On note un taux élevé de résistance aux traitements dans la leucémie myéloïde aiguë (LMA). Cette résistance peut être associée aux altérations de TP53. Les « ubiquitin specific peptidases » (USP) sont impliquées dans plusieurs cancers mais leurs rôles ne sont pas élucidés dans les LMA. L’analyse de l’expression génique par RT-PCR quantitative de 21 USP et des gènes de l’axe USP7-MDM2-TP53-CDKN1A dans 111 échantillons de LMA a montré une dérégulation de USP44, USP1, USP28 et CDKN1A dans respectivement 72%, 44%, 25% et 42% des cas. CDKN1A, une cible importante de TP53, pourrait avoir un rôle dans la résistance au traitement. Nous avons développé un modèle expérimental pour évaluer la réponse des cellules leucémiques à la doxorubicine et au nutlin 3, un modulateur non génotoxique de TP53, selon l’expression initiale de CDKN1A. Ce travail préliminaire suggère que certains membres de la famille des USP et CDKN1A pourraient représenter de nouvelles cibles thérapeutiques dans les LMA. / There is a high rate of drug resistance in acute myeloid leukemia (AML) which may be associated with TP53 alterations. The « Ubiquitin specific peptidases » (USP) are involved in several cancers but their roles in AML are not elucidated. Gene expression analysis of 21 USP and genes of the USP7-MDM2-TP53-CDKN1A axis by quantitative RT-PCR in 111 AML samples, showed a deregulation of USP44, USP1, USP28 and CDKN1A in respectively 72%, 44%, 25% and 42% of cases. CDKN1A, an important TP53 target, may have a role in treatment resistance. We have developed an experimental model to assess the response of leukemic cells to doxorubicin and nutlin 3, a non genotoxic TP53 modulator, in relation to the CDKN1A expression level. This preliminary work suggests that some members of the USP family and CDKN1A could represent novel therapeutic targets in AML.

No description available.

A Leucemia Mielóide Crônica (LMC) é uma neoplasia mieloproliferativa resultante da expansão clonal de células mielóides positivas para o cromossomo Philadelphia. A patogênese da LMC está associada à expressão do oncogene BCR-ABL1, que codifica a proteína Bcr-Abl com constitutiva atividade da tirosina quinase, promovendo a mieloproliferação exacerbada e a resistência à apoptose das células leucêmicas. Os pacientes com LMC são tratados principalmente com inibidores de tirosina quinase (TKI), mas a resistência aos inibidores e a refratariedade tem sido relatada em alguns pacientes na fase crônica e na maioria dos pacientes em fases avançadas da doença. Assim sendo, continua a ser de suma importância a elucidação da patogênese da LMC e a busca de novos alvos terapêuticos, como os membros da via de sinalização Hippo e reguladores do ciclo celular, da família Aurora quinase. O presente estudo quantificou o nível de expressão de genes que codificam componentes da via de sinalização Hippo (MST1, MOB1B, MOBKL1B, LATS1, LATS2, YAP e TAZ) e Aurora quinases A e B em: 1) pacientes com LMC em diferentes fases da doença, resistentes ou sensíveis à terapia com mesilato de imatinibe (MI), em indivíduos saudáveis e 2) linhagens celulares HL-60, HL-60.Bcr- Abl tratadas com TKI (imatinibe, dasatinibe e nilotinibe), KCL22 e LAMA84 resistentes e sensíveis ao MI. Os níveis de expressão dos genes alvo foram correlacionados com o índice de prognóstico de Sokal. Os principais resultados revelaram que há alteração nos genes MST1, MOB1B, MOBKL1B, LATS1, LATS2, TAZ, AURKA e AURKB em pacientes com LMC em relação aos controles. Não houve correlação entre o índice de Sokal e a expressão gênica dos genes da via Hippo, MST1, MOB1B, MOBKL1B, LATS1, LATS2 e TAZ, assim como os genes Aurora quinases A e B. Pacientes com LMC em fases avançadas apresentaram maiores valores de expressão dos genes TAZ e AURKB, comparado aos pacientes na fase crônica. Os pacientes resistentes ao TKI apresentaram as expressões dos genes MST1, TAZ e AURKB, significativamente mais elevadas, comparado aos pacientes sensíveis ao MI. Os resultados dos estudos em linhagens celulares indicaram principalmente que a expressão do gene LATS1 pode ser modulada pela atividade de tirosina quinase Bcr-abl e que o oncogene BCR-ABL1 induz a expressão de AURKA, AURKB, LATS1 e TAZ. Em conjunto os dados obtidos revelam que a alteração da expressão dos genes da família Aurora quinase, A e B, e dos genes que codificam proteínas da via Hippo contribui para a patogênese e progressão da LMC. O desenvolvimento de fármacos e/ou a identificação de marcadores tumorais para a via de sinalização Hippo e família Aurora quinase, podem otimizar o tratamento da LMC, aumentando a susceptibilidade das células leucêmicas a apoptose e levando a um melhor