No description available.

The aims of this thesis were to examine: (1) plasma and cellular pharmacokinetics of daunorubicin and its major metabolite daunorubicinol in patients with acute leukaemia, and the relationships between pharmacokinetics, patient response and the presence of P glycoprotein; (2) actions of the multidrug resistance reversing agents cyclosporin A and trifluoperazine, at clinically achievable concentrations, on daunorubicin accumulation and retention in human leukaemia cell lines and patients with acute leukaemia; and (3) effect of daunorubicin on the cell membrane of both sensitive and resistant cell lines, with and without the multidrug resistance reversing agents. Twenty-seven patients with acute leukaemia received daunorubicin as part of induction therapy. The plasma and cellular levels of daunorubicin and its metabolite daunorubicinol were determined using HPLC. There were no significant differences between patients who went into complete remission (12 out of 23) compared to those who did not respond for any of the plasma pharmacokinetic parameters. There was a significant difference in the cellular daunorubicin and daunorubicinol area under the concentration-time curve between responders and non responders (p less than 0.02), as well as in cellular Cmax, cellular clearance and cellular volume of distribution. Eleven patients were P glycoprotein positive and 10 P glycoprotein negative (no sample available for 2 patients). There was no correlation between patient response and the presence of P glycoprotein; nor a correlation between the cellular concentration of daunorubicin or daunorubicinol and P glycoprotein. Patients responding to chemotherapy had higher cellular daunorubicin and daunorubicinol compared to non responders. In contrast to in vitro studies, overexpression of P glycoprotein was not the reason for the lower cellular daunorubicin levels. Cyclosporin A was capable of increasing both cellular accumulation and retention in the drug resistant CEM/VLB and HL 60/ADR cell lines, but not in the drug sensitive CEM and HL 60 cell lines. Trifluoperazine had no effect in any of the four cell lines. In contrast to the cell line findings, only the combination of cyclosporin A and trifluoperazine were able to increase both accumulation and retention in the blast cells of patients at initial presentation. The multidrug resistant reversing agents alone had no effect in increasing accumulation or retention in the blast cells of P glycoprotein positive patients, nor patients in relapse. The cell line studies show that at clinically relevant concentrations only cyclosporin A is capable of increasing daunorubicin accumulation in both the drug resistant P glycoprotein positive (VLB) and P glycoprotein negative (ADR) cell lines. Thus, cyclosporin A does not work only by inhibiting the actions of P glycoprotein. Trifluoperazine was unable to reverse drug resistance at clinically relevant concentrations in either cell lines or patient blast cells. However, the combination of cyclosporin A and trifluoperazine increased accumulation in patient blast cells at initial presentation, suggesting that these agents may be more useful in patients at initial presentation than relapse. Daunorubicin was immobilised by linking it to poly vinyl alcohol and the effect of immobilised-daunorubicin was studied on the four cell lines above. The immobilised-daunorubicin was able to decrease cell growth in the drug sensitive HL 60 cell line but not in the drug resistant VLB or ADR cell lines. Poly vinyl alcohol itself was cytotoxic to the CEM cell line. The multidrug resistance reversing agents cyclosporin A and trifluoperazine were only capable of increasing cytotoxicity in the HL 60 cell line, with no effect in the drug resistant VLB or ADR cell lines.

