Alcohol: a recently identified risk factor for breast cancer

Acknowledgements: I thank Dr. Barbour Warren, arriet Richardson and Alison James for their helpful input.

Role of hypoxia and hypoxia-inducible factor-1 in tumour immune escape

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor

Disequilibrium between coagulation and fibrinolysis can lead to severe haemostatic disorders such as thrombosis and hemophilia. Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFI may also mediate connections between coagulation and inflammation. Studies have associated high plasma TAFI levels with risk for thrombotic diseases. Interestingly, steroid hormones, such as estrogen and progestogens used in hormone replacement therapy or oral contraceptive preparations, have been shown to affect plasma TAFI levels. Regulation of the expression of the gene encoding TAFI, CBP2, is likely an important determinant of the role of the TAFI pathway in vivo; this concept motivated the investigations described in this thesis. In Chapter 2, the results of my research lead to the identification of key transcription factors regulating CPB2. Specifically, we described the binding of NF-Y and HNF-1 to the CPB2 promoter. NF-Y was shown to be an important factor for the basal CPB2 promoter activity. Binding of HNF-1 is essential for the activity of the promoter and is potentially responsible for the liver specific expression of CPB2. In Chapter 3, we set to investigate the effect of female sex hormone on hepatic expression of CPB2. We demonstrated that the levels of TAFI protein secreted from cultured hepatoma cells (HepG2) are decreased by 17beta-estradiol and progesterone. The change in protein expression was paralleled by decreases in CPB2 mRNA abundance and promoter activity. Deletion analysis of the CPB2 promoter indicated that the genomic effects of estrogen and progesterone are likely mediated via a non-classical mechanism. In Chapter 4, we evaluated the effects of various inflammatory mediators on expression of the gene encoding mouse TAFI (Cpb2). Our results showed that Cpb2 mRNA abundance and promoter activity are up-regulated by inflammatory mediators IL-1beta, IL-6, and TNFalpha. We also showed that TNFalpha mediates its effect via the binding of NFkB. Additionally, our results suggest that TNFalpha promotes the binding of NFkB to the promoter by increasing its translocation to the nucleus. The NFkB site is not conserved between human and mouse and may explained the different responses to inflammation observed in vivo.

A Quantitative and Mechanistic Assessment of Activated Thrombin-Activatable Fibrinolysis Inhibitor and its Role in Pathological Bleeding and Thrombosis

The coagulation and fibrinolytic systems are linked by the thrombin-thrombomodulin complex which regulates each system through activation of protein C and TAFI, respectively. We have used novel assays and techniques to study the enzymology and biochemistry of TAFI and TAFIa, to measure TAFI activation in hemophilia A and protein C deficiency and to determine if enhancing TAFI activation can improve hemostasis in hemophilic plasma and whole blood. We show that TAFIa not TAFI attenuates fibrinolysis in vitro and this is supported by a relatively high catalytic efficiency (16.41μM-1s-1) of plasminogen binding site removal from fibrin degradation products (FDPs) by TAFIa. Since the catalytic efficiency of TAFIa in removing these sites is ~60-fold higher than that for inflammatory mediators such as bradykinin it is likely that FDPs are a physiological substrate of TAFIa. The high catalytic efficiency is primarily a result of a low Km which can be explained by a novel mechanism where TAFIa forms a binary complex with plasminogen and is recruited to the surface of FDPs. The low Km also suggests that TAFIa would effectively cleave lysines from FDPs during the early stages of fibrinolysis (i.e. at low concentrations of FDPs). Since individuals with hemophilia suffer from premature fibrinolysis as a result of insufficient TAFI activation we quantified TAFI activation in whole blood from hemophilic subjects. Both the rate of activation and the area under the TAFI activation time course (termed TAFIa potential) was determined to be reduced in hemophilia A and the TAFIa potential was significantly and inversely correlated with the clinical bleeding iii phenotype. Using a novel therapeutic strategy, we used soluble thrombomodulin to increase TAFI activation which improved the clot lysis time in factor VIII deficient human plasma and hemophilic dog plasma as well as hemophilic dog blood. Finally, we briefly show in a biochemical case study that TAFI activation is enhanced in protein C deficiency and when afflicted individuals are placed on Warfarin anticoagulant therapy, TAFI activation is reduced. Since TAFIa stabilizes blood clots, this suggests that reducing TAFI activation or inhibiting TAFIa may help restore blood flow in vessels with pathological thrombosis.

Role of Stromal Cell-Derived Factor-1 in Neoangiogenesis in Endometriosis Lesions

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.

Elucidation of the interactions between the transcription factor E2A and the transcriptional co-activator CBP/p300