prognóstico da doença / Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from clonal expansion of myeloid cells positive for the Philadelphia chromosome. The CML pathogenesis is associated with BCR-ABL1 oncogene expression, which encodes the Bcr-Abl protein with a constitutive tyrosine kinase activity, leading to leukemic cell high proliferation and resistance to apoptosis. CML patients are mainly treated with tyrosine kinase inhibitors (TKI), but some of CML patients in chronic phase are resistant and in advanced phases are refractory to TKI. Thus, it is still relevant to elucidate the CML pathogenesis and seek to new therapeutic targets, including the Hippo signaling pathway members and cell cycle regulatory genes such as those encoding the Aurora kinase family. The present study quantified the RNA expression level of genes encoding components from the Hippo cell signaling pathway (MST1, MOB1B, MOBKL1B, LATS1, LATS2, YAP, and TAZ) and Aurora kinase A and B in: 1) CML patients at different stages of the disease, in CML patients resistant or sensitive to imatinib mesylate therapy, healthy individuals and 2) in cell lines HL-60, HL-60.Bcr-Abl treated with TKI (imatinib mesylate, dasatinib and nilotinib), KCL22 and LAMA84 resistant and sensitive to IM. The RNA expression levels of the target genes were also correlated to the CML Sokal\'s prognostic score values. The main results revealed that there are alterations in the genes MST1, MOB1B, MOBKL1B, LATS1, LATS2, TAZ, AURKA and AURKB in patients with CML in relation to the controls. There was no correlation between the Sokal index and the gene expression of the Hippo, MST1, MOB1B, MOBKL1B, LATS1, LATS2 and TAZ genes, as well as the Aurora kinase genes A and B. Patients with advanced phase CML had higher values of expression of the TAZ and AURKB genes, compared to the patients in the chronic phase. Patients resistant to TKI had significantly higher MST1, TAZ and AURKB gene expression compared to MI-sensitive patients. The results of the studies in cell lines indicated primarily that the expression of the LATS1 gene can be modulated by the Bcr-abl tyrosine kinase activity and the BCR-ABL1 oncogene induces the expression of AURKA, AURKB, LATS1 and TAZ. Together, the data show that altered expression of Aurora kinase family genes, A and B, and genes coding for Hippo pathway proteins contribute to the pathogenesis and progression of CML. The development of drugs and/or identification of tumor markers for the Hippo signaling pathway and the Aurora kinase family can optimize CML treatment by enhancing the susceptibility of leukemic cells to apoptosis and leading to a better disease prognosis

Leucemia mieloide crônica (LMC) é uma neoplasia hematológica maligna associada à atividade tirosinoquinase da oncoproteína BCR-ABL1. A maioria dos casos de LMC é tratada com sucesso com inibidores tirosinoquinase de BCR-ABL1, mas uma porcentagem significativa de pacientes desenvolve resistência ao fármaco. Estudos recentes indicam que a célula-tronco leucêmica é resistente ao tratamento com imatinibe. A identificação de outras proteínas que cooperam com as vias de sinalização BCR-ABL1 podem indicar novos alvos terapêuticos. Os substratos do receptor de insulina (IRS) têm emergido como proteínas importantes na fisiopatologia de neoplasias sólidas e hematológicas. Um inibidor farmacológico de IGF1R-IRS1/2, NT157, foi desenvolvido e mostrou resultados promissores em estudos pré-clínicos com tumores sólidos. A associação constitutiva de IRS1 com BCRABL1 e o efeito antineoplásico resultante do silenciamento espefício de IRS1 em células K562 BCR-ABL1+ suportam a hipótese deste trabalho. O objetivo do presente estudo foi investigar o efeito da inibição farmacológica de IGF1R-IRS1/2 no fenótipo de células hematopoéticas leucêmicas BCR-ABL1+ utilizando células primárias, células K562 e modelos murinos. IRS1, mas não IRS2, apresentou-se menos expresso em amostras de medula óssea de pacientes com diagnóstico de LMC quando comparadas às amostras de células hematopoéticas normais (p<0,0001). NT157 reduziu a formação de colônias de células primárias de pacientes com LMC, mas não de células hematopoéticas de indivíduos saudáveis. Em células K562, o tratamento com o inibidor farmacológico de IGF1R-IRS1/2, NT157, reduziu a viabilidade e proliferação celular e induziu apoptose (p<0,05), inibiu a fosforilação de IGF1R, STAT3, STAT5, 4EBP1, P70S6K e ERK1/2, aumentou a expressão dos genes supressores de tumor CDKN1A, FOS e JUN e reduziu a expressão dos oncogenes MYC e BCL2 (p<0,05); a inibição específica de IRS1 através de lentivírus, mas não de IRS2, reduziu a viabilidade celular (p<0,05). Em células murinas Ba/F3 BCR-ABL1 e BCRABL1T315I, o inibidor farmacológico de IGF1R-IRS1/2, NT157, induziu apoptose e reduziu ativação de ERK1/2 in vitro. Na dose de 50mg/kg/dia, NT157 intraperitoneal e/ou imatinibe por gavagem falharam em reduzir o crescimento tumoral in vivo em modelo de tumor alográfico induzido por células Ba/F3 BCR-ABL1 e BCR-ABL1T315I. Em modelo animal de leucemia induzido por transplante de células hematopoéticas transduzidas com BCR-ABL1: (i) o tratamento com NT157 100mg/kg intraperitoneal, 3 vezes por semana, combinado com imatinibe 100mg/kg/dia por gavagem, reduziu significativamente o peso do baço, e preveniu a perda de peso comparado com veículo (p<0,05); apenas a monoterapia com imatinibe prolongou a sobrevida dos animais, (ii) o tratamento com NT157 70mg/kg intraperitoneal, 3 vezes por semana, e/ou imatinibe 70mg/kg/dia por gavagem não teve impacto no peso do baço e na variação do peso corporal; o tratamento com imatinibe em monoterapia ou combinado com NT157 prolongou a sobrevida dos animais (p<0,05). Em conclusão, o inibidor farmacológico de IGF1R-IRS1/2 representa uma droga potencialmente eficaz no tratamento da LMC, especialmente em casos de resistência com mutação BCR-ABL1 T315I. A avaliação da eficácia do inibidor farmacológico NT157 em modelos animais de LMC exige ajustes nos modelos murinos utilizados no presente estudo, melhor entendimento da farmacodinâmica e farmacocinética do composto e ajustes no esquema terapêutico utilizado. / Chronic myeloid leukemia (CML) is a hematological malignancy associated with the tyrosine kinase activity of the BCR-ABL1 oncoprotein. Most cases of CML are successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Recent studies indicate that leukemic stem cell is resistant to imatinib treatment. The identification of other proteins that cooperate with the BCR-ABL1 signaling pathway may indicate novel therapeutic targets. Insulin receptor substrates (IRS) have emerged as important proteins in the pathophysiology of solid and hematological neoplasms. A pharmacological inhibitor of IGF1R-IRS1/2, NT157, has been developed and shown promising results in preclinical studies with solid tumors. The constitutive association of IRS1 with BCR-ABL1, and the antineoplastic effects resulting from IRS1-specific silencing in K562 BCR-ABL1+ cells, support the hypothesis of this work. The aim of the present study was to investigate the effect of pharmacological inhibition of IGF1R-IRS1/2 on the phenotype of BCR-ABL1+ leukemia cells, using primary cells, K562 cell line and murine models. IRS1, but not IRS2, was downregulated in bone marrow samples from CML patients compared to bone marrow cells from healthy donors (p<0.0001). NT157 reduced colony formation of primary cells from CML patients but not from healthy donors. In K562 cells, treatment with the pharmacological inhibitor IGF1R-IRS1/2, NT157, reduced cell viability and proliferation, induced apoptosis (p<0.05), inhibited the phosphorylation of IGF1R, STAT3, STAT5, 4EBP1, P70S6K and ERK1/2, increased expression of tumor suppressor genes CDKN1A, FOS and JUN, and reduced expression of oncogenes MYC and BCL2 (p<0.05); IRS1 silencing mediated by lentivirus, but not IRS2, reduced cell viability (p<0.05). In murine Ba/F3 BCRABL1 and Ba/F3 BCR-ABL1T315I cells, the pharmacological inhibitor of IGF1R-IRS1/2, NT157, induced apoptosis and reduced ERK1/2 activation in vitro. At 50mg/kg/day, NT157 intraperitoneally and/or imatinib orally, failed to reduce tumor burden in vivo in an allographic tumor model induced by Ba/F3 BCR-ABL1 and Ba/F3 BCR-ABL1T315I cells . In an animal leukemia model induced by transplantation of BCR-ABL1-transduced hematopoietic cells : (i) treatment with NT157 100mg/kg intraperitoneally, 3 times per week, combined with imatinib 100mg/kg/day per gavage, significantly reduced spleen weight, and prevented weight loss compared to vehicle (p<0.05); only imatinib monotherapy prolonged survival (p<0.05), (ii) treatment with NT157 70 mg/kg intraperitoneally, 3 times per week, and/or imatinib 70 mg/kg/day per gavage had no impact on spleen weight and body weight; treatment with imatinib alone or combined with NT157 prolonged survival (p<0.05). In conclusion, the pharmacological inhibitor of IGF1R-IRS1/2 represents a potential effective drug in CML therapy, especially in cases of resistant BCR-ABL1 T315I mutation. The evaluation of the NT157 pharmacological efficacy in CML animal models requires adjustments in the murine models used in the present study, better understanding of the pharmacodynamics and pharmacokinetics of the compound and adjustments in the therapeutic scheme used.

Fosfoetanolamina sintética (Pho-s) é um monoéster análogo à fosfoetanolamina da membrana celular fosforilada artificialmente, com propriedades antiinflamatórias e apoptóticas para vários tipos de células tumorais humanas e murinas. Neste projeto foram avaliados os efeitos antitumorais in vitro da Pho-s e da formulação lipossomal DODAC/Pho-s na linhagem tumoral de leucemia mielóide crônica humana (K-562), em comparação ao modelo resistente a múltiplas drogas K-562 Lucena (MDR+). Os efeitos de citotoxicidade da Pho-s na linhagem tumoral K-562 e K-562 Lucena (MDR+) foram avaliados pela viabilidade celular utilizando o teste da Sulforodamina B, e os valores da IC50% obtidos foram de 43.1 mM e 145.9 mM, respectivamente após 24 horas de tratamento. O tratamento com o carreador DODAC vazio nas células K-562 e K-562 Lucena (MDR+) a IC50% foi de 0,0003mM e 0,008mM respectivamente, e com o tratamento com a formulação lipossomal DODAC/Pho-s a IC50% obtida respectivamente de, 0,56 mM e 0,31 mM. A viabilidade celular foi determinada a exclusão pelo azul de tripan 0,2%, em sistema automatizado Vi-Cell e foram significativas as diminuições da viabilidade celular em comparação ao grupo controle não tratado nas diversas concentrações e em diferentes períodos de tempo de tratamento. As alterações nas distribuições nas populações celulares nas fases do ciclo celular determinadas por citometria de fluxo mostraram aumento do DNA fragmentado (Sub/G1) em 12 e 24 horas de tratamento. Atividade apoptótica das células tumorais pela expressão da Anexina V/PI, no estágio de apoptose inicial, apoptose tardia e necrose foram quantificadas em citometria de fluxo, o tratamento com Pho-s, quando comparado ao grupo controle K-562 (40 e 80 mM) e K-562 Lucena (MDR+) (146 e 292 mM) ocorreu aumento significativo do número de células apoptóticas e em menor percentual o número de células necróticas. O tratamento com a Pho-s em célula leucêmica K-562 e K-562 Lucena (MDR+) tratadas, respectivamente com 40 e 80 mM e 146 e 292 mM mostraram que independente da expressão do fenótipo de resistência há uma redução significativa no potencial elétrico mitocondrial, analisado pelo o ensaio da rodamina-123. Os marcadores de controle e progressão do ciclo celular e da apoptose, mostraram efeitos moduladores da Pho-s dependentes da p53 na expressão da moléculas pró-apoptóticas. Esse conjunto de informações demonstrou os efeitos apoptóticos da Pho-s e da formulação lipossomal nas células tumorais independentemente do perfil molecular de resistência (MDR+), o que possibilita dizer que esse composto possui significativo potencial terapêutico nesse grupo de leucemias / Synthetic phosphoethanolamine (Pho-s) is a monoester analogous to the phosphoethanolamine which composes the membrane of an artificially phosphorylated cell, with anti-inflammatory and apoptotic properties for various types of human and murine tumor cells. In this project, we evaluated in vitro antitumor effects of Pho-s and the DODAC/Pho-s liposomal formulation in the human chronic myeloid leukemia (K-562) tumor line in comparison with the K-562 Lucena (MDR+). The effects of cytotoxicity of Pho-s on K-562 and K-562 Lucena (MDR +) tumor cells lines were evaluated by cell viability using the Sulforhodamine B test, and the IC50% values obtained were 43.1 mM and 145.9 mM after 24 hours of treatment, respectively. Treatment with the empty DODAC carrier on the K-562 and K-562 Lucena (MDR+) at IC50% cells was 0.0003 mM and 0.008 mM, and the treatment with the DODAC/Pho-s liposomal formulation obtained results of 0.56 mM and 0.31 mM, respectively. Cell viability was determined by 0.2% trypan blue exclusion in automated Vi-Cell system and the decreases in cell viability were significant in comparison to the untreated control group at various concentrations and different treatment time periods. Alterations in the distributions of cell populations in the cell cycle phases determined by flow cytometry showed increment of fragmented DNA (Sub/G1) in 12 and 24 hours of treatment. Apoptotic activity of tumor cells by the expression of Annexin V/PI, in the early apoptosis, late apoptosis phase and necrosis stage were quantified in flow cytometry, the treatment with Pho-s, when compared to the control group K-562 (40 and 80 mM) and K-562 Lucena (MDR +) (146 and 292 mM) demonstrated that there was a significant increase in the number of apoptotic cells and, in a lower percentage, the number of necrotic cells. Treatments with Pho-s in leukemic cell K-56 with 40 and 80 mM and in K-562 Lucena (MDR+) with 146 and 292 mM showed that independent of the expression of the resistance phenotype there is a significant reduction in the electrical potential of the mitochondrial membrane, analyzed with the rhodamine-123 assay. Control and progression markers of cell cycle and apoptosis showed p53-dependent modulating effects on the expression of pro-apoptotic molecules. This set of information demonstrated the apoptotic effects of Pho-s and liposomal formulation on tumor cells independently of the molecular resistance profile (MDR+), which makes it possible to say that this compound has significant therapeutic potential in this group of leukemias

INTRODUÇÃO: O impacto do aumento de expressão do fator de crescimento endotelial no curso da Leucemia Mielóide Crônica (LMC) ainda é desconhecido, porém há relatos de que estes pacientes apresentam maior densidade vascular em medula óssea do que em indivíduos saudáveis, principalmente em crise blástica. Outro fator recentemente associado ao aumento da angiogênese é a expressão anormal da proteína hsecurina, que, por sua vez, inibi uma protease denominada separase, responsável pela separação das cromátides irmãs durante a anáfase da mitose. Por esses motivos, quantificamos células endoteliais circulantes e VEGF em portadores de LMC como marcador de angiogênese e expressão do gene hsecurina. MÉTODOS: Realizamos análise prospectiva e consecutiva de uma coorte de 31 pacientes com LMC em fase crônica ao diagnóstico, 23 em crise blástica, 30 em fase acelerada, atendidos no ambulatório de Hematologia da FMUSP e 50 indivíduos saudáveis, doadores de plaquetas por aférese, para quantificação da porcentagem de células endoteliais circulantes e subtipos pelo método de citometria de fluxo no laboratório de Imunopatologia HC/FMUSP. Desta coorte 25 pacientes em fase crônica, 14 em crise blástica, 26 em fase acelerada e 32 indivíduos saudáveis foram analisados para os genes hsecurina e VEGF por PCR quantitativo em tempo real. RESULTADOS: A mediana da porcentagem das células