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The promyelocytic leukaemia protein (PML) is a tumour suppressor initially identified in acute promyelocytic leukaemia (APL). In APL, PML and the retinoic acid receptor alpha (RARalpha) genes are fused as a consequence of the translocation t(15;17). The product of the chimeric gene is the oncogenic PML-RARalpha protein. The PML gene encodes multiple nuclear and cytoplasmic isoforms. PML nuclear isoforms (nPML) are the main components of the PML nuclear bodies (PML-NBs), sub-nuclear structures involved in the modulation of essential cellular players including the tumour suppressor p53. Nuclear PML has been intensively studied, while, the role of cytoplasmic PML remains poorly understood. Increasing evidence indicates that PML could bear cytoplasmic functions in both physiological and pathological settings. This study aims to gain more insights into the function of PML and PML-RARalpha cytoplasmic pool of proteins. Recently, two missense mutations resulting in truncated PML cytoplasmic protein (Mut PML) have been identified in aggressive APL cases. We found that Mut PML alters the structure and the function of the PML-NB mainly through the cytoplasmic relocation of nPML. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive and apoptotic functions. In the cytoplasm, Mut PML interacts and stabilizes PML-RARalpha, thus potentiating its block of RA-induced transcription and differentiation. A mutant of PML-RARalpha (Delta2) accumulating in the cytoplasm is able to inhibit RA-dependent transcription and differentiation, suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we found that Delta2 expression blocks G-CSF-dependent myeloid differentiation and causes partial transformation of primary haematopoietic progenitor cells.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O esqueleto quinona foi identificado como um importante grupo farmacofÃrico para atividade citotÃxica de vÃrios compostos utilizados na clÃnica, constituindo ainda uma das maiores classes de agentes anticÃncer. A β-lapachona à uma das quinonas mais estudadas nos Ãltimos anos. Suas aplicaÃÃes mais importantes estÃo relacionadas Ãs aÃÃes contra vÃrias cÃlulas tumorais. Seu derivado, nor-β-lapachona (composto 1), tem atividade anticÃncer similar. Assim, o objetivo desse trabalho foi avaliar o mecanismo de aÃÃo envolvido na citotoxicidade do composto 1 e de seu derivado nitrofenilamino (composto 2) em cÃlulas leucÃmicas HL-60. Inicialmente foi investigado o efeito desses compostos na viabilidade de cÃlulas HL-60 apÃs 24 horas de incubaÃÃo, mostrando CI50 de 2,92 e 0,48 μM para os compostos 1 e 2, respectivamente. Acerca da seletividade celular, o composto 1 mostrou forte seletividade para cÃlulas tumorais, enquanto que o seu derivado foi apenas parcialmente seletivo. Estudos feitos em cÃlulas HL-60 indicaram que o composto 2 induz morte celular por apoptose como mostrado pelas mudanÃas morfolÃgicas, fragmentaÃÃo do DNA, despolarizaÃÃo da membrana mitocondrial e externalizaÃÃo da fosfatidilserina. Ambos compostos aumentaram a geraÃÃo de espÃcies reativas de oxigÃnio (EROs). Nossos resultados sugerem que a introduÃÃo do radical nitrofenilamino na posiÃÃo 3 do anel furano aumenta a citotoxicidade da nor--lapachona em cÃlulas HL-60. Esses achados apontam para o potencial dessas quinonas sintÃticas como modelo para a produÃÃo de novos compostos com propriedades anticÃncer. / The quinone moiety has been identified as an important pharmacophoric group for cytotoxic activity of several compounds clinically used, constituting just one of the major classes of anticancer agents. β-lapachone is one of the most studied quinones in the last years. Its most important applications are related to its action against several cancer cells. Its derivative, nor-β-lapachone (compound 1), has similar anticancer activity. Thus, the aim of this work was to evaluate the mechanism of action involved in nor--lapachone and its nitrophenylamino derivative (compound 2) cytotoxicity in a leukemia cells model of HL-60 cell line. Initially it was investigated the effect of both the compounds on HL-60 cells viability after 24 hours of incubation, showing IC50 values of 2.92 and 0.48 μM to compounds 1 and 2, respectively. Considering the selectivity, compound 1 showed strong selectivity to cancer cells, while its derivative was only partially selective. Studies performed in HL-60 cells, after 24 hours, indicated that compound 2 induces cell death by apoptosis as showed by morphological changes, DNA fragmentation, mitochondrial membrane depolarization and phosphatidylserine externalization. Both tested compounds increased generation of reactive oxygen species (ROS). Our results suggest that a nitrophenylamino radical introduction at position 3 of the furane ring enhances the cytotoxicity of nor--lapachone in HL-60 cell line. These findings highlight the potential of these synthetic quinones as prototypes molecules to produce new compounds with anticancer properties.

