Thesis (D.Tech.: Clinical Technology)-Durban Institute of Technology, 2005 xxiv, 235 leaves ; ill. ; 30 cm / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They ex¬ert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancer-promoting metabo¬lites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunomodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL –1, IL-6, IL-8, IL-10 and TNF-) were determined using ELISA kits.

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Thesis (D.Tech.: Clinical Technology)-Durban Institute of Technology, 2005 xxiv, 235 leaves ; ill. ; 30 cm / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They ex¬ert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancer-promoting metabo¬lites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunomodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL –1, IL-6, IL-8, IL-10 and TNF-) were determined using ELISA kits.

Radiotherapy is the use of ionising radiation to induce localised DNA damage to cancerous tissues, leading to cell death and disease control. In order to maximise tumour growth control and to limit damage of the healthy surrounding tissues and the consequent side effects for the patient, molecular determinants of tumour radioresistance are investigated as potential clinical targets. A high-throughput siRNA colony formation assay screen in HeLa cervical carcinoma cells previously published by our laboratory identified modulators of radiosensitivity. From the list CSF1R, EPHB2, GAK and TOPK, were selected and validated. TOPK (T-LAK cell-originated protein kinase, also known as PDZ-binding kinase, PBK) was selected for further investigation because it is overexpressed in most malignancies but not in normal tissues, apart from testis and placenta. Knockdown of TOPK was shown to induce radiosensitisation in a panel of cancer cell lines with no significant effects on normal cells. A role for TOPK in the cell cycle response to ionising radiation (IR) was discovered in HCT116 colorectal cancer cells, with alterations in the G₁/S and G₂/M checkpoints. Furthermore, immunoprecipitation experiments identified a physical interaction between TOPK and CDKN1A (p21) at 8 hours after IR. Apoptosis and the number of multinucleated cells were significantly increased in TOPK depleted cells exposed to IR, suggesting the possibility of aberrant mitosis and mitotic catastrophe in these cells. High TOPK expression in early breast cancer patients was shown to be associated with poor recurrence-free survival. In addition, immunohistochemistry (IHC) analysis on samples from prostate cancer patients identified a strong correlation between high levels of TOPK and poor clinical response to radiotherapy. In order to facilitate future in vivo experiments, an HCT116 shRNA stable knockdown cell line was developed and two commercially available TOPK inhibitors were tested and optimised. Taken together, these data suggest that TOPK is a molecular determinant of radiosensitivity with a great potential for future clinical applications.

The design of the proposed study was to administer R-FB to 72-day old Min/+ mice for up to 42 days. In order to capture the process of tumor reduction, animals were necropsied at various time points. At each time point animals were evaluated for tumor loads and presence of apoptotic cells along the small intestine. Studies have shown that when R-flurbiprofen (R-FB) is administered in the Min/+ mouse model it can cause the prevention and regression on intestinal tumors.