endoteliais circulantes foi de 0, 0146% em LMC em crise blástica e 0,0059% no grupo controle, p < 0,01 às custas das células endoteliais maduras (p < 0,01). A mediana de células endoteliais circulantes em crise blástica foi de 0, 0146%, superior à da fase acelerada (0,0059%), p < 0,01 com predomínio de células endoteliais maduras (p < 0,01). Em relação à expressão do gene VEGF observamos aumento estatisticamente significativo nas fases crônica (p < 0,01), acelerada (p < 0,01) e crise blástica (p = 0,04). Encontramos aumento significativo da expressão do gene hsecurina na crise blástica da doença, com mediana de 0,390 em relação aos grupos controle com mediana de 0,125 (p < 0,01) e fase acelerada, com mediana de 0,230 (p = 0,04). Os pacientes na fase crônica da doença apresentaram mediana de 0,260 e p = 0,03 quando comparados com o grupo controle. CONCLUSÃO: Observamos neste estudo que a quantificação de CEC é uma ferramenta útil para predizer e identificar precocemente a progressão da LMC para fase blástica, diferentemente da variável VEGF que foi elevado em todas as fases da doença. A expressão do gene hSecurina na fase crônica da doença foi significantemente alta, demonstrando provável relação com a elevação da taxa de proliferação celular. Entretanto, estudos complementares do gene hSecurina deverão ser realizados na crise blástica da LMC, para entendermos com precisão o real significado nesta fase da doença. / INTRODUCTION: The impact of the increased expression of vascular endothelial growth factor in the course of chronic myeloid leukemia (CML) is still unknown, but there are reports that those patients have higher vascular density in bone marrow than healthy individuals, particularly in blast crisis. Another factor recently associated with increased angiogenesis is the abnormal expression of protein hSecurin, which, in turn, inhibits a protease called separase, responsible for the separation of sister chromatids during the anaphase of mitosis. For these reasons, we quantified circulating endothelial cells and VEGF in patients with CML as a marker of angiogenesis and hSecurin gene expression. METHODS: We performed a prospective analysis of consecutive cases in a cohort of 31 patients with CML in chronic phase at diagnosis, 23 in blast crisis, 30 in accelerated phase who attended the outpatient Hematology FMUSP ward, and 50 healthy subjects, platelet apheresis donors, for quantification of the percentage of circulating endothelial cells and subtypes through the flow cytometry method, at HC/FMUSP Immunopathology laboratory. In this cohort, 25 patients in chronic phase, 14 in blast crisis, 26 in accelerated phase, and 32 healthy subjects were tested for the genes VEGF and hSecurin by quantitative real-time PCR. RESULTS: The median percentage of circulating endothelial cells was 0.0146% in CML in blast crisis and 0.0059% in the control group, p <0.01 at the expense of mature endothelial cells (p <0.01). The median circulating endothelial cells in blast crisis was 0.0146% higher than in accelerated phase (0.0059%), p <0.01 with predominance of mature endothelial cells (p <0.01). Regarding the expression of the VEGF gene, a statistically significant increase was observed in chronic phase (p <0.01), accelerated (p <0.01) and blast crisis (p = 0.04). We found a significant increase in hSecurin gene expression in blast crisis disease, with a median of 0.390 compared to control groups, with a median of 0.125 (p <0.01) and accelerated phase, with a median of 0.230 (p = 0.04). Patients with chronic disease had a median of 0.260 and p = 0.03 compared with the control group. CONCLUSION: In this study, we observed that the quantification of CPB is a useful tool to predict and identify the early progression of CML to blast phase, unlike the VEGF variable, which was elevated in all stages of the disease. The expression of hSecurin gene in chronic phase was significantly higher, demonstrating a likely relationship with the increased cell proliferation rate. However, further studies of hSecurin gene should be made in the blastic crisis of CML to understand precisely the real meaning at this stage of the disease.

A técnica de análise de sobrevivência tem sido muito utilizada por pesquisadores na área de saúde. Neste trabalho foi usada uma distribuição em análise de sobrevivência recentemente estudada, chamada distribuição exponencial generalizada. Esta distribuição foi estudada sob todos os aspectos: para dados completos e censurados, sob a presençaa de covariáveis e considerando sua extensão para um modelo multivariado derivado de uma função cópula. Para exemplificação desta nova distribuição, foram utilizados dados reais de câncer (leucemia mielóide aguda e câncer gástrico) que possuem a presença de censuras e covariáveis. Os dados referentes ao câncer gástrico tem a particularidade de apresentar dois tempos de sobrevida, um relativo ao tempo global de sobrevida e o outro relativo ao tempo de sobrevida livre do evento, que foi utilizado para a aplicação do modelo multivariado. Foi realizada uma comparação com outras distribuições já utilizadas em análise de sobrevivência, como a distribuiçãoo Weibull e a Gama. Para a análise bayesiana adotamos diferentes distribuições a priori para os parâmetros. Foi utilizado, nas aplicações, métodos de simulação de MCMC (Monte Carlo em Cadeias de Markov) e o software Winbugs. / Survival analysis methods has been extensively used by health researchers. In this work it was proposed the use a survival analysis model recently studied, denoted as generalized exponential distribution. This distribution was studied in all respects: for complete data and censored, in the presence of covariates and considering its extension to a multivariate model derived from a copula function. To exemplify the use of these models, it was considered real cancer lifetime data (acute myeloid leukemia and gastric cancer) in presence of censored data and covariates. The assumed cancer gastric lifetime data has two survival responses, one related to the total lifetime of the patient and another one related to the time free of the disease, that is, multivariate data associated to each patient. In these applications there was considered a comparative study with standard existing lifetime distributions, as Weibull and gamma distributions.For a Bayesian analysis we assumed different prior distributions for the parameters of the model. For the simulation of samples of the joint posterior distribution of interest, we used standard MCMC (Markov Chain Monte Carlo) methods and the software Winbugs.

