Statement of Problem Telomere dynamics in the three-dimensional (3D) space of the mammalian nucleus plays an important role in the maintenance of genomic stability. However, the telomere distribution in 3D nuclear space of normal and tumor cells was unknown when the study was initiated. Methods Telomere fluorescence in situ hybridization (FISH) and 3D molecular imaging, deconvolution, and analysis were used to investigate telomere organization in normal, immortalized and tumor cells from mouse and human cell lines, and primary tissues. Results Telomeres are organized in a non-overlapping manner and in a cell-cycle dependant fashion in normal cells. In the late G2 phase of cell cycle, telomeres are assembled into a flattened sphere that is termed the telomeric disk In contrast, the telomeric disk is disrupted in the tumor cells. Moreover, telomeric aggregates (TAs) are found in tumor cells. Conditional c-Myc over-expression induces telomeric aggregation leading to the onset of breakage-bridge-fusion cycles and subsequent chromosomal abnormality. Conclusions Telomeres are distributed in a nonrandom and dynamic fashion in the 3D space of a normal cell. Telomeric aggregates are present in cells with genomic instability such as tumor cells and cells with deregulation of c-Myc. Consequently, TA can be a useful biomarker for research in cancer and other disease processes.

The transcription factor c-Myc is an early downstream target of oestrogen action in breast cancer cells in culture and it has been speculated that aberrant c-Myc expression may mediate antioestrogen resistance. However, studies of c-Myc protein expression as either a prognostic or predictive marker in human breast cancer have been limited and contradictory, as have been studies of c-Myc expression during breast cancer evolution. In order to assess the relationship between c-Myc protein expression and outcome from breast cancer, a representative cohort of 292 women with invasive ductal carcinoma (IDC) and linked clinicopathological data was assembled and tissue microarrays (TMA) generated from the archived breast cancer specimens. Detailed assessments of the expression of cyclin D1, cyclin E, p21WAF1/Cip1 and p27Kip1 were also conducted and analysed in relation to c-Myc expression using immunohistochemistry. Changes in c-Myc protein expression in a TMA model of breast cancer evolution were also conducted. Finally the cell-cycle effects of low-level constitutive c-Myc expression and high-level inducible c-Myc expression were evaluated in MCF-7 cells in vitro. Key novel results obtained were that c-Myc protein expression changed from preferentially nuclear to preferentially cytoplasmic during the evolution of breast cancer. In women with early invasive breast carcinoma, a "high-risk" cytoplasmic predominant c-Myc expression pattern was defined (~13% of cases) that independently predicted for poor outcome generally, among ER positive cases and in ER postive cases treated with endocrine therapy. In vitro studies confirmed that c-Myc overexpression was associated with resistance to the anti-proliferative effects of anti-oestrogens with persistence of both cyclin D1-cdk4 and cyclin E-cdk2 activities in the face of anti-oestrogen treatment. Further novel findings were that high cyclin D1 expression (upper 10% of expressors) was an independent predictor of poor outcome among ER positive breast cancer cases. Amongst ER + PR positive cases, both "high-risk" c-Myc expression and high level cyclin D1 expression were independent predictors of poor outcome. In summary, these data indicate that aberrant expression of the cell cycle proteins c-Myc and cyclin D1 may result in poor breast cancer outcomes in hormone receptor positive breast cancer and reinforces the importance of the cell cycle as a potential site of therapeutic manipulation in endocrine-resistant breast cancer.