L’inhibition, par p16INK4a, de la progression du cycle cellulaire est considérée comme liée à un arrêt de la progression en phase G1 du à l’inhibition de l’activité de CDK4/6. Nous montrons que l’expression ectopique de p16INK4a dans trois lignées cellulaires malignes, p16-/- et pRb+/+, issues de tissus différents, provoque un allongement de la durée de la phase S et du cycle cellulaire total. L’ensemble de nos travaux sur p16INK4a sauvage et son mutant p16G101W indique que p16INK4a induit un allongement de la phase S i) indépendamment de l’origine tissulaire des cellules analysées et ii) en partie lié aux conséquences de l’inhibition de l’activité de CDK4/6 et peut-être des MAP-kinases. Dans sa localisation nucléaire, p16INK4a interviendrait dans la régulation du cycle cellulaire indépendamment de sa liaison à CDK4. L’expression de CDK1 est inhibée par p16INK4a dans les trois lignées analysées. Dans les cellules MCF7 et U87, cette inhibition est post-transcriptionnelle, médiée par la région 3’non traduite de l’ARNm de CDK1, et est associée à une modification de l’équilibre d’expression, tissu-spécifique, des microARN régulant potentiellement CDK1. Nous démontrons que CDK1 est une cible de miR- 410 et miR-650 induits par p16INK4a et le rôle de l’inhibition de la voie pRb/E2F par p16INK4a dans l’induction de miR-410. Ainsi, p16INK4a régule l’expression des gènes à différents niveaux en modifiant l’équilibre fonctionnel des facteurs de transcription et, en conséquence, des miARN. / The inhibition of cell cycle progression by p16INK4a, have been considered to result from arrest in G1 phase due to inhibition of CDK4/6 activity. We show that ectopic expression of p16INK4a in three human malignant cell lines, p16-/- and pRb +/ +, derived from different tissues, led to an increase in the length of S phase and of the entire cell cycle. Our studies using wild-type p16INK4a and p16G101W mutant indicated that p16INK4a induces a lengthening of S phase i) independently of tissue origins and ii) partly linked to the inhibition of CDK4/6 activity and possibly MAP-kinases. In the nucleus, p16INK4a may intervene in regulating the cell cycle independently of binding to CDK4. The expression of CDK1 is inhibited by p16INK4a in three cell lines analyzed. In MCF7 and U87cells, this inhibition is post-transcriptional, mediated by the 3' non translated region of CDK1 mRNA, and is associated with changes in the balance of the expression of microRNAs, which regulate potentially CDK1. We demonstrate that CDK1 is a target of miR-410 and miR-650, both induced by p16INK4a and the role of the inhibition of pRb/E2F pathway by p16INK4a in the induction of miR-410. Thus, p16INK4a regulates gene expression at different levels by modifying the functional balance of transcription factors and, consequently, the microRNAs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cervical cancer is the second most common cancer among women worldwide and one of the most prevalent female cancers in Brazil. In premalignant and malignant lesions of the uterine cervix the p16INK4a protein, which participates in cell cycle control, exhibits a dramatic increase in its expression possibly due to the presence of human papillomavirus (HPV) oncoproteins. Two polymorphisms in the p16 gene, p16INK4a 540C>G and p16 580C> T, are located in the 3' untranslated region (3'UTR), which is involved in the post-transcriptional regulation of gene expression. The aim of this study was to investigate possible associations between the p16 500C>G and p16 540C>T polymorphisms and the developing of cervical neoplasias and/or lesion severity, considering the expression levels of the p16INK4a protein in cervical lesions, and some classic risk factors for cervical cancer, including HPV infection. A total of 567 women residents in Rio de Janeiro was selected, 319 with abnormal cervical cytology results (case group), and 248 with no previous history of cervical cytological changes (comparison group). Peripheral blood samples from all participants were used for molecular analysis of the polymorphisms p16 500C>G and p16 540C>T, which was performed by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) using the restriction enzymes MspI and HaeIII, respectively. The expression of p16 in 137 biopsies of women belonging to the group of cases was assessed by immunohistochemistry. The detection of HPV DNA in cervical cells was performed in all samples of the comparison group and in 194 samples of the case group by a PCR-based method using two primers pairs, MY09/MY11 and GP05+/GP06+. TheINK4a two study groups are in Hardy-Weinberg equilibrium. The genotype distributions for p16 500C>G and p16 