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa clonal caracterizada pela presença do cromossomo Philadelphia e o oncogene BCR-ABL1. Este oncogene codifica a oncoproteína Bcr-Abl com atividade tirosina-quinase constitutiva. A proteína Bcr-Abl é responsável pela resistência das células leucêmicas a apoptose. Atualmente, os pacientes com LMC são tratados com os inibidores de tirosina-quinase - mesilato de imatinibe, dasatinibe e nilotinibe. Apesar de o tratamento ser eficiente, pacientes em fases avançadas e mesmo na fase crônica da doença, apresentam resistência à terapia. Desta forma, novos fármacos devem ser investigados para melhorar o tratamento da LMC. As L-aminoácido oxidases (LAAOs) têm sido descritas como substâncias citotóxicas e indutoras de apoptose. Assim, o principal objetivo do presente estudo foi investigar o potencial antitumoral da LAAO isolada da serpente Calloselasma rhodostoma (CR-LAAO) nas células Bcr-Abl positivas. Avaliou-se a citotoxicidade da CR-LAAO nas linhagens HL-60 (linhagem Bcr-Abl negativa), HL-60.Bcr-Abl, K562 e KCL22 (linhagens Bcr-Abl positivas) e nas células mononucleares (MNC) de sangue periférico de indivíduos saudáveis, na presença ou ausência da catalase. Para investigar os mecanismos da ação citotóxica da CR-LAAO, realizou-se os ensaios de indução de apoptose por meio da quantificação das percentagens de núcleos hipodiplóides e anexina V-FITC, nas linhagens celulares e nas células MNC de indivíduos saudáveis e pacientes com LMC. Avaliou-se também os níveis de expressão das caspases 3, 8 e 9, o potencial de membrana mitocondrial, danos no DNA e o efeito apoptótico da toxina combinada com os inibidores de tirosina-quinase nas linhagens Bcr-Abl positivas. Além disso, investigamos se a CR-LAAO foi capaz de modular a expressão dos apoptomiRs miR-15a, miR-16, miR-145, miR-26a, hsa-let-7d, miR-142-3p, miR-29c, miR-146a, miR-21, miR-130a e miR-130b, assim como das proteínas pro- e anti-apoptóticas Bak, Bax, Bid, Bim, A1, Bcl-2, c-Flip, Ciap-2 e Mcl-1 nas linhagens Bcr-Abl positivas. Nossos resultados mostraram que o efeito citotóxico da CR-LAAO foi mais potente nas linhagens Bcr-Abl positivas em relação às células MNC de indivíduos saudáveis, e está associado ao peróxido de hidrogênio produzido durante a reação enzimática da CR-LAAO. Demonstrou-se também que a CR-LAAO induziu apoptose nas linhagens Bcr-Abl postivas testadas e nas células MNC de pacientes com LMC na fase crônica da doença. Em todas as linhagens celulares detectou-se danos no DNA, perda do potencial de membrana e ativação das caspases 3, 8 e 9. A percentagem de apoptose aumentou quando as células HL-60.Bcr-Abl foram tratadas com a CR-LAAO combinada com os inibidores de tirosina-quinase. A CR-LAAO modulou a expressão dos apoptomiRs miR-15a, miR-16, miR-145, miR-26a, hsa-let-7d, miR-142-3p, miR-29c, miR-21, miR-130a e miR-130b e de possíveis proteínas alvos nas linhagens Bcr-Abl positivas. Sendo assim, os resultados obtidos sugerem que a CR-LAAO apresenta uma ação antitumoral capaz de destruir as células leucêmicas / Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the presence of Philadelphia chromosome and BCR-ABL1 oncogene. This oncogene encodes the Bcr-Abl tyrosine kinase (TK) which presents a constitutive activity. The Bcr-Abl is responsible for leukemic cells resistance to apoptosis. The CML patients are currently treated with tyrosine kinase inhibitors (TKI) - imatinib mesylate, dasatinib and nilotinib. Although TKI are efficient for CML treatment, patients in advanced phases and even in chronic phase of the disease present resistance to therapy. Thus, potential new drugs must be investigated to improve the CML treatment. The L-amino acid oxidases (LAAOs) have been described as cytotoxic and apoptosis-inducing substances. Here, we investigated the LAAO from Calloselasma rhodostoma (CR-LAAO) antitumoral potential against Bcr-Abl positive cells. We evaluated the CR-LAAO cytotoxic effect against HL-60 (Bcr-Abl negative cell line), HL-60.Bcr-Abl, K562, KCL22 (Bcr-Abl positive cell lines) and the peripheral blood mononuclear cells (PBMC) from healthy subjects, in the presence or absence of catalase. To investigate the mechanisms underlying the CR-LAAO cytotoxic action, we performed the apoptosis induction assays through the hypodiploid nuclei and annexin-V quantification in the cell lines and PBMC from healthy subjects and CML patients. We also evaluated the levels of caspases 3, 8 and 9 expression, the mitochondrial membrane potential, DNA damage and the apoptotic effect of CR-LAAO combined with TKI on Bcr-Abl positive cells. In addition we investigated if CR-LAAO was capable of modulating the apoptomiRs miR-15a, miR-16, miR-29c, hsa-let-7d, miR-145, miR-146a, miR-21, miR-130a, miR-130b, miR-142-3p and miR-26a, the pro- and anti-apoptotic proteins (Bak, Bax, Bid, Bim, A1, Bcl-2, c-Flip, Ciap-2 and Mcl-1 expression in HL-60, HL-60.Bcr-Abl, K562 and KCL22 cells. Our results showed that the CR-LAAO cytotoxic effect was more potent in Bcr-Abl positive cell lines than in PBMC from healthy subjects and it is linked to hydrogen peroxide produced during the enzymatic action of CR-LAAO. It was also demonstrated that CR-LAAO was capable of inducing apoptosis in Bcr-Abl positive cell lines and CML patient\'s cells in chronic phase of the disease. In all tested cell lines, the loss of mitochondrial membrane potential, DNA damage and caspases 3, 8 and 9 activation were detected. The apoptosis percentage was improved when HL-60.Bcr-Abl cells were treated with CR-LAAO combined with TKI. The CR-LAAO modulated the apoptomiRs miR-15a, miR-16, miR-145, miR-26a, hsa-let-7d, miR-142-3p, miR-29c, miR-21, miR-130a and miR-130b expression as well the predict target proteins levels on Bcr-Abl positive cells. Thus, our results suggest that CR-LAAO presents an antitumoral action capable of destroying the CML cells.