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As cordiaquinonas sÃo naftoquinonas meroterpenÃides isolados de plantas pertencentes ao gÃnero Cordia com vÃrias atividades biolÃgicas descritas, incluindo atividades antifÃngica, larvicida e citotÃxica. O objetivo deste trabalho foi avaliar o potencial anticÃncer de uma cordiaquinona isolada das raÃzes da planta Cordia leucocephala. O presente estudo avaliou o potencial citotÃxico da cordiaquinona J em vÃrias linhagens de cÃlulas tumorais e normais pelo teste do MTT e seu possÃvel mecanismo de aÃÃo. A cordiaquinona J mostrou valores de CI50 variando de 4,6 a 6,8 μM em cÃlulas leucÃmicas e 33,6 a 37 μM em cÃlulas normais, apÃs 24 horas de incubaÃÃo. Nas cÃlulas HL-60 foi observado induÃÃo de apoptose preferencialmente pela via extrÃnseca. A induÃÃo do dano ao DNA observado pelo tratamento com a cordiaquinona J atravÃs do ensaio do cometa foi associado com a ativaÃÃo de proteÃnas quinases da via ATM/ATR. O dano ao DNA, assim como a ativaÃÃo das proteÃnas quinases da via ATM/ATR foi visualizado em cÃlulas HL-60, mas nÃo em cÃlulas normais. Estes efeitos em HL-60 podem estar relacionados com a depleÃÃo da expressÃo proteica de glutationa e de c-myc observados. O potencial anticÃncer foi confirmado in vivo atravÃs da inibiÃÃo do tumor sarcoma-180 em 72,5% apÃs o tratamento com 50 mg/kg de cordiaquinona J. O prÃ-tratamento tanto das cÃlulas quanto dos animais com N-acetil-L-cisteina inibiu todos os efeitos observados in vitro e in vivo reforÃando o papel da geraÃÃo das espÃcies reativas de oxigÃnio na atividade antitumoral da cordiaquinona J. / Cordiaquinones are meroterpenoid naphtoquinones from plants belonging to the genus Cordia with several described biological activities, including antifungal, larvicidal and cytotoxic effects. The aim of this study was to evaluate the anticancer potential of a cordiaquinone isolated from the roots of Cordia leucocephala plant. The present study evaluated the cytotoxic potential of cordiaquinone J in several tumor and normal cell lines by MTT assay and its possible mechanism of action. The study of the mechanism of action of cordiaquinones L and M, in human leukemia cells (HL-60) showed induction of cell death by apoptosis, and these effects were related to the induction of oxidative stress. Then the study was continued only with the cordiaquinone J. The cordiaquinone J showed IC50 values ranging from 4.6 to 6.8μM in leukemia cells and 33.6 to 37 μM in normal cells, after 24 hours of incubation. In HL-60 cells was observed apoptosis induction preferentially by extrinsic pathway. The induction of DNA damage by cordiaquinone J observed by comet assay was associated with activation of protein kinases of ATM/ATR pathway. The DNA damage, as well as activation of protein kinases via the ATM / ATR was observed in HL-60 cells but not in normal cells. These effects in HL-60 cells may be related to the depletion of protein expression of glutathione and c-myc observed. The anticancer potential was confirmed in vivo through inhibition of sarcoma-180 tumor by 72.5% after the treatment with 50 mg/kg of cordiaquinone J. The pre-treatment of cells or animals with N-acetyl-L-cysteine abolished most of the in vitro and in vivo observed effects, reinforcing the role of reactive oxygen species generation in cordiaquinone J activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Cancer cells display increased metabolic demands to support their proliferation and biosynthetic needs. It has been extensively shown in cancers, that amino acids have functions beyond the role of mRNA translation. The breadth of functions makes amino acid restriction an effective strategy for cancer therapy; hence an important line of research involves targeting amino acid acquisition and metabolism therapeutically. Currently, asparagine depletion via L-Asparaginase in acute lymphoblastic leukemia (ALL) remains the only clinically approved therapy to date. In the first project, we showed that ALL cells are auxotrophic