Throughout society’s continual war against cancer, we have attempted pharmacological intervention only to find that tumors develop modes of resistance. It is well known that genetics play an integral role in cancer. Technological advances have greatly improved our ability to study cancer biochemistry beyond the genome by measuring changes in the expression and activity of RNA, proteins, and lipids in experimental models and human patients. As our techniques and technology to perform cancer research progresses, it is becoming more evident that cancer cells develop stress tolerance mechanisms at multiple levels within the central dogma, including altering mRNA expression, enzyme concentrations, and functional activity of cellular proteins and lipids. In the first chapter, I review previous discoveries demonstrating the importance of metabolic reprogramming in cancer cells and how shifts in metabolic pathways contribute to cancer progression and therapeutic challenges. I discuss how mass spectrometry is a multifunctional research tool that can be used to identify global shifts in gene expression, identify oncogenic roles of specific metabolites and corresponding metabolic pathways, conduct enzyme activity assays, and understand the effects of drugs on cell signaling and metabolic flux through specific pathways. While metabolic reprogramming is a complex and multifaceted concept, the following chapters focus on two specific stress tolerance pathways of lipid and protein metabolism we have shown to significantly promote cancer cell evolution, proliferation, and drug resistance in models of human pancreatic and colon cancer. I describe novel mass spectrometry-based lipidomics and proteomics methods we developed to measure and determine the biological impact of these pathways in each model. I discuss the contributions we have made toward increasing general knowledge of metabolic reprogramming networks in cancer and how they may be targeted in more specific and effective manners to sensitize cancers to therapeutic drugs. Specifically, the second chapter entails our study of a pro-survival lipid metabolic pathway driven by the sphingolipid modifying enzyme sphingosine kinase in a panel of differentially reprogrammed pancreatic cancer subclones. The third chapter describes our novel kinetic proteomics approach to identify how the cellular degradation system autophagy is used to selectively remodel the proteome of colon tumor cells in a xenograft mouse model of colon cancer. Lastly, I discuss how these and other projects completed during my graduate work lay a foundation for ongoing research to further our fundamental understanding of cancer metabolism and treatment development.

