 signalling, provided direction for future work and highlight the potential of CD38 as a therapeutic target in CLL.

B cell chronic lymphocytic leukaemia (B-CLL) is the most common form of adult leukaemia in the western world, characterised by the slow accumulation of small mature B lymphocytes in the circulating blood. Disease progression is prolonged and patients may survive for more than 20 years, however it ultimately remains incurable with conventional chemotherapy. Regulation of gene expression is a complex process and exerted at multiple steps in the expression pathway, including translation. Translational regulation has not previously been studied in B-CLL yet may be important as there are numerous examples of translation de-regulation in tumorigenesis. Results obtained suggest that a reduced level of protein synthesis was occurring in B-CLL cells. Furthermore, cDNA microarray analysis was performed providing a gene expression profile at the level of translation, which correlates with the biology of the disease. Internal ribosome entry could be a possible mechanism of up-regulation utilised, and evidence is provided to suggest this may be the case. Potential IRES activity was observed for three genes, found to be translationally up-regulated in B-CLL cells by the cDNA microarray analysis, under normal and serum starvation conditions. The microarray data provides some potential new targets for treatment. In particular, the up-regulation of the angiotensin II type 1 receptor may influence survival of B-CLL cells. Initial evidence suggests that its ligand angiotensin II could influence B-CLL cell survival. Median survival of B-CLL patients is 10 years, however disease progression is variable; some patients will survive for many years while others have a more aggressive form and die within a couple of years. Progression has been observed to correlate with the mutational status of the immunoglobulin heavy chain variable (IgVH) genes. Differences in polysome association for the two subtypes were determined, using the microarray data obtained, identifying a number of genes that may be significant to the biology of the disease.

Escherichia coli are usually regarded as a harmless human colonic flora. However, pathogenic strains of E. coli have been associated with infections that could range from infected mucosal surfaces by intestinal pathogenic E. coli to the more severe cases of disseminated infections throughout the body by the extraintestinal pathogroups. The main focus of this project was to investigate the genomic contents of pathogenic bloodstream infection (BSI)-associated E. coli strains. This is because the genome contents of the E. coli BSI-associated isolates have not been well studied, with only few reports indicating that the pathogenincity of these strains could be attributed to horizontally acquired DNAs known as genomic islands (GEIs). The genomic contents of 10 clinical BSI-associated E. coli strains, isolated at the Leicester Royal Infirmary were investigated in this study. The first approach used to investigate the genomic contents of these strains was by interrogating the downstream ends of tRNA genes for their GEI contents by the sequential PCR strategy tRIP-PCR (tRNA interrogation for pathogenicity islands) followed by the SGSP-PCR (single genome specific primer-PCR). In this approach the flanking regions of the tRNA sites were used to first screen the tRNA genes for their GEIs followed by amplifying the boundaries of the identified GEIs. In the second approach termed Microarray-Assisted mobilome Prospecting (MAmP), the physical genome size of the tested strains obtained by the pulsed-field gel electrophoresis (PFGE) is compared to the sum total of the bits of the genome detected or visualized by the array. The difference between the two measurements is used to estimate the size of the novel, non-microarray-represented mobile genome (mobilome) present in the tested strains. Remarkably, despite only studying 10 E. coli strains, associated with a single disease type the tRIP-PCR method has identified at least 3 GEIs that contain novel sequences, and 46 GEIs, resembling uropathogenic E. coli CFT073-like entities. One particular strain E105 had 13 tRNA sites occupied with GEIs. On the other hand, an average novel, non-microarray-borne mobilome of (219 kb /strain) was obtained by the MAmP which, corresponds with previous studies. The strategies used in this study had proved successful in addressing and identifying mobilome-rich strains. Therefore, using such approaches in combination with whole genome sequencing progects could prioritize the strains and the genomic regions that need to be sequenced. Such prioritization would avoid sequencing of hundreds of isolates to identify their novel gene pool and would reduce the cost of genomic sequencing. Moreover, applying such approaches for the identification of new virulence genes and/or pathogenic mechanisms could lead to significant improvements in the treatment of E. coli infections.