for asparagine and rely on exogenous sources for this non-essential amino acid. However, sensitivity to L-Asparaginase therapy is mitigated by the expression of the enzyme asparagine synthetase (ASNS), involved in de novo asparagine biosynthesis. We showed that this adaptive response requires two essential steps; demethylation of the ASNS promoter and recruitment of activating transcription factor 4 (ATF4) to the promoter to drive ASNS transcription. Our follow-up study in ALL cells showed that asparagine bioavailability (through de novo biosynthesis or exogenous sources) is essential to maintain the expression of the critical oncogene c-MYC. c-MYC is a potent transcription factor and is dysregulated in over 60% of cancers, including hematopoietic malignancies. We showed that this regulation by asparagine is primarily at the translation level and c-MYC expression is rescued only when exogenous asparagine is available or when cells can undertake de novo biosynthesis. At the biochemical level, asparagine depletion also causes an induction of ATF4 mediated stress response and suppression of global translation mediated by decreased mammalian target of rapamycin complex 1 (mTORC1) activity. However, we found that neither inhibition of the stress response or rescuing global translation rescued c-MYC protein expression. We also extended this observation to c-MYC-driven lymphomas using cell lines and orthotopic in vivo models. We showed that genetic inhibition of ASNS or pharmacological inhibition of asparagine production can significantly limit c-MYC protein and tumor growth when environmental asparagine is limiting. Overall, our work shows an essential role for asparagine in lymphoid cancers and has expanded on the usage of L-Asparaginase to resistant leukemias and lymphomas.

<p>c-Myc is one of the most important oncogenes. G-quadruplex DNA secondary structure formed in the proximal promoter region of c-Myc functions as a transcription silencer and is targetable by small molecules. Therefore, the c-Myc promoter G-quadruplex (MycG4) is an attractive anticancer drug target. Protein recognition of MycG4 is essential for its transcriptional regulating. Nucleolin was discovered as a major MycG4 binding protein in 2009. It shows a remarkably higher binding affinity for MycG4 over its known substrate NRE_RNA and overexpression of nucleolin represses the activity of the c-Myc promoter. However, little is known about its molecular recognition of MycG4. Here, we use X-ray crystallography combined with other biochemical and biophysical methods to understand how nucleolin recognizes MycG4. Nucleolin is a 77 kD protein with a modular organization. The four RNA-binding domains (RBD) of nucleolin are the minimal domains for high affinity binding with MycG4. We show that nucleolin prefers the c-Myc parallel G-quadruplex with a 6-nt central loop (Myc161) that is the thermodynamically favored conformation. Using a custom G4 DNA microarray, we optimized the MycG4 sequence with over 10-fold increased binding affinity to nucleolin. Fabs are widely used tools to facilitate crystallization and we have discovered Fabs that specifically bind the nucleolin-MycG4 complex using a phage display screening. This approach enabled us to obtain crystals of the nucleolin-MycG4-Fab ternary complex diffracted at 2.6 Å and we determined the crystal structure. In the structure, the parallel MycG4 is very well-defined with two K⁺ between the three G-treads. The central 6-nt loop residue protrude from the G4-core and extensively recognized by the nucleolin. Only RBD1 and RBD2 of nucleolin are seen in the crystal structures and interact extensively with the 6-nt central loop and 5′-flanking of MycG4. The binding surface and area of the globular MycG4 by nucleolin is much more extensive than NRE_RNA and involves an extra binding site. Fab binds to both RBD1 and 3′-end of MycG4 to stabilize the complex. The well-defined partial RBD2-3 linker and a cavity close to the 1-nt T19 loop suggest that the missing RBD3 likely binds the 3^rd loop of MycG4. This structure is the first MycG4-protein complex structure. It will help understand MycG4 and nucleolin interactions and the development of MycG4 targeted cancer therapeutics. This structure also provides novel insights into how proteins recognize the globular G-quadruplexes, highlighting the potential of G-quadruplexes as a platform for multivalent interactions such as with multiple tandem RBDs.</p>