Angiogênese é o termo utilizado para descrever o crescimento de novos vasos sanguíneos a partir dos já existentes. Vários estudos demonstraram a densidade microvascular (DMV) como um fator prognóstico nas leucemias, em particular, leucemia promielocítica aguda (LPA). Este subtipo de leucemia corresponde a cerca de 20 a 25% das leucemias mielóides agudas nos países latino-americanos e apresenta características clínicas, morfológicas e biológicas peculiares. A halofuginona (HF), originalmente descrita como um agente antifúngico apresenta capacidade de inibir o crescimento tumoral e formação de vasos em modelos animais de tumores sólidos. Estudos realizados por nosso grupo demonstraram que a HF inibiu a secreção do VEGF e a proliferação de linhagens celulares de LPA. Desta forma, o presente trabalho teve por objetivo determinar o potencial antiproliferativo e antiangiogênico da HF em um modelo experimental in vivo da LPA e avaliar os mecanismos subjacentes a sua ação. Primeiramente, a análise do ciclo celular em células NB4 tratadas com HF apresentou significativa diminuição da proliferação celular (2,093 ± 0,304 vs. 41,21 ± 3,25), juntamente com um aumento significativo da apoptose (12,53 ± 1,53 vs. 21,95 ± 0,79; p=0,0007). Por meio da técnica de Real Time Array foi possível identificar dois grupos de genes associados a apoptose celular diferencialmente expressos em células tratadas com HF: TNF, TNFRSF9, TNFTSF10B, CD40, FAS, CASP10, CASP8 e CASP3, sugerindo que a HF induz a via extrínseca de apoptose. A análise in vivo da HF foi realizada em camundongos NOD/SCID previamente irradiados e transplantados com células leucêmicas PML-RAR murinas. Camundongos tratados com HF, por 21 dias após o transplante não apresentaram remissão molecular, determinada pela amplificação do gene PML-RARA por PCR, porém foi observada menor infiltração leucêmica em relação aos camundongos não tratados (Leucócitos: 4,2 ± 3,89 vs. 20,6 ± 21,9; p<0.0001); Hemoglobina: 12,0 ± 1,40 vs. 9,6 ± 1,67; p<0.0001; e Plaquetas: 932,0 ± 122,5 vs. 552,0 ± 83,2; p<0.001 respectivamente) e um menor peso relativo do baço (0,006 vs. 0,012, p=0,0415). Ademais, a contagem diferencial e imunofenotipagem da medula óssea evidenciaram menor porcentagem de células mielóides imaturas (16,88 ± 6,27 vs. 44,06 ± 27,06). A HF também foi capaz de inibir a fosforilação de SMAD2 e consequentemente bloquear a via do TGF- em células NB4. No entanto, animais leucêmicos apresentaram menor nível sérico de TGF- em relação aos saudáveis e tratados (475,58 vs. 1.378,45/1.146,82 pg/mL; p<0,0001), sugerindo que o blasto leucêmico produz esta citocina e a diminuição de células leucêmicas resultou em diminuição dos níveis séricos de TGF-. A HF não aumentou a sobrevida dos animais leucêmicos e a elevação das enzimas hepáticas sugeriu que o tratamento foi hepatotóxico. Por fim, com relação à angiogênese, a análise da expressão gênica mostrou que o tratamento com HF inibiu a expressão de VEGF e EGF e o estudo por imunohistoquímica de seções da medula óssea evidenciou menor expressão VEGF (30 vs. 80%, p=0,0227), porém não houve diminuição da DMV. O conjunto desses resultados mostrou que a angiogênese é um importante alvo terapêutico na LPA, e que apesar da toxicidade, a HF apresenta potencial antileucêmico, tanto por conta de seus efeitos antiproliferativos e próapoptóticos, quanto por sua capacidade de inibir a produção de fatores próangiogênicos. / Angiogenesis is the term used to describe the growth of new blood vessels from the existing ones. Several studies have demonstrated the microvascular density (MVD) as a prognostic factor in leukemia, particularly acute promyelocytic leukemia (APL). This subtype of leukemia corresponds to 20-25% of acute myeloid leukemia in Latin America and presents clinical, morphological and biological peculiar characteristics. The halofuginone (HF) originally described as an antifungal agent has ability to inhibit tumor growth and vessel formation in animal models of solid tumors. Our group demonstrated that HF inhibits the VEGF secretion and cell proliferation in APL cell lineages. Thus, this study aimed to determine the antiproliferative and antiangiogenic potential of HF in an experimental model of APL in vivo and evaluate the mechanisms underlying its action. First, the cell cycle analysis in NB4 cells treated with HF showed a significant decrease in cell proliferation (2.093 ± 0.304 vs. 41.21 ± 3.25), along with a significant increase in apoptosis (12.53 ± 1.53 vs . 21.95 ± 0.79, p = 0.0007). Through Real Time Array it was possible to identify two groups of apoptosis associated genes differentially expressed in HF treated cells: TNF, TNFRSF9, TNFTSF10B, CD40, FAS, CASP10, CASP8 and CASP3, suggesting that HF induces apoptosis by extrinsic pathway. In vivo analysis of HF was performed in irradiated NOD/SCID mice transplanted with murine PML-RAR leukemic cells. Mice treated with HF for 21 days after transplantation showed no molecular remission as determined by amplification of PML-RARA gene by PCR, however minor leukemic infiltration was observed compared to untreated mice (leukocytes: 4.2 ± 3.89 vs . 20.6 ± 21.9, p <0.0001), hemoglobin: 12.0 ± 1.40 vs. 9.6 ± 1.67, p <0.0001, and Platelets: 932.0 ± 122.5 vs. 552.0 ± 83.2, p <0.001 respectively) and a lower relative weight of spleen (0.006 vs. 0.012, p = 0.0415). Furthermore, the differential count and immunophenotyping of bone marrow showed a lower percentage of immature myeloid cells (16.88 ± 6.27 vs. 44.06 ± 27.06). The HF was also able to inhibit the SMAD2 phosphorylation and consequently block the TGF- pathway in NB4 cells. However, leukemic animals presented lower serum TGF- compared to the healthy and treated (475.58 vs. 1378.45/1146.82 pg / mL, p <0.0001), suggesting that the leukemic blasts produces this cytokines and the decrease in leukemic cells resulted in decreased serum levels of TGF-. The HF did not increase the survival of leukemic animals and elevated liver enzymes suggested that the treatment was hepatotoxic. Finally, regarding angiogenesis, gene expression analysis showed that the HF treatment inhibited the expression of VEGF and EGF and the study by immunohistochemistry of sections of bone marrow showed less VEGF expression (30 vs. 80%, p = 0 0227), but there was no decrease in the MVD. Taken together, these results showed that angiogenesis is an important therapeutic target in APL, and despite the toxicity, HF has antileukemic potential, for both the antiproliferative and proapoptotic effects, and also for its ability to inhibit the production of proangiogenic factors.

Ping Zhang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 131-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

Thesis (M.Sc.). / Written for the Faculty of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Angiogênese é o termo utilizado para descrever o crescimento de novos vasos sanguíneos a partir dos já existentes. Vários estudos demonstraram a densidade microvascular (DMV) como um fator prognóstico nas leucemias, em particular, leucemia promielocítica aguda (LPA). Este subtipo de leucemia corresponde a cerca de 20 a 25% das leucemias mielóides agudas nos países latino-americanos e apresenta características clínicas, morfológicas e biológicas peculiares. A halofuginona (HF), originalmente descrita como um agente antifúngico apresenta capacidade de inibir o crescimento tumoral e formação de vasos em modelos animais de tumores sólidos. Estudos realizados por nosso grupo demonstraram que a HF inibiu a secreção do VEGF e a proliferação de linhagens celulares de LPA. Desta forma, o presente trabalho teve por objetivo determinar o potencial antiproliferativo e antiangiogênico da HF em um modelo experimental in vivo da LPA e avaliar os mecanismos subjacentes a sua ação. Primeiramente, a análise do ciclo celular em células NB4 tratadas com HF apresentou significativa diminuição da proliferação celular (2,093 ± 0,304 vs. 41,21 ± 3,25), juntamente com um aumento significativo da apoptose (12,53 ± 1,53 vs. 21,95 ± 0,79; p=0,0007). Por meio da técnica de Real Time Array foi possível identificar dois grupos de genes associados a apoptose celular diferencialmente expressos em células tratadas com HF: TNF, TNFRSF9, TNFTSF10B, CD40, FAS, CASP10, CASP8 e CASP3, sugerindo que a HF induz a via extrínseca de apoptose. A análise in vivo da HF foi realizada em camundongos NOD/SCID previamente irradiados e transplantados com células leucêmicas PML-RAR murinas. Camundongos tratados com HF, por 21 dias após o transplante não apresentaram remissão molecular, determinada pela amplificação do gene PML-RARA por PCR, porém foi observada menor infiltração leucêmica em relação aos camundongos não tratados (Leucócitos: 4,2 ± 3,89 vs. 20,6 ± 21,9; p<0.0001); Hemoglobina: 12,0 ± 1,40 vs. 9,6 ± 1,67; p<0.0001; e Plaquetas: 932,0 ± 122,5 vs. 552,0 ± 83,2; p<0.001 respectivamente) e um menor peso relativo do baço (0,006 vs. 0,012, p=0,0415). Ademais, a contagem diferencial e imunofenotipagem da medula óssea evidenciaram menor porcentagem de células mielóides imaturas (16,88 ± 6,27 vs. 44,06 ± 27,06). A HF também foi capaz de inibir a fosforilação de SMAD2 e consequentemente bloquear a via do TGF- em células NB4. No entanto, animais leucêmicos apresentaram menor nível sérico de TGF- em relação aos saudáveis e tratados (475,58 vs. 1.378,45/1.146,82 pg/mL; p<0,0001), sugerindo que o blasto leucêmico produz esta citocina e a diminuição de células leucêmicas resultou em diminuição dos níveis séricos de TGF-. A HF não aumentou a sobrevida dos animais leucêmicos e a elevação das enzimas hepáticas sugeriu que o tratamento foi hepatotóxico. Por fim, com relação à angiogênese, a análise da expressão gênica mostrou que o tratamento com HF inibiu a expressão de VEGF e EGF e o estudo por imunohistoquímica de seções da medula óssea evidenciou menor expressão VEGF (30 vs. 80%, p=0,0227), porém não houve diminuição da DMV. O conjunto desses resultados mostrou que a angiogênese é um importante alvo terapêutico na LPA, e que apesar da toxicidade, a HF apresenta potencial antileucêmico, tanto por conta de seus efeitos antiproliferativos e próapoptóticos, quanto por sua capacidade de inibir a produção de fatores próangiogênicos. / Angiogenesis is the term used to describe the growth of new blood vessels from the existing ones. Several studies have demonstrated the microvascular density (MVD) as a prognostic factor in leukemia, particularly acute promyelocytic leukemia (APL). This subtype of leukemia corresponds to 20-25% of acute myeloid leukemia in Latin America and presents clinical, morphological and biological peculiar characteristics. The halofuginone (HF) originally described as an antifungal agent has ability to inhibit tumor growth and vessel formation in animal models of solid tumors. Our group demonstrated that HF inhibits the VEGF secretion and cell proliferation in APL cell lineages. Thus, this study aimed to determine the antiproliferative and antiangiogenic potential of HF in an experimental model of APL in vivo and evaluate the mechanisms underlying its action. First, the cell cycle analysis in NB4 cells treated with HF showed a significant decrease in cell proliferation (2.093 ± 0.304 vs. 41.21 ± 3.25), along with a significant increase in apoptosis (12.53 ± 1.53 vs . 21.95 ± 0.79, p = 0.0007). Through Real Time Array it was possible to identify two groups of apoptosis associated genes differentially expressed in HF treated cells: TNF, TNFRSF9, TNFTSF10B, CD40, FAS, CASP10, CASP8 and CASP3, suggesting that HF induces apoptosis by extrinsic pathway. In vivo analysis of HF was performed in irradiated NOD/SCID mice transplanted with murine PML-RAR leukemic cells. Mice treated with HF for 21 days after transplantation showed no molecular remission as determined by amplification of PML-RARA gene by PCR, however minor leukemic infiltration was observed compared to untreated mice (leukocytes: 4.2 ± 3.89 vs . 20.6 ± 21.9, p <0.0001), hemoglobin: 12.0 ± 1.40 vs. 9.6 ± 1.67, p <0.0001, and Platelets: 932.0 ± 122.5 vs. 552.0 ± 83.2, p <0.001 respectively) and a lower relative weight of spleen (0.006 vs. 0.012, p = 0.0415). Furthermore, the differential count and immunophenotyping of bone marrow showed a lower percentage of immature myeloid cells (16.88 ± 6.27 vs. 44.06 ± 27.06). The HF was also able to inhibit the SMAD2 phosphorylation and consequently block the TGF- pathway in NB4 cells. However, leukemic animals presented lower serum TGF- compared to the healthy and treated (475.58 vs. 1378.45/1146.82 pg / mL, p <0.0001), suggesting that the leukemic blasts produces this cytokines and the decrease in leukemic cells resulted in decreased serum levels of TGF-. The HF did not increase the survival of leukemic animals and elevated liver enzymes suggested that the treatment was hepatotoxic. Finally, regarding angiogenesis, gene expression analysis showed that the HF treatment inhibited the expression of VEGF and EGF and the study by immunohistochemistry of sections of bone marrow showed less VEGF expression (30 vs. 80%, p = 0 0227), but there was no decrease in the MVD. Taken together, these results showed that angiogenesis is an important therapeutic target in APL, and despite the toxicity, HF has antileukemic potential, for both the antiproliferative and proapoptotic effects, and also for its ability to inhibit the production of proangiogenic factors.

Acute promyelocytic leukemia (APL) is a clinically distinct and rare type of acute myeloid leukemia and represents an oncologic emergency. Even rarer is the incidence of APL in pregnancy with less than 60 cases described in the literature. A 33-year-old pregnant female at 34 week gestation presented to hospital with reports of abdominal pain. On admission she was found to have acute onset pancytopenia with a WBC count of 1.2, Hemoglobin of 9.7g/dl, and platelet count of 26000. Initial history, exam, and investigations including a peripheral smear, coagulation panel, liver function, vitamin b12 and folate levels did not reveal possible etiology of pancytopenia. Given worsening pancytopenia, bone marrow biopsy was done which showed 58% promyelocytes and 11% blasts with numerous Auer rods present. Cytogenetics showed abnormal female karyotype with t(15:17) and FISH analysis revealed PML/RARA fusion in 76.5% of analyzed cells. The above findings were diagnostic of APL. After multidisciplinary discussion with high risk obstetrics physician, it was decided to immediately induce labor for immediate initiation of treatment of APL. She had a prolonged labor requiring aggressive blood product support and initiation of All trans retinoic acid (ATRA) before delivery given concerns of coagulopathy. Induction treatment with Arsenic trioxide (ATO) was started the day after her delivery. Repeat bone marrow biopsy on day 24 showed complete morphologic remission. Shortly thereafter, she started cycle 1 of consolidation with ATRA and arsenic trioxide. APL is characterized by a translocation between chromosome 15 and 17. Coagulopathy is a pathognomonic feature of this leukemia and often the reason for high mortality in early course of disease. APL when treated with ATRA and ATO, has excellent remission rate and 99% overall survival at 2 years. APL in pregnancy is associated with increased risk of preterm delivery, perinatal mortality, and miscarriage. Following pregnancy, there is an increased risk of bleeding, infection, or placental abruption. ATRA, one of the pillars around which treatment of APL revolves, is highly teratogenic during the first trimester and has low risk later in pregnancy. Treatment is directed by the trimester of pregnancy. Termination of pregnancy or treatment with single agent conventional chemotherapy is preferred in the first trimester whereas treatment with ATRA prior to delivery and use of chemotherapy after delivery is the preferred approach in the 2nd and 3rd trimester. This case is an example of individualized approach with a multidisciplinary team need in the setting of scarce data.