Chronic Lymphocytic Leukaemia (CLL) remains incurable and novel treatments are urgently required to combat this disease. The proteasome inhibitor bortezomib induces high levels of apoptosis in vitro in CLL patient samples, but clinical trials have been discouraging. In this study, bortezomib induced apoptosis in all CLL samples tested at nanomolar concentrations. However, incubation of CLL cells with red blood cells (RBCs) reduced the activity of bortezomib. These data imply that RBC uptake may reduce the activity of bortezomib in vivo. TNF-related apoptosis inducing ligand (TRAIL) is an attractive cancer therapy because of its selectivity towards tumour cells. TRAIL and agonistic mAbs to two TRAIL receptors (TRAIL-R1 and TRAIL-R2) are in clinical trials. CLL cells are resistant to TRAIL but can be sensitised by pre-treatment with histone deacetylase inhibitors (HDACi). HDACi sensitised CLL cells to preparations of TRAIL that induce apoptosis through TRAIL-RI but not through TRAIL-R2. In contrast, K562 cells, when pre-treated with HDACi, responded to preparations of TRAIL that induce apoptosis through TRAIL-R2. To confirm this, TRAIL receptor-selective mutants were generated and tested for specificity in cell lines. CLL cells, pre-treated with depsipeptide, responded only to the TRAIL-R1 mutant. These data confirm that CLL cells can be sensitised to TRAIL induced apoptosis primarily through TRAIL-R1 and that clinical trials in CLL should focus on HDACi in combination with preparations of TRAIL that induce apoptosis through TRAIL-R1. Recent publications suggest that internalisation of CD95 and TNFRI are required for induction of apoptosis. TRAIL-internalisation has not been widely studied. In this study, TRAIL-induced DISC formation was shown to occur at the plasma membrane, suggesting that TRAIL signalling is unlike other members of the TNF superfamily (CD95L and TNFa). Inhibiting TRAIL internalisation with hyperosmotic solution did not inhibit apoptosis induction, suggesting that internalisation is not important for TRAIL activity.

The thesis is in two parts. The first concerns a laboratory study of human lymphoma cells and their interactions with reticulo-endothelial cells. Electron microscopy and tissue culture studies indicated that macrophages and lymphocytes were in close contact with tumour cells but no cytocidal effect was noted in the intact tissue. A time lapse film of lymph nodes in culture shows that when ceils are teased, out in a monolayer, the tumour cells become susceptible to direct attack by lymphocytes and tissue macrophages. Lymph node cultures of lymphoma liberate chemotactic factors to the supernatant which attract eosinophils, monocytes and leucocytes. The eosinophil chemotactic factor was found in high concentrations only in lymph nodes involved with Hodgkin's disease. Also in the supernatants were found two factors which depressed host cells. After incubation of suoernatants with lymphocytes, their subsequent ability to transform was depressed and after incubation with normal monocytes, subsequent chemotaxis was inhibited. Peripheral blood monocytes from lymphoma patients were significantly less mobile than age-sex match controls in a chemotaxis assay. The clinical part of the thesis describes firstly a search, for space-time clusters in the occurrence of lymphomas in the South East of Scotland over a period of eleven years. Fourteen clusters were found from centralised computer records but only one of these survived closer scrutiny. Secondly a cohort of one hundred consecutive patients with lymphoma has been followed for two to seven years. Overall survival of 68°/o is considerably superior to retrospective studies which took place before 1970. Improvement in survival is due to rigorous staging, more appropriate radiotherapy and the advent of effective chemotherapy. Several serum parameters such as low albumin, low immunoglobulin M and low serum a macroglobulin independently worsened the prognosis. This information taken with the findings of the laboratory studies suggest that immunodeficiency in lymphoma patients is accompanied by a poor prognosis and maybe due to release of tumour specific factors. Such high risk patients should be managed by intensive immunological support including removal of such factors and restoration to high protein levels prior to or along with cytoreductive therapy.

No description available.