The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an essential signaling mechanism that might arrest cancer proliferation. Pin1 is the first phosphorylation-dependent PPIase enzyme to be discovered. The Pin1 regulatory mechanism, acting on other mitotic proteins in vivo and in vitro, remains largely unknown. For the study of Pin1 function, two types of assays were used to identity ligands for Pin1: (1) The Enzyme-Linked Enzyme Binding Assay (ELEBA) for the identification of WW domain ligands, (2) a catalytic assay to identified inhibitors of Pin1 catalytic activity. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain from chemical libraries. By using the ELEBA, a pSer-Pro peptidomimetic library of 315 ligands was screened, identifying three promising ligands cis-D2, O2, and M18. Competitive Kd values for cis-D2, O2, and M18 were determined to be 263 ± 6.4, 206 ± 3.4, and 130 ± 3.0μM, respectively. Furthermore, we screened the pSer-Pro peptidomimetic library using a Pin1 discontinuous-catalytic assay to identify inhibitors of Pin1. Ligands D20 and K7 were identified to decrease more than 90% of the Pin1 catalytic activity. To investigate the nature of the Pin1 interaction with c-Myc, we synthesized and characterized four peptides corresponding to the c-Myc sequence. These peptides were used in NMR isomerization studies of Pin1 by our collaborator Dr. Jeffry Peng (University of Notre Dame). Preliminary work shows that Pin1 binds and isomerizes the Ac–LLPpTPPLSPS–NH₂ peptide at the cMyc pThr58 position. Finally, we measured a secondary kinetic isotope effect (2º KIE) to study the Pin1 catalytic mechanism of proline isomerization. The ratio of kH/kD for unlabeled and [d₃]Ser-labeled substrate gave a SKIE value of 1.34 ± 0.01. The normal 2º KIE value indicates that carbonyl-serine hybridization is not changing from sp² to sp³. This result supports substrate analogue inhibitor studies, and previous solvent and SKIE results on Pin1, that suggest a twisted amide mechanism assisted by a transient hydrogen bond in the transition state. / Ph. D.

As neoplasias de cabeça e pescoço constituem um importante problema de saúde pública devido à alta incidência e alguns tipos estão associados a fatores comportamentais como consumo de álcool e tabaco. Apesar desses dados, a doença, especialmente em sua fase inicial, pode ser curada e alguns tipos podem ser prevenidos. Portanto, existe a necessidade de identificar e validar novos biomarcadores em câncer de cabeça e pescoço com aplicação em prognóstico e seleção de terapias mais adequadas. Neste sentido, o objetivo deste trabalho foi validar o perfil de três proteínas, SET, hnRNP K e MARK3 em tumores de cabeça e pescoço, e verificar a potencial aplicação como marcadores de diagnóstico e prognóstico em HNSCC, bem como propor um papel para estas proteínas na tumorigênese. Foram analisadas 22 amostras de tumores de cabeça e pescoço por western blotting (WB) e 96 amostras (91 tumores, 4 biópsias e 1 controle) dispostas em duplicata em lâmina de tissue microarray, obtidas no Brasil e cedidas pelo Grupo GENCAPO, por imunohistoquímica (IHC). Os dados obtidos foram correlacionados com todos os parâmetros clínicos e patológicos e com prognóstico do paciente com HNSCC por um período de 48 meses. Os resultados obtidos por WB e IHC mostraram acúmulo e fragmentação da SET e acúmulo nuclear e citoplasmático da hnRNP K nos tumores comparado a respectiva margem