E2A is a transcription factor that plays a particularly critical role in lymphopoiesis. The chromosomal translocation 1;19, disrupts the E2A gene and results in the expression of the fusion oncoprotein E2A-PBX1, which is implicated in acute lymphoblastic leukemia. Both E2A and E2A-PBX1 contain two activation domains, AD1 and AD2, which comprise conserved ΦxxΦΦ motifs where Φ denotes a hydrophobic amino acid. These domains function to recruit transcriptional co-activators and repressors, including the histone acetyl transferase CREB binding protein (CBP) and its paralog p300. The PCET motif within E2A AD1 interacts with the KIX domain of CBP/p300, the disruption of which abrogates the transcriptional activation by E2A and the transformative properties of E2A-PBX1. The generation of a peptide-based inhibitor targeting the PCET:KIX interaction would serve useful in further assessing the role of E2A and E2A-PBX1 in lymphopoiesis and leukemogenesis. An interaction between E2A AD2 and the KIX domain has also been recently identified, and the TAZ domains of CBP/p300 have been shown to interact with several transcription factors that contain ΦxxΦΦ motifs. Thus the design of an inhibitor of the E2A:CBP/p300 interaction requires the full complement of interactions between E2A and the various domains of CBP/p300 to be elucidated. Here, we have used nuclear magnetic resonance (NMR) spectroscopy to determine that AD2 interacts with KIX at the same site as PCET, which indicates that the E2A:KIX interaction can be disrupted by targeting a single binding site. Using an iterative synthetic peptide microarray approach, a peptide with the sequence DKELQDLLDFSLQY was derived from PCET to interact with KIX with higher affinity than the wild type sequence. This peptide now serves as a lead molecule for further development as an inhibitor of the E2A:CBP/p300 interaction. Fluorescence anisotropy, peptide microarray technology, and isothermal titration calorimetry were employed to characterize interactions between both TAZ domains of CBP/p300 and the PCET motif and AD2 of E2A. Alanine substitution of residues within PCET demonstrated that the ΦxxΦΦ motif is a key mediator of these interactions, analogous to the PCET:KIX interaction. These findings now inform future work to establish possible physiological roles for the E2A:TAZ1 and E2A:TAZ2 interactions.

A FUNCTIONAL, COMPARATIVE AND CLINICAL ANALYSIS OF SPERM-BORNE OOCYTE ACTIVATING FACTOR, PAWP

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.

Analysis of parameters of haemostasis in pre-eclamptic, normotensive pregnant and non-pregnant women.

Pregnancy is characterized by a state of heightened coagulation, which is exacerbated in pathological conditions such as pre-eclampsia (PET). PET is further associated with abnormal maternal inflammation and increased circulating microparticles (MP); however, a mechanistic link between these pathological features has never been established. It is proposed in this thesis that abnormal maternal inflammation is causally linked to pro-coagulant trophoblast MP shedding via a mechanism mediated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF), thereby contributing to maternal coagulopathies associated with PET. Using thromboelastography (TEG) and standard laboratory tests, haemostatic function was evaluated in PET and normotensive subjects at delivery and post-partum. Furthermore, the effects of the menstrual cycle and oral contraceptive (OC) use on haemostatic function were assessed in non-pregnant subjects in order to understand their influence on post-partum haemostasis. Plasma TNF and pro-coagulant MP levels were evaluated in the pregnant subjects. Using chorionic villi explants from human term placentas, MPs were quantified after TNF administration. The pro-coagulant potential of placental MPs was evaluated by TEG by spiking whole-blood with medium containing MPs from chorionic villi. TEG identified increased whole-blood coagulability in PET subjects at delivery, demonstrating its increased sensitivity over standard laboratory tests at identifying haemostatic alterations associated with PET. Haemostatic alterations were normalized by six weeks post-partum. TEG also identified cyclic haemostatic variations associated with OC use. Chorionic villi treated with TNF (1 ng/ml) shed significantly more MPs than untreated placentas. MPs from chorionic villi increased the coagulability of whole-blood. Together, results provide evidence supporting the concept that abnormal maternal inflammation is causally linked to the development of maternal coagulopathies in pregnancy complications. Moreover, TEG may be superior to standard laboratory tests in evaluating haemostasis in pregnant and non-pregnant subjects.

Glycemic State Regulates Brain-Derived Neurotrophic Factor Responsiveness of Neurons in the Paraventricular Nucleus of the Hypothalamus

Brain derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and binds to the tropomyosin-related kinase B (TrkB) receptor. Like other neurotrophic factors, BDNF is involved in the development and differentiation of neurons. Recently, studies have suggested important roles for BDNF in the regulation of energy homeostasis. The paraventricular nucleus (PVN) is critical for normal energy balance contains high levels of both BDNF and TrkB mRNA. Studies have shown that microinjections of BDNF into the PVN increase energy expenditure, suggesting BDNF plays a role in energy homeostasis through direct actions in this hypothalamic nucleus. We used male Sprague-Dawley rats to perform whole-cell current-clamp experiments from PVN neurons in slice preparation. BDNF was bath applied at a concentration of 2nM and caused depolarizations in 54% of neurons (n = 25; mean change in membrane potential: 8.9 ± 1.2 mV), hyperpolarizations in 23% (n = 11; mean change in membrane potential: -6.7 ± 1.4 mV), while the remaining cells tested were unaffected. Previous studies showing effects of BDNF on γ-aminobutyric acid type A (GABAA) mediated neurotransmission in PVN led us to examine if these BDNF-mediated changes in membrane potential were maintained in the presence of tetrodotoxin (TTX) sodium channel blocker (N = 9; 56% depolarized, 22% hyperpolarized, 22% non-responders) and bicuculline (GABAA antagonist) (N = 12; 42% depolarized, 17% hyperpolarized, 41% non-responders), supporting the conclusion that these effects on membrane potential were postsynaptic. We also evaluated the effects of BDNF on these neurons across varying physiologically relevant extracellular glucose concentrations. At 10 mM 23% (n = 11; mean: -6.7 ± 1.4 mV) of PVN neurons hyperpolarized in response to BDNF treatment, whereas at 0.2 mM glucose, 71% showed hyperpolarizing effects (n = 12; mean: -6.3 ± 2.8 mV). Our findings reveal that BDNF has direct impacts on PVN neurons and that these neurons are capable of integrating multiple sources of metabolically relevant input. Our analysis regarding glucose concentrations and their effects on these neurons’ response to other metabolic signals emphasizes the importance of using physiologically relevant conditions for study of central pathways involved in the regulation of energy homeostasis.