published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The small GTPases function as molecular switches to control diverse signaling cascades. The mammalian Rit and Rin, along with Drosophila Ric, comprise an evolutionarily conserved subfamily of the Ras-related GTPases. Previous studies using cultured cell models suggested that Rit was involved in the control of cell proliferation, transformation, neuronal differentiation, morphogenesis, and cell survival, but the principal physiological function of Rit remained uncharacterized. To address this outstanding question, we employed a genetic approach, engineering a Rit knockout mouse. Using this animal model, we demonstrate a central role of Rit in governing cell survival in a p38-dependent fashion. Primary mouse embryonic fibroblasts (MEFs) derived from Rit-/- mice display increased apoptosis and selective disruption of MAPK signaling following oxidative stress. These deficits include a reduction in ROS-mediated stimulation of a novel p38-MK2-HSP27 signaling cascade, which appears to act upstream of the mTORC2 complex to control Akt-dependent cell survival. In the adult brain, proliferation of stem cells within the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), provide a lifelong supply of new neurons. Adult neurogenesis appears critical for learning and memory and is altered in animal models of brain injury and neurological diseases. Thus, a greater understanding of the regulation of adult neurogenesis will provide insight into its myriad physiological roles but also to the development of therapeutic strategies for the treatment of injury and the progression of brain diseases. Here we find that Rit plays a central role in governing the survival of hippocampal neurons in response to oxidative stress. Importantly, using a controlled cortical impact model of traumatic brain injury (TBI), we show that Rit acts to protect newborn immature neurons within the SGZ of the DG from apoptosis following TBI. Finally, studies indicate that Rit plays a significant role in directing IGF-1 signaling, a key neurotrophin known to promote neurogenesis and to protect neurons against apoptotic stress. Together, these studies establish Rit as a critical regulator of a p38 MAPKdependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress, including the survival of newborn hippocampal neurons in the traumatically injured brain.

Parthenolide (PN), a major active component of the traditional herbal medicine feverfew, has been shown to have anti-inflammatory and anti-tumor properties. More remarkably, the cytotoxicity of PN seems selective to tumor cells but not their normal cell counterparts. In the present study, we investigate whether and how PN selectively enhances tumor sensitivity to radiation therapy by using prostate cancer cells LNCaP, DU145 and PC3, as well as normal prostate epithelial cells PrEC. Our study demonstrates that inhibition of NF-κB pathway and suppression of its downstream target MnSOD are common mechanisms for the radiosensitization effect of PN in prostate cancer cells. The differential susceptibility to PN in two radioresistant cancer cells, DU145 and PC3, is due, in part, to the fact that in addition to NF-κB inhibition, PN activates the PI3K/Akt pro-survival pathway in both cell lines. The presence of PTEN in DU145 cells enhances the radiosensitization effect of PN by suppression of the steady state level of activated p-Akt. We also demonstrate that PN selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. PN causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H2DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, PN activates NADPH oxidase leading to a decrease in the level of reduced thioredoxin, activation of PI3K/Akt and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets, antioxidant enzyme MnSOD and catalase. Importantly, when combined with radiation, PN further increases ROS levels in PC3 cells, while it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing GSH level. Overall, our data support the concept that increasing oxidative stress in cancer cells, which are already under high constitutive oxidative stress, will lead to cell death, while the same stress may allow normal cells to maintain redox homeostasis through adaptive response. Thus, modulating cell redox status may be a novel approach to efficiently and selectively kill cancer cells.

<p>Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an <i>in vitro</i> cell model. The major findings were as follows:</p><p>i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4. </p><p>ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways. </p><p>iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection.</p><p>iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3 </p><p>v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.</p>