cirúrgica e tecido normal. A hnRNPK mostrou valor prognóstico sendo associada a sobrevida global do paciente. A proteína c-Myc e a sua forma fosforilada foram analisadas nas amostras de tumores e suas respectivas margens cirúrgicas devido a sua relação com SET, PP2A e hnRNP K. Os resultados mostraram acúmulo da c-Myc fosforilada e total nas amostras tumorais, o que coincidiu com aumento de SET e hnRNP K. Com relação à proteína MARK3, observou-se sua redução no tumor e menor sobrevida livre de doença. Foi realizado ensaio de RNA de interferência (RNAi) contra hnRNP K e SET em linhagem de carcinoma oral (HN13). A redução da proteína SET por RNAi levou a redução significativa da hnRNP K, enquanto a hnRNP K gerou menor efeito na proteína SET, sugerindo um efeito regulatório na expressão ou manutenção da hnRNP K pela SET na célula tumoral. A interferência contra a hnRNP K também reduziu a proliferação celular tumoral. Em conclusão, o aumento da proteína SET está associado à desmoplasia em HNSCC e pode ser um potencial marcador específico para essa condição. hnRNP K e MARK3 podem servir como potenciais marcadores em HNSCC e ajudar a identificar um subgrupo de pacientes com pobre prognóstico. A hnRNPK exerce efeito positivo na proliferação da célula tumoral. SET e hnRNP K podem atuar como fatores oncogênicos favorecendo o aumento de c-Myc. / The head and neck cancers constitute a major public health problem due to the high incidence and some types are associated with behavioral factors such as consumption of alcohol and tobacco. Despite these data, the disease, especially in its early stage can be cured and some types can be prevented. Therefore, there is a need to identify and validate new biomarkers in head and neck cancer, with applications in prognosis and selection of therapies most appropriate. Accordingly, the objectives of this study were validation of the profile of three proteins, SET, hnRNP K and MARK3 in tumors of head and neck, and verify the potential application as markers for diagnosis and prognosis in HNSCC, and suggest a role for these proteins in tumorigenesis. We analyzed 22 samples of head and neck tumors by western blotting (WB) and 96 samples (91 tumors, 4 biopsies and 1 control) arranged in duplicate in the tissue microarray slide, obtained in Brazil and assigned by the GENCAPO Group, by immunohistochemistry (IHC). The data were correlated with all clinical and pathological parameters and prognosis of patients with HNSCC for a period of 48 months. The results obtained by WB and IHC showed the SET accumulation and fragmentation and hnRNP K nuclear and cytoplasmic accumulation in tumor compared to the surgical margin and normal tissue. The hnRNPK prognostic value has been associated with overall survival of patients. The c-Myc protein and its phosphorylated form were analyzed in tumor and surgical margins samples due to its relationship with SET, PP2A and hnRNP K. The results showed accumulated total and phosphorylated c-Myc in tumor samples, which was coincided with increase in SET and hnRNP K. Regarding the protein MARK3 was observed its reduction in tumor and lower disease-free survival. RNA interference (RNAi) against hnRNP K and SET were performed in oral squamous cell carcinoma line (HN13). SET protein reduction by RNAi led to significant reduction of hnRNP K, and hnRNP K showed a minor effect on SET protein, suggesting a regulatory effect on expression or maintenance of hnRNP K by SET in tumor cells. Interference against hnRNP K also reduced tumor cell proliferation. In conclusion, increased SET protein is associated with desmoplasia in HNSCC and may be a potential specific marker for this condition. hnRNP K and MARK3 can serve as potential markers in HNSCC and help identify a subgroup of patients with poor prognosis. The hnRNPK must act a positive effect on cell proliferation of the tumor. SET and hnRNP K may act as oncogenic factors contributing for c-Myc activity.