Les cellules Natural Killer (NK) sont jouent un rôle essentiel dans la surveillance des hémopathies malignes. Cependant, les cellules tumorales développent des mécanismes immunosuppresseurs pour échapper à l'immunité cellulaire NK. Ainsi, le maintien ou l'amélioration des performances des cellules NK sont considérés comme des défis majeurs. Les objectifs de cette partie étaient d'évaluer l'impact de la perfusion de cellules NK activées sur la récupération et la biologie des cellules NK circulantes après l'allo-SCT. Des doses croissantes de cellules NK activées par IL-2 ex-vivo ont été perfusées chez des patients atteints de tumeurs malignes hématologiques 3 mois après allo-SCT. Nos résultats ont montré une fréquence plus élevée des cellules NK dans la périphérie des patients traités. Bien que le phénotype immature soit remarquable peu après le traitement, les cellules NK circulantes, présentaient un état d'activation avec un profil de maturation amélioré après 6 mois de traitement. Nous avons également constaté que l'expression des récepteurs NK activateurs (NKG2D, NKp30 et NKp46) augmentait sur les cellules NK circulantes des patients. De plus, ces cellules ont montré une augmentation significative de la capacité de dégranulation ainsi que de la sécrétion de cytokines (IFN-Ƴ et TNF-α) tout au long de l'étude. Ces différences ont notamment été observées chez les patients ayant reçu des doses plus importantes de cellules NK activées. En conclusion, nous supposons que la perfusion de fortes doses de cellules NK activées ex-vivo pourrait être associée à l'amélioration du phénotype et des fonctions des cellules NK au cours de la reconstitution immunitaire après allo-SCT. / Natural killer (NK) cells are effector lymphocytes of the innate immune system that have the ability to kill transformed cells. They play a critical role in hematological malignancies surveillance, however, tumor cells develop immunosuppressive mechanisms to escape NK cell-mediated killing. So, maintaining or improving NK cell performance is considered a major challenge. Our goals are to evaluate the impact of activated NK cells infusion on the recovery and biology of circulating NK cells post allo-SCT.Three different doses (dose 1: 106 NK cell/Kg, n=3; dose 2: 5x106 NK cell/Kg, n=7; dose 3: >5x106 NK cell/Kg, n=6) of ex-vivo IL-2 activated NK cells were infused into patients with hematological malignancies after all-SCT. Our results showed higher frequency of NK cells in the periphery of patients treated with larger doses of activated NK cells. Although the notable immature phenotype shortly after treatment, the circulating NK cells in patients receiving larger doses of activated NK cells displayed more activation status with improved maturation profile after 6 months of treatment. We also found that the expression of activating NK receptors (NKG2D, NKp30, and NKp46) augmented on circulating CD56dim NK cells of patients receiving larger doses of activated NK cells. Moreover, these cells showed a significant increase in degranulation capacity as well as cytokine secretion (IFN-Ƴ and TNF-α) throughout study period. In conclusion, we hypothesize that infusion of high-dose of ex-vivo activated NK cells could be associated with improvements of NK cell phenotype and function during immune reconstitution after allo-SCT.

Das BCL-2 Gen wurde als Onkogen der t (14;18)(q32;q21)-Translokation bei follikulären Non- Hodgkin-Lymphomen identifiziert. Die biologische Wirkung des BCL-2 Proteins liegt in der Hemmung der Apoptose. Bei der AML wird eine vermehrte BCL-2 Expression und eine dem- entsprechend verminderte Apoptose bei unreifen malignen myeloischen Vorläuferzellen gefun- den. Diese Krankheit ist teilweise auch chemoresistent. Goldstandard der Induktionstherapie bei AML ist eine Kombination aus Ara-C und Idarubicin, welche Doppel- und Einzelstrang- brüche der DNA induzieren. Apoptose der Leukämiezellen wird durch Schädigung der DNA ausgelöst. BCL-2 kann die Zellen durch Hemmung der Apoptose schützen, indem es die Cy- tochrom-C-Freisetzung blockiert. Darüber hinaus befinden sich die BCL-2- überexprimierenden Zellen in der G0-Phase und sprechen dabei schlecht auf die Chemothera- pie an. Deshalb stellt BCL-2 den Leukämiezellen "doppelten" Schutz zur Verfügung. BCL-2 spielt somit eine wichtige Rolle bei der Chemoresistenz. Ob ein Therapieprotokoll in der Be- handlung der AML effektiv ist, schlägt sich in der Kinetik der zunehmenden oder abnehmen- den BCL-2-Transkripte nieder. Zur Kontrolle des BCL-2-Transkriptes ist die quantitative PCR der qualitativen PCR überlegen. Die Quantifizierung dieses Transkriptes wurde mittels Echtzeit-Fluoreszenz-PCR realisiert. Bei der Echtzeit-Fluoreszenz-PCR wird die Reaktion im geschlossenen Reaktionsgefäß durchge- führt, sodass die Gefahr von Kontamination minimiert werden kann. Da keine Post-PCR Schrit- te nötig sind, wird die Überprüfung zahlreicher Proben durch ein 96-well-Format innerhalb eines Laufes ermöglicht. Die Echtzeit-Fluoreszenz-PCR garantiert ihre Spezifität durch eine spezifi- sche Sonde-Zielsequenz-Bindung und erlaubt eine exakte Quantifizierung der BCL-2- Transkriptzahl. In der vorliegenden Arbeit wurde die BCL-2-Expression in 53 AML-Fällen mittels Echtzeit- Fluoreszenz-PCR untersucht. Das ?-Actin Gen wurde als Referenzgen benutzt. Für die BCL-2- Expression wurde eine Ratio aus der Transkriptzahl des BCL-2 Gens und des ?-Actin Gens ge- bildet. Bei 53 AML-Fällen, die den sieben AML-Subtypen zugeordnet werden konnten(FAB M0-M7), konnte eine BCL-2-Expression nachgewiesen werden. Trotz der unterschiedlich hohen BCL-2-Expression bei diesen Patienten, ergab sich keine signifikante Korrelation zwischen der BCL-2-Expression und den FAB-Subtypen. Außerdem wurde die BCL-2-Expression in T- Zellen, B-Zellen und Granulozyten aus 5 AML-Patienten nachgewiesen. Die BCL-2-Expression wurde nicht von den Subpoplationen der mononukleären Zellen wie z.B. T-Zellen, B-Zellen, Granulozyten beeinflusst. Bei sieben Patienten wurden Proben im Verlauf untersucht. Dabei korrelierte eine hohe oder ansteigende BCL-2-Expression mit einem Rückfall der AML. Die Anzahl der untersuchten Proben im Verlauf ist jedoch zu klein, um definitive Schlußfolgerungen zu ziehen. Eine prospektive Untersuchung von größeren Patientenzahlen erscheint sinnvoll. / The bcl-2 oncogene was discovered by virtue of its association with the translocation, t(14;18) (q32;q21), observed in most follicular lymphomas. The bcl-2 protein is a 26 kDa integral membrane protein which functions by enhancing cell viability through the inhibition of apoptotic death. Acute myeloid leukemia is a lethal malignant disease characterized by an abnormal proliferation and differentiation of myeloid progenitor cells. The bcl-2 oncogene contributes to leukemogenesis by prolonging the life span of defected progenitor cells. Although the expression of bcl-2 in blast cells of acute myeloid leukemia is heterogeneous, a significant proportion of blast cells are shown to have high bcl-2 levels. The highest bcl-2 levels are found in cells that grow autonomously in vitro and also in blast cells expressing the CD34 surface antigen. These groups of AML patients are tranditionally the ones in which the prognosis is poor, because most of the chemotherapeutic agents like cytosine-arabinoside (Ara-C) exert their effect by triggering apoptosis. The high level of the bcl-2 gene that inhibits apoptosis is implicated in the resistance of AML blast cells to chemotherapy and leads to unfavorable prognosis. In this study, a real time fluorescence PCR assay was used to monitor the expression of the bcl-2 transcript in the therapeutic course of AML patients. By applying this rapid new developed quantitative method, the changes of the bcl-2 transcript with chemotherapy can help to evaluate the efficacy of therapeutic interventions in AML. The real time fluorescence PCR has many advantages over traditional measures. First, the assay is extremely rapid because post-PCR processing steps are unnecessary. All relevant data are collected real time during the course of a 2h PCR cycle program; data analysis can be completed in less than 10 min. Second, the assay from reaction set-up to data collection and analysis is a closed-tube system, which reduces the risk of false positive resulting from PCR product carry- over contamination and eliminates variation from additional pipetting steps. Finally, the real time fluorescence PCR is highly specific for the gene target of interest. Here the expression levels of the bcl-2 gene were measured in 53 patients with acute myeloid leukemia and normalized by ?-actin, a house-keeping gene expression as endogenous reference. The bcl-2/?-actin ratio from the 53 patients with AML was various, but not related to FAB subtypes. And also, this transcript ratio was not affected by mononucleated cell types. The samples from seven patients were measured to evaluate the association between the bcl-2 expression and the responsiveness of AML patients to the chemotherapy. The high or gradual elevation of the bcl-2 expression demonstrated the loss of effect in update-therapy protocol and the relapse in AML patients. Although the amount of samples are not large enough to reach the final conclusion, it is of significance that a number of patients will be analyzed in the future.