The studies presented in this dissertation establish the dynamics of Eleven nineteen Lysine-rich leukemia (ELL) family of elongation factors during B cell differentiation and provide a description of ELL3 function in B cells. The transition from a mature naïve B cells into an activated B cell is dependent on a large increase in transcriptional output, which is followed by focused expression on secreted immunoglobulin upon terminal differentiation into plasma cell. While ELL family members have previously been implicated in alternative splicing at the immunoglobulin heavy chain locus in plasma cells, their presence and function prior to differentiation is currently not known. However, the use of elongation factors has been implied by the finding of mostly paused RNA polymerase II in the genome of naïve B cells. In the first study, the expression of transcriptional elongation factor ELL3 is shown to be restricted to activated B cells and B cell lymphomas. All three family members were characterized in B cell lymphoma cell lines, genome wide expression, microarray analysis and primary B cell stimulus. The expression of ELL3 was induced upon activation of B cells concurrently with family member ELL. In addition, the abundant expression of ELL3 was restricted to GC derived B cell lymphoma cell lines. While the expression of ELL is maintained, the expression of ELL3 is diminished and ELL2 is up-regulated in terminally differentiated plasma cells. The expression of master regulator of terminal plasma cell differentiation PRDM1 was inverse correlated with that of ELL3. To further establish PRDM1s role in regulating the ELL family member dynamics, global binding was assessed in plasma cell lines. Chromatin immunoprecipitation followed by quantitative PCR was utilized to identify direct association of PRDM1 at exclusively the ELL3 loci. Ectopic expression of PRDM1 in B cells down regulated the expression of ELL3. Furthermore, two consensus PRDM1 binding sites were defined at the ELL3 loci, which mediate significant repression of the promoter activity. Collectively, these experiments indicate that PRDM1 mediates the switch from ELL3 in B cells to ELL2 in plasma cells. The data presented in the final chapter aimed at defining a function for ELL3 in the cells that express it most abundantly, which are B cell lymphoma cell lines. Transient depletion of ELL3 in a Burkitt’s lymphoma cell line resulted in a diminished proliferation rate due to a severe disruption of DNA replication and its regulators minichromosome maintenance proteins. Additionally, compromised cell division and mitotic regulators were observed along with increased DNA damage and cell death. The data presented here demonstrate a key role for ELL3 in the proliferation and survival of B cell lymphomas and positions ELL3 as an attractive therapeutic target against B cell lymphoma’s with a germinal center origin.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Monika Šmídlová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Effect of selected cytostatics for the treatment of leukemia on the activity of human carbonyl reducing enzymes Key words: reductases, leukemia, cytostatics, inhibition Anthracycline antibiotics, especially daunorubicin, are widely used for the treatment of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Although the efficacy of these drugs is high, treatment is still limited due to cardiotoxicity and tumor cell resistance to anthracyclines. Mechanisms that contribute to the formation of anthracycline resistance include metabolic biotransformation (reduction) to less efficient secondary alcohols. The reduction is calatyzed by carbonyl reducing enzymes belonging to aldo-keto reductase (AKR) and short chain dehydrogenase/reductase (SDR) superfamilies. The discovery of AKR and SDR inhibitors could help to overcome anthracycline resistance and also reduce cardiotoxicity caused by these drugs. The aim of the diploma thesis was to find out whether all-trans-retinoic acid, cyclophosphamide, cytarabine, cladribine and prednisolone are able to inhibit anthracycline reductases AKR1A1, AKR1B10, AKR1C3, AKR7A2...