Células Y-1 apresentam o gene K-ras amplificado, o que resulta em altos níveis de expressão da proteína codificada por este gene. Este fato faz com que células Y-1 apresentem níveis cronicamente altos de K-Ras-GTP. Além disso, estas células apresentam uma relativa desregulação da transição G0&#8594;Gl&#8594;S, a qual é caracterizada por uma porcentagem de células entrando na fase S do ciclo celular na condição carenciada; e também, por um afrouxamento na regulação de Myc, o qual apresenta níveis basais significantes de mRNA e proteína. Para verificar se existe uma relação entre K-Ras-GTP elevado e os níveis basais de Myc e a desregulação na transição G0&#8594;Gl&#8594;S, células Y-1 foram transfectadas com uma forma dominante negativa de H-ras, H-ras Asn-17 (RasN 17). Os transfectantes resultantes também foram utilizados para verificar o papel de Ras na transdução do sinal iniciado por FGF-2 e ACTH. Com estes clones foi possível verificar uma redução nos níveis de ativação de K-Ras na condição carenciada, e com isso ficou claro que FGF-2 e ACTH são capazes de induzir a ativação de K-Ras, porém com cinética diferentes: uma ativação tardia e lenta para FGF-2, e rápida e transiente para ACTH. Com a redução nos níveis de Ras-GTP, verificamos uma concomitante redução no basal da proteína c-Myc e também no basal de entrada em S, indicando que existe uma correlação entre estes fatores. Além disso, os clones Yl-RasN17 foram determinantes para mostrar que em células Y-1 a presença de Akt/PKB constitutivamente ativada é conseqüência dos níveis cronicamente elevados de K-Ras-GTP (Forti et al, 2002). / Abstract not available.

ACTH é o hormônio trófico que estimula a esteroidogênese, promove o crescimento e a manutenção do córtex adrenal. Porém, em linhagens adrenocorticais, assim como em culturas primárias, ACTH inibe a proliferação celular. A linhagem Y-1 de células adrenocorticais de camundongo tem as seguintes respostas a ACTH: aumento da esteroidogênese, arredondamento celular, bloqueio do ciclo celular em G1 e indução dos proto-oncogenes fos e jun. Esta linhagem também responde muito Sem a FGF2, um protótipo da família dos FGFs (Fibroblast Growth Factors) que regula diferenciação e proliferação de diversos tipos celulares, sendo estimulada a transitar pelas fases G0&#8594;G1&#8594;S do ciclo celular. ACTH antagoniza este efeito de FGF2, inibindo parcialmente a entrada em S induzida por FGF2. Este projeto buscou compreender o papel de c-Myc no controle do ciclo celular de Y-1, com ênfase nos efeitos de ACTH e FGF2 na expressão e atividade de c-Myc. Mostramos que os dois principais controles da expressão de c-Myc em Y-1 são transcrição e degradação da proteína, sendo a concentração de c-Myc o único controle sobre o sistema Myc/Max/Mad, uma vez que a expressão de Max e de Mad-1 , Mad-4 e Mxi é constitutiva em células Y-1. FGF2 induz a expressão de c-Myc através da indução da transcrição e aumento da estabilidade da proteína de forma totalmente dependente da via de Erk-MAPK. ACTH, por outro lado, não interfere com a transcrição de c-myc, mas promove fortemente a degradação da proteína, dependentemente da via de PKA. Utilizando um sistema de transfecção transiente, transfectamos uma quimera da proteína c-Myc com o receptor de estrógeno, MycER. Quando ativada por tamoxifen, a quimera migra para o núcleo e reverte a ação anti-mitogênica de ACTH sobre FGF2, porém, não tem efeito sobre células carenciadas tratadas ou não com ACTH apenas. Em conclusão, o antagonismo entre ACTH e FGF2 no controle da transição G0&#8594;G1&#8594;S do ciclo celular de Y-1 pode ser explicado pelas suas ações antagônicas sobre a estabilidade da proteína c-Myc. / ACTH is the trophic hormone that stimulates steroidogenesis, promotes growth and maintenance of the adrenal cortex. However, in adrenal cell lines, as well as in primary cultures, ACTH inhibits cell proliferation. ACTH effects on Y-1 cells are: increasing in steroidogenesis, cell rounding, cell cycle blocking in G1 phase and induction of fos and jun proto-oncogenes expression. Y-1 cell line displays a robust response to FGF2, a member from the FGFs family (Fibroblast