A Proinflammatory Cytokine Inhibits P53 Tumor Suppressor Activity

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis

8-Chloroadenosine induces apoptosis in human coronary artery endothelial cells through the activation of the unfolded protein response

© 2019 The Authors Infiltration of leukocytes within the vessel at sites of inflammation and the subsequent generation of myeloperoxidase-derived oxidants, including hypochlorous acid, are key characteristics of atherosclerosis. Hypochlorous acid is a potent oxidant that reacts readily with most biological molecules, including DNA and RNA. This results in nucleic acid modification and the formation of different chlorinated products. These products have been used as biomarkers of inflammation, owing to their presence in elevated amounts in different inflammatory fluids and diseased tissue, including atherosclerotic lesions. However, it is not clear whether these materials are simply biomarkers, or could also play a role in the development of chronic inflammatory pathologies. In this study, we examined the reactivity of different chlorinated nucleosides with human coronary artery endothelial cells (HCAEC). Evidence was obtained for the incorporation of each chlorinated nucleoside into the cellular RNA or DNA. However, only 8-chloro-adenosine (8ClA) had a significant effect on the cell viability and metabolic activity. Exposure of HCAEC to 8ClA decreased glycolysis, and resulted in a reduction in ATP, with a corresponding increase in the chlorinated analogue, 8Cl-ATP in the nucleotide pool. 8ClA also induced sustained endoplasmic reticulum stress within the HCAEC, which resulted in activation of the unfolded protein response, the altered expression of antioxidant genes and culminated in the release of calcium into the cytosol and cell death by apoptosis. Taken together, these data provide new insight into pathways by which myeloperoxidase activity and resultant hypochlorous acid generation could promote endothelial cell damage during chronic inflammation, which could be relevant to the progression of atherosclerosis

The desmoplastic response : mechanisms of tumour-induced fibrogenesis

The main concern of this thesis is with desmoplasia - a process in which excessive connective tissue is deposited in a neoplasm. This is a common phenomenon in neoplasia but one whose mechanisms are poorly understood. To study the process, I used a human malignant melanoma cell line (UCT-Mel 7) that was established in this laboratory and that, when injected into athymic mice, gave rise to tumours that showed a number of interesting features. Firstly, the tumour induced a marked desmoplastic response as evidenced by a high content of hydroxyproline in tumour lysates, intense staining for reticulin in sections of the tumour and infiltration of the tumour by host mesenchymal cells. Secondly, the desmoplasia was associated in UCT-Mel 7-derived tumours with an unusual phasic pattern of growth that was related to the in vitro passage number of the melanoma cells. On occasions, murine tumours developed at the site of inoculation of human tumour cells. I have identified 2 possible mechanisms by which UCT-Mel 7 cells could have induced the desmoplastic response: either the tumour cells could have exerted their effect indirectly, i.e. via macrophages, or they could have stimulated the host's stromal cells directly. UCT-Mel 7 cells were shown to be chemotactic for mouse macrophages and human foreskin fibroblasts were stimulated, in a dose-dependent manner, to synthesize increased amounts of collagen when co-cultured with mouse peritoneal exudate cells. Stimulation could only be effected by direct cell:cell contact; medium conditioned by macrophages was not effective. The amount of stimulation was not dependent on the state of activation of the peritoneal cells nor on the strain of mouse used. Tumour cells were also found to act directly. Co-culture of UCT-Mel 7 cells and fibroblasts resulted in increased collagen synthesis by the fibroblasts. Increased synthesis of the protein was reflected in an increase in the amount of collagen mRNA. UCT-Mel 7 cell stimulated in a dose-dependent manner with an absolute requirement for intimate cell:cell contact with the fibroblasts. DNA synthesis was not required. Dexamethasone, retinoic acid and the tumour promoter, phorbol myristate acetate, had significant primary effects on fibroblast collagen synthesis but did not modify the response to melanoma cells. Indomethacin, however, had a minimal primary effect upon the fibroblasts but significantly augmented the melanoma cell effect. The nature of the stimulatory cell:cell contact is still uncertain. The gap junction inhibitor, α-glycyrrhetinic acid, did not diminish the melanoma cell effect. Preliminary findings suggested that cell-surface proteoglycans may be important. Removal of the proteoglycans with the inhibitor of proteoglycan assembly, 4-methylumbelliferyl-β-D-xyloside, abrogated the melanoma cell:fibroblast interaction. Recombinant basic fibroblast growth factor did. not seem to be involved in the desmoplastic response. It was of incidental interest to note that this compound inhibited fibroblast collagen synthesis in a manner that was augmented by the concomitant addition of heparin. A surprising finding was the production of a potent inhibitor of collagen synthesis by superinduced cells of the mouse macrophage cell line, P388D₁. This inhibitor has not been fully characterised

The role of low-density lipoprotein modification by mpo-derived oxidants in atherosclerosis

THE ROLE OF LOW-DENSITY LIPOPROTEIN MODIFICATION BY MPO-DERIVED OXIDANTS IN ATHEROSCLEROSIS Myeloperoxidase (MPO) is a peroxidase generated by leukocytes at sites of inflammation. Excessive production of MPO-derived oxidants is linked to the onset of inflammatory diseases. Atherosclerosis is characterised by the build up of lipid laden macrophages (foam cells) within large arteries. Oxidative modification of low-density lipoprotein (LDL) is reported to be a possible pathway that contributes to this mechanism. Although studies have defined different sources of LDL oxidation that occur in vivo, the effect of the MPO oxidant, hypothiocyanous acid (HOSCN) on LDL modification has not been comprehensively assessed. In this study, the contribution of HOSCN to LDL modification was comprehensively assessed and compared to that observed with hypochlorous acid (HOCl) and cyanate (OCN-). HOCl and HOSCN were shown to modify both the protein and lipid component of LDL, with HOSCN contributing to significant lipid peroxidation, while HOCl mainly led to apoB100 modification. OCN- exposure resulted to protein carbamylation, which is the outcome of Lys modification. The contribution of HOSCN, HOCl and OCN--modified LDL to macrophage dysfunction was also studied. In this case, increased uptake of lipids were detected when modified LDL were exposed to macrophages, though the effects of HOSCN- and OCN--modified LDL were not as significant as HOCl-modified LDL. Furthermore, the reduction in lysosomal enzyme activity was also detected after exposure of macrophages to modified LDL. The outcomes observed in this study contribute to the knowledge regarding the role of MPO and SCN-‘–derived oxidants in the development of atherosclerosis. Although MPO is an independent risk factor and a prognostic predictor of cardiovascular events, the data found in the present study is consistent with SCN- having a protective effect, which resulted in preventing foam cell formation and modulating the extent of LDL modification induced by MPO

Studies of the content and distribution of glutathione and glutathione S-transferases in cells

The Glutathione S-transferases (GSTs; EC 2.5.1.18) are a multigene family of dimeric isoenzymes which catalyse a variety of reactions utilizing the tripeptide glutathione (GSH; γ-Glu-Cys-Gly). Many of these, such as the conjugation of GSH to xenobiotic and endogenous electrophiles and the reduction of organic hydroperoxides, are involved in cellular detoxification and the GSTs are of particular interest for their anti-carcinogenic action. This thesis is concerned with the application of a variety of techniques to investigate the content and intracellular distribution of GSH and GSTs in rat liver, in cell lines derived from rat liver (IAR20 & IAR6.1) and mouse fibroblasts (Balb 3T3), and in two human tumour cell lines (HeLa & MCF7). Of particular interest was the controversial presence of the GSTs in the nucleus, which may be linked to their ability to protect against chemical carcinogenesis. The GSH and GST isoenzyme contents of these five cell lines were determined and the subcellular distribution of the GSTs was investigated using immunocytochemistry. Some evidence for a nuclear localization was obtained. However, analysis of the intracellular distribution of GSTs in rat liver using subcellular fractionation was inconclusive. Monochlorobimane (MCB), which undergoes GST-catalysed conjugation to GSH to yield a fluorescent product (MB-SG), was tested in conjunction with flow cytometry as a possible means of measuring cellular GSH. The GST enzyme kinetics were first analysed and considerable isoenzyme specificity towards MCB was observed. It was predicted that the kinetics of MCB conjugation in the above cell lines would vary according to their GSH and GST isoenzyme contents, and this was confirmed using flow cytometry. The implications of this for the use of MCB to measure cellular GSH are discussed. MCB-stained cell lines and primary rat hepatocytes were examined by fluorescence microscopy. Fluorescence was observed over the whole cell, but was often brighter over the nucleus. However, when the MB-SG conjugate was microinjected into the cytoplasm of primary rat hepatocytes, it diffused rapidly through the cell and into the nucleus, producing a very similar pattern of fluorescence to that observed using MCB. Therefore, MCB-staining may not indicate the true intracellular distribution of GSH and the GSTs

A study of non-steroidal anti-inflammatory drugs on human neutrophil toxic oxygen metabolite production

A study was carried out on the effect of a wide range of nonsteroidal anti-inflammatory drugs (NSAIDs) on the receptor- and postreceptor-mediated respiratory burst in human neutrophils. Superoxide (O2-) - which can give rise to toxic oxygen metabolites known to cause tissue damage - was measured spectrophotometrically by the reduction of ferricytochrome C. It was found that the NSAIDs fell into three categories: 1) those that increased production, 2) those that had no effect and 3) those that decreased production. These findings could have clinical relevance for the therapy of chronic inflammatory diseases such as rheumatoid arthritis. In order to investigate the mode of action of those drugs which potentiated 01 release, a pharmacological study of the transduction mechanisms of the respiratory burst was undertaken. A number of cyclooxygenase and 5-lipoxygenase inhibitors had no effect on the stimulated response, implying that the enhancing effect of the NSAIDs was independent of their effect on arachidonic metabolism. Putative "specific" inhibitors of the diacylglycerol (DAG) metabolizing enzyme, DAG kinase, gave similar results to those obtained with the potentiating NSAIDs (with the exception of RAF), indicating that the NSAID effect could involve increased DAG/protein kinase C (PKC) activity. The role of the DAG/PKC pathway in the respiratory burst was addressed using a novel series of potent PKC inhibitors reported to be selective for PKC over other protein kinases. It was found that these agents inhibited 01 production induced by both receptor and postreceptor stimuli, and also in activation sequences that are reported to be Ca2+-independent. Differences in the order of potency of the PKC inhibitors within the range of stimuli used indicated that there might be different PKC isoenzymes, differentially affected by the inhibitors. The potentiating NSAIDs were tested for an effect on isolated PKC, but none were found to directly activate the enzyme. Inhibition by wortmannin of the response to some stimuli may suggest a role for the phospholipase D pathway of DAG generation. This study supports a role for the DAG/PKC pathway in signal- transduction for the neutrophil respiratory burst