The mammalian testis is divided into two distinct compartments which carry out its principal functions. Spermatogenesis occurs within the seminiferous tubules and androgen biosynthesis primarily occurs in the interstitial space. Both these processes are entirely dependent upon the two major testicular somatic cell populations - the Sertoli and Leydig cells respectively. In human males, testicular spermatogenic and endocrine function declines during the ageing process. Of particular significance is the reported age-related decrease in Leydig cell androgen production as androgens have been suggested to play a crucial role in supporting lifelong general health in men, with low circulating testosterone linked to an increased risk of developing chronic age-related cardiometabolic diseases. However, the relationship between ageing, testicular function and disease is not fully understood, impeding the development of novel therapeutic strategies to treat age-related testicular dysfunction. In one set of studies undertaken herein, a series of novel mouse models of premature ageing were utilised to begin to dissect the process of age-related testicular degeneration. Firstly, a novel knockout-first conditional allele of a previously reported premature-ageing model driven by Cisd2 (CDGSH Iron Sulphur Domain 2) deficiency was validated and the testicular phenotype characterised and compared to that of naturally aged mice at 18-months of age. Histological analyses revealed premature testicular atrophy at 6-months of age in CISD2 deficient mice, consistent with observations of the naturally aged testis. Circulating testosterone was significantly lower in CISD2-deficient mice compared to wild-type controls at 6-months of age and the luteinising hormone/testosterone ratio was significantly elevated, indicative of compensated Leydig cell failure. mRNA expression of key genes involved in androgen production were also significantly reduced in the CISD2-deficient testis, pointing to Leydig cell dysfunction in this model of premature aging. Next, Cre/LoxP technology was used to delete Cisd2 from specific testicular cell populations to determine which cell types control/support Leydig cell function during the ageing process. Testosterone production was unaffected when Cisd2 was disrupted in either the Leydig cell population or Sertoli cell population. These observations suggest that disruption to the testicular microenvironment in which Leydig cells reside, rather than intrinsic Leydig cell ageing, may play a significant role in age-associated Leydig cell dysfunction. A second set of studies were carried out to investigate the role of leukemia inhibitory factor (LIF) signalling in the maintenance of testicular function. LIF is a pleiotropic cytokine belonging to the interleukin-6 family. In the rodent testis, LIF is expressed in fetal life and adulthood; the peritubular myoid cells thought to be the main site of production. Given their anatomical location within the testis, LIF produced by peritubular myoid cells may act on both intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, LIFR is expressed in germ cells, Sertoli cells, Leydig cells as well as testicular macrophages suggesting that LIF may be a key paracrine regulator of testicular function. However, the precise role of LIF/LIFR signalling in the testis is largely unknown. As such, models of testicular cell-specific Lifr deletion were generated using Cre/LoxP technology. Analysis of these novel models of conditional LIFR ablation revealed that LIFR is dispensable in germ cells for normal spermatogenesis. However, LIFR ablation from Sertoli cells resulted in a progressive degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules. In a final set of studies, a rat model of Leydig cell ablation-regeneration was used to determine the regenerative capacity of human adipose-derived perivascular stem cells (hAd-PSC) as a potential therapy for testicular dysfunction. Following ethane dimethanesulphonate (EDS) mediated Leydig cell ablation, primary hAd-PSCs, cultured with or without LH, IGF-1, PDGFBB, T3 and ITS supplement, were transplanted into the rat testis and Leydig cell regeneration was monitored via serial measurements of circulating luteinising hormone (LH) and testosterone. Overall, hAd- PSCs had no impact on the recovery of circulating testosterone levels. However, when pre-cultured with the cocktail of hormone/growth factor supplements, the LH spike induced by the removal of testosterone negative feedback was dampened, suggesting the transplanted cells may promote Leydig cell regeneration. Whether these cells differentiate into Leydig cells, or simply provide paracrine support to the regenerating Leydig cells remains to be determined. Although Ad-PSCs may enhance regeneration kinetics, the transplanted cells were undetectable in the testis 5 weeks post transplantation suggesting they may not survive in the context of long term xenogeneic transplantation.