Growth Factors), which regulates differentiation and proliferation in many cell types, being induced to enter G0&#8594;G1&#8594;S phases of fhe cell cycle upon FGF2 stimulation. ACTH antagonizes FGF2 effect, partially inhibiting cell cycle progression stimulated by FGF2. This project aimed to investigate c-Myc role in Y-1 cell cycle control, with emphasis on ACTH and FGF2 effects on its expression and activity control. We have shown that there are two main controls of c-Myc expression in Y-1 cells, transcription and protein stability. c-Myc concentration regulates the system Myc/Max/Mad, once Max and also Mad-1, Mad-4 and Mxi expression is constitutive in Y-1 cells. FGF2 induces c-Myc expression by increasing its transcription rate and stabilizing the protein in an Erk-MAPK pathway dependent manner. ACTH, on the other hand, does not control c-myc transcription but promotes a strong degradation of the protein through the PKA pathway. Using a transient transfection system, we were able to express MycER, a chimera of c-Myc and estrogen receptor in Y-1 cells. When activated by tamoxlfen, MycER is translocated to cell nucleus, where it abolishes the anti-mitogenic effect of ACTH over FGF2. However, it has no effect on cell cycle progression of serum starved cells treated or not with ACTH only. In conclusion, their antagonist effects on c-Myc protein stability can explain the antagonist effects of ACTH and FGF2 on the control of G0&#8594;G1&#8594;S transition of Y-1 cell cycle.

Les cyclooxygénases (COXs) sont une famille d'enzymes impliquées dans la biosynthèse des prostaglandines. COX-2 est la forme inductible qui est induite pendant l'inflammation et qui est surexprimée dans plusieurs cancers. Plusieurs évidences suggèrent que COX-2 joue un rôle dans la prolifération cellulaire et l'apoptose. Ces évidences concernent surtout les tumeurs solides et les mécanismes impliqués ne sont pas complètement connus et surtout pour les cancers d'origine hématopoïétique. Pour notre étude, nous avons étudié l'effet d'inhibiteurs de COX-2 (nimésulide, NS-398 et célécoxib) sur la prolifération et l'apoptose de lignées cellulaires leucémiques et lymphoblastiques, Hel, Jurkat, Raji et U937. Nous avons montré que les différents inhibiteurs de COX-2 diminuent la prolifération des différentes lignées cellulaires. Les cellules U937 sont apparues comme les cellules les plus sensibles à ces inhibiteurs alors que les cellules K562 étaient les plus résistantes. Nous avons montré que cette modulation correspond à une accumulation des cellules en phase G0/G1 du cycle cellulaire, accompagnée d'une diminution précoce de l'expression de c-Myc et d'une augmentation de l'expression de marqueurs de différenciation dans les cellules U937 (CD15) et Hel (CD41a et CD61). Dans la deuxième partie de ce projet, nous avons étudié les effets des différents inhibiteurs de COX-2 sur l'apoptose induite par différents agents chimiothérapeutiques dans nos modèles cellulaires. Nous avons ainsi montré que les inhibiteurs de COX-2 inhibent fortement l'apoptose induite par plusieurs agents chimiothérapeutiques. Nous avons démontré que la prévention de l'apoptose se situe avant l'activation de Bax et de Bak. Par ailleurs, cet effet est caractérisé par une incapacité des agents chimiothérapeutiques à déclencher un stress apoptotique. Toutes nos données ont donc démontré un effet anti-apoptotique des inhibiteurs de COX-2 sur l?apoptose intrinsèque vs l'apoptose extrinsèque à un stade précoce de l'induction de l'apoptose. Ces données suggèrent des précautions quant à l'utilisation des inhibiteurs de COX-2 en combinaison avec la chimiothérapie. Dans la troisième partie de notre projet, nous avons étudié la combinaison des inhibiteurs de COX-2 avec la curcumine, une substance naturelle connue pour ses propriétés antitumorales. Nos travaux ont montré que la curcumine seule conduit à une accumulation des cellules U937 en phase G2/M du cycle cellulaire, suivie d'une induction d'apoptose. Cependant, le prétraitement des cellules U937 avec du célécoxib à des concentrations non-apoptogéniques contrecarre l'apoptose induite par la curcumine, suggérant ainsi