6,742 research outputs found
FUCA1 is induced by wild-type p53 and expressed at different levels in thyroid cancers depending on p53 status
Fucose residues of cell surface glycans, which play important roles in growth, invasion and metastasis, are added by fucosyltransferases (FUTs) and removed by α-L-fucosidases (FUCAs). By the differential display method, we isolated a 3' non-coding region of α-L-fucosidase-1 (FUCA1) (a gene coding for the lysosomal fucosidase-1 enzyme) as a wild-type p53-inducible gene: 18S and 20S FUCA1 mRNA species were induced in Saos-2 cells transfected with a temperature-sensitive p53 mutant at the permissive temperature. By microarray analyses of thyroid cancer biopsy samples, FUCA1 RNA expression levels were found to be lower in anaplastic thyroid cancer samples (ATCs), while they were higher in papillary thyroid cancer samples (PTCs) and in normal thyroid tissues. Since most ATCs were reported to carry the mutated form of p53, while PTCs carry mostly the wild-type form of p53, it is likely that FUCA1 expression levels are regulated, at least in part, by the p53 status in thyroid cancers. In order to better understand the role played by FUCA genes in thyroid tumorigenesis, we examined the clonogenic potential in vitro of thyroid cell lines transfected with either FUCA1 or FUCA2 (the latter gene coding for a secreted, non-lysosomal enzyme). We found that α-L-fucosidases did not suppress grossly cell growth. Contrary to what we observed with the expression of FUCA1, the FUT8 expression levels were found high in ATCsbut lower in PTCs and normal thyroid tissues. Taken together, these results suggest the possibility that the higher fucose levels on cell surface glycans of aggressive ATCs, compared to those of less aggressive PTCs, may be at least in part responsible for the more aggressive and metastatic phenotype of ATCs compared to PTCs, as the expression levels of FUCA1 and FUT8 were inversely related in these two types of cancers. Fucose residues of cell surface glycans, which play important roles in growth, invasion and metastasis, are added by fucosyltransferases (FUTs) and removed by α-L-fucosidases (FUCAs). By the differential display method, we isolated a 3' non-coding region of α-L-fucosidase-1 (FUCA1) (a gene coding for the lysosomal fucosidase-1 enzyme) as a wild-type p53-inducible gene: 18S and 20S FUCA1 mRNA species were induced in Saos-2 cells transfected with a temperature-sensitive p53 mutant at the permissive temperature. By microarray analyses of thyroid cancer biopsy samples, FUCA1 RNA expression levels were found to be lower in anaplastic thyroid cancer samples (ATCs), while they were higher in papillary thyroid cancer samples (PTCs) and in normal thyroid tissues. Since most ATCs were reported to carry the mutated form of p53, while PTCs carry mostly the wild-type form of p53, it is likely that FUCA1 expression levels are regulated, at least in part, by the p53 status in thyroid cancers. In order to better understand the role played by FUCA genes in thyroid tumorigenesis, we examined the clonogenic potential in vitro of thyroid cell lines transfected with either FUCA1 or FUCA2 (the latter gene coding for a secreted, non-lysosomal enzyme). We found that α-L-fucosidases did not suppress grossly cell growth. Contrary to what we observed with the expression of FUCA1, the FUT8 expression levels were found high in ATCs but lower in PTCs and normal thyroid tissues. Taken together, these results suggest the possibility that the higher fucose levels on cell surface glycans of aggressive ATCs, compared to those of less aggressive PTCs, may be at least in part responsible for the more aggressive and metastatic phenotype of ATCs compared to PTCs, as the expression levels of FUCA1 and FUT8 were inversely related in these two types of cancers
In vitro assessment of the oncogenic potential of novel MTC-associated germline RET mutations
Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2013Thyroid carcinoma is the most frequent endocrine malignancy. Medullary thyroid carcinoma (MTC) is one of the thyroid tumors and represents about 5 to 8% of all cases. It arises from C cells (parafollicular cells), in contrast with the other thyroid tumors that arise from follicular cells. MTC can occur either in a sporadic or hereditary form, being the latter associated to multiple endocrine neoplastic type 2 (MEN2) and familial MTC (FMTC). RET (rearrangement during transfection) encodes a membrane receptor tyrosine kinase that, when mutated, can originate MTC. The aim of this project was to evaluate, through in vitro functional experimental approaches, the oncogenic potential of two newly identified RET germinal variants detected in two MTC patients from our institution: RET C515T (RET missense alteration TGCTGG at codon 515 - Cys515Trp) and RET T636M (RET missense alteration ACGATG at codon 636 - Thr636Met. By expressing the novel RET variants in non-transformed NIH 3T3 mouse fibroblasts, and comparing their effect with wild-type RET, we were able to observe an increase of cell growth and proliferation, loss of contact inhibition and an increase in cell migration rate. All these are common characteristics of tumoral cells, indicating that these new RET variants hold some relevant transforming potential. This transforming potential is, nevertheless, low when compared to that of RET MEN2A- causing mutation, C634R, which is consistent with the mild phenotype, observed in the patients of this study, characterized by late onset and low aggressiveness. The functional characterization of these two new variants not only contributes to the understanding of the molecular mechanisms behind the development of MTC in these families, but also provides useful information to optimize the diagnostic and clinical management of hereditary MTC.Após um quarto de século de grandes avanços, a investigação na área da oncologia tornou-se rica e complexa, mostrando assim que o cancro é uma doença que envolve mudanças dinâmicas no genoma. Muitas evidências indicam que a tumorigénese em humanos é um processo com várias etapas, que reflete não só as alterações no genoma, mas também que levam a uma transformação progressiva de células normais em células derivadas altamente malignas. Carcinoma é qualquer tumor maligno que provenha de células epiteliais, e que pode originar metástases. Existem inúmeros tipos de carcinoma, sendo os carcinomas endócrinos um deles, abrangendo uma enorme variedade, tanto a nÃvel do fenótipo como do genótipo. Os carcinomas da tiróide são o tipo de carcinomas endócrinos mais frequentes, e podem ser divididos em dois grandes grupos: os carcinomas da tiróide que provêm de células foliculares e os carcinomas da tiróide que provêm de células parafoliculares. Os carcinomas medulares da tiróide (CMT) representam cerca de 5 a 8% dos carcinomas da tiróide. Estes desenvolvem-se a partir de células C (células parafoliculares), enquanto os outros tipos de tumores desenvolvem-se a partir das células foliculares. Os carcinomas medulares podem ocorrer de forma esporádica (cerca de 75% do total dos casos) ou hereditária (cerca de 25%), sendo esta última associada a sÃndrome de neoplasia endócrina múltipla de tipo 2 (NEM2), subdividida em NEM2A e NEM2B, e CMT familiar (CMTF). Os carcinomas medulares da tiróide possuem uma enorme variedade, que podem ir de tumores com bom prognóstico, a carcinomas altamentes agressivos, com uma elevada taxa de mortalidade. Mutações no proto oncogene RET (Rearranjado durante a transfeção) estão na origem do desenvolvimento do carcinoma medular da tiróide. Este proto oncogene codifica para um recetor membranar com atividade de tirosina-cinase e está envolvido em várias vias de sinalização que controlam processos como a sobrevivência e proliferação celular, migração e angiogénese, entre outros. Os casos de CMTF estão geralmente associados a mutações pontuais a nÃvel germinal no gene RET que podem ser mutações que afetam a região extracelular ou o domÃnio de tirosina-cinase deste recetor. Nos casos onde não existe qualquer mutação na proteÃna RET, as cisteÃnas extracelulares formam ligações de bissulfito intramoleculares, estabilizando o recetor na sua conformação monomérica. O domÃnio das cisteÃnas é altamente conservado e tem um papel importante na manutenção da estrutura secundária e terciária do domÃnio extracelular do RET, através destas ligações de bissulfito. Quando ocorre uma mutação que leva à perda de um resÃduo de cisteÃna esta torna-se livre para formar uma ligação intermolecular com outro receptor mutado, ocorrendo assim a dimerização e fosforilação da proteÃna RET, tornando-a ativa, mesmo na ausência de ligandos. Assim, mutações ativadoras neste gene conduzem a uma ativação não-regulada destas vias, levando assim ao surgimento do fenótipo tumoral. Este trabalho tem como objectivo a realização de estudos funcionais in vitro para duas variantes germinais do proto-oncogene RET, não descritas na literatura, associadas a dois casos distintos de carcinoma medular da tiróide (referidos como paciente A e paciente B), tentando assim avaliar seu o grau de patogenicidade. A paciente A, sem evidência clÃnica na famÃlia de sÃndrome NEM2A/2B ou CMTF, foi submetida, aos 79 anos, a tiroidectomia total devido a um CMT unifocal. O teste genético ao gene RET identificou uma variante heterozigótica no codão 636 (ACGATG) no exão 11, que resulta na substituição do aminoácido treonina por metionina na região extracelular da proteÃna RET, designada por RET T636M. Apenas um membro da famÃlia desta paciente foi testado, a filha de 50 anos que apresentava bócio multinodular, tendo-se verificado ser negativa para esta variante. A paciente B tinha 60 anos à data do diagnóstico, tendo sido submetida a tiroidectomia total devido a CMT multifocal. O teste genético ao gene RET revelou uma variante germinal presente em homozigotia no exão 8, codão 515 (TGCTGG), que leva à substituição de uma cisteÃna por um triptofano no domÃnio extracelular da proteÃna RET, designada por RET C515T. O facto dos pais desta paciente serem primos em primeiro grau levou provavelmente a esta variante se apresentar em homozigotia. Esta variante foi também encontrada, apesar de em heterozigotia, em oito de dez familiares estudados. Nenhum destes apresentou qualquer sintoma sugestivo de CMT ou NEM2, exceto uma prima que também veio a desenvolver CMT aos 63 anos. O potencial oncogénico de uma variante pode ser estimado através de diversos ensaios funcionais in vitro, avaliando-se assim sua capacidade de transformação induzida pela mutação comparativamente com o controlo normal, sem mutação. Para estes estudos tem-se em conta as caracterÃsticas especÃficas das células tumorais, que as distinguem das células normais, não transformadas. Para tal, utiliza-se normalmente a linha celular de fibroblastos de ratinho NIH 3T3, comercialmente disponÃvel, sendo analisado o efeito da expressão exógena do péptido mutante nestas células. Antes de se dar inicio aos ensaios funcionais, efetuou-se a análise funcional in silico para prever o efeito patogénico de cada variante. Os ensaios funcionais incluÃram o ensaio de formação de focos, migração, crescimento e ciclo celular. A expressão exógena do gene RET nas células transfetadas foi monitorizada e quantificada pela técnica de western-blott. O resultado destes estudos funcionais revelou que as novas variantes do gene RET, RET C515T e RET T636M, em comparação com o efeito induzido pelo RET selvagem, levavam um aumento do crescimento e proliferação celular, à perda de inibição por contacto e um aumento da migração celular. Todas estas caracterÃsticas são comuns à s células tumorais, levantando-se assim a hipótese de que estas novas variantes podem possuir um potencial oncogénico relevante. O potencial transformante destas novas variantes foi, no entanto, inferior ao observado para a mutação RET C634R, causadora do fenótipo NEM2A e utilizada como controlo positivo neste estudo. Com base nos estudos funcionais in vitro, estas novas variantes apresentam um baixo potencial oncogénico, resultados estes consistentes com o fenótipo pouco agressivo observado em ambas as pacientes deste estudo. Apesar da variante RET T636M não envolver diretamente um resÃduo de cisteÃna, poderá induzir a uma alteração conformacional na proteÃna que poderá afetar uma ligação intramolecular localizada na vizinhança deste codão. Isto poderá ter um impacto na estrutura da proteÃna RET, bem como na sua ativação. Curiosamente, esta variante está localizada na vizinhança de certos codões que, quando mutados, estão associados a fenótipos mais agressivos. Por outro lado, a variante RET C515W envolve diretamente um resÃduo de cisteÃna, podendo a sua perda ter consequências diretas na ativação do recetor RET. IndivÃduos com mutações pouco patogénicas no gene RET podem necessitar de uma segunda mutação germinal ou somática para desenvolver um fenótipo mais agressivo, que leve à expressão clÃnica da doença. Esta hipótese foi levantada para a paciente B que possuÃa a variante em homozigotia, mas foi descartada quando se descobriu que uma prima, que possuÃa a mesma variante embora em heterozigotia, veio igualmente a desenvolver CMT, sugerindo que apenas um alelo mutado é suficiente para a manifestação da doença. Fatores externos como o estilo de vida de cada indivÃduo ou do próprio ambiente em que este se encontra inserido, adicionado ao desenvolvimento tardio da doença, bem como à sua baixa penetrância, são fatores que provavelmente contribuem para as variações na expressão clÃnica da doença, sendo assim difÃcil de perceber o efeito da mutação só por si. Apesar de serem necessários estudos funcionais adicionais, a caracterização destas duas novas variantes contribui, não só, para o conhecimento dos mecanismos moleculares inerentes ao desenvolvimento de CMT nestas famÃlias, como também para a optimização do diagnóstico e o aconselhamento clinico de CMT hereditário
A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues
An investigation into the role of autoimmunity in vitiligo
Vitiligo is an acquired depigmenting disorder characterised by the loss of functional melanocytes from the cutaneous epidermis. A role for autoimmunity is supported by the presence of circulating antibodies and T lymphocytes which react against melanocyte antigens in patients with vitiligo. The identification and characterisation of these autoantigens will improve understanding of the immune response in vitiligo, and may allow development of better therapies and diagnostic tools. Candidate immunoregulatory genes may predispose to vitiligo. However, this study failed to find an association between vitiligo and a polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4), although the polymorphism increases the likelihood of autoimmune endocrinopathy patients developing vitiligo. The epitopes on melanocyte-specific antigens tyrosinase and Pmel17 which are recognised by antibodies in vitiligo patient sera were identified by molecular mapping. Multiple regions of tyrosinase and at least two domains on PmeI17 were identified as B cell epitopes. Sequence analysis revealed that the tyrosinase epitopes are likely to be cross-reactive with tyrosinase-related proteins but that the antibody response to Pmel17 is distinct. Antibody reactivity to a melanocyte protein, MelanA, targeted by a cellular immune response in vitiligo and melanoma was investigated by immunoblotting and radioimmunoassay. No MelanA-specific antibodies were isolated suggesting that either it is not a target of the humoral immune response in vitiligo, or that antibody reactivity was not detectable by the methods used. To identify novel vitiligo autoantigens, a melanoma cDNA expression library was constructed in a phage-display cloning system and immunoscreened with vitiligo patient IgG. Several possible autoantigens were enriched by this technique, including proteins previously characterised as autoantigens in other disorders. Additionally, humoral reactivity was identified to a protein with a possible role in pigmentation, the melanin-concentrating hormone receptor 1 (MCHR1). MCHR1-specific antibodies were detected in 16.4% (9/55) of vitiligo patients but not in other diseases or healthy control subjects. The study demonstrates the usefulness of phage-display for further autoantigen identification in vitiligo
Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine
Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h-and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking
Genomic analysis of the function of the transcription factor gata3 during development of the Mammalian inner ear
We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKB beta, a dramatic increase in Akt1/PKB alpha protein and relocation of Akt1/PKB alpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome
Receptor Gone Rogue: Investigating Localization of a Cancerous Thyroid Hormone Receptor
Thyroid hormone receptors play a crucial role in regulating differentiation, growth and development in response to thyroid hormone, and mutations in these receptors can have severe medical consequences ranging from endocrine dysfunction to cancer. Tumors of patients with hepatocellular carcinoma (HCC) display a high incidence of mutant thyroid hormone receptors (TRs), and one such mutant is TRα1 (K74E, A264V). The binding partners and gene targets of this mutant have been characterized, but the role of intracellular localization in the pathogenesis of TRα1 (K74E, A264V) has not yet been determined. Here, it was observed that the mutant receptor has a tendency to aggregate when transfected into HeLa (human) cells. Fluorescence microscopy was used to determine the extent of this aggregation. The data showed that the TR mutant displays a significantly higher frequency of nuclear and cytosolic aggregates. Furthermore, it induces a significantly higher amount of nuclear and cytosolic aggregates in the wild type TR when coexpressed in the cell, and this effect increases with increasing amounts of transfected mutant-containing plasmid. These results highlight a potential dominant-negative of TRα1 (K74E, A264V) effect as it pertains to localization and offer a new layer of understanding to the altered activity of this mutant TR during the development of cancer
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