Exposure of cells to hydrogen peroxide can cause oxidative damage to cellular constituents including lipids, protein, and DNA. At elevated concentrations, hydrogen peroxide can trigger cell death by apoptosis or necrosis. Apoptotic cell death can be prevented by overexpression of the oncoprotein Bcl-2. The exact mechanism by which Bcl-2 blocks cell death is controversial. Some researchers believe that Bcl-2 possesses antioxidant properties that protect cells from apoptosis. The purpose of this thesis was to assess oxidative stress and apoptosis following hydrogen peroxide exposure in Jurkat T cells overexpressing Bcl-2. One of the major objectives was to ascertain whether or not Bcl-2 overexpression elevated the antioxidant capacity of Jurkat T cells to provide protection from oxidant-induced cell death. Hydrogen peroxide treated Jurkat cells became apoptotic at moderate levels of oxidant (25-100 uM H2O2), and necrotic at higher doses (greater than 200 uM H2O2). Bcl-2 overexpression prevented caspase activation and cell death at the apoptotic doses of H2O2, but not the necrotic doses. Caspase inhibition studies demonstrated that Bcl-2 overexpression provided a greater level of resistance from H2O2-induced cell death than the broad-spectrum caspase inhibitor z-VAD.fmk. A systematic study was carried out examining the antioxidant status of Jurkat cells overexpressing Bcl-2. Several Bcl-2 transfectants were utilised for the study, so that any differences seen could be correlated to the level of Bcl-2 expression. Surprisingly, there were no statistically significant differences among the Bcl-2 transfectants for any of the antioxidant enzymes. Jurkat cells overexpressing Bcl-2 exhibited the same level of oxidative damage to lipids and protein in response to H2O2 exposure as the parental Jurkat cells. Interestingly, Jurkat cells overexpressing Bcl-2 continued to grow in culture after H2O2 exposure, despite harboring damage to cellular constituents. Consistent with these results, H2O2 treated Jurkat cells overexpressing Bcl-2, which failed to undergo apoptosis, were more prone to genomic instability. Together, these findings suggest that Bcl-2 overexpression protects Jurkat cells from H2O2-induced cell death by blocking apoptosis. Jurkat cells overexpressing Bcl-2 were no better at detoxifying oxidants and showed the same level of oxidative damage following H2O2 exposure. As a result, the overexpression of Bcl-2 considerably enhanced the mutagenicity of H2O2.

Glyco-oxidation is linked to the pathophysiology of diabetes and diabetic complications. The process of glyco-oxidation generates reactive dicarbonyls, which form adducts on arginine residues in distributions throughout the proteome that are site-specific depending on the protein microenvironment. Dicarbonyl adducts are thus markers for glyco-oxidative stress. Various approaches using mass spectrometry permits the identification, localization, and quantification of these dicarbonyl adducts. Using MG as a model dicarbonyl, a shotgun proteomics approach identified the sites for modification of major plasma proteins. Thirty five sites on seven abundant plasma proteins were found, and investigation into the microenvironment surrounding the target arginine sites revealed a neighboring charged residue motif where adjacent residues were either negatively or positively charged. One of the sites identified was R257 in HSA, which is located in the important drug binding site I. We validated drug site I as a target for MG modification by the adaptation of two assays to monitor the effect of MG modification. MG significantly decreases the rate of hydrolysis of PGE2 in drug site I, and induces the displacement of prodan from drug site I. Molecular modeling of warfarin docking at drug site I with the MG-modified R257 resulted in significantly decreased binding and change in binding orientation. The oxidation products of susceptible residues methionine, tryptophan, and cysteine were evaluated using MRM of oxidized HSA peptides. Oxidation of methionine gave the M+16 single oxidized product, and M329 in HSA was the most responsive site. Oxidation of the sole W214 tryptophan produced the W+32 double oxidation product, and oxidation of C34 produced the C+48 triple oxidation product. MG, 3DG, and glucosone were evaluated for propensity to modify 12 HSA sites based on MRM of dicarbonyl modified HSA. Dicarbonyl modification was independent of arginine solvent accessibility. In a clinical study using nephropathy as an endpoint, sites of oxidation and modification of HSA by MG, 3DG, and glucosone were quantified by MRM. The most important variable among diabetic subjects was metformin use, and subjects taking metformin had significantly reduced markers for glyco-oxidation. These findings may be useful in the development of new diabetes therapies that aim to ameliorate glyco-oxidative stress.

Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin inhibitors. I hypothesize that the anti-tumor effect of mTOR inhibitors in mantle cell lymphoma is mediated by an increase in manganese superoxide dismutase (MnSOD) protein expression and accumulation of hydrogen peroxide (H₂O₂). Findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H₂O₂, and is necessary for the full growth inhibitory effect of rapamycin. Furthermore, over-expression of MnSOD elevated the level of H₂O₂ and increased sensitivity to MnSOD. Treatment with rapamycin resulted in a loss of serine 473 phosphorylation of AKT and increased levels of MnSOD were found to be due to inhibition of the mTORC2 complex. These results are the first to suggest that long term treatment of MCL cells with rapamycin inhibits the mTORC2 complex. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of mTOR inhibitors, we may be able to identify additional predictive markers to improve the therapeutic value, or study drug combinations that will enhance the effect of ROSinduced cytotoxicity. A retrospective study utilizing samples from lymphoma patients receiving standard anthracycline-based therapies, identified single nucleotide polymorphisms in oxidative stressrelated genes associated with survival. Individuals carrying minor allele SNPs in myeloperoxidase (MPO) and an aldo-keto reductase (AKR1C3) were found to be associated with shorter time to disease progression and death. This data suggest that some patients may benefit from a different therapy than the current standard of care and that regulation of the redox environment plays a role in aggressive lymphoma treatment response.

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

Diabetes, which is characterized by elevated plasma glucose, can have a devastating effect on peripheral nerves frequently leading to the clinical symptoms of neuropathy. Diabetic autonomic neuropathy (DAN) results from damage to autonomic nerves, and the most troubling forms of DAN often lead to cardiovascular abnormalities and premature death. Despite the prevalence of DAN and the impact to quality and life expectancy, the precise mechanisms underlying these pathologies are poorly understood. Recently, a new model for the onset of DAN was proposed where hyperglycemia-induced oxidative stress inactivates nicotinic acetylcholine receptors (nAChRs), the main receptor driving autonomic synaptic transmission at sympathetic ganglia. This inactivation leads to the depression of synaptic transmission, and consequently triggers the onset of autonomic neuropathy in diabetic mice. However, the source and pathways contributing to the elevation of reactive oxygen species (ROS) and oxidative stress remained unclear. In recent years it has been shown that the accelerated formation of advanced glycation end products (AGEs) and activation of their receptor (RAGE) in diabetes play a major role in the induction of oxidative stress in sensory nerve damage. Thus we hypothesized that the activation and up-regulation of RAGE during high glucose conditions is a major source of ROS production in sympathetic neurons leading to the inactivation of nAChRs and autonomic malfunction. In this thesis we show for the first time that RAGE is expressed in cultured sympathetic neurons and is also up-regulated during high glucose conditions. Our results further demonstrate that direct RAGE activation by its natural ligands leads to an increase in cytoplasmic ROS which in turn induces the inactivation of nAChRs in sympathetic neurons. We also report that high glucose-induced ROS generation and subsequent inactivation of nAChRs is prevented in sympathetic neurons from RAGE knock-out mice. The results of this dissertation suggest RAGE to be a pivotal source of ROS production leading to the functional deficits observed in sympathetic neurons during high glucose conditions.

Ozone therapy is an alternative form of therapy that has gained attention in the last couple of years. It is believed that O3 may exert a stimulatory effect on the antioxidant defence and immune systems and may therefore be effective in the treatment of ischemic disorders. diabetes mellitus. AIDS and other diseases. On the other hand. it is well known that O3 is a reactive molecule that is toxic to the pulmonary system. Therefore. there remains scepticism regarding its use as a form of therapy. In order to shed some light on this. the effects of ozone autohemotherapy (O3-AHT) on the energy homeostasis of white blood cells were investigated. The possible protective effects of the plasma antioxidant defence system during O3-AHT, were also investigated. Venous blood from six apparently healthy human donors was collected in heparin. In one aliquot a precise volume of blood was mixed with an equal volume of O2/O3 gas mixture containing 20 or 80 μg/ml O3 for 20 minutes. In the other aliquot, the plasma was washed out and the cells resuspended in a buffered phosphate solution. The buffered blood cells were treated with the same concentrations of O3. Control samples was either not treated or treated with a corresponding volume of O2 . Various biochemical analyses were done on the whole blood and buffered cells to determine the oxidant/antioxidant status, cell viability, apoptosis and mitochondrial function. The higher concentration of O3 increased oxidative stress and caused death of white blood cells. Antioxidant enzyme (catalase, glutathione reductase and glutathione peroxidase) activity and the plasma antioxidant capacity decreased, whereas superoxide dismutase levels increased slightly. Exposure to O3 also increased caspase 3/7 activity. A decrease in mitochondrial function was measured by a decrease in ATP levels and an increase in NADH/NAD+ ratio. Complex IV of the respiratory chain was almost completely inhibited by both O3 concentrations. These results indicated that the death of white blood cells was probably through apoptosis. These effects were more evident in the absence of plasma antioxidants. Therefore. high concentrations of O3 were damaging to the cells, but this effect was lessened by antioxidants present in plasma. In view of the results, the use of O3 as a therapy needs to be reconsidered. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.