The aims of this thesis were to examine: (1) plasma and cellular pharmacokinetics of daunorubicin and its major metabolite daunorubicinol in patients with acute leukaemia, and the relationships between pharmacokinetics, patient response and the presence of P glycoprotein; (2) actions of the multidrug resistance reversing agents cyclosporin A and trifluoperazine, at clinically achievable concentrations, on daunorubicin accumulation and retention in human leukaemia cell lines and patients with acute leukaemia; and (3) effect of daunorubicin on the cell membrane of both sensitive and resistant cell lines, with and without the multidrug resistance reversing agents. Twenty-seven patients with acute leukaemia received daunorubicin as part of induction therapy. The plasma and cellular levels of daunorubicin and its metabolite daunorubicinol were determined using HPLC. There were no significant differences between patients who went into complete remission (12 out of 23) compared to those who did not respond for any of the plasma pharmacokinetic parameters. There was a significant difference in the cellular daunorubicin and daunorubicinol area under the concentration-time curve between responders and non responders (p less than 0.02), as well as in cellular Cmax, cellular clearance and cellular volume of distribution. Eleven patients were P glycoprotein positive and 10 P glycoprotein negative (no sample available for 2 patients). There was no correlation between patient response and the presence of P glycoprotein; nor a correlation between the cellular concentration of daunorubicin or daunorubicinol and P glycoprotein. Patients responding to chemotherapy had higher cellular daunorubicin and daunorubicinol compared to non responders. In contrast to in vitro studies, overexpression of P glycoprotein was not the reason for the lower cellular daunorubicin levels. Cyclosporin A was capable of increasing both cellular accumulation and retention in the drug resistant CEM/VLB and HL 60/ADR cell lines, but not in the drug sensitive CEM and HL 60 cell lines. Trifluoperazine had no effect in any of the four cell lines. In contrast to the cell line findings, only the combination of cyclosporin A and trifluoperazine were able to increase both accumulation and retention in the blast cells of patients at initial presentation. The multidrug resistant reversing agents alone had no effect in increasing accumulation or retention in the blast cells of P glycoprotein positive patients, nor patients in relapse. The cell line studies show that at clinically relevant concentrations only cyclosporin A is capable of increasing daunorubicin accumulation in both the drug resistant P glycoprotein positive (VLB) and P glycoprotein negative (ADR) cell lines. Thus, cyclosporin A does not work only by inhibiting the actions of P glycoprotein. Trifluoperazine was unable to reverse drug resistance at clinically relevant concentrations in either cell lines or patient blast cells. However, the combination of cyclosporin A and trifluoperazine increased accumulation in patient blast cells at initial presentation, suggesting that these agents may be more useful in patients at initial presentation than relapse. Daunorubicin was immobilised by linking it to poly vinyl alcohol and the effect of immobilised-daunorubicin was studied on the four cell lines above. The immobilised-daunorubicin was able to decrease cell growth in the drug sensitive HL 60 cell line but not in the drug resistant VLB or ADR cell lines. Poly vinyl alcohol itself was cytotoxic to the CEM cell line. The multidrug resistance reversing agents cyclosporin A and trifluoperazine were only capable of increasing cytotoxicity in the HL 60 cell line, with no effect in the drug resistant VLB or ADR cell lines.

