CD38 is a transmembrane glycoprotein with both ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase activities; it is also known as a cell surface receptor. CD38 utilizes NAD(P) as a substrate to produce the second messengers, Nicotinic acid adenine dinucleotide phosphate (NAADP) and Cyclic adenosine diphosphate ribose (cADPR). CD38 has been implicated in several diseases. For instance, in chronic lymphocytic leukemia (CLL), it is known as a poor prognostic marker and as a disease modifier. Also, abundant data are available on the receptor functions of CD38 in CLL. However, the aim of the work described in this thesis was to investigate the enzymatic functions of CD38 in leukemia. The work also addresses the question of why CD38+ subset leukemia patients are characterised by poor outcome. It has been postulated that CD38 is the major NADase in cells, and that knocking it down increases NAD levels significantly. Thus, it was hypothesized that NAD levels might be depleted and result in detrimental consequences on cell physiology when CD38 is significantly expressed. Also, it was suggested that a similar linkage might be also present in leukemia, contributing to poor outcome. To test this hypothesis, a human leukaemia cell line (HL60) was used as a convenient model that differentiates into CD38+ cells when stimulated using all-trans retinoic acid (ATRA). It is shown that CD38 is expressed extracellularly and intracellularly in the differentiated cells, as evaluated by qPCR, FACS, Western blotting and the NGD cyclization assay. However, one of the major consequences of the early expression of CD38 (at 3 h) was a substantial depletion of intracellular NAD+ levels that was apparent by 4 h after treatment with ATRA. These novel data suggest a major role for CD38 as a main regulator of NAD during the differentiation. The main role of CD38 in degradation of NAD was confirmed by using a CD38 inhibitor (kuromanin). Interestingly, the drop in NAD+ levels during the differentiation was reversed after treatment with kuromanin. Furthermore, the CD38 homologue, CD157, and other NAD-consuming enzymes (PARP and SIRT) were all investigated, and it was found that there are no substantial roles of all these enzymes on the NAD+ degradation during the differentiation. In contrast, qPCR results for NAD-biosynthesis enzymes during the differentiation process showed a significant rise in indolamine 2,3-dioxygenase (IDO) mRNA expression, with lesser increases in nicotinamide nucleotide adenyltransferase (NMNAT) and nicotinamide phosphoribosyl transferase (NAMPT) mRNA levels. The consequences of low NAD levels on cell metabolism were also assessed; the results show a reduction in lactate production and glutathione levels with an elevation of TBARS levels. However, the NAD+:NADH ratio remained relatively constant. Moreover, the effects of low NAD levels on DNA repair and cell death were also investigated in response to DNA damage caused by UVB. Preliminary findings show that, in CD38+ cells, there is a resistance to apoptotic cell death. Additionally, CD38 expression was also investigated in leukaemia cells, and was found to be regulated in response to hypoxic environment, or the change in NAD+ levels following FK866, kuromanin and NAD+ application. Altogether, these studies raise the possibility that the impact of CD38 enzymatic function on NAD levels and the negative consequences on NAD(P)-dependent processes might play an important role in the poor prognosis in CD38+ leukemia patients.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of immature nonfunctional highly proliferative hematopoietic cells in the blood, due to a blockage in myeloid differentiation at various stages. Since the success of the differentiation agent, All-trans retinoic acid (ATRA), in the treatment of acute promyelocytic leukemia (APL), much effort has gone into trying to find agents that are able to differentiate AML cells and specifically the leukemic stem cell (LSC). CD44 is a cell surface receptor that is over-expressed on AML cells. When bound to anti-CD44 monoclonal antibodies (mAbs), this differentiation block is relieved in AML cells and their proliferation is reduced. The molecular mechanisms that AML cells undergo to achieve this reversal of their apparent phenotype is not fully understood. To this end, we designed a study using quantitative phosphoproteomics approaches aimed at identifying differences in phosphorylation found on proteins involved in signaling pathways post-treatment with CD44-mAbs. The Rho family of GTPases emerged as one of the most transformed pathways following the treatment with CD44-mAbs. The P21 activated kinase 2(PAK2), a target of the Rho family of GTPases, was found to be differentially phosphorylated in AML cells post-treatment with CD44-mAbs. This protein has been found to possess a role similar to that of a switch that determines whether the cell survives or undergoes apoptosis. Beyond confirming these results by various biochemical approaches, our study aimed to determine the effect of knock down of PAK2 on AML cell proliferation and differentiation. In addition, over-expression of PAK2 mutants using plasmid cloning was also explored to fully understand how levels of PAK2 as well as the alteration of specific phospohorylation sites could alter AML cell responses to CD44-mAbs. Results from this study will be important in determining whether PAK2 could be used as a potential therapeutic target for AML once its levels are altered.