The aims of this thesis were to examine: (1) plasma and cellular pharmacokinetics of daunorubicin and its major metabolite daunorubicinol in patients with acute leukaemia, and the relationships between pharmacokinetics, patient response and the presence of P glycoprotein; (2) actions of the multidrug resistance reversing agents cyclosporin A and trifluoperazine, at clinically achievable concentrations, on daunorubicin accumulation and retention in human leukaemia cell lines and patients with acute leukaemia; and (3) effect of daunorubicin on the cell membrane of both sensitive and resistant cell lines, with and without the multidrug resistance reversing agents. Twenty-seven patients with acute leukaemia received daunorubicin as part of induction therapy. The plasma and cellular levels of daunorubicin and its metabolite daunorubicinol were determined using HPLC. There were no significant differences between patients who went into complete remission (12 out of 23) compared to those who did not respond for any of the plasma pharmacokinetic parameters. There was a significant difference in the cellular daunorubicin and daunorubicinol area under the concentration-time curve between responders and non responders (p less than 0.02), as well as in cellular Cmax, cellular clearance and cellular volume of distribution. Eleven patients were P glycoprotein positive and 10 P glycoprotein negative (no sample available for 2 patients). There was no correlation between patient response and the presence of P glycoprotein; nor a correlation between the cellular concentration of daunorubicin or daunorubicinol and P glycoprotein. Patients responding to chemotherapy had higher cellular daunorubicin and daunorubicinol compared to non responders. In contrast to in vitro studies, overexpression of P glycoprotein was not the reason for the lower cellular daunorubicin levels. Cyclosporin A was capable of increasing both cellular accumulation and retention in the drug resistant CEM/VLB and HL 60/ADR cell lines, but not in the drug sensitive CEM and HL 60 cell lines. Trifluoperazine had no effect in any of the four cell lines. In contrast to the cell line findings, only the combination of cyclosporin A and trifluoperazine were able to increase both accumulation and retention in the blast cells of patients at initial presentation. The multidrug resistant reversing agents alone had no effect in increasing accumulation or retention in the blast cells of P glycoprotein positive patients, nor patients in relapse. The cell line studies show that at clinically relevant concentrations only cyclosporin A is capable of increasing daunorubicin accumulation in both the drug resistant P glycoprotein positive (VLB) and P glycoprotein negative (ADR) cell lines. Thus, cyclosporin A does not work only by inhibiting the actions of P glycoprotein. Trifluoperazine was unable to reverse drug resistance at clinically relevant concentrations in either cell lines or patient blast cells. However, the combination of cyclosporin A and trifluoperazine increased accumulation in patient blast cells at initial presentation, suggesting that these agents may be more useful in patients at initial presentation than relapse. Daunorubicin was immobilised by linking it to poly vinyl alcohol and the effect of immobilised-daunorubicin was studied on the four cell lines above. The immobilised-daunorubicin was able to decrease cell growth in the drug sensitive HL 60 cell line but not in the drug resistant VLB or ADR cell lines. Poly vinyl alcohol itself was cytotoxic to the CEM cell line. The multidrug resistance reversing agents cyclosporin A and trifluoperazine were only capable of increasing cytotoxicity in the HL 60 cell line, with no effect in the drug resistant VLB or ADR cell lines.

Made available in DSpace on 2016-08-19T18:16:04Z (GMT). No. of bitstreams: 1 Dissertacao Nivania.pdf: 1096637 bytes, checksum: 1906816bc4fae34d973336171b5bd415 (MD5) Previous issue date: 2012-07-30 / Background: The recent WHO classification incorporates immunophenotypic and cytogenetic characteristics that have prognostic impact. Several studies have attempted to identify clinical, biological and laboratory findings that correlate with prognosis, in order to incorporate them within the risk classification system used to define the therapeutic strategy. This work was done since there is no study in our state correlating the clinical and laboratory variables to specific immunological as predictive factors in the treatment of patients with acute leukemia. Objective: To evaluate the predictive value of different markers in PB (peripheral blood) and BM (bone marrow) in the induction period of treatment and disease-free survival in patients with acute leukemia. Methods: We evaluated 110 patients diagnosed with acute leukemia by