Giloma derive from glial cell, which is the most common malignant and deadly primary tumor that affects the brain and nervous system, and the possible causes are not fully understood. Glioma cells are highly invasive, and can spread to distant area of the brain, this invasive behavior makes complete tumor debulking virtually impossible. Glioma even resists to high dose of radiotherapy and chemotherapy, the prognosis of malignant glioma remains dismal and the estimated median survival time is 12¡Ð15 months. The previous studies showed that the interaction of hyaluronan (HA), the abundant component of the ECM in the adult central nervous system, with cell-surface receptors, CD44 is able to mediate motility, tumor formation and multidrug resistance of glioma. In addition, the interacted between HA and CD44, that could up-regulate glioma HA production. But the effect of hyaluronan synthases (HAS) expression in this regulation mechanism was not described clearly. In this study, the HAS expression was a target gene in the rat glioma cell line¡ÐC6 on the conditions of HA addition or cd44 gene silence, respectively. The results showed that HA addition increased the HAS expression, and cd44 gene silence caused the less expression of HAS, and which could restored by HA addition. Futher, the HA addition could prolong cell proliferation , decrease the expression of the CD44 and GFAP, the astrocyte differentiation marker, and increase brain tumor stem cell marker¡Ðnestin expression, and this result could reappear by the cd44 gene silence alone. However, instead the stemness of cell, the cell toward differentiation and proliferation by HA addition after the cd44 gene silence. From those results, the interaction between HA and CD44 could exist the positive feedback to trigger the HA production, and HA could regulate cells proliferation and differentiation by interaction with CD44 in the glioma cells.

Tissues are not solely composed of cells. The extracellular matrix is important for the cell well-being and cell-cell communication. The glycosaminoglycan hyaluronan (HYA) is a widely distributed extracellular matrix (ECM) component. The molecule has prominent physicochemical properties, foremost viscoelastic and osmotic, but participates in many biological processes such as cell migration, proliferation, tissue turnover, wound healing and angiogenesis. HYA is synthesised by either of three different hyaluronan-synthesising enzymes, HAS1-3, and its main ligand is the transmembrane receptor CD44. In the heart and vessels the matrix components are of great importance for endurance and elasticity which are prerequisites for a normal function. The aims of the study were to describe the distribution of HYA and its receptor CD44 in normal cardiovascular tissue and to investigate the ECM composition in myocardial hypertrophy. Normal conditions were studied in a rat model. These studies showed that the tunica adventitia in almost all vessels stained strongly for HYA. The expression in the tunica intima and media on the venous side, differed between the vessels and was almost absent on the arterial side. In the adult animals only minute amounts of CD44 were detected. The expression of both HYA and CD44 was increased in newborn rats. In the heart HYA was unevenly distributed in the interstitium. Strong HYA-staining was seen in the valves and in the adventitia of intramyocardial vessels. Almost no CD44-staining was observed. Notably, there was no obvious difference between newborn and adult animals. In an experimental rat model of pressure-induced cardiac hypertrophy the mRNA-levels of HAS1, HAS2, CD44, basic Fibroblast Growth Factor (FGF-2) and Fibroblast Growth Factor Receptor-1 (FGFR-1) were elevated on day 1 after aortic banding. HAS2, CD44 and FGFR-1 were at basal levels on day 42. The HYA-concentration was significally elevated on day 1. HYA was detected in the interstitium by histochemistry and CD44 was detected mainly in and around the intramyocardial vessels. The HYA-staining was increased in myectomi specimens from patients with HCM compared to controls. HYA was detected in the interstitium, in fibrous septas and in the adventitia of intramyocardial vessels. No CD44 was detected in HCM or in control specimens. Our results indicate that HYA and CD44 play an active role in the maturing vessel tree and that the ECM content of HYA is increased in experimental myocardial hypertrophy and human hypertrophic cardiomyopathy.

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

Le potentiel de motilité des cellules de l’organisme est d’une importance capitale pour plusieurs processus assurant son homéostasie. Toutefois, une perte de contrôle dans cette habilité peut entraîner des conséquences dramatiques, comme cela est observé chez les cellules malignes de différents cancers. Les glioblastomes démontrent une capacité d’invasion phénoménale qui explique en grande partie le faible pronostic associé à ces cancers du système nerveux central. Deux éléments, sur lesquels reposent cette particularité, ont attiré notre attention, soit l’interaction entre le récepteur d’adhésion CD44 et l’acide hyaluronique ainsi que les phénomènes de contraction du corps cellulaire. L’acide hyaluronique est un très grand polysaccharide fortement présent dans le cerveau et dont les tumeurs ont la propension d’en enrichir leur environnement. Parallèlement, son principal récepteur membranaire, le CD44, est surexprimé dans nombres de cancers et contribuerait de manière importante à l’invasion des glioblastomes. L’implication mécanique du CD44 dans le déplacement cellulaire, bien que suggéré par plusieurs études, doit toutefois être encore confirmée. La matrice extra-cellulaire de l’encéphale présente très peu d’espaces à travers lesquels les cellules malignes peuvent s’immiscer. Ces cellules doivent donc se déformer afin de pouvoir s’introduire dans des interstices souvent inférieurs au diamètre du corps cellulaire. Il a été démontré, que pour ce faire, le corps cellulaire est compressé par l’action de l’acto-myosine d’une manière dépendante de RocK. Nous avons voulu évaluer si la stabilité mécanique du couple HA/CD44 était capable d’assurer la transmission de forces de traction et s’il était possible d’observer, avec une haute résolution, les mouvements de contraction du corps cellulaire. Pour ce faire, nous avons employé des techniques innovatrices dérivées des nanosciences afin de sonder ces événements sur des cellules de glioblastomes en culture. Les résultats obtenus révèlent que le CD44 présente effectivement plusieurs caractéristiques similaires aux intégrines telles qu’une affinité pour son ligand qui est dépendante de son association au cytosquelette d’actine, un renforcement potentiel de son ancrage au réseau d’actine ainsi qu’une distribution déterminée à la surface de la cellule. Les expériences visant à investiguer les processus de contraction des glioblastomes ont permis l’observation de mouvements oscillatoires de quelques nanomètres seulement sur leur surface dorsale. Ces fluctuations sont attribuables à l’action de l’actomyosine et nécessite la présence fonctionnelle de RocK. De plus, une analyse fréquentielle a démontré une périodicité similaire aux cycles de protrusion et de rétraction qui avaient été observés sur le lamellipode de fibroblastes. Cette thèse présente en détail ces observations et décrit leurs implications en physiologie cellulaire.

The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [82]-96).

Würzburg, Univ., Diss., 2000. / Zsfassung in dt. Sprache. - Auch als CD-Rom.