Made available in DSpace on 2014-08-13T14:50:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-04Bitstream added on 2014-08-13T18:00:11Z : No. of bitstreams: 1 000764397.pdf: 797747 bytes, checksum: 6a89fbc6b7371005e020d9f2b9ab35ec (MD5) / A Leucemia Linfocítica Crônica (LLC) é uma neoplasia de linfócitos B maduros, de curso clínico heterogêneo, com alguns pacientes necessitando de tratamento imediato e outros vivendo sem tratamento por décadas. Por essa característica, se faz necessária a identificação de marcadores prognósticos acessíveis e de valor clínico. O objetivo principal foi avaliar retrospectivamente os indicativos prognósticos de progressão da doença (ZAP-70, CD38, ploidia de DNA e Fase S) em pacientes portadores de LLC, frente a sua evolução clínica. Como objetivos secundários, foram comparadas as metodologias de análise de ZAP-70 por citometria de fluxo(CF) ISO Método e Análise em Tubo Único, assim como as técnicas de análise de conteúdo de DNA pelo modelo matemático (ModFit LT™) e estratégia de gating boleano (Infinicyt™). Na cauística foram incluídos 27 pacientes que possuíam disponíveis os dados clínicos e laboratoriais em estudo e que concordaram e preencheram o termo de consentimento livre e esclarecido. A avaliação da situação clínica foi feita uma única vez, e assim como os resultados das expressões de CD 38 e ZAP 70, foi realizada através de revisão de prontuário médico. A análise do conteúdo de DNA por CF foi avaliada pelos dois métodos supracitados, a partir de arquivos do laboratório de citometria de fluxo da instituição. Como resultados, não se observou relação das expressões de ZAP-70 e CD38 com o estado clínico dos pacientes. Em relação à análise de índice de DNA e Fase S, não houve diferença estatística (p=0,69 e p=0,08 respectivamente utilizando-se os softwares ModFit LT™ e Infinicyt™). Também foi encontrado que os dados obtidos na análise de DNA indicam a possibilidade de utilização da Fase S na avaliação prognóstica dessa série de pacientes. Todos os três fatores estudados são considerados prognósticos. Não houve relação dos marcadores estudados com a evolução ... / The Chronic Lymphocytic Leukemia (CLL) is a mature B lymphocytes malignancy, on heterogeneous clinical course, where some patients require immediate treatment and others live for decades without treatment. For this peculiarity, it’s required the identification of prognostic markers and clinical value. The main objective was to retrospectively, evaluate the prognostic indicator of the disease progression (ZAP- 70, CD38, DNA ploidy and S phase) in patients under CLL, facing the clinical evolution. As secondary purposes, methodologies were compared to ZAP- 70 analysis by flow cytometry (FC) ISO Method and Single-Tube Analysis, as well as technical analysis of DNA content by the mathematical model (ModFit LT ™) and boolean gating principle (Infinicyt ™ ). On the registration of the cases observed, there were included 27 patients under the clinical and laboratory data who had available and filled in the term consenting agreement. The evaluation of the clinical situation was made only once, and the results of expressions and CD38 and ZAP 70, were held by reviewing medical records. The analysis of DNA content by CF was evaluated by the above two methods files from a laboratorial flow cytometry institution. As a result, no relation of the expressions of CD38 and ZAP- 70 with the clinical status of the patients was observed. Concerning the analysis of DNA index and S phase, there wasn’t any statistical difference (p = 0.69 and p = 0.08 respectively using the ModFit LT ™ and Infinicyt ™ software ). It was also found that the data obtained in the analysis of DNA indicates the possibility of using the S phase in the prognostic evaluation of those patients. Conclusion: all the three factors studied are considered prognostic. There was no relation of the markers studied to the clinical course of patients. To correlate these data with clinical follow-up of those patients, it is necessary a longer period of time