In recent years, magnetic nanowires (NWs) have been widely used for their therapeutic potential in biomedical applications. The use of iron (Fe) NWs combines two important properties, biocompatibility and remote manipulation by magnetic fields. In addition the NWs can be coated and functionalized to target cells of interest and, upon exposure to an alternating magnetic field, have been shown to induce cell death on several types of adherent cells, including several cancer cell types. For suspension cells, however, using these NWs has been much less effective primarily due to the free-floating nature of the cells minimizing the interaction between them and the NWs. Leukemic cells express higher levels of the cell surface marker CD44 (Braumüller, Gansauge, Ramadani, & Gansauge, 2000), compared to normal blood cells. The goal of this study was to functionalize Fe NWs with a specific monoclonal antibody towards CD44 in order to target leukemic cells (HL-60 cells). This approach is expected to increase the probability of a specific binding to occur between HL-60 cells and Fe NWs. Fe NWs were fabricated with an average diameter of 30-40 nm and a length around 3-4 μm. Then, they were coated with both 3-Aminopropyl-triethoxysilane and bovine serum albumin (BSA) in order to conjugate them with an anti-CD44 antibody (i.e. anti-CD44-iron NWs). The antibody interacts with the amine group in the BSA via the 1-Ethyl-3-3-dimethylaminopropyl-carbodiimide and N-Hydroxysuccinimide coupling. The NWs functionalization was confirmed using a number of approaches including: infrared spectroscopy, Nanodrop to measure the concentration of CD44 antibody, as well as fluorescent-labeled secondary antibody staining to detect the primary CD44 antibody. To confirm that the anti-CD44-iron NWs and bare Fe NWs, in the absence of a magnetic field, were not toxic to HL-60 cells, cytotoxicity assays using XTT (2,3-Bis-2-Methoxy-4-Nitro-5-Sulfophenyl-2H-Tetrazolium-5-Carboxanilide) were performed and resulted in a high level of biocompatibility. In addition, the internalization of the coated NWs have been studied by coating them with a pH dependent dye (pHrodoTM Red) that showed a signal once the NWs were internalized by the cell.

La polyarthrite rhumatoïde est une maladie auto-immune qui touche environ 1% de la population mondiale. Elle entraîne une inflammation synoviale et une destruction de l’architecture articulaire. L’articulation rhumatoïde est un milieu très inflammatoire qui est infiltré par des cellules telles que les neutrophiles, les macrophages, les monocytes, les lymphocytes B et T ; par des protéines telles que des cytokines, des chimiokines, des molécules d’adhésion, par des complexes immuns, et par des auto-anticorps comme les facteurs rhumatoïdes qui sont un marqueur spécifique de la maladie et qui permettent d’établir un pronostic sur son évolution. De plus, dans l’articulation rhumatoïde, il y aura formation d’un pannus rhumatoïde avec érosion progressive du cartilage et de l’os. Les ostéoclastes sont les cellules principalement impliquées dans cette érosion osseuse. Afin d’effectuer des études in vitro, les ostéoclastes sont généralement obtenus à partir de monocytes isolés de sang humain, ce qui rend leur étude complexe car les variations sont très grandes d’un donneur de sang à l’autre. Nous avons utilisé la lignée cellulaire d’origine myéloïde HL60 pour obtenir des ostéoclastes et avons caractérisé ces cellules HL60 « osteoclast-like » dans tous les aspects de leur fonction et de leur activation. Nous avons aussi montré que les facteurs rhumatoïdes pouvaient agir directement sur les ostéoclastes pendant leur différenciation et pendant la résorption osseuse. Cette étude nous a permis d’amorcer la compréhension du rôle possible des facteurs rhumatoïdes sur la résorption osseuse dans la polyarthrite rhumatoïde.