Les mutations héréditaires dans le gène BRCA2 sont associées à une forte susceptibilité au développement du cancer du sein et de l’ovaire. La protéine suppresseur de tumeur BRCA2 est essentielle pour préserver l’intégrité des chromosomes après endommagement de l’ADN. BRCA2 est impliquée dans la recombinaison homologue (RH), une voie fiable de réparation des cassures de l’ADN. BRCA2 exerce aussi un rôle pendant la mitose afin d’assurer un point de contrôle et une division cellulaire correcte. Bien que le rôle de BRCA2 dans la RH soit bien établi, la littérature décrive une restauration partielle de la fonction de RH dans des cellules ne possédant pas le site de liaison à l’ADN en C-terminal (CT-DBD), ce que nous a encouragé à voir s’il existait un domaine secondaire de liaison à l'ADN. L'analyse in silico a révélé un domaine zf-PARP putatif dans la région N-terminale. Normalement, ce type de domaine s’associe à l’ADN, ce que nous a porté à l’examiner. En utilisant des fragments purifiés de la partie N-terminale comprenant le site putatif dans des analyses de changements de mobilité électrophorétique, nous avons montré une activité de liaison à l’ADN. En comparaison avec le CT-DBD canonique, le site de liaison à l’ADN en N-terminal (NT-DBD) manifeste une affinité plus forte pour divers substrats et contrairement du CT-DBD il est capable de s’associer à l’ADN à double brin. En utilisant des tests d’échange de brin, nous avons également montré que le NT-DBD peut stimuler la fonction de recombinaison de RAD51. De plus, des variantes faux-sens dans le NT-DBD trouvé chez les patients atteints de cancer du sein ont montré une activité réduite d’association à l’ADN et une stimulation diminuée de l’activité de RAD51 ce qui implique que ces amino-acides sont importants pour les deux fonctions. Ce travail révèle un nouveau sitede liaison à l’ADN, ce qui contrairement au CT-DBD est capable de s’associer à l’ADN double-bras(db) et stimuler l’activité de recombinaison de RAD51. Nous proposons que le NT-DBD positionne RAD51 à la jonction entre ADNdb et ADNsb, ce qui facilite le chargement de RAD51 sur l’ADN recouvert de RPA. Cette activité pourrait promouvoir la RH pendant la réparation des cassures de l’ADN (von Nicolai, C et al., 2016, under revision).Afin de définir la prévalence des mutations de NT-DBD pour la prédisposition au cancer, nous avons sélectionné des variants faux-sens non-classifiés (variants of unknown clinical significance), identifiés dans des familles à risque élevé de développer un cancer du sein. Nous avons effectué des tests afin d’étudier l’impact de ces variants sur la fonction de BRCA2 dans la RH et la mitose. Certains de ces variants ont conduit à une hypersensibilité aux agents endommageant l’ADN et aux inhibiteurs de PARP, caractéristique d’une RH défectueuse alors qu’un de ces variants était compétent pour la réparation. Tous les variants ont induit une duplication normale des centrosomes, mais la cytokinèse était défectueuse. Ce phénotype suggère un défaut dans la formation du midbody et de l’abscission. Cette étude aidera à classifier les VUS dans le NT-DBD et facilitera la consultation génétique pour des individus. BRCA2 est un médiateur de la RH dépendante de RAD51. Son homologue méiotique, DMC1, partage structure et fonction similaire et s’associe à BRCA2. Néanmoins, la pertinence fonctionnelle de cette interaction reste élusive. Nous avons montré que BRCA2 interagit avec DMC1 au travers des répétitions BRC et promeut la formation de molécules d'adhérence. Cet effet stimulant est dû au renforcement de la liaison de DMC1 à l’ADN. BRCA2 complet et fonctionnel était surtout capable de stimuler l’activité d’échange de brin de DMC1, ce qui confirme les résultats obtenus avec les répétitions BRC. Nos résultats identifient BRCA2 comme une protéine de médiation de la recombinaison méiotique et renforcent le rôle des répetitions BRC dans cette fonction (Martinez, von Nicolai, et al., PNAS, 2016). / Germline mutations in the BRCA2 gene lead to high susceptibility to the development of breast and ovarian cancer. The tumor suppressor protein BRCA2 is essential for preserving chromosome integrity after DNA damage emerging from endogenous or exogenous sources. BRCA2 functions in Homologous Recombination (HR), the most reliable pathway to repair DNA double strand breaks. BRCA2 exerts its tumor suppressor role also at several stages during mitosis where it ensures checkpoint control and proper cell division.Although the function of BRCA2 in HR is well established, evidence from the literature describing a partial restoration of HR function in cells lacking the C-terminal DNA binding domain (CT-DBD) brought us to test the hypothesis of a secondary DNA binding domain in BRCA2.In silico analysis of the protein revealed a putative zinc finger-PARP domain in exon 10 of the N-terminal region. This type of domain usually binds DNA which prompted us to examine this activity in vitro. Using purified N-terminal fragments comprising the putative DNA binding domain in electrophoresis mobility shift assay we demonstrated the DNA binding activity of the N-terminus of BRCA2. When compared to the canonical CT-DBD, the N-terminal DNA binding domain (NT-DBD) exhibits stronger affinity for various DNA substrates and unlike the CT-DBD, it can also associate with dsDNA. Using a DNA strand exchange assay we also showed that the NT-DBD stimulates the recombination function of RAD51. In addition, BRCA2 missense variants in the NT-DBD found in breast cancer patients showed reduced dsDNA binding and decreased stimulation of RAD51 recombination activity on dsDNA/ssDNA containing substrates, implying that these residues are important for both functions. This work revealed a novel DNA binding domain in the N-terminus of BRCA2 that, in contrast to the CT-DBD, can associate with dsDNA and promote RAD51 recombination activity. We propose that the NT-DBD positions RAD51 at the ssDNA/dsDNA junction facilitating RAD51 loading onto the RPA-coated ssDNA. This activity may promote HR in DSB repair and in daughter strand gap repair (von Nicolai, C et al., 2016 submitted).To define the relevance of NT DBD on cancer predisposition, we selected several missense variants of unknown clinical significance (VUS) found in families at high risk to develop breast cancer located in this region. We used in vitro and in vivo functional assays to study the impact of the mutations on BRCA2 function in HR and mitosis. Some of the variants exhibited hypersensitivity to DNA damaging agents and PARP inhibitors, a hallmark of defective HR while one variant was proficient in repair. All variants showed normal centrosome duplication, but exhibited delayed or failed cytokinesis. This phenotype suggests a defect of the variants in midbody formation and abscission as a consequence of impaired BRCA2 function. It remains to be established if the defects in HR and cytokinesis are related. In the future, this study will help to classify VUS in the NT-DBD and facilitate genetic counselling of individuals carrying these mutations.BRCA2 is a mediator protein in RAD51-dependent HR. Its meiotic counterpart, DMC1, shares similar structure and function and binds BRCA2. However, the functional relevance of this interaction remained elusive. In this work, we showed that through the BRC repeats, BRCA2 interacts with DMC1 and promotes joint molecule formation. This stimulatory effect is due to the enhancement of DMC1 assembly on ssDNA. Importantly, full-length BRCA2 also stimulated the DNA strand exchange activity of DMC1, confirming the results with the isolated BRC repeats. Our results identify BRCA2 as a mediator of meiotic recombination and underline the role of the BRC repeats on this function (Martinez, von Nicolai, et al., 2016, PNAS).

No description available.

Pancreatic cancer (PC) remains a significant, unresolved issue because of its complex genetic blueprint and lack of reliable detection markers. The purpose of this study was to examine the possible correlation between tobacco use, gender, and age in the etiopathogenesis of PC and other cancer types with a shared-gene association (CTSG-A). The unified paradigm of cancer causation was used to understand the pathopoiesis mechanism of smoking and shared genes in PC. A cross-sectional study was performed using secondary data from the cancer survivorship module of the 2014 Behavioral Risk Factor Surveillance System survey. Results of ordinal logistic regression analyses indicated no correlation between smoking and prevalence of PC and CTSG-A, but gender and age were significant predictors. Gender has a statistically significant effect on the prediction of PC/ CTSG-A induction and promotion. Increased probability of developing the disease was found as the person reach the age between 62 and 69 years of age. Findings may enhance the understanding of environmental, genetic, and biodemographic interactions in disease evolution (induction, promotion, and expression periods). Findings may also be used to promote population health and improve health behaviors for individuals in vulnerable, high-risk groups.

Triple-negative breast cancers (TNBCs) are highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. Combination therapy that includes targeting the murine double minute 2 (Mdm2) signaling axis offers a promising approach. Protein-protein interaction inhibitors such as Nutlin-3a block the binding of key signaling molecules such as p53, p73α, and E2F1 to the hydrophobic pocket of Mdm2 and can lead to activation of cell-death signaling pathways. Since clinical trials for TNBC are evaluating the DNA damaging agent carboplatin, the objective of this thesis was to evaluate the therapeutic potential and mechanism of action of combination carboplatin and Nutlin-3a to treat TNBC. In TNBC cell lines with a mutant p53 background, we determined if modulation of Mdm2 function in the context of carboplatin-mediated DNA damage resulted in a synergistic inhibition of cell growth. Several ratios of carboplatin:Nutlin-3a were strongly synergistic in increasing cell death, with combination indices of 0.5 and lower. Mechanistic studies indicated that drug sensitivity and Mdm2 expression were dependent on p73. Mdm2 localized to a larger degree in the chromatin fraction isolated from cells treated with the combination treatment consistent with observations by others that Mdm2 binds to the Mre11/Rad50/Nbs1 complex, inhibits the DNA damage response, and increases drug sensitivity. In vivo efficacy experiments were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. For assessment of baseline tumor burden and randomization, fluorescent imaging of E2-Crimson expressing TMD231 cells was performed. Following Nutlin-3a and carboplatin combination treatment, there was a statistically significant reduction in primary tumor volume as well as lung metastases with significantly increased probability of survival compared to Vehicle and single drug treatments (p<0.001). While there was a decrease in bone-marrow cellularity, this did not lead to bone-marrow aplasia, and body weights recovered to normal levels within 7 days post-treatment. The present studies demonstrate the promise of Mdm2 as a therapeutic target in combination with conventional therapy, increase our understanding of how to potentiate DNA damage in cancers, and may lead to new clinical therapies for triple-negative primary and metastatic breast cancer.