que ce type de combinaison ne serait pas une bonne stratégie dans les cellules hématopoïétiques. L'utilisation chronique des inhibiteurs de COX-2 peut être associée à des effets secondaires importants consécutifs à l'inhibition de l'activité de COX-2. Dans la dernière partie de notre projet, nous avons démontré que le 2,5 diméthyl-célécoxib (DMC), un analogue du célécoxib qui n'inhibe pas l'activité de COX-2, induit une diminution de la prolifération cellulaire et induit l'apoptose des cellules U937 et K562. Par ailleurs, ces effets sont plus importants que ceux observés avec le célécoxib. Par conséquent, ce composé a démontré de meilleures propriétés antitumorales et représente une voie thérapeutique prometteuse contre les leucémies. Tous nos résultats soutiennent donc l'idée que les inhibiteurs de COX-2 présentent les effets anti-tumoraux les plus efficaces que lorsqu'ils sont administrés seuls. Les effets observés avec le DMC suggèrent que ce composé pourrait représenté une voie alternative aux inhibiteurs de COX-2 en thérapie anti-cancéreuse. / Cyclooxygenases (COXs) are a family of enzymes, which catalyze the rate-limiting step in prostaglandin biosynthesis. COX-2 is the inducible isoform, upregulated during inflammation and overexpressed in various cancers. There are evidences of a role for COX-2 in cell proliferation and apoptosis especially in solid tumors, whereas little is known for cancers of hematopoietic origin. In our study, we analyzed the effect of COX-2 inhibitors (nimesulide, NS-398 and celecoxib) on cell proliferation and apoptosis of a panel of leukemic and lymphoblastic cell lines, Hel, Jurkat, K562, K562, Raji and U937. We found that the different inhibitors slow down cell proliferation in the different hematologic cell lines tested. U937 cells appeared as the most sensitive, whereas K562 were the most resistant to this effect. We provide evidence that this modulation corresponds to an accumulation of the cells in G0/G1 paralleled by an early downregulation of c-Myc and the expression of cell type-specific differentiation markers in U937 (CD15) and Hel (CD41a and CD61). In the second part of our study, we investigate the effect of COX-2 inhibitors on apoptosis induced by chemotherapeutic agents in our cell models. We demonstrated that COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We demonstrated an early prevention of apoptotic signaling, prior to Bax/Bak activation. The preventive effect is associated with an impairment of the ability of chemotherapeutic agents to trigger their apoptogenic stress. Altogether, our results demonstrate an anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at early steps of apoptosis commitment. These results suggest cautions in the use of COX-2 inhibitors with chemotherapy. In the third part of our project, we investigated the combination of COX-2 inhibitors with curcumin, a natural product known for its anti-tumor properties. Our findings show that curcumin alone leads to an accumulation of U937 cells in G2/M phase of cell cycle, followed by an induction of apoptosis. However, the pretreatment of U937 cells with celecoxib at non-apoptogenic concentrations, counteracted curcumin-induced apoptosis, thus showing that this combination is not a good anti-cancer strategy in our cell models. The chronic use of COX-2 inhibitors can be associated with severe side effects due to the inhibition of COX-2 enzyme. In the last part of our project, we demonstrated that 2,5 dimethyl-celecoxib (DMC), a structurally analogue of celecoxib, which is not able to inhibit COX-2 activity, induces an inhibition of cell proliferation and an induction of apoptosis in U937 and K562 cells. These effects are stronger than those observed with celecoxib. Thus, this compound demonstrated better anti-tumor properties and may represent a promising therapeutic approach against leukemia. Altogether, our study supports the idea that COX-2 inhibitors display anti-tumor effects in our cell models, but only when administrated alone. The effects observed with DMC suggest that this compound may represent an alternative approach to COX-2 inhibitors in cancer therapy.