Le point de départ du projet de recherche rapporté dans le présent mémoire a été la synthèse dans notre laboratoire, d'une nouvelle famille de dérivés du 2P-piperazino-5a-androstane-3a, 17P-diol. Un premier criblage des produits synthétisés utilisant les cellules de la leucémie myéloïde aiguë HL-60, a révélé que huit de ces produits avaient une activité anti-leucémique certaine. Nous avons alors commencé par confirmer l'activité antiproliferative de ces produits sur les cellules HL-60, ensuite nous avons exploré leur potentiel antiprolifératif sur huit autres lignées cancéreuses ainsi que leur sélectivité d'action par rapport à deux lignées normales. Les produits sélectionnés ont inhibé la prolifération cellulaire de sept lignées cancéreuses avec des IC50 variant de 0.3 à 6.4 _iM. De plus, ils étaient faiblement toxiques sur les lignées normales. Ces résultats nous ont encouragés à entreprendre l'étude du mécanisme d'action de cette nouvelle famille d'aminostéroïdes sur les cellules leucémiques HL-60. C'est ainsi que trois produits ont été sélectionnés, leur effet sur le cycle cellulaire et sur la différenciation des cellules HL-60 a été déterminé et leur cytotoxicité par induction de l'apoptose a été confirmée. Une étude plus poussée, utilisant l'un des produits, a montré que l'apoptose induite par cette famille d'aminostéroïdes est initiée par l'activation catalytique des caspases. Afin d'étendre l'étude des relations structure-activité de ces produits, on a effectué la synthèse et le criblage de leurs analogues pregnanes. Les trois produits les plus intéressants ont inhibé la prolifération des cellules HL-60 avec des valeurs IC50 de l'ordre de 1.9 \iM, tout en étant faiblement toxiques envers les lymphocytes normaux (IC5_= 10-31 \iM). Une étude préliminaire de leur mécanisme d'action a montré qu'il serait similaire à celui de leurs analogues androstanes.

No description available.

Orientadores: Carmen Verissima Ferreira, Hiroshi Aoyama / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T13:30:15Z (GMT). No. of bitstreams: 1 Cavagis_AlexandreDonizetiMartins_D.pdf: 4238254 bytes, checksum: af81f06637a0dca49e3ab3fabe00d10d (MD5) Previous issue date: 2005 / Resumo: O objetivo central deste trabalho foi avaliar a modulação de MAPKs em células de leucemia humana induzidas a apoptose e diferenciação. No capítulo 1, estudou-se o efeito da tetrahidroxiquinona (THQ), uma molécula que pode participar de um ciclo redox formando radicais semiquinona e, conseqüentemente, espécies reativas de oxigênio (EROs). A citotoxicidade da THQ, mediada pela formação de EROs, foi revertida por tratamento das células com glutationa e N-acetil-L-cisteína. A produção de EROs ocorreu concomitan-temente com a apoptose das células HL60 através da via mitocondrial e com redução na atividade de várias moléculas anti-apoptóticas da via de sobrevivência celular, inclusive da proteína quinase B (PKB). A transfecção das células HL60 com a PKB levou à diminuição da citotoxicidade dependente de EROs gerada pela THQ. No capítulo 2, demonstrou-se que a análise de um arranjo simultâneo de 1176 substratos com seqüências de consenso de diferentes quinases permitiu identi-ficar a via de sinalização das MAPKs como principal alvo da violaceína, um pigmento com propriedades antitumorais in vitro produzido pela Chromobacterium violaceum do rio Amazonas e que induz diferenciação de células HL60 em monócitos e granulócitos. Os resultados também indicaram que a análise do quinoma empregando arranjos metabólicos é uma ferramenta promissora na elucidação do modo de ação de fármacos em nível molecular. O capítulo 3 corresponde a um artigo de revisão em português sobre a riboflavi-na, componente do complexo vitamínico B2 que participa de importantes eventos bioquímicos, como reações redox envolvendo transferência de um ou dois elétrons, e também agindo como fotossensibilizador. Este artigo mostra que o comportamento peculiar e multifuncional da riboflavina permite que ela atue como nucleófilo ou eletrófilo e também descreve a associação deste composto com diferentes doenças como o câncer e doenças cardiovasculares / Abstract: The principal aim of this work was to assess the MAPKs family status in human leukaemia cells induced to undergo apoptosis and differentiation. In chapter one, the effect of tetrahydroxyquinone (THQ), a highly redox active molecule which can participate of a redox cycle with semiquinone radicals leading to the formation of reactive oxygen species (ROS), was investigated in HL60 cells. THQ caused substantial ROS formation followed by cytotoxicity that was sensitive to glutathione and N-acetyl-L-cysteine. Furthermore, ROS production coincided with HL60 cell apoptosis through the mitochondrial pathway and was followed by reduced activity of various anti-apoptotic survival molecules, including the protein kinase B pathway. Importantly, transfection of protein kinase B into HL60 cells leading to an artificial increase in protein kinase B activity inhibited ROS-dependent cytotoxicity. In chapter two, peptide arrays of 1176 different kinase consensus substrates unambiguously identified the MAP kinase pathway as a major target for violacein, the anti-leukemic purple-colored pigment produced by Chromobacterium violaceum from the Amazon River that stimulates HL60 cells to differentiate into monocytes and granulocytes. The results also indicate that kinome profiling using metabolic arrays is a promising and suitable tool for studying the molecular mechanisms of drug action. Chapter three is a review article in Portuguese about riboflavin, a component of the vitamin B2 complex, that has important roles in biochemistry, especially in redox reactions due to its ability to participate in both one- and two-electron transfers as well as acting as photosensitizer. This article describes the peculiar and multifunctional behavior of riboflavin which allows it to take part in several biochemical pathways both as nucleophile and electrophile. Moreover, the association of this vitamin with different diseases, including cancer and cardiovascular diseases, is also discussed. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Made available in DSpace on 2014-07-29T16:11:51Z (GMT). No. of bitstreams: 1 Dissertacao - Wanessa Machado Andrade - 2011.pdf: 1952980 bytes, checksum: e95aa49d2fb71af8883a1b476f7d219a (MD5) Previous issue date: 2011-08-30 / Na busca por novos agentes antineoplásicos os produtos de origem natural têm um papel de destaque, uma vez que cerca de 25% dos fármacos prescritos no mundo são obtidos de plantas e aproximadamente 49% dos fármacos desenvolvidos nas últimas três década foram obtidos a partir de produtos naturais. Eugenia dysenterica DC. pertence à família Myrtaceae e é conhecida p pula mente c m cagaita , sendo empiricamente utilizada no Brasil para o tratamento de distúrbios gastrointestinais e diabetes. Neste trabalho foram investigados a citotoxicidade e os mecanismos de indução de apoptose do extrato padronizado hidroalcóolico das folhas da E. dysenterica DC. sobre células leucêmicas HL60 e K562, atividade antitumoral in vivo em camundongos com tumor ascítico de Ehrlich (TAE), bem como a atuação do extrato frente a células basais, como fibroblastos e eritrócitos, além do perfil de toxicidade oral aguda em camundongos. Os estudos de citotoxicidade foram realizados por meio dos ensaios de exclusão do azul de tripano e redução do tetrazólio (MTT). Os mecanismos de indução de apoptose celular foram investigados por microscopia de luz e de fluorescência e por citometria de fluxo. A investigação do potencial antitumoral in vivo foi feito através da curva de sobrevida utilizando camundongos portadores do TAE tratados com diferentes doses do extrato padronizado hidroalcóolico das folhas da E. dysenterica DC. Os ensaios de segurança foram realizados pelo método de incorporação do corante vermelho neutro em células 3T3 (fibroblastos de camundongos), potencial hemolítico utilizando sangue de carneiro e toxicidade oral aguda de acordo com o guia OECD 423 T xicidade O al Aguda de Classe , 2001. Os dados foram analisados pelo teste t de Student e curva de Kaplan Meier para a análise da curva de sobrevida. As médias foram consideradas estatisticamente significativas quando P < 0,05. O extrato da E. dysenterica DC. foi citotóxico para as células leucêmicas HL60 e K562. A análise morfológica das células K562 indicou que o tratamento com o extrato hidroacóolico da planta induziu morte celular por apoptose. A apoptose foi constatada também pelo aumento na geração de espécies reativas de oxigênio, pela redução do potencial de membrana mitocondrial, pela externalização da fosfatidilserina, pela saída de citocromo c da mitocôndria, pela modulação de Bax/Bcl2, pela eduçã de NF&#1178;B e pela ativação das caspases 3 e 8. O extrato da planta também apresentou citotoxicidade concentração-dependente frente às células basais 3T3, porém essa citotoxicidade foi cerca de 7 e 2 vezes menor que para as células leucêmicas HL60 e K562, respectivamente. O extrato não provocou hemólise e nem causou toxicidade nos camundongos tratados com a dose de 2000 mg/Kg, apresentando toxicidade classe 5. A partir da análise dos resultados foi possível concluir que o extrato padronizado hidroalcóolico das folhas da E. dysenterica DC. apresenta atividade citotóxica e indutora de apoptose em células K562 e HL60, atividade antitumoral in vivo em camundongos com TAE, além de apresentar perfil de segurança in vitro e in vivo.

The maturation of lamin A is completed by the endoproteolytic cleavage of its farnesylated precursor protein, prelamin A. In the absence of this cleavage, prelamin A can neither give rise to lamin A nor assemble into the nuclear lamina. We call the enzyme which catalyzes this endoproteolytic step the 'prelamin A endoprotease'. In this study, we begin characterization of the regulation of prelamin A endoprotease. In particular, we address the question as to whether prelamin A endoprotease activity is constitutive in cells or responds to expression of prelamin A. To do this, we compared the activity of this novel endoprotease in cells which express prelamin A with those that do not. Our data shows that the enzymatic activity of prelamin A endoprotease is enhanced by the expression of prelamin A.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr. Publié aussi en version électronique.