Resumo: Estudo retrospectivo com a finalidade de avaliar o impacto da introdução de um regime alternativo de condicionamento para transplantes de medula óssea alogênicos com doadores aparentados, totalmente compatíveis, portadores de Leucemia Mielóide Crônica, em Fase Crônica. No período de agosto de 1996 a julho de 2008, foram incluídos 158 pacientes, analisados em dois diferentes momentos: o primeiro inclui os pacientes transplantados antes de 2004, quando 72(45%) deles, foram tratados com a combinação Busulfano e Ciclofosfamida (BUCY). Neste período inicial os dados demonstraram melhores resultados nos pacientes abaixo de 30 anos. No segundo período (após 2004) o regime de condicionamento era orientado em função da idade do receptor, condições clínicas, e pelo histórico individual de comorbidades. Com um total de 86 pacientes, transplantados até o final da análise, 44(28%) deles receberam a combinação BUCY e 42(27%) indivíduos foram alocados para o protocolo envolvendo Busulfano e Fludarabina (FLUBU). As informações do grupo transplantado antes de 2004 mostram forte influência da idade do receptor, acima ou abaixo de 30 anos, na Sobrevida Global (p=0, 005), na Sobrevida Livre de Doença (p=0, 001) e na Mortalidade não Relacionada à Recaída (p=0,01). Outra observação nessa população foi a ocorrência da Doença Enxerto contra o Hospedeiro crônica, aos cinco anos, mais freqüentemente observada entre os pacientes que receberam Células Progenitoras Periféricas (92% vs 49% quando foi utilizada a Medula Óssea como fonte, p= 0, 004). Após 2004 os dois grupos de tratamento foram muito heterogêneos em relação a sua composição etária, com idade mediana superior no grupo que recebeu FLUBU (45 (28 a 54) anos vs. 28 (16 a 51) anos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This is a retrospective study that aims to assess the impact of the introduction of an alternative conditioning regimen in stem celltransplants with allogeneic fully matched related donors, in patients with Chronic Myeloid Leukemia in Chronic Phase. From August 1996 to July 2008 we included 158 patients, tested during two different periods: the first period includes patients transplanted before 2004, when 72 (45%) of them were treated with the combination of Busulfan and Cyclophosphamide (BUCY). These data demonstrated that the outcome was better among patients below the age of 30 years. The second period comprised all patients transplanted after 2004. In this second period the choice of the conditioning regimen was based on recipient age, clinical condition and the individual history of comorbidities . With a total of 86 patients transplanted by the end of the analysis, 44 (28%) of them received the BUCY combination and 42 (27%) patients were allocated to the protocol with Busulfan and Fludarabine (FLUBU). For the group transplanted before 2004 a strong influence of recipient age (above or below 30 years old) on Overall Survival (p = 0.005), Disease-free Survival (p = 0.001) and Mortality Related to Transplantation (p = 0.01) could be demonstrated. Graft Versus Host Disease, at five years, was most frequently observed among patients receiving Peripheral blood Progenitor Cells (92% vs 49%) when compared with the use of Bone Marrow as a stem cell source, p = 0.004). After 2004 the two treatment groups were very heterogeneous in relation to their age structure, with older patients receiving FLUBU (45 (28 to 54) years vs 28 (16 to 51), p < 0.0001). Although we analyzed two distinct age groups , the results were comparable in terms of Overall Survival at 3 years (p = 0.49) in Disease-free Survival at 3 ... (Complete abstract click electronic access below) / Orientador: Belinda Simões / Coorientador: Ana Lúcia Coradazzi / Coorientador: Celso Arrais / Banca: Lígia Niero-Melo / Banca: Júlio Cesar Voltarelli / Mestre

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-01Bitstream added on 2014-06-13T19:29:04Z : No. of bitstreams: 1 duarte_pd_me_botfm.pdf: 611993 bytes, checksum: ccf0323dc58e760455c77b8af2abcf44 (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / Fundação Amaral Carvalho / Leucemia Linfóide Crônica (LLC) é uma neoplasia maligna de linfócitos B maduros com aspecto monomórfico, que se acumulam em tecidos linfóides secundários, medula óssea e sangue periférico. O diagnóstico é realizado com base em achados clínico-morfológicos e imunofenotípicos. Os pacientes com LLC são estratificados conforme o estadiamento clínico para definição de terapêutica; atualmente a descoberta de marcadores prognósticos, como ZAP-70, trouxe novas perspectivas ao tratamento, principalmente no estádio precoce. Os objetivos deste estudo foram: avaliar a eficácia da pesquisa da ZAP-70 em biópsia de medula óssea (BMO) e em inclusão do coágulo, utilizando-se a técnica de imuno-histoquímica; avaliar possíveis correlações