immunophenotyping. In PB were analyzed: the percentage of blasts, leukocyte count and platelets and hemoglobin, and percentage of blasts in BM, all at diagnosis and after treatment in order to evaluate the therapeutic response. Results: Of 110 patients 61.82% were male. Most (80.9%) was framed in the age group of children and adolescents. As for the subgroups of acute leukemias 61.82% of ALL patients had type B, 12.73% of ALL type T and 25.45% of AML. The percentage of blasts in BM and PB for each of the sub groups of leukemia during induction treatment showed no significant difference on the other hand, in a comparative study between the subgroups of leukemia, there was a difference in their standard of presentation to diagnosis. For T-ALL platelet count on the initial induction period (D0) showed significant difference for patients who relapsed after the induction period. Regarding AML, patients with lower hemoglobin on D0 showed a significant tendency to relapse after the induction period. The recovery of platelet count to LLA B at the last day of the induction period of the treatment to values above 100,000/mm³ is not pointed to the recurrence of the disease follow-up period. Conclusion: The percentage of blasts in BM and PB not shown to have predictive value in respect of reference of each of these groups of leukemia in the induction phase of treatment. Platelet counts below 100.000/mm3 in T-ALL seem to D0 have predictive value for recurrence of disease after the induction period of treatment. For AML, patients with lower Hb determination on D0 were more likely to relapse after the induction period. The CD10 antigen expression at diagnosis of the disease should be a marker of good prognosis for remission induction phase of treatment. For ALL B, platelet counts above 100.000/mm3 in the D29 demonstrated predictive value for maintenance of non-recurrence of disease after D29. / Introdução: Seguindo os conceitos da classificação da OMS para as leucemias agudas, vários estudos tentam identificar características clínicas, biológicas e laboratoriais que se correlacionam com o prognóstico, objetivando incorporá-las dentro do sistema de classificação de risco usado para definir a estratégia terapêutica. Este trabalho foi realizado posto não haver nenhum estudo em nosso estado correlacionando as características clínicas e laboratoriais específicas às variáveis imunológicas como fatores preditivos no tratamento de pacientes com leucemias agudas. Objetivo: Avaliar o valor preditivo de diferentes marcadores em SP (sangue periférico) e MO (medula óssea) no período de indução do tratamento e sobrevida livre de doença em pacientes com leucemias agudas. Metodologia: Foram avaliados 110 pacientes com diagnóstico de leucemia aguda por imunofenotipagem. Em SP foram analisados: o percentual de blastos, a contagem de leucócitos e plaquetas e dosagem de hemoglobina; e o percentual de blastos em MO, todos ao diagnóstico e após o tratamento, a fim de avaliar a resposta terapêutica. Resultados: Dos 110 pacientes 61,82% eram do sexo masculino. A maioria (80,9%) estava enquadrada na faixa etária infanto-juvenil. Quanto aos subgrupos de leucemias agudas 61,82% dos pacientes eram portadores de LLA tipo B, 12,73% de LLA tipo T e 25,45% de LMA. O percentual de blastos em MO e SP para cada um dos sub grupos de leucemia, no período de indução do tratamento não demonstrou diferença significativa, por outro lado, em estudo comparativo entre os sub grupos de leucemia, houve diferença no seu padrão de apresentação ao diagnóstico. Para as LLA -T a contagem de plaquetas no dia inicial do período de indução (D0) demonstrou diferença significativa para os pacientes que apresentaram recidiva após o período de indução. Em relação às LMA, os pacientes com menor dosagem de hemoglobina no D0 apresentaram significativa tendência à recidiva após o período de indução. A recuperação da contagem de plaqueta para LLA B no último dia do período de indução do tratamento a valores acima de 100.000/mm³ apontou para a não recidiva da doença no período de acompanhamento. Conclusão: O percentual de blastos em SP ou MO não demonstraram ter valor preditivo em relação à remissão de cada um destes grupos de leucemias na fase de indução do tratamento. A contagem de plaquetas abaixo de 100.000/mm3 nas LLA - T ao D0 parecem ter valor preditivo para recidiva da doença após o período de indução do tratamento. Para as LMA, os pacientes com menor dosagem de Hb no D0 apresentaram maior tendência à recidiva após o período de indução. A expressão do antígeno CD10 ao diagnóstico da doença deva ser um marcador de bom prognóstico para remissão da doença na fase de indução do tratamento. Para as LLA B, contagens de plaquetas acima de 100.000/mm3 ao D29 demonstraram valor preditivo para manutenção de não recidiva da doença após D29.

Made available in DSpace on 2016-08-19T18:16:01Z (GMT). No. of bitstreams: 1 ELDA PEREIRA NORONHA.pdf: 2725549 bytes, checksum: 8726d2a693daed2c3b58abc727f064a3 (MD5) Previous issue date: 2010-07-07 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / Acute leukaemia is the most common type of cancer in childhood. Its diagnosis depends on immunophenotyping, which enables identification of the lineage, the grade of maturation, and the identification of markers with prognostic value. Assessment of the incidence of leukaemia subtypes worldwide has shown important variations in relation to geographical distribution, sex, age, ethnicity and socio-economic conditions. The objective of this work was to determine the immunophenotypic profile and the frequency, in different age groups, of subtypes of acute leukaemia in patients treated at the oncology center of reference Instituto Maranhense de Oncologia Aldenora Bello, in São Luís, Maranhão, and to study, in children with acute lymphoid leukaemia (ALL), the relationship between the expression of CD34 and the expression of aberrant phenotypes and prognostic factors. The diagnosis of acute leukaemia was obtained based on blood count, myelograms, cytochemical tests and immunophenotyping by flow cytometry. Monoclonal antibodies were used against T antigens (CD1a, CD2, CD3, CD4, CD5, CD7 and CD8), B antigens (CD10, CD19, CD22, CD79a and IgM), antigens of myeloid (CD13, CD14, CD33, CD64, CD117, MPO), erythroid (alpha-glycophorin), and platelet (CD61 and CD41a) differentiation, non-specific lineage antigen (CD45) and precursor cell antigens (CD34, HLA-DR). Acute leukaemia was classified according to the French-American-British (FAB) classification criteria and those of the European Group for the Immunological Characterisation of Leukaemias (EGIL). Seventy cases of de novo acute leukaemias were analysed over the period from September 2008 to January 2010, of which 31.4% were in adults and 68.6% in children. Among the adult patients 22.7% were diagnosed with ALL and 77.3% with acute myeloid leukaemia (AML), with the AML M0 subtype being most frequent. In children, 77.1% of the patients were diagnosed with ALL, and 18.7% with AML, with the AML M4 subtype the most frequent, and 4.2% with acute biphenotypic leukaemia (ABL). Among ALL, in children, B-ALL represented 72.9% of the cases and T-ALL 27.1%. The peak incidence of ALL was between 1 and 4 years of age. The most frequent subtypes of B-ALL were BII-ALL (pre-pre-B, common B), followed by the subtype BIII-ALL (pre-B). Among ALL and AML there was anomalous expression in 45.2% and 26.9% of cases, respectively. In ALL among children no statistically significant difference was found between the groups with and without anomalous expression in relation to the haematological parameters and the response to treatment. The expression of CD34 was negatively correlated with the number of leucocytes and percentage of blasts in peripheral blood. Furthermore, the expression of CD34 in B-ALL appeared to be associated with characteristics of better prognosis, while in T-ALL the opposite was observed. The antibodies used were sufficient to classify the cases immunologically. The use of immunophenotyping to diagnose acute leukaemias in our state enabled the diagnosis of minimally differentiated cases of AML (AML M0), as well as detection of the increased frequency of T-ALL in our population, suggesting that there may be differences in the prevalence of the FAB subtypes of AML, as well as the subtypes of ALL, in different regions of Brazil. / A leucemia aguda é o tipo de câncer mais comum na infância. Para o seu diagnóstico é indispensável a utilização da imunofenotipagem, que permite definir a linhagem, o grau de maturação, e a identificação de marcadores com valor prognóstico. A avaliação da incidência dos subtipos de leucemias no mundo tem mostrado variações importantes em relação à distribuição geográfica, sexo, idade, raça e condições sociais. Este trabalho objetivou determinar o perfil imunofenotípico e a freqüência, em diferentes faixas etárias, dos subtipos de leucemias agudas de pacientes tratados no centro oncológico de referencia Instituto Maranhense de Oncologia Aldenora Bello em São Luís-Maranhão; e estudar, em crianças com Leucemia Linfóide Aguda (LLA), a relação da expressão do CD34 e de fenótipos aberrantes com fatores prognósticos. O diagnóstico das leucemias agudas foi feito com base no hemograma, mielograma, provas citoquímicas e imunofenotipagem por citometria de fluxo. Utilizou-se anticorpos monoclonais contra antígenos T (CD1a, CD2, CD3, CD4, CD5, CD7 e CD8), antígenos B (CD10, CD19, CD22, CD79a e IgM) , antígenos de diferenciação mielóide (CD13, CD14, CD33, CD64, CD117, MPO), eritróide (alfa-glicoforina), plaquetário (CD61 e CD41a), antígeno de linhagem não específica (CD45) e antígenos de células precursoras (CD34, HLA-DR). As leucemias agudas foram classificadas de acordo com os critérios da classificação Franco-Americana-Britânica (FAB) e do Grupo Europeu para Caracterização Imunológica das Leucemias (EGIL). Analisou-se 70 casos de leucemias agudas de novo no período de setembro de 2008 a janeiro de 2010, dos quais 31,4% eram em adultos e 68,6% em crianças. 22,7% dos pacientes adultos foram diagnosticados como LLA e 77,3% como leucemia mielóide aguda (LMA), sendo o subtipo LMA M0 o mais freqüente. Em crianças, 77,1% dos pacientes foram diagnosticados como LLA, 18,7% como LMA, sendo mais freqüente o subtipo LMA M4 e 4,2% como leucemia bifenotípica aguda (BAL). Entre as LLA, em crianças, a LLAB representou 72,9% dos casos e a LLA T 27,1%. O pico de incidência da LLA foi entre 1 e 4 anos. Os subtipos de LLAB mais freqüente foram LLABII (pré-pré-B, B comum), seguido do subtipo LLA BIII (pré-B). Na LLA e LMA houve expressão anômala em 45,2% e 26,9% dos casos, respectivamente. Na LLA, em crianças, não se encontrou diferença estatisticamente significante, entre os grupos com e sem expressão anômala, em relação aos parâmetros hematológicos e resposta ao tratamento. A expressão do CD34 apresentou-se com correlação negativa com o número de leucócitos e porcentagem de blastos em sangue periférico. Pode-se observar que a expressão do CD34 na LLAB parece estar associada a características de melhor prognóstico, já na LLAT observa-se o contrário. Os anticorpos utilizados foram suficientes para classificar imunologicamente os casos. A utilização da imunofenotipagem para o diagnóstico de leucemias agudas em nosso estado permitiu diagnosticar casos de LMA minimamente diferenciadas (LMA M0), bem como as LLAT ocorridas com elevada freqüência em nossa população, sugerindo que podem haver diferenças na prevalência dos subtipos FAB da LMA, assim como dos subtipos de LLA, em diferentes regiões do Brasil.

CXCR4, récepteur de la chimiokine SDF-1 (stromal cell-derived factor 1) joue un rôle capital dans l’hématopoïèse normale mais aussi dans la biologie de la cellule leucémique. Ce récepteur est exprimé à la surface des blastes et participe à « l’ancrage » de la cellule souche leucémique (CSL) au sein de la niche médullaire. Les interactions de la CSL avec le micro-environnement sont source de signaux de survie et de résistance à l’apoptose. La première partie de ce travail correspond à deux analyses en cytométrie en flux de l’expression de CXCR4 et de molécules d’adhérence sur des échantillons diagnostiques de LAM (leucémie aiguë myéloïde). Ce travail confirme la valeur pronostique péjorative de l’expression de CXCR4 et propose un modèle de stratification pronostique des patients, en fonction de leur phénotype d’adhérence. La deuxième partie s’intéresse à l’identification de potentielles cibles thérapeutiques dans un modèle de LAL à chromosome Philadelphie, pathologie au pronostic sombre malgré les progrès thérapeutiques liés aux ITK (inhibiteurs de tyrosine kinase). L’inhibition de CXCR4 par l’AMD3100 permet de potentialiser l’efficacité de l’aracytine et du dasatinib dans un modèle de co-culture stromale avec la lignée SUPB15. Une deuxième piste de ciblage thérapeutique de la LAL Phi+ est l’inhibition de la protéine chaperone HSP90. Une expression forte de HSP90 (dans les LAL Phi+ par rapport aux LAL Phi-) s’associe à une plus grande cytotoxicité du 17-AAG. En conclusion, CXCR4 est un récepteur clé de la cellule leucémique. L’étude de son niveau d’expression permet des stratifications pronostiques des patients et son blocage en fait une cible thérapeutique prometteuse / CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. The first part of this work consists of two flow cytometry analyses of CXCR4 and adhesion molecules expression in patients with AML (acute myeloid leukaemia) at diagnosis. The results confirm that CXCR4 expression is associated with poor prognosis and this work proposes to stratify patients, according to their adhesive phenotype, in order to establish risk-adapted strategies. The second part deals with the identification of potential therapeutic targets in a model of ALL with chromosome Philadelphia. Despite therapeutic improvements with the ITK (tyrosine kinase inhibitors) era, long term survival remains poor. The inhibition of CXCR4 by the AMD3100 enhances the sensitivity of SUPB15 cell line to cytarabine and dasatinib therapy in a model of stromal co-culture. A second way of therapeutic targeting of the ALL Phi + is the inhibition of the heat-shock protein HSP90. High percentage of HSP90-positive cells (in Ph+ ALL samples) is associated with high sensitivity to 17-AAG. In conclusion, CXCR4 appears as a key receptor of the leukaemic cell. The analysis of its level of expression allows prognostic stratifications and its blockade represents a promising therapeutic target

No description available.

Les leucémies aiguës myéloïdes (LAM) sont des proliférations malignes de progéniteurs bloqués lors de la différenciation myéloïde. Le caryotype des blastes leucémiques identifie 3 groupes pronostiques distincts. Parmi les LAM à risque cytogénétique favorable, figurent les leucémies aiguës promyélocytaires (LAP), et celles avec inv(16) ou t(8;21). Les micro-ARNs sont des acteurs clef de l’hématopoïèse et sont aussi impliqués dans la leucémogénèse des LAM. Ils sont très stables dans le sérum et sont utilisés comme biomarqueurs dans les cancers. Le but de cette thèse était d’évaluer si une caractérisation pangénomique des micro-ARNs sériques permettait de distinguer ces 3 types de LAM entre elles ainsi que les LAM avec caryotype normal (NK-AML); d’identifier des micro-ARNs circulants fortement surexprimés dans les NK-AML par rapport à des sujets sains, pour une utilisation ultérieure comme marqueurs de maladie résiduelle; et de mieux préciser le pronostic des NK-AML. Ainsi, nous avons identifié une signature sérique spécifique des LAP liée à une dérégulation des micro-ARNs localisés dans la région DLK1-DIO3 soumise à l’empreinte, en 14q32. Ces micro-ARNs, dont l’origine était le blaste leucémique, étaient corrélés aux facteurs pronostiques connus des LAP. Par ailleurs, deux micro-ARNs, miR-10a-3p et miR-196b-5p, distinguant les NK-AML des LAM avec inv(16) ou t(8 ;21) ont été montré surexprimés dans les NK-LAM avec une mutation de NPM1 et/ou de FLT3-ITD. Enfin l’expression de ces deux micro-ARNs est corrélée à la dérégulation transcriptionnelle et à la méthylation de l’ADN affectant les gènes HOX et TALE. En conclusion, cette étude des micro-ARNs sériques ouvre un nouveau champ d’exploration à visée pronostique dans les LAM. / Acute myeloid leukaemia (AML) is a malignant proliferation of progenitors blocked during myeloid differentiation. The karyotype of the leukemic blasts identified three distinct prognostic groups. Among the favourable risk cytogenetics AML include acute promyelocytic leukaemia (APL), and those with inv (16) or t (8; 21). Micro-RNAs are key players in hematopoiesis and are also involved in leukemogenesis of AML. They are very stable in serum and used as biomarkers in cancers. The aim of this thesis was to evaluate whether a genome-wide characterization of serum micro-RNAs possible to distinguish these three types of AML them as well as AML with normal karyotype (NK-AML); identify micro-RNAs circulating highly overexpressed in NK-AML compared to healthy subjects, for later use as markers of residual disease; and to better define the prognosis of NK-AML. Thus, we have identified a specific serum signing of LAP related to a deregulation of micro-RNAs located in the DLK1-DIO3 under the imprinting, in 14q32. These micro-RNAs whose origin was the leukemic blasts were correlated with known prognosis factors of APL. In addition, two micro-RNAs, miR-10a-3p and miR-196b-5p, distinguishing the NK-AML with inv (16)-AML or t (8; 21)-AML have been shown overexpressed in NK-AML with mutation NPM1 and / or FLT3-ITD. Finally the expression of these two micro-RNAs correlates withtranscriptional deregulation and DNA methylation affectingTALE and HOX genes. In conclusion, this study of serum microRNAs opens a new field of exploration to assess the prognosis in AML.

Différentes translocations génomiques sont fréquemment associées à l'apparition de leucémies myéloïdes aiguës (LMA). Ces translocations génomiques résultent de l’assemblage de deux gènes conduisant à la production d'une protéine de fusion. C'est le cas de la translocation t (3; 5) (q25.1; q34) impliquant le suppresseur tumoral NPM et l'oncogène MLF1 donnant naissance à la protéine de fusion NPM-MLF1. Généralement, les gènes impliqués dans ces translocations contrôlent la croissance cellulaire, la différenciation ou la survie cellulaire. Cependant, pour NPM-MLF1 les causes du gain ou de la perte de fonction associée à la translocation demeurent inconnues car nous ne savons pas comment cette translocation peut favoriser ou participer à l'avènement de la LMA. Le but de ce travail est d’analyser le rôle de NPM-MLF1 dans le cancer et d’examiner comment son activité contribue à la leucémie en faisant des études d’interactions protéine/protéine. En effet, l’étude de la fonction d’une protéine implique souvent de connaître ses partenaires d’interactions. Pour ce faire, la technique de double hybride dans la souche de levure AH109 a été utilisée. Tout d’abord, les ADN complémentaires (ADNc) de MLF1, NPM1 et de NPM-MLF1, MLF1-Like (une partie de MLF1 de l’acide aminé 94 à 157) normaux et mutés du domaine MTG8-Like constitué des acides aminés (a.a.) 151 à 164 de MLF1 (excepté NPM) ont été clonés dans un vecteur d'expression de levure pGBKT7. Les ADNc de GFI-1, mSin3A, PLZF, HDAC1 et HDAC3 ont été clonés dans le plasmide pGADT7 de façon à créer des protéines de fusion synthétiques avec le domaine de liaison à l'ADN et de trans-activation de la protéine GAL4. Le plasmide pGBKT7 possède un gène TRP1 et pGADT7 un gène LEU2 qui permettent la sélection des clones insérés dans la levure. Aussi, le pGBKT7 a un épitope c-myc et pGADT7 un épitope HA qui permet de voir l’expression des protéines par buvardage de type Western. Après la transformation des levures les interactions protéine/protéine ont été observées en vérifiant l’expression des gènes rapporteurs HIS3, LacZ, MEL1, ADE2 de la levure en utilisant des milieux de sélection YPD/-Leu/-Trp, YPD/-Leu/-Trp/-His, YPD/-Leu/-Trp/-His/-Ade, YPD/-Leu/-Trp/+ X-Gal, YPD/-Leu/-Trp/ + X-α-Gal. Ensuite, les interactions trouvées par double-hybride ont été vérifiées dans les cellules érythroleucémiques K562 par immuno-précipitation (IP) de protéines suivies de buvardages Westerns avec les anticorps appropriés. NPM-MLF1, MLF1, MTG8, MLF1-Like surexprimés dans les cellules K562 ont été clonés dans le plasmide pOZ-FH-N. pOZ-FH-N possède un récepteur IL-2 qui permet de sélectionner les cellules qui l’expriment ainsi qu’un tag Flag-HA qui permet de voir l’expression des protéines par buvardage-Western. Les résultats du double-hybride suggèrent une interaction faible de NPM-MLF1 avec HDAC1, HDAC3 et mSin3A ainsi qu’une interaction qui semble plus évidente entre NPM-MLF1 et PLZF, GFI-1. NPM interagit avec GFI-1 et mSin3A. Aussi, MLF1 et MLF1-Like interagissent avec HDAC1, HDAC3, GFI-1, PLZF mais pas avec mSin3A. Les IP suggèrent que NPM-MLF1 interagit avec HDAC1, HDAC3, mSin3A et PLZF. MLF1 et MLF1-Like interagissent avec HDAC1, HDAC3 et mSin3A. L’interaction de NPM-MLF1 avec GFI-1, MLF1 et MLF1-Like avec PLZF et GFI-1 n’a pas encore été vérifiée par IP. Ainsi, nos observations permettent de suggérer que NPM-MF1, MLF1 et NPM pourraient jouer un rôle dans la transcription et la régulation de l’expression de certains gènes importants dans l’hématopoïèse et une variété de processus cellulaires parce qu’ils interagissent avec différents corépresseurs. En déterminant les partenaires protéiques de MLF1, NPM et NPM-MLF1, leurs fonctions et comment NPM-MLF1 influence et modifie le fonctionnement cellulaire normal; il sera possible de renverser le processus de LMA favorisé par la t (3; 5) NPM-MLF1 par la technologie d’interférence à l’ARN. / Abstract Different genomic translocations are frequently associated with the development of acute myeloid leukemia (AML). Genomic translocations can result in the fusion of two genes leading to the formation of a fusion protein. This is the case of the T (3; 5) (q25.1; q34) translocation that implicates the tumour suppressor NPM1 and the oncogene MLF1, giving rise to the fusion protein NPM-MLF1. Generally the genes implicated in these translocations control cell growth, differentiation and survival. However, for NPM-MLF1 the reasons behind the gain or loss of function associated with the translocation are still unknown as we still ignore how this translocation can enhance or take part in the AML development. The goal of my master degree project was to analyse in part the role of NPM-MLF1 in cancer and to examine how its activity can contribute in leukemia through protein/protein interaction assays. The usual study of a protein function implicates the investigation of interacting partners. For this purpose, we used the yeast AH109 to conduct a two-hybrid screen assay. The MLF1, MLF1-Like (amino acid 94 to 157 of MLF1), NPM1 and NPM-MLF1 cDNAs, normal and mutated in the MTG8-Like domain from the amino acid (a.a.) 151 to 164 of MLF1 (with the exception of NPM1) were cloned into the yeast expression vector pGBKT7. The GFI-1, mSin3A, PLZF, HDAC1 and HDA3 cDNAs were cloned into the vector pGADT7. These clones were developed to creat synthetic fusion proteins with the DNA binding or trans-activation domain(s) of the protein Gal4. The pGBKT7 vector contains the TRP1 gene and the pGADT7 the LEU2 gene. These genes were used for the selection of the yeast clones transformed with the plasmids mentioned above. In addition, the pGBKT7 vector has c-myc-tag and the PGADT7 vector the HA-tag allowing the assessment of protein expression through Western Blot analysis. After yeast transformation, the protein/protein interaction were studied while verifying the expression of the reporter genes HIS, LacZ, MEL1, ADE while using the following selective medias YPD/-Leu/-Trp, YPD/-Leu/-Trp/-His, YPD/-Leu/-Trp/-His/-Ade, YPD/-Leu/-Trp/+ X-Gal, YPD/-Leu/-Trp/ + X-α-Gal. The interactions determined by the two-hybrid screening were verified in the erythroleukemic cells K562 using immuno-precipitation (IP) of the proteins followed by western blot using the appropriate antibodies. To achieve this, NPM-MLF1, MLF1, ETO, MLF1-Like cDNAs were cloned into the pOZ-FH-N vector that possess an IL2 receptor, which allows the selection of the positive transformed clones in the cell and a Flag-HA tag that permit the verification of protein expression through Western-blot. The two-hybrid screen results suggest that NPM-MLF1 interacts with HDAC1, HDAC3, PLZF, GFI and mSin3A. NPM interacts with GFI-1 and mSin3A. This has not been yet verified using the IP method. As in the case of MLF1, MLF1-Like interacts with HDAC1, HDAC3, GFI-1 and PLZF. However, no interaction was observed with Sin3A. The IP experiments suggest that NPM-MLF1 interacts with HDAC1, HDAC3, mSin3A and PLZF. MLF1 and MLF1-Like interact with HDAC1, HDAC3 and mSin3A. The interaction of NPM-MLF1 with GFI1 as well as MLF1 and MLF1-Like with PLZF and GFI-1 are not yet verified by IP. Therefore, our observations led to the suggestion that NPM-MLF1, MLF1 and NPM can play a role in the transcription and the regulation of the expression of certain genes that are important for hematopoiesis and a variety through the determination of the protein partners of MLF1, NPM and NPM-MLF1, their functions and how NPM-MLF1 influence/modify the normal cellular function, and by focusing on this study, it might become possible to reverse the AML process that is by the t(3;5) NPM-MLF1 while using the RNA interference technology.

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