540C>T and the distributions of haplotype combinations in the two groups were not statistically different. The analysis of the subgroup HSIL+cancer (cases with high-grade intraepithelial lesion or invasive carcinoma) compared to the subgroup HSIL (cases with low-grade squamous intraepithelial lesions) revealed a significant difference in the distribution of haplotype combinations (p = 0.036) and marginal differences in the genotype distributions for p16 500C>G (p = 0.071) and p16 540C>T (p = 0.051). The p16 540G allele, in heterozygosis or homozygosis (OR = 1.91, 95% CI = 1.08-3.37), and the haplotype combination p16 500C-540C 500G-540C (OR = 2.34, 95% CI = 1.202-4.555) showed to be associated with the severity of cervical lesions. On the other hand the p16 580T/T genotype (OR = 0.25, 95% CI = 0.08-0.79), and the haplotype combination p16 500C-540T 500C-540T (OR=0.27, 95% CI = 0.088-0.827) exhibited a protective effect against the development of higher grade cervical lesions. Interaction analyses between the p16 polymorphisms and the p16 protein expression or the HPV infection were compromised by the reduced number of analyzed samples. No interaction was observed between the studied polymorphisms and the classical risk factor for cervical cancer. Our data point out the importance of the p16 gene polymorphisms as severity markers in cervical neoplasia. INK4aINK4aINK4a. / O câncer de colo do útero é o segundo carcinoma mais frequente em mulheres no mundo e um dos cânceres femininos mais incidentes no Brasil. Em lesões pré-malignas e malignas do colo uterino, a proteína p16INK4a, que participa do controle do ciclo celular, apresenta um aumento considerável de sua expressão, devido possivelmente à presença de oncoproteínas do papilomavírus humano (HPV). Dois polimorfismos no gene p16INK4a, p16 500C>G e p16 540C>T, estão localizados na região 3 não traduzida (3UTR), que está envolvida na regulação pós-transcricional da expressão gênica. O objetivo deste estudo foi avaliar possíveis associações entre os polimorfismos p16 500C>G e p16 540C>T e o desenvolvimento de neoplasias cervicais e/ou a severidade das lesões, considerando os níveis de expressão da proteína p16INK4a nas lesões cervicais e certos fatores de risco clássicos para o câncer cervical, incluindo a infecção pelo HPV. Para isso, foram selecionadas 567 mulheres residentes no Rio de Janeiro, 319 com citologia cervical alterada (grupo de casos) e 248 sem história prévia de alteração citológica do colo uterino (grupo de comparação). Amostras de sangue periférico de todas as participantes foram utilizadas na análise molecular dos polimorfismos p16 500C>G e p16 540C>T através da técnica de PCR-RFLP (reação em cadeia da polimerase - polimorfismo de comprimento de fragmento de restrição), usando as enzimas de restrição MspI e HaeIII, respectivamente. A expressão da proteína p16INK4a em 137 biópsias de mulheres pertencentes ao grupo de casos foi avaliada por imunohistoquímica. A detecção de DNA do HPV em células cervicais foi feita em todas as amostras do grupo de comparação e em 194 amostras do grupo de casos pela técnica de PCR, usando dois pares de oligonucleotídeos, MY09/MY11 e GP05+/GP06+. Os dois grupos de estudo se encontram em equilíbrio de Hardy-Weinberg. As distribuições genotípicas para p16 500C>G e p16 540C>T e as distribuições de combinações haplotípicas nos dois grupos não apresentaram diferenças significativas. A análise do subgrupo HSIL+câncer (casos com lesão intraepitelial de alto grau ou carcinoma invasivo) em comparação com o subgrupo LSIL (casos com lesão intraepitelial de baixo grau) revelou diferença significativa entre as distribuições das combinações haplotípicas (p = 0,036) e diferenças marginais entre as distribuições genotípicas para p16 500C>G (p = 0,071) e p16 540C>T (p = 0,051). O alelo p16 540G, em heterozigose ou homozigose (OR = 1,91, IC 95% = 1,08-3,37), e a combinação haplotípica p16 500C-540C 500G-540C (OR = 2,34, IC 95% = 1,202-4,555) mostraram-se associados com a severidade da lesões cervicais. Já o genótipo p16 540T/T (OR = 0,25, IC 95% = 0,08-0,79), e a combinação haplotípica p16 500C-540T 500C-540T (OR = 0,27, IC 95% = 0,088-0,827) exibiram papel protetor contra o desenvolvimento de lesões mais severas. As análises de interação entre os polimorfismos de p16INK4a e a expressão de p16 ou a infecção pelo HPV foram comprometidas pelo número reduzido de amostras analisadas. Não se observou qualquer interação entre os polimorfismos estudados e os fatores de risco clássicos para o câncer de colo uterino. Nossos resultados apontam para a importância dos polimorfismos do gene p16INK4a como marcadores de severidade da neoplasia cervical.

Submitted by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-29T15:10:17Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Livia Gusmao.pdf: 1628245 bytes, checksum: 42849e5f87bd042f59660a228ded7164 (MD5) / Approved for entry into archive by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-29T15:10:28Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Livia Gusmao.pdf: 1628245 bytes, checksum: 42849e5f87bd042f59660a228ded7164 (MD5) / Made available in DSpace on 2017-09-29T15:10:28Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Livia Gusmao.pdf: 1628245 bytes, checksum: 42849e5f87bd042f59660a228ded7164 (MD5) Previous issue date: 2013 / Hospital Icaraí / O líquen escleroso vulvar está envolvido em uma das vias da carcinogênse da vulva ligada à neoplasia intraepitelial vulvar diferenciada. A metilação da região promotora do DNA é a principal alteração epigenética pela qual um gene é inativado em seres humanos. A metilação do gene P16INK4a, que é um supressor de tumor e atua como inibidor da cinase dependente da ciclina, tem sido descrita como um evento precoce na carcinogênese vulvar. A metilação do gene TIMP-2, que atua como regulador de metaloproteinases, tem sido descrito como marcador de matriz extracelular. Esta pesquisa tem como objetivo estudar a presença da metilação dos genes P16INK4a e TIMP-2 no líquen escleroso vulvar e avaliar a associação das variáveis idade, doença de tireóide, tabagismo, uso de hormônio e prurido vulvar com a metilação dos referidos genes. Trata-se de um estudo transversal, onde foram analisadas 32 amostras obtidas por biópsia de pacientes com líquen escleroso vulvar. As amostras foram submetidas à extração do DNA por meio da técnica do fenol:clorofórmio e à avaliação da metilação dos genes P16INK4a e TIMP-2 pela modificação química do DNA pelo método do bissulfito. O DNA modificado foi submetido à PCR e a visualização do produto pelo gel de poliacrilamida. O estudo da associação de cada uma das variáveis com a metilação de ambos genes não mostrou significado estatístico. Notou-se que 39% (11/28) das amostras exibiram metilação somente para o gene TIMP-2, e nenhuma para o gene P16INK4a isoladamente, enquanto 32% (9/28) apresentaram metilação em ambos genes de forma simultânea. A análise da associação da metilação entre ambos genes mostrou significado estatístico (p=0,0292). Esses resultados sugerem que a associação da metilação entre os genes P16INK4a e TIMP-2 possa promover instabilidade genômica, podendo funcionar como marcador na evolução da doença. Estudos futuros sobre alterações moleculares da matriz extracelular, que por precederem as alterações morfológicas, talvez possam funcionar como sinalizador, individualizando as pacientes com maior risco de evolução do líquen escleroso vulvar para a NIV diferenciada e/ou câncer de vulva / The vulvar lichen sclerosus is involved in one of the pathways of vulvar carcinogenesis linked to differentiated vulvar intraepithelial neoplasia. The methylation of the promoter region of the DNA is the main epigenetic modification in humans in which a gene is inactivated. Methylation of P16INK4a gene, which is a tumor suppressor and acts as an inhibitor of cyclin-dependent kinase, has been described as an early event in carcinogenesis vulva. Methylation of TIMP-2 gene, which acts as a regulator of metalloproteinases, has been described as a marker of extracellular matrix. This research aims to study the presence of methylation of the P16INK4a gene and TIMP-2 in vulvar lichen sclerosus and evaluate the association of age, thyroid disease, smoking, hormone use and vulvar itching with methylation of these genes. It is a cross-sectional study, which analyzed 32 samples obtained by biopsy from patients with vulvar lichen sclerosus. The samples were subjected to DNA extraction by using the technique of phenol: chloroform and evaluation of methylation of the p16INK4a gene and TIMP-2 by chemical modification of DNA by the method of bisulfite. The modified DNA was subjected to PCR and visualization of the product by polyacrylamide gel electrophoresis. The association of each variable in the methylation of both genes showed no statistical significance. It was noted that 39% (11/28) samples showed methylation only to TIMP-2 gene, and none only for the P16INK4a gene, while 32% (9/28) exhibited methylation on both genes simultaneously. The analysis of the association between methylation of both genes showed statