No description available.

Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma subtype with heterogeneous clinical outcome and a significant number of patients still die of their disease. Characterising lymphoma patients’ autoantibody repertoires represent one approach to improve the understanding of their disease biology. Our hypothesis was that characterisation of antigens eliciting humoural immune responses in lymphoma patients may provide insights into mechanisms of lymphomagenesis and identify novel diagnostic/therapeutic targets. HIP1R was validated as a novel B-NHL autoantigen by immunoblotting with patients’ sera. No response was identified to the related HIP1 protein. Consistent with this finding, more widespread expression of HIP1R, compared to HIP1, was observed in lymphoma cell lines. Expression studies, at both transcript (qRT-PCR and analysis of microarray datasets) and protein levels (blotting and immunolabelling), identified abundant HIP1R in normal B cells and low level expression in a poor prognosis activated B-cell (ABC)-like DLBCL subtype. Upregulation of HIP1R expression was observed in activated non-malignant B cells at both transcript and protein levels, suggesting that downregulation of HIP1R expression in ABC-DLBCL might be a disease-related process. Despite its potential for DLBCL subtyping, HIP1R protein expression was not statistically significantly associated with patients’ survival in a series of 256 DLBCL. Short FOXP1 isoforms were identified as one mechanism repressing HIP1R transcription in an ABC-DLBCL cell line. Phenotypic analysis of HIP1R-depleted B-cell lymphoma cells indicated that HIP1R silencing could increase the surface levels of B-cell receptor (BCR) components such as IgM and CD79b. HIP1R represents a novel lymphoma autoantigen and cell-of-origin marker for distinguishing germinal centre- versus ABC-DLBCL subtypes. As ABC-DLBCL survival is dependent on chronic BCR signalling, future studies will address whether HIP1R silencing plays a fundamental role in disease pathogenesis by promoting BCR signalling.

Despite improved treatments for Non-Hodgkin’s Lymphoma (NHL) and Multiple Myeloma (MM), most patients eventually relapse and these diseases remain largely incurable. This has precipitated recent research into more clinically relevant in vitro models to enable development of more effective therapies. We have validated and standardised two in vitro primary culture models using tumour samples derived from patients with NHL, Chronic Lymphocytic Leukaemia (CLL) and MM. Several novel findings have been demonstrated. In vitro sensitivity of primary NHL cells cocultured in a CD40L model predicted clinical response to bortezomib in patients receiving the drug in a phase II trial. In vitro sensitivity correlated with CD40 expression, identifying a potential surrogate biomarker for response to bortezomib. The novel HDAC inhibitor, UCL67022 was 10-fold more potent than vorinostat in NHL and produced synergy when combined with bortezomib. UCL67022 maintained its potency in primary MM samples grown in an HS-5 stromal model. It modulated cytokine secretion resulting in downregulation of cytokine-induced signalling pathways (JAK/STAT3). A novel Hsp90 inhibitor, KW-2478 maintained activity in the HS-5 model and enhanced the activity of bortezomib and melphalan. Hsp70 was identified as a potential surrogate biomarker to monitor the combinatorial effect in future clinical trials. A highly synergistic and schedule-dependent cytotoxic effect occurred when primary MM cells were pre-treated with melphalan followed by bortezomib, with important implications for future clinical trial design. IL-6, IL-8 and VEGF levels correlated with resistance to bortezomib and melphalan and were associated with activation of JAK/STAT, MAPK and PI3K/Akt signalling pathways. Antibody neutralization of IL-6, IL-8 and VEGF resulted in restoration of drug sensitivity. We have therefore demonstrated the ability of primary culture models to predict response to chemotherapy, to identify therapeutically beneficial novel agents and to enable study of tumour microenvironmental interactions responsible for drug resistance in patients with haematological malignancies.

The unfolded protein response (UPR) is a complex signalling pathway activated in response to endoplasmic reticulum stress. In recent years, the UPR has been implicated in cancer and chemosensitivity, particularly in solid tumours. This thesis investigated the potential value of targeting the UPR as a novel therapeutic approach in haematological malignancies using a panel of cell lines representing AML, lymphoma and myeloma. The UPR was constitutively active in these haematological cancer cell lines, with differential activation of key UPR proteins both in the panel and between the panel, peripheral blood mononuclear cells and the colorectal cancer cell line HT-29. A number of strategies were used to modulate the UPR and study chemosensitivity. Minimally toxic concentrations of the ER stress inducer thapsigargin protected cells from cytotoxic agents, with a reduction in antiproliferative drug effect. The activity of the novel small molecule versipelostatin, reported to downregulate the ER molecular chaperones GRP78 and GRP94, was also investigated, with the downregulation previously reported in solid tumour cell lines (Park et al. 2004) confirmed in HT-29 cells, but not observed in the haematological cell lines studied (although versipelostatin was an effective cytotoxic agent at low micromolar concentration). Combination experiments with the chemical chaperone 4-phenylbutyric acid (PBA) resulted in a small increase in apoptosis when PBA was combined with ER stress inducers. However, PBA also showed HDAC inhibitory activity at the concentrations used. Finally, siRNA mediated silencing of GRP78 and GRP94 in THP1 (AML) and U266 (myeloma) cells resulted in a decrease in the targeted protein, but showed only minimal effects on chemosensitivity. In conclusion, the UPR is activated in these haematological cancer cell lines and plays a complex role in chemosensitivity. In contrast to previous reports in solid tumour cells, modulating the UPR in these haematological malignancies had only a modest effect on chemosensitivity.

[Truncated abstract] Every year thousands of individuals suffer from thrombotic related complications that in some cases can be fatal and every year millions of women take some form of hormonal contraceptive. In some cases, there is a cause and effect relationship between the two as users of the combined oral contraceptive pill have an increased risk of developing a thrombotic event. Increased circulating levels of oestrogen cause a prothrombotic shift in the coagulation cascade resulting from upregulation of several procoagulant proteins and a decrease of key anticoagulant proteins. One of the most oestrogen sensitive anticoagulants is Protein S (PS), a product of the PROS1 gene. PS acts as a cofactor to activated protein C (aPC) and the PS-aPC complex serves to downregulate clot formation by deactivating the tenase and prothrombinase complexes via proteolytic cleavage of activated factors VIII and V, respectively. As such, low PS levels are associated with an increased risk of developing thrombotic disorders such as pulmonary embolism, stroke or coronary thrombosis and deep vein thrombosis. During pregnancy when oestrogen levels increase, a steady decline in PS is evident in the early weeks of gestation and continues to decrease to below the normal range in the 2nd trimester, remaining there until post-partum. In addition, reduced free and total PS levels are observed in users of the combined oral contraceptive (COC) pill that contains an oestrogen and a progestin. Interestingly, users of 3rd generation COCs have significantly greater reductions of PS than do 2nd generation COC users. The difference between the two forms is the type of progestin, not the oestrogen, which is predominantly ethinyl oestradiol in the majority of commercially available preparations. At present, a mechanism to describe the relationship between oestrogen and/or progesterone associated with the observed in vivo changes in the levels of PS has not been identified. The aim of this thesis was to define the molecular mechanisms involved in the regulation of PS expression by oestrogen and progesterone. In this study, a Combined Single-stranded conformational analysis and Heteroduplex Analysis (CSHA) iv methodology was optimised for screening both PROS1 DNA and mRNA for the detection of mutations. '...' This may explain why users of 3rd generation COCs display a greater reduction in circulating PS levels compared to 2nd generation users. To investigate potential PS interactions with other proteins that could be hormonally regulated, a yeast-2-hybrid (Y-2-H) screen was performed using the PS molecule as a 'bait' against molecules derived from liver and bone marrow cDNA libraries. A clone that contained a portion of another haemostatic protein, Protein Z (PZ) was isolated and confirmed via sequencing. As no full length PZ clones were identified, a second Y-2-H screen was performed once again using the PS molecule as bait and the PZ molecule as the fish. Interaction between the two proteins was shown to be possible via the successful growth of colonies on triple knock out selective media and by positive ß-galactosidase activity.

No description available.

CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the alpha2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the alpha2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis.

CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the alpha2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the alpha2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis.

Background Several case reports and studies have alluded to an increased prevalence of venous thrombosis in human immunodeficiency virus positive (HIV-positive) patients. Although a relationship between HIV infection and thrombotic disease has been suggested, the mechanisms predisposing to thrombosis have not been fully elucidated. Aim A prospective study, to determine possible coagulation factor abnormalities that could explain the predisposition to thrombosis in HIV-infected African (Black) patients, was undertaken. Method African (Black) patients, with acute upper segment deep vein thrombosis (DVT) confirmed by duplex ultrasound, were enrolled. Patients who had recognisable risk factors such as recent surgery, pregnancy or malignancy, were excluded. After informed consent, blood samples were taken for baseline tests as well as a thrombophilia screen. The control group comprised known HIV-positive African (Black) patients without DVT. Patients with DVT who were found to be HIV-negative were also analysed. Analysis was done in 2 parts: HIV-positive patients with and without thrombosis and HIV-positive and negative patients with thrombosis were compared. Results Part A: HIV-positive patients with and without thrombosis Of the 77 patients with DVT, 50 patients tested HIV-positive. These 50 patients (HIV-positive DVT-arm), as well as 56 controls (HIV-positive, no DVT), were enrolled into the study. The groups were well matched with regard to age, sex and cluster designation 4 (CD4) count. On univariate analysis, significant findings in the DVT-arm were a history of active tuberculosis on treatment, low protein C levels and a positive qualitative D-dimer, whereas on multivariate analysis, only tuberculosis and an elevated D-dimer proved to be significant. Part B: HIV-positive and negative patients with thrombosis There were 20 HIV-negative patients with DVT who met our inclusion criteria Limited assessment was done on this group owing to unavailability of some data. The mean age of the HIV positive DVT group was significantly lower than the HIV-negative group with DVT (31.78 vs. 41.45 years; p=0.005). There was no significant difference in the prevalence of tuberculosis between the HIV-positive and HIV-negative patients with thrombosis (p = 0.269). Mean protein C levels were reduced in the HIV-positive group and normal in the HIV-negative group. They were significantly lower in the HIV-positive patients compared to the negative group (p=0.02). Conclusion The findings of the study suggest a relationship between HIV, its complications and DVT. Although this study confirms HIV infection as a risk factor for thrombosis, clear pathogenetic mechanisms remain to be elucidated. In our population, tuberculosis appears to be an important risk factor predisposing patients to the development of DVT, both in the HIVpositive and negative population. Further studies will need to be done to confirm this hypothesis. / Thesis (MMed)-University of KwaZulu-Natal, Durban, 2006.