The inherited bone marrow failure syndrome Fanconi Anaemia (FA) is caused by mutations in any one of the multiple FANC genes, which encode proteins that collaborate in the FA/BRCA DNA damage response pathway. FA is characterised by extreme predisposition to acute myeloid leukaemia (AML). AML in FA is associated with typical chromosomal aberrations involving gains of the long arm of chromosome 3 (3q gains). These are linked to overexpression of the oncogene ecotropic viral integration site 1 (EVI1). Based on this clinical observation, the hypothesis that EVI1 confers leukaemic transformation, in particular in the context of FA, was tested. Mouse embryonic stem cells with either functional or disrupted FA/BRCA-pathway were used to model normal and FA-associated embryonic haematopoiesis, and the effect of EVI1 overexpression was assessed in this model. EVI1 functions were also investigated with respect to protein interactions, focusing on the interaction with the co-repressor C-terminal binding protein 1 (CtBP1), in the context of genotoxic stress. To study this, in vitro haematopoietic differentiation assays, flow cytometry, mass spectrometry, immunoprecipitations and immunofluorescence were employed. In vitro haematopoietic differentiation using mouse embryonic stem cells with defective Fanca was successfully developed and applied. The analysis revealed that EVI1 overexpression in haemangioblast-like cells prevented the generation of haematopoietic precursors through endothelial to haematopoietic transition. Studies into EVI1 protein interaction dynamics showed that DNA damage-induced phosphorylation of EVI1 modifies interaction with the co-repressor CtBP1. This interaction was demonstrated to be partially required for the EVI1-induced block of the development of haematopoietic precursors using the mESC-based model. An EVI1-mediated modulation of the FA phenotype characteristic G2-arrest and of the FA-associated embryonic haematopoiesis was not demonstrated. This study contributes to the understanding of the function of the EVI1 oncogene in normal and FA-associated haematopoiesis and the DNA damage response. FA-associated haematopoiesis and leukaemogenesis can be further studied using the embryonic haematopoiesis model developed here, and further studies can build on the data generated with respect to EVI1.

Since its isolation in 2004, the research landscape around graphene and other 2D materials has expanded rapidly and now encompasses fields as diverse as electronic engineering and drug delivery. For biomedical applications, one of the most desirable properties of the graphene family of nanomaterials (GFNs) is their 2D geometry; the high surface area to volume ratio that is characteristic of nanomaterials is taken to its extreme in a material that can be viewed as being entirely surface. This particular property alongside the versatility with which they may be functionalised both makes GFNs well positioned to function as the foundation of highly tailored and multifunctional therapeutics platforms. In this project, two GFN types, namely pristine graphene and graphene oxide, were prepared to form suspensions suitable for application to therapeutics delivery. Firstly, experiments using four essential amino acids with pristine graphitic material were undertaken to assess whether graphene flakes could be suitably exfoliated and suspended using sonication in the presence of aqueous solutions of these biocompatible molecules. A positive correlation was found between the hydrophobicity of the amino acid and the presence of one or more aromatic rings in the amino acid, and the efficacy of exfoliation both in terms of concentration achieved in suspension and flake thinness. However, the system itself was found to be highly complex, both with regards to the sonication used to exfoliate the graphitic flakes, and the interactions between the amino acids and the flakes. These considerations limited the wider applicability of this form of graphene preparation for therapeutics delivery applications. Secondly, work was performed on graphene oxide (GO), a GFN far more studied in the literature, but notoriously heterogeneous. Therefore much of the work completed focused on its characterisation. A combination of established and novel fluorescence-based characterisation methods were used to fully characterise three preparations of GO, before preliminary experiments were undertaken to test their interactions with cell components. The work showed that the inherent fluorescence of GO can be exploited to improve suspension characterisation; raster image correlation spectroscopy (RICS) was used to measure the apparent hydrodynamic radii of the flakes and flow cytometry was used to provide insight into the interactions between GO flakes and serum components. Preliminary cellular experiments confirmed that flow cytometry could be also employed to assess particular graphene characteristics in the context of cell culture, demonstrating the relatively low toxicity of PEGylated GO compared to unfunctionalised GO. Finally, as the therapeutics target for this project was leukaemia, a targeting ligand was designed and synthesised that could bind to CXCR4 - a receptor that is overexpressed on CLL B-cells, as well as many other cancer types. The ligand was synthesised such that it could easily be attached to GO, however its molecular structure is flexible enough that it can be attached to a number of different therapeutics materials. It was confirmed using both competition and functional assays that the molecule was antagonistic, and was able to deliver a conjugated fluorescent molecule specifically to the CXCR4 receptors on primary CLL B-cells. The work presented in this thesis illustrates the complexity that affects the use of GFNs in biomedicine, but also confirms the potential for their future development. The field is still young, and therapeutics delivery is likely to benefit from advances in the preparation of pristine graphene, and from methods to minimise the heterogeneity of GO. These steps will support a route towards clinical application. In addition, as the field of 2D materials expands, other materials with enviable surface area to volume ratios may come to the fore. Furthermore, this thesis has shown the value of exploring novel approaches to the characterisation of GFNs, and has identified approaches that may be exploited to improve applications of GFNs in biomedicine. Additionally, the aim of using GFNs as a platform for a multifunctional therapeutics delivery vehicle was developed with regards to the attractive CXCL12/CXCR4 axis, which is relevant in a large number of disease states including over 20 cancers, by demonstrating a flexible targeting ligand that could be used to exploit the CXCR4 receptor as a drug delivery target.

Abstract Gelatinases (MMP-2 and MMP-9) play a key role during invasion and metastazising of malignant cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumours. However little is known about their role in hematological malignancies. In the present work, gelatinase expression and its clinicopathological correlations were studied with immunohistochemical staining in 10 cases representing normal bone marrow aspirate smears, 123 cases representing diagnostic bone marrow samples of patients with different leukaemias (35 AML, 7 CLL, 6 CML, 75 ALL), 67 diagnostic paraffin-embedded lymph node biopsies from patients with Hodgkin's lymphoma and 57 biopsies from patients with non-Hodgkin's lymphomas. The lymphoma samples were also stained with factor VIII antibody to evaluate the extent of new vessel formation and the non-Hodgkin's lymphoma cases also with tissue inhibitor of metalloproteinases -1 (TIMP-1) antibody. CLL did not express either of the MMP enzymes, while CML in the chronic phase expressed strongly both of the enzymes. In ALL, gelatinase expression was weak and detectable in pediatric cases in only 12.7% and in the adults in 65% of the cases. In adult ALL, MMP-2 expression correlated strongly with an extramedullary and invasive pattern of disease presentation. In AML MMP-2 positivity had markedly favorable prognostic and predictive power. In lymphoma studies, no correlations could be detected between gelatinase expression and the clinical parameters of invasion. MMP-9 positivity was related to the presence of B symptoms, which difference was statistically significant in Hodgkin's lymphoma. In Hodgkin's lymphoma, strong MMP-9 expression also implicated decreased neovascularization. In both lymphoma types, strong MMP-9 expression correlated with unfavorable prognosis, which difference was statistically significant in non-Hodgkin's lymphomas and remained as a tendency in Hodgkin's lymphoma. MMP-2 had statistically significant association with a favorable prognosis in Hodgkin's lymphoma. Combination of the results of both stainings further increased prognostic power. All together these findings implicate that gelatinases could be used as prognostic tools in AML and lymphomas albeit this needs to be verified in larger materials.

Philosophiae Doctor - PhD / Epidemiological studies have highlighted that leukaemia can be considered as the most prominent malignancy after radiation exposure during childhood. The lifetime risk on radiation-induced leukaemia for a given dose is 3 – 5 times higher for children compared to adults. The high risk at a young age is related to the elevated sensitivity of the red bone marrow where haematopoietic stem and progenitor cells (HSPCs) are located. HSPCs self-renewal capacity and long-life span increase their susceptibility to DNA damage accumulation, making them a major target of radiation-induced carcinogenesis. Proton beam therapy (PBT) is increasingly used to treat paediatric brain tumours due to its dose sparing properties compared to conventional X-ray based radiotherapy.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukaemia (CLL). The importance of this signalling pathway identifies it as a prime therapeutic target; however, the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. This article discusses the role of NF-κB in CLL as a common crossroad for pathways promoting drug resistance in CLL. We provide the background on how this pathway contributes to both spontaneous and drug-induced apoptosis. Potential new avenues to regulate this pathway in CLL are also discussed.

M.Med.(Haematology), Faculty of Health Sciences, University of the Witwatersrand, 2008 / This study aimed to identify the relevance of these prognostic features in the modern treatment era in South African children. A retrospective analysis of the presentation clinical and laboratory features and treatment outcomes of all children treated for Precursor B cell ALL at the Johannesburg Hospital was performed. Between January 1997 and May 2007, 100 children were reviewed. Clinical features (age, race and gender) emerged as significant prognostic variables. Laboratory features (white cell count and genetic features) lacked significance. Early morphologic response on day 15 identified a subgroup associated with a favourable outcome. However the presence of > 5% blasts was not significantly predictive of relapse or death at this time point. Minimal residual disease (MRD) detection by modified immunoglobulin gene rearrangement and flow cytometry techniques did not improve the predictive value of the morphological assessment. In a low resource setting, the challenge is to design cost effective MRD detection methods to improve the identification of patients at risk for relapse.

Children treated for acute lymphoblastic leukemia (ALL) are immunodeficient and therefore at an increased risk of infection and cancer recurrence. Natural killer (NK) cells are a subset of lymphocytes that are very efficient at combatting infections and cancer; however, children treated for ALL have impaired NK cell number and function. Exercise has the potential to bolster NK cell number and function, at least in healthy children and adults. Limited evidence suggests exercise may also have beneficial effects on NK cells in children treated for cancer. However, these previous exercise immunology studies in children with cancer have yielded low sample sizes. Therefore, the aim of this study was to assess the: 1a) feasibility, 1b) acceptability and 1c) safety of performing an exercise intervention in children with ALL. The secondary objectives were to assess the 2a) effects of acute exercise on NK cell number, function and receptor expression in children receiving maintenance therapy for ALL compared to healthy children, as well as to 2b) assess how the NK response changes over 4 months of therapy, and to 2c) assess the link between physical activity and NK cell number and function at rest in children receiving maintenance therapy for ALL. Children undergoing maintenance therapy for ALL (n=4) were recruited from McMaster Children’s Hospital, and healthy sex and pubertal-status matched children (n=4) were recruited from the Hamilton community. ALL patients completed a total of 3 exercise visits, occurring monthly after their regularly scheduled chemotherapy session. At each exercise visit, children were asked to complete 30 minutes of continuous biking, followed by 1 hour of rest. Blood samples were drawn at rest prior to exercise (PRE), immediately after exercise (POST) and 1 hour into recovery (REC). Healthy children only completed one exercise visit. During recovery, participants were asked to complete a physical activity enjoyment scale (PACES) questionnaire and a structured interview in order to assess exercise acceptability and to gauge participant feedback on study components, respectively. Participants were outfitted with an accelerometer to track physical activity levels between visits. Feasibility was assessed by tracking recruitment statistics, study completion rates and exercise completion rates. Acceptability of accelerometer wear was assessed by tracking accelerometer wear and log rates. Safety was assessed by tracking adverse events. All parameters were reported using descriptive statistics. We approached 22 patients to participate, and 4 children completed the study (100% completion rate) out of a goal of 15. Primary deterrents to participation were that patients and families did not want to extend time spent at the hospital or had time restrictions and that patients were uncomfortable with blood collection methods. Exercise was feasible (94% exercise completion rate), acceptable (4.2 ± 0.38 out of 5 PACES score), and safe. Accelerometer wear rates (61.9% (range 3.7-100.0%)) and log completion rates (69.0% (25.9-100.0)) were moderate. Exercise transiently increased NK cell number and function in healthy children and some children with ALL. There were no patterns in the change of the NK cell response to acute exercise over time. We were unable to assess the link between physical activity and NK cells due to a paucity of data. This study cautiously suggests that exercise is a feasible, acceptable and safe intervention that may increase NK cell number and function in children treated for ALL. / Thesis / Master of Science in Medical Sciences (MSMS) / Children treated for leukemia have weak immune systems, making them more susceptible to developing infections and cancer recurrence. Natural killer cells are a special immune cell that is very effective at combatting cancer and infections; however, children treated for leukemia have very low amounts of natural killer cells and they do not function well. Exercise is a simple way to boost the immune system in healthy adults and children, by increasing the number and function of natural killer cells. We don’t know what effect exercise has on natural killer cells in children with leukemia. Previous studies looking at the effects of exercise on the immune system of children with cancer have not been able to recruit enough children to participate. Therefore, it is also important to investigate why children with cancer may not want to participate in exercise studies looking at immune function. The main goals of this thesis were to assess how likely we are to recruit enough children being treated for leukemia to participate in a study looking at how exercise changes natural killer cells, if our participants enjoyed being part of this study, and how safe exercise is for children being treated for leukemia. We also wanted to learn about how natural killer cells respond to exercise in children being treated for leukemia. We found that most of the children and families that decided not to participate in our study felt they did not have time, and the second most common reason for not participating was because the children experienced anxiety surrounding blood draws for the study. The children that decided to participate in the study enjoyed the exercise and being in the study. We also found that the exercise was safe. Finally, we saw that exercise was able to increase natural killer cell numbers and function in some, but not all, children treated for leukemia. The results of this study suggest that exercise may be a realistic and safe way to improve immune function in some children with leukemia.

The tumour microenvironment plays a key role in tumour progression. In this thesis acute myeloid leukaemia (AML) was used as a model system to investigate the interplay between stromal and cancer cells. AML is a heterogeneous clonal disorder of haematopoietic undifferentiated progenitor cells or 'blast cells', which accumulate in the bone marrow and lead to the reduced output of crucial haematopoietic elements. Due to its heterogeneity (at least in part), treatment of the disease has not witnessed great innovation in the past 30 years. The bone marrow microenvironment (BMM) has a key role in the haematological malignancies contributing to the survival of leukaemic blasts. Relapse in AML occurs because of residual disease and evidence suggests that this resistance is facilitated through leukaemic cells ability to reside in BMM niches. To understand the precise role of the BMM in AML progression and therefore target any supportive mechanisms requires knowledge of how AML cells communicate with their microenvironment. In the work presented in this thesis I undertook a multi-proteomic approach that utilised liquid chromatography tandem mass spectrometry (LC-MS/MS) to assess the interplay between AML and BMM cell signalling. This thesis shows the results of a secretomic analysis of stromal cell lines, which identified a previously uncharacterized panel of six stromal secreted proteins (BMP-1, CSF-1, CTGF, HGF, S100-A4 and S100-A11) that support primary AML cell survival and proliferation in culture. Comparison of AML cell signalling (using global phosphoproteomic methods) following treatment with the newly identified growth factors revealed that these signalling proteins elicit multi-nodular activation of signalling networks with known anti-apoptotic activity. Consistent with the cell signalling proteomics data, cell viability studies as a function of pharmacological kinase inhibitor treatment determined that the sensitivity of AML to targeted kinase inhibitors was modulated by the supportive stromal conditioned media. To investigate heterotypic signalling between cell populations, AML/stromal cell co-cultures were designed, tested and optimised. These studies identified additional activated pathways in AML cells that were only present when AML cells had physical interaction with stroma. Complementary analysis of the stromal cells which had been first cultured with AML cells revealed that despite heterogeneity there is an emerging stromal phospho-proteomic signature that is different in BMM independent AML cells vs BMM interactive AML cells. Collectively these findings evidence the influence that the BMM can have on AML signalling. Although evidence for the influence of BMM in modulating AML resistance to standard chemotherapy exists, this study highlights specific BMM components that contribute to the ability of AML cells to circumvent current treatments based on kinase targeted drugs. These observations have implications for designing future therapies for AML.

Thesis (M.Sc. (Biochemistry))-- University of Limpopo, 2001 / Refer to document