1,721,002 research outputs found
Oncogene c-KIT: promoter region and downstream effectors as novel therapeutic targets
Full text linkAn attractive anticancer strategy deals with the impairment of the functions of selected genes relevant for cell proliferation. Among them an interesting target is represented by KIT. c-KIT - V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog - is a tyrosine kinase receptor involved in the regulation of cell survival and proliferation as well as in progression of some tumors. Its expression has been confirmed to be crucial for leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor (MCT), is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model.
In this work we explore two strategies to suppress the cell growth activity of this oncogene.
The first one was based on the downregulation of gene expression by induction of non-canonical DNA structures at promotorial level. Indeed, in this region of the human oncogene KIT two guanine-rich sequences (KIT1_man and KIT2_man) were identified. Here, we characterized the canine KIT promoter region in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results demonstrate that the conformational features of the canine KIT1 sequence are substantially shared with the human one whereas an altered distribution among several folded conformations characterizes the two isoforms identified in dog-derived KIT2 sequences.
These structural diversities identified KIT1 and KIT2 as two potential distinct targets, which means that G-4 ligands can be designed to preferentially bind human/dog sequences. Thus, an “in house” library of compounds belonging to different chemical scaffolds (i.e., anthracene, anthracenedione, indole, quinoline, isoquinoline, fluorenone and heterocyclic diamidines derivatives) was screened for the recognition of these particular folding. Three promising G4-ligands, belonging to anthracenedione (Bal1,5 – Bapl2,6) and anthracene (Bis1,8) families, were selected and different techniques (Fluorescent Intercalator Displacement, Fluorescence melting studies, CD titrations, Surface Plasmon Resonance and Polymerase Stop Assay) were applied to fully dissect their interaction with KIT-related sequences. Our results showed that Bal1,5 is an efficient G-4 binder towards all the tested sequences, while Bapl2,6 and Bis1,8 preferentially recognize KIT2 sequences. Interestingly, a parallel level of cytotoxicity and suppression of KIT production was observed in cells treated with Bal1,5.
In the second approach, downstream effectors of c-KIT pathway were considered as putative therapeutic targets. In particular we focused on a cytoplasmic tyrosine kinase FER.
During the third year of my PhD I spent 7 months at Centre de Recherche en Cancérologie de Marseille (France) in the Dr P. Dubreuil’s team. In this period we worked to confirm a potential tumor suppressor role of FER. For this purpose mouse embryonic fibroblasts MEFs wild type and knockout for fer immortalized with SV40 were used. Removing fer, an uncontrolled growth was observed for KO MEFs in the contact inhibition test. However, at the moment a detailed description of the molecular events was not designed. Indeed no defined differences between molecular mechanisms, related to protein complexes involved in cell adhesion mechanisms, were observed between wt and KO MEFs.
In conclusion, present results suggest that G-quadruplex structures at the promotorial level represent a promising therapeutic target in order to prevent oncogene KIT transcription. As far as FER is concerned, further studies are necessary to better define its possible tumor-suppressor role
Olha pra mim! inclusão/exclusão e violências nas memórias de estudantes de um curso de Pedagogia: Caroline Kern ; orientadora, Ana Maria Borges de Sousa, coorientadora, Silvia Zanatta Da Ros
Full text linkTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Educação, Programa de Pós-graduação em Educação, Florianópolis, 2014.Esta tese pretendeu evidenciar determinados contextos que revelam violênciasvivenciadas no decorrer da escolarização (ensino fundamentale médio) por estudantes de um curso de pedagogia e que apontam paraprocessos de inclusão/exclusão escolar. Considerando essa questão inicial,foi importante problematizar os contextos de violências escolaresrelatados por esses estudantes, estabelecendo a relação entre o espaço intramurosda escola e a sociedade; discutir as questões centrais que se entrelaçamnos discursos enunciados dos sujeitos de pesquisa e entre eles eaqueles constantes nos dispositivos jurídicos; contribuir para maior aprofundamentoe abrangência dos estudos que envolvem inclusão/exclusão eviolências, cotejando os dispositivos jurídicos que fundamentam as políticasnacionais de educação inclusiva com os relatos escritos em memoriaisdescritivos da vida escolar dos referidos estudantes. A pesquisa, decunho qualitativo, pretendeu visibilizar as vozes dos sujeitos de pesquisa,constituindo um diálogo em que seus relatos fossem escutados/ouvidos/sentidos em acordo com a posição alteritária bakhtiniana. Os memoriaisda vida escolar, fonte documental, compuseram o corpus de análise juntoa outros relatos obtidos no exercício das atividades docentes da pesquisadorano ensino superior e cotejados com os dispositivos jurídicos(Estatuto da Criança e do Adolescente, Lei n. 8069 de 13 de julho de1990, e Constituição Federal da República Federativa/1988) que amparamos pressupostos teóricos das políticas públicas de educação inclusivano país. Tais documentos orientaram o tema geral desta tese, qual seja:?Agredidos? e ?agressores? no espaço intramuros da escola não se opõemum ao outro, mas constituem um mesmo processo, no qual as violênciase a inclusão/exclusão se fazem conteúdo na/da escola. Conteúdo que étambém constituído e constituidor de contextos sociais mais amplos (relaçõessociais). O estudo partiu do pressuposto de que as violências compõema tríade com a inclusão/exclusão, lugar onde a questão central desteestudo se coloca. Pretendeu-se visibilizar as contradições escolares queforjam violências e que se distanciam e se aproximam das diretrizes edos dispositivos da denominada educação inclusiva, dispositivos essesque, paradoxalmente, constroem processos de exclusão. Como o conceitode violências encontra diferentes significados em acordo com o tempohistórico, as condições sociais e a cultura, as violências foram aqui compreendidascomo algo que fundamenta e representa as relações existentesnesta sociedade, em que, ao fundamentá-las, são base necessária aosprocessos de inclusão/exclusão escolar. A problematização das chamadasviolências na ?temporalidade? presente é mediada pela compreensão deque o sujeito cartesiano, à parte das relações sociais, é ou não violentoem si/para si. A violência estrutural do âmago da sociedade capitalista,enquanto produto social, como expressão dos pilares necessários à sustentaçãodo todo social e fundamentando-o dilata esses pilares. Comoreferencial de base, assumiram-se os pressupostos do materialismo históricodialético e algumas aproximações com a teoria bakhtiniana, pormeio de conceitos que auxiliaram a compreensão dos processos escolaresem que inclusão/exclusão e violências estiveram evidenciadas.Esta tesis pretendió evidenciar determinados contextos que revelan violenciasvividas en el correr de la escolaridad (enseñanza fundamental ymedia) por estudiantes de un curso de graduación en pedagogía y queapuntan a procesos de inclusión/exclusión escolar. Considerando esacuestión inicial, fue importante problematizar los contextos de violenciaescolares relatados por esos estudiantes, estableciendo la relación entreel espacio intramuros escuela y la sociedad; discutir las cuestionescentrales que se entrelazan en los discursos mencionados de los sujetosde investigación y entre ellos y aquellos constantes en los dispositivosjurídicos; contribuir para una mayor profundidad y abarcadura de losestudios que envuelven inclusión/exclusión y violencias, cotejando losdispositivos jurídicos que fundamentan las políticas nacionales de educacióninclusiva con los relatos escritos en memoriales descriptivos de lavida escolar de los referidos estudiantes. La investigación de cuño cualitativopretendió visibilizar las voces de los sujetos de investigación constituyendoun diálogo en que sus relatos fueran escuchados/oídos/sentidosen acuerdo con la posición alteritaria bakhtiniana. Los memoriales dela vida escolar, fuente documental, compusieron el ?corpus? de análisisjunto a otros relatos obtenidos en el ejercicio de las actividades docentesde la investigadora en la enseñanza superior y cotejados con los dispositivosjurídicos (Estatuto del Niño y del Adolescente Ley n.8069 del 13de julio de 1990, Constitución Federal de la República Federativa/1988)que amparan las hipotéticas teorías de las políticas públicas de educacióninclusiva en el país. Tales documentos orientaron el tema general de estatesis, cual sea: ?Agredidos y agresores? en el espacio intramuros de la escuelano se oponen uno a otro, pero constituyen un mismo proceso, en elcual las violencias y la inclusión/exclusión se convierten en el contenidoen/de la escuela. Contenido que es también constituido y constituidor decontextos sociales más amplios (relaciones sociales). El estudio partió dela suposición de que las violencias componen la tríada con la inclusión/exclusión, lugar donde la cuestión central de este estudio se coloca. Sepretendió visibilizar las contradicciones escolares que crean violenciasy que se distancian y se aproximan de las directrices y de los dispositivosde la denominada educación inclusiva, esos dispositivos que contradictoriamenteconstruyen procesos de exclusión. Como el conceptode violencias encuentra diferentes significados concordando con el tiempohistórico, las condiciones sociales y la cultura, las violencias fueronaquí entendidas como algo que fundamenta y representa las relacionesexistentes en esta sociedad, en que, al fundamentarlas, son base necesariaa los procesos de inclusión/exclusión escolar. La problemática delas llamadas violencias en la ?temporalidad? presente y mediada por lacomprensión de que el sujeto cartesiano, además de las relaciones sociales,es o no violento en si/para si. La violencia estructural de la esenciade la sociedad capitalista siendo ella producto social como expresión delos pilares necesarios para la sustentación del todo social y fundamentándolo,dilata esos pilares. Como referencia se asumen las hipótesis delmaterialismo histórico dialéctico y algunas aproximaciones con la teoríabakhtiniana, por medio de los conceptos que auxiliaron la comprensiónde los procesos escolares en que inclusión/exclusión y violencias estuvieronevidenciadas
G-rich sequences within proto-oncogene KIT promoter region as targets for anticancer therapy.
No full textIn last decades the role of non-canonical DNA structures as switching on/off mechanism of gene transcription has been extensively investigated. In particular, downregulation of gene expression by induction of G-quadruplex DNA structures at promotorial level is an attractive anticancer strategy. In humans, two guanine-rich sequences (h_kit1 and h_kit2) were identified in the promotorial region of the proto-oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, lung carcinomas, which are often associated to c-kit mis-regulation. Here we screeneed libraries of G-quadruplex binders to identify novel hits highly selective for the G-quadruplexes of KIT promoter. To provide the biological rationale for the molecular and cellular mechanisms of action of this novel therapeutic approach, a full characterization of target selectivity, DNA complex formation and cellular effects has been performed.
To ultimately provide the required knowledge to convert the identified hits into new species-specific targeted drugs, we searched for a robust translational model too. Interestingly, the most common skin cancer in domestic dog, mast cell tumor, is also linked to a mutation and/or to an over-expression of c-kit. Moreover, a general conserved promotorial sequence between the two species has been confirmed, thus supporting dog as an excellent animal model. In particular, the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified and, in comparison with the human counterpart, both of them showed an altered distribution among several folded conformations. Here we compared our results on human and canine cells, in order to provide a more comprehensive description of the physiological pathways supported by c-kit mis-regulation in carcinogenesis and to highlight the similarities/differences between the two species
Evaluation of epigenetic mechanisms regulating HOXD10, FGFR2 and ITIH5 gene expression in canine B-cell lymphoma: an in vitro approach.
No full textIntroduction. Despite B-cell Lymphoma (BCL) is the most common canine hematological cancer, there is still a lack of knowledge on molecular events contributing to its development and progression. Aim of this work was to investigate the epigenetic regulation of HOXD10, FGFR2 and ITIH5 genes, whose promoters were recently shown to be methylated, by genome-wide methylation profiling, in canine diffuse large BCL. To understand the role of methylation in silencing aforementioned genes, the CLBL-1 cell line was treated with two hypomethylating drugs (HDs). Owing to complexity of epigenetic mechanisms, histone deacetylase inhibitors (HDACis), alone or in combination with HDs, were also tested.
Materials and methods. CLBL-1 cells were incubated with two HDs (azacytidine and decitabine), alone or in combination with HDACis (valproic acid, trichostatin and vorinostat), at concentrations corresponding to their IC50 and IC20 values (Alamar blue test), respectively. Then, gene methylation status and mRNA levels were measured using methyl sensitive PCR (MSP) and quantitative Real Time PCR (qPCR).
Results. MSP showed an overall decrease of gene methylation status following the incubation with both HDs; meantime, qPCR highlighted a reversion of gene expression, confirming a methylation-dependent gene silencing mechanism. Interestingly, a higher pattern of gene re-expression was observed following the exposure to HDs combined with HDACis (and, mostly, with valproic acid). Moreover, HDACis alone increased the expression of FGFR2, demonstrating the important contribution of histone deacetylation, above methylation, in the regulation of this gene
Targeting canine c-KIT promoter by a candidate DNA G-quadruplex ligand
No full textIntroduction
G-quadruplexes (G-quad) are DNA secondary structures formed by stacked G-tetrads frequently located in telomeres and promoter regions of proto-oncogenes. Recently, two G-rich sequences, canine kit1 (d_kit1) and kit2 (d_kit2), folding into G-quad, have been identified in canine c-KIT promoter. Accordingly, the aim of the present study was to test a possible small molecule inhibitor known to decrease significantly target gene expression by stabilizing KIT1 and KIT2 G-quad structures.
Materials and Methods
The constitutive gene expression of c-KIT and other proto-oncogenes (BCL2, VEGFα, VEGFR2, KRAS, TERT) mRNA was measured in canine mast cell tumor (MCT) cell line C2 by quantitative RT-PCR (qPCR). Therefore, the G-quad ligand 50% inhibitory concentration (IC50) was determined by using the Alamar Blue cytotoxicity test. Finally, its time- and dose-dependent transcriptional effects upon c-KIT and other target genes were evaluated by using qPCR.
Results
In C2 cell line, target genes were shown to be constitutively expressed and measurable up to 96 hours of culture. The IC50 value of G-quad ligand was shown to be 1,37 μM and, at lower cytotoxic concentration, it significantly down-regulated c-KIT mRNA levels (after 24h hours of incubation). A similar effect, although of low intensity, was noticed for BCL2.
Conclusions
The G-quad candidate ligand seems to be a promising candidate for targeting KIT-dependent tumors such as MCT. However, such an assumption needs to be confirmed with further molecular studies. Comparative studies about the efficacy of this and other G-quad ligands in blocking human kit G-quadruplex are currently underway.
Supporting grants: University of Padua (CPDA114388
A G-quadruplex binding compound down-regulates KIT, BCL2 and PDGFRβ expression in human gastric carcinoma cell line
No full textINTRODUCTION
Proto-oncogene KIT, a tyrosine kinase receptor, is involved in the initiation and progress of human tumors, e.g. gastrointestinal stromal tumors, melanoma, mastocytosis and acute leukemia. The stabilization of DNA secondary structures called G-quadruplexes, located in promoter regions of KIT and other proto-oncogenes, could result in a block of gene transcription.
MATERIALS AND METHODS
An “in house” library of compounds were screened for the recognition of two KIT G-quadruplex regions. Three positive hits were tested in a human gastric carcinoma cell line (HGC27). The 50% inhibitory concentration (IC50) was measured by using the Alamar Blue cytotoxicity test. The effects of these ligands upon mRNA of KIT and other proto-oncogenes, PDGFRβ and BCL2 were evaluated after 6, 12 and 24 hours of incubation and by using two concentrations for each ligand.
RESULTS
Compound 1 was the most effective ligand in stabilizing the two KIT G-quadruplex regions. The IC50 value was obtained for compounds 1 and 2, while it was not determinable for the third ligand (>10 μM). Overall, compound 1 was the most effective in inhibiting proto-oncogene transcription; it lowered KIT and PDGFRβ mRNA expression in a dose-dependent manner, while a time- and concentration-dependents down-regulation was observed for BCL2.
CONCLUSIONS
Compound 1 might represent a promising candidate for targeting KIT-dependent neoplasia, and such an assumption needs to be confirmed with further molecular studies. Comparative studies about the efficacy of these ligands in another human cell line are currently underway.
REFERENCES
Bejugam M., Sewitz, S., Shirude P. S., Rodriguez R., Shahid R. & Balasubramanian S. (2007). Trisusbstituted isoalloxazines as a new class of G-quadruplex binding ligands: small molecule regulation of c-kit oncogene expression. Journal of the American Chemical Society, 129(43), 12926-12927.
Gunaratnam M., Swank S., Haider S. M., Galeasa K., Reszka A. P., Beltran M., Cuenca F., Fletcher J. A. & Neidle S. (2009). Targeting human gastrointestinal stromal tumor cells with a quadruplex-binding small molecule. Journal of Medicinal Chemistry, 52(12), 3774-3783
Characterization of the promoter region of the proto-oncogene c-kit in canine mast cell tumour
No full textG-quadruplex structures at telomeric and promotorial sequences represent promising chemotherapeutic targets. Small molecules can bind and stabilize already formed G-quadruplexes or can induce their formation from G-rich sequences. In order to select efficient and selective G-quadruplex ligands, the conformational equilibria of the nucleic acid need to be characterized.
In this work we are interested in the promotorial sequence of c-kit, a proto-oncogene which over expression or gain of function mutations characterizes human cancers, such as gastrointestinal stromal tumors (GIST), melanoma and mastocytosis. In humans, this region contains two G-rich sequences, c-kit1 and c-kit2. The G-quadruplex structures assumed by these two sequences have been extensively studied by means of X-ray crystallography and NMR. The stabilization of these secondary structures resulted in a suppression of gene transcription. In the domestic dog, c-kit over expression or mutations have been found in cutaneous mast cell tumor (MCT), representing the most common (7%-21%) canine skin tumor.. Therefore, we expect that dog could be a robust model to have a better insight into c-kit abnormalities in cancer and into the role of potential c-kit G-quadruplex ligand in anticancer chemotherapy. To support this, the canine upstream promotorial sequence was cloned and sequenced in both healthy and MCT-suffering dogs. Our results identified two putative G-quadruplex sequences in the canine promotorial region. Thus, we compared them with the human ones. In particular, large attention was devoted to clarify the conformational equilibria occurring in physiologically relevant conditions by means of spectroscopic and electrophoretic techniques.
This work represents the required preliminary step for a better understanding of MCT biology, progression and treatment as well as to assess dogs as a translational model for the many c-kit related human tumours
Characterization of the promoter region of the proto-oncogene c-Kit in canine mast cell tumour
No full textIn medicinal chemistry, G-quadruplex (G4) represents a promising chemotherapeutic target. The unveiling of telomerase role in cell cycle progression and the defi nition of its mechanism of action
prompted the search of small molecule able to block it by promoting G4 formation at the telomere ends. More recently, the role of conformational equilibria of G-rich sequences as modulator of gene expression has been assessed and opened new perspectives. In particular, several oncogene promoters
were found to contain G-rich sequences and have been considered as targets for anticancer therapy.
Among them, we were interested in c-kit, due to its widespread relevance in tumorigenesis and tumor maintenance. Interestingly, two distinct G-rich sequences were identifi ed in c-kit promoter and both are now structurally characterized in their G4 conformation.
The knowledge of the target is defi nitely a great advantage to the identifi cation and/or optimization of new ligands directed towards these genomic portions. However, we also need solid models to assess the pharmacological effi ciency of potential new drugs. In this context dog can represent a remarkable
translational animal for human cancer.
Indeed, mast cell tumour (MCT) is the most common (7% to 21%) cutaneous skin tumour of dogs and, interestingly, the MCT aggressive behaviour, (which results in poor outcome) is often
characterized either by an overexpression or a mutational status of c-Kit. All this makes c-kit an important target for dog chemotherapy. Furthermore, c-kit mutations occurring in dog MCTs are similar to those found in human cancers, such as gastrointestinal stromal tumors (GIST), melanoma
and mastocytosis. Therefore, canine MCT could represent a proper disease model to evaluate the functional consequences of c-kit abnormalities in cancer and the role of c-kit inhibitors in antitumor chemotherapy. To validate such an assumption, we started a detailed characterization of the promoter region of
c-kit in dogs. The sequence of canine upstream promotorial sequence was cloned and sequenced in both healthy and MCT-suffering dogs. Then, the canine sequences were compared to the human ones. In particular, large attention was devoted to clarify the conformational equilibria occurring in physiologically relevant conditions. This work represents the required preliminary step for a better understanding of MCT biology,
progression and treatment as well as to export this knowledge in the many c-Kit related human tumours
As políticas para a educação especial integrada em Cabo Verde: um estudo sobre os processos de escolarização e profissionalização de pessoas cegas
Full text linkDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Educação. Programa de Pós-Graduação em Educação.A década de 90 do século XX foi marcada fundamentalmente pela disseminação de relatórios, declarações e documentos oficiais de governos e organismos internacionais que resultaram, em parte, das discussões realizadas no âmbito das conferências promovidas pelas agências multilaterais dentre as quais se destaca a UNESCO. As proposições apresentadas sinalizaram para a "Educação para Todos", cujas manifestações, na particularidade dos objetivos desta pesquisa, são as escolas inclusivas. Considerando que os processos de exclusão continuam se materializando não só de diversos modos e estratégias como também em diversos setores e espaços sociais, analisa-se, nesta dissertação, as políticas para a Educação Especial Integrada em Cabo Verde, arquipélago pertencente ao Continente Africano. Especificamente, investigam-se os processos de escolarização e profissionalização de pessoas cegas. Os dados e a análise realizada evidenciam, entre vários aspectos, que as políticas para a Educação Especial Integrada, do ponto de vista dos organismos governamentais, foram implementadas no arquipélago recentemente e, no momento, os trabalhos desenvolvidos voltam-se mais para a garantia da escolarização obrigatória. Assim, a questão da profissionalização figura como uma preocupação, mas não como prioridade, uma vez que a realidade caboverdiana não é diferente daquela dos demais países do Terceiro Mundo no que diz respeito aos altos índices de desemprego. Ao se analisar particularmente os processos de escolarização/profissionalização de pessoas cegas, constatou-se que estes se deram predominantemente no âmbito do voluntarismo e do pioneirismo de pessoas que, de vítimas da exclusão, se tornaram autores e atores decisivos de ações inclusivas na educação e no trabalho. Portanto, essas ações e práticas individuais anteciparam-se não só em relação às proposições teórico-legais como também à implementação da Educação Especial Integrada em Cabo Verde. Como decorrência disto, a investigação realizada demonstra, por um lado, que os referidos processos ocorreram por conta de iniciativas particulares e, por outro, evidencia que essas iniciativas/práticas resultaram na criação de ONGs que, somadas com as ingerências de organismos multilaterais e outros fatores, fizeram com que as autoridades governamentais propusessem políticas públicas como resposta às demandas e pressões da sociedade civil e de grupos específicos
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