deste marcador na BMO com evolução clínica e risco de progressão; correlacionar a imuno-expressão da ZAP- 70 com o arcabouço reticulinico das BMO; avaliar se este arcabouço possui correlação com o prognostico da doença e, por fim, traçar um perfil epidemiológico dos pacientes com LLC atendidos no Hospital das Clinicas da Faculdade de Medicina de Botucatu (FMB). Foram selecionados 153 pacientes com LLC, atendidos no ambulatório do Serviço de Hematologia da FMB-UNESP, de 1980 a 2008, e 9 pacientes assistidos no Serviço de Onco-Hematologia do Hospital Amaral Carvalho-Jaú, no período de 2000 a 2008, com o mesmo diagnóstico, perfazendo 162 casos. Destes, 79 possuíam prontuários que foram revisados, bem como dados morfológicos da BMO e AMO. Foi realizada a pesquisa da ZAP- 70 pela técnica de imuno-histoquímica nas amostras parafinadas de BMO e inclusão do coágulo. Observou-se que 55,7% dos pacientes eram do sexo masculino; 86,1% de etnia branca; a mediana de idade foi 65 anos; relação homem:mulher de 1,2:1. 40,5% dos pacientes tiveram diagnóstico por achado incidental e 73,4%, já com adenopatia secundária à admissão. 17,2%... / Chronic lymphocytic leukemia (CLL) is a malignant neoplasm consisting of mature monomorphic B lymphocytes that accumulate in secondary lymphoid tissues, bone marrow and peripheral blood. Diagnosis is based on clinical, morphological and immunophenotypic findings. CLL patients are categorized according to clinical aspects for treatment management and currently, certain tools, such as prognostic marker ZAP- 70, have determined new treatment perspectives, especially for patients in early stages. The study aimed to evaluate the efficiency of ZAP-70 investigation in bone marrow biopsies (BMBs) and inclusion coagulate using immunohistochemistry; identify possible correlations of this marker in clinical evolution and risk of progression; correlate the immunoexpression of ZAP-70 with BMB reticulin network; evaluate whether this network correlates with disease prognosis and; outline an epidemiological profile of patients with CLL attended in the Clinics Hospital of Botucatu Faculty of Medicine (FMB). The sample consisted of 153 CLL patients attended at hematology outpatient clinic of the FMB-UNESP, from 1980 to 2008, and 9 CLL patients attended at the oncohematology service of the Amaral Carvalho Hospital, Jaú, from 2000 to 2008, totaling 162 cases. In 79 cases, medical records including BMB and bone marrow aspirate (BMA) morphological data were reviewed. Immunohistochemistry for ZAP-70 was conducted on paraffinated samples of BMB and inclusion coagulate. Observation revealed that: 55.7% were male, 86.1% white, with a median age of 65 years-old; 40.5% of cases were diagnosed by incidental findings in routine blood smears; 73.4% were diagnosed with peripheral adenomegaly on admission. 17.2% were diagnosed in the early stages and 15% of cases showed prolonged remission. BMB and BMA morphology revealed that 94.8% of patients presented increased cellularity... (Complete abstract click electronic access below)

Thesis (MTech (Medical Technology))--Cape Technikon, 1996. / Haematological defects observed in clonal deletions of mtDNA and the inhibition of mitochondrial function by benzene and chloramphenicol, suggest a role for mtDNA in the pathogenesis of radiation - induced preleukaemia (MDS). The fact that leukaemia cells contain abnormal mitochondria and abnormally structured mtDNA, makes it reasonable to assume mtDNA mutations could be central to the pathogenesis of both MDS and leukaemia. It was decided to examine MDS patients for the presence of mtDNA length mutations (dimers and cocantameres). Such topological forms have already been reported in the literature in association with human leukaemia. These steric considerations suggest that mtDNA dimers are probably non-functional due to supercoiling. Thus, it was felt that a progressive accumulation of non-functional dimers in the haematopoietic compartment could account for many of the clinical features associated with MDS. Transmission electron microscopy was used to examine haematopoietic mtDNA in the bone marrow of six patients with MDS. Abnormal mtDNA dimer formation was found in all instances. The proportional number of these dimers were found to roughly correlate with the Myeloid/ Erythroid cell ratio in the bone marrow, and it appeared likely that the dimers were generated in the myeloid compartment during early MDS.

Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...

Apoptosis serves as a natural barrier to cancer development, and the resistance to apoptosis represents one of the key capabilities acquired during tumor development or progression. Impairment of the intrinsic apoptotic pathway exemplifies one of the established mechanisms of constitutive or acquired drug resistance. As most of the currently used cytotoxic drugs initiate tumor cell death by direct or indirect triggering of the intrinsic apoptotic pathway, impairment of the intrinsic pathway is associated with therapy failure. Targeting of the death receptors, however, enables induction of apoptosis even in the chemotherapy resistant cancer cells. TRAIL is a death ligand belonging to the TNFα superfamily that specifically kills tumor cells while sparing healthy tissues. Much enthusiasm has been generated for TRAIL as a highly promising targeted anti-cancer agent. However, many primary tumors have been shown to be TRAIL resistant. In attempt to overcome such an intrinsic TRAIL resistance a wide array of agents have been shown to sensitize tumor cells to TRAIL. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. In this study we analyzed the sensitivity of diverse hematologic malignancies to TRAIL-induced apoptosis and measured the...

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os bufodienolÃdeos sÃo esterÃides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da famÃlia Bufonidae utilizado na medicina chinesa. Os bufodienolÃdeos possuem grande variedade de atividades biolÃgicas, incluindo atividades antineoplÃsicas. Em relaÃÃo à atividade antitumoral, os bufodienolÃdeos tem demonstrado inibir o crescimento de vÃrias linhagens de cÃlulas cancerÃgenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotÃxico e genetÃxico de seis bufodienolÃdeos em seis linhagens tumorais humanos, trÃs linhagens murinas normais e cÃlulas mononucleadas do sangue perifÃrico (CMSP) humano. Todos os seis bufodienolÃdeos foram citotÃxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 ÂM. Os bufodienolÃdeos testados nÃo apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de aÃÃo envolvido. Uma sequÃncia de experimentos in vitro foram realizados utilizando-se a linhagem leucÃmica HL-60. As cÃlulas foram tratadas em diferentes concentraÃÃes da amostra hellebrigenina (0,03, 0,06 e 0,12 ÂM) por 24 horas. A viabilidade das cÃlulas (nÃmero de cÃlulas viÃveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o nÃmero de cÃlulas reduziu a partir da menor concentraÃÃo (0,03 ÂM) testada e a porcentagem de cÃlulas com membrana integra reduziu a partir da concentraÃÃo 0,06 ÂM. A anÃlise morfolÃgica por citometria de fluxo revelou aumento de cÃlulas com padrÃo apoptÃtico a partir da concentraÃÃo de 0,06 ÂM. Jà a anÃlise do conteÃdo nuclear, nos mostrou aumento de fragmentaÃÃo de DNA sub-G1 indicativo de apoptose e acÃmulo de cÃlulas na fase G2/M a partir das concentraÃÃes de 0,03 e 0,06 ÂM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalizaÃÃo da fosfatidilserina, despolarizaÃÃo mitocondrial, ativaÃÃo da caspase iniciadora 8 e consequente ativaÃÃo das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotÃxico por induÃÃo de mais de uma via apoptÃtica. Hellebrigenina nÃo foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberraÃÃes cromossÃmicas em CMSP. Por meio dos estudos de docking molecular foi possÃvel predizer a ligaÃÃo entre hellebrigenina e topoisomeraseII&#945; humana, resultado compatÃvel com a possÃvel inibiÃÃo dessa enzima. De forma geral, os resultados apontam o potencial citotÃxico do bufodienolÃdeo hellebrigenina / Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frogâs skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolÃdeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 ÂM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 ÂM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 ÂM) tested and the percentage of cells with reduced membrane integrity from 0.06 ÂM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 ÂM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 ÂM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseII&#945;, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin