Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 4 uppsatser.

Le cancer est une maladie multifactorielle définie par un développement rapide de cellules anormales. Les cellules malignes acquièrent des avantages compétitifs qui permettent une croissance et prolifération anormales, grâce à un large spectre de changements génétiques et épigénétiques conduisant à des changements majeurs dans les profils de transcriptome et protéome et ainsi des modifications dans des voies de signalisation, le trafic intracellulaire et le métabolisme. Des nombreuses voies cellulaires ont été étudiées dans le contexte du cancer, y compris la signalisation, la migration, la perte de la polarité cellulaire apico-basale et l'adhésion cellulaire, cependant très peu est connu sur les altérations au niveau des organelles. Cette thèse a comme objectif d'identifier des altérations dans les compartiments intracellulaires et d'étudier leurs corrélations avec la progression du cancer. Dans la culture cellulaire classique, l'étude systématique de l'organisation du positionnement relatif des organelles est difficile en raison des fortes hétérogénéités morphologiques des cellules. Pour contourner ce problème, nous avons utilisé l’innovante technique des micro-patrons combinée à des cartes de densité des organelles. Après une analyse systématique de différentes lignées cellulaires représentant différents grades du cancer de la vessie, nous avons identifié des changements dans le positionnement de plusieurs organelles. Le changement de position le plus important a été observé pour les lysosomes, dont la distribution était plus périphérique dans les cellules représentant des grades plus avancées du cancer de la vessie. Ceci suggère que le positionnement des lysosomes pourrait être potentiellement important dans la progression du cancer. Par conséquent, nous avons cherché à caractériser l'impact de l’altération des lysosomes sur le comportement des cellules transformées. Nous avons constaté que les changements dans le positionnement des lysosomes jouent un rôle dans l'invasion des cellules cancéreuses de la vessie. En effet, le transport antérograde des lysosomes est en corrélation avec l’invasion 3D, contrairement au transport rétrograde qui corrèle avec une diminution de l’invasion cellulaire. Enfin, nous avons étudié les mécanismes moléculaires par lesquels les altérations du lysosome ont un impact sur l'invasion cellulaire. / Cancer is a multifactorial disease defined by a rapid development of abnormal cells. Malignant cells acquire competitive advantages for growth and proliferation through a big spectrum of genetic and epigenetic changes leading to major changes in the transcriptome and proteome profiles and thus to alterations in multiple signaling pathways, intracellular trafficking and metabolism. Although many cellular pathways have been studied in the context of cancer, including signaling, migration, loss of apical-basal cell-polarity and cell adhesion, little is known about cancer-related alterations on the sub-cellular, organelle level. This PhD thesis aimed to identify alterations in intracellular compartments and to study how these changes correlate with cancer progression. In classical culture, the systematic study on the organization and relative positioning of organelles is challenging because of the strong morphological cell-to-cell variations. To overcome this problem, we used innovative micro-patterning technique in combination with quantitative, probabilistic mapping of cell organelles. Using a systematic analysis of different cell lines representing different stages of bladder cancer, we identified several changes in the positioning of organelles. The most striking phenotype was revealed by lysosomes, whose distribution was more peripheral in cells representing higher grades of bladder cancer. This suggested that lysosome positioning could be potentially relevant in cancer progression. Therefore, we aimed to characterize the impact of lysosome alteration on cell behavior in transformed cells. We found that changes in lysosome positioning played a role on bladder cancer cell invasion. Indeed, anterograde transport of lysosomes correlate with 3D invasion behavior, contrary to retrograde transport that correlated with decreased cell invasion. Finally, we studied about the molecular mechanisms by which lysosome alterations impact cell invasion.

Malignant melanoma arises from the pigment producing melanocytes in epidermis and is the most aggressive type of skin cancer. The incidence of malignant melanoma is increasing faster than any other type of cancer in white population worldwide, with a doubling rate every 10-20 years. So far, the only identified external risk factor for malignant melanoma is UV exposure. Elimination of photodamaged cells by apoptosis (programmed cell death) is essential to prevent tumor formation. Melanocytes are considered relatively resistant to apoptosis, however, the regulation of apoptosis in melanocytes is still unknown. The aim of this thesis was to investigate the apoptotic process following ultraviolet (UV) irradiation in primary cultures of human melanocytes. Focus was on regulation of mitochondrial stability by Bcl-2 family proteins and the possible participation of lysosomal proteases, cathepsins. UV irradiation activated the mitochondrial pathway of apoptosis, leading to cytochrome c release, caspase activation, and nuclear fragmentation. No change in protein expression of Bax and Bcl-2 was observed in response to UV. Instead, translocation of the Bcl-2 family proteins from cytosol to mitochondia was important in the regulation of survival and death of melanocytes. The findings further demonstrated permeabilization of the lysosomal membrane to occur early in the apoptotic process, resulting in cathepsin release into the cytosol. The cathepsins were potent pro-apoptotic mediators and triggered apoptosis upstream of Bax translocation and mitochondrial membrane permeabilization. In response to both heat and UV irradiation, there was a marked increase in expression of stress-induced heat shock protein 70 (Hsp70), which inhibited apoptosis by binding lysosomal and mitochondrial membranes and counteracting the release of cathepsins and cytochrome c. Furthermore, UV irradiation activated c-jun N-terminal kinase (JNK), which triggered apoptosis upstream of cathepsins release from the lysosomes. In addition, JNK mediated apoptosis through phosphorylation of pro-apoptotic Bim, which was released from anti-apoptotic Mcl-1, by UV induced Mcl-1 depletion. This thesis illustrates that permeabilization of mitochondria and lysosomes and release of their constituents to the cytosol participates in UV induced apoptosis signaling in human melanocytes in vitro. The process is regulated by a complex network of pro- and anti-apoptotic proteins, exerting their effects through intracellular translocation and alteration of protein expression.

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases predominantly affecting infants and children, which in some cases can present into adulthood. There are fourteen genes comprising the 13 known subtypes of NCLs (CLN1-CLN8, CLN10-CLN14; CLN9 has been reclassified as CLN4). The NCL diseases share common molecular and clinical features, including cellular accumulation of autofluorescent storage material, characteristic histological findings (curvilinear inclusions, fingerprint profiles, and granular osmophilic deposits), markedly low brain weight, seizures, blindness, motor dysfunction and behavioral disabilities. Though the functions of the CLN proteins are not fully understood, they are mainly localized to the lysosomal compartment and autophagic pathway. Previous works have focused on understanding the individual functions of the CLN proteins. However, there is little research examining the interactions between CLN proteins and their involvement in neurogenesis. The CLN proteins also show involvement in various other signaling pathways, notably the mTOR and p53 pathways, and may therefore have implication as important signaling molecules during development and aging. In this thesis, I outline a variety of interactions between CLN proteins, as well as their role in lysosome formation and autophagy. I further examine the involvement of these proteins in lysosomes of microglia, and potential functions of microglia during neurogenesis in childhood and adulthood. I hypothesize that the CLN proteins are likely involved in a common pathway which is highly regulated during neurogenesis through microglial release of pro-inflammatory molecules. Though these diseases are incurable, enzyme replacement shows promise as a treatment for NCL; cerliponase alpha (BioMarin Pharmaceuticals) is the first and only FDA-approved enzyme replacement treatment for CLN2 disease. Future in-depth investigation of protein-protein interactions as well as their involvement in signaling pathways during development is necessary in order to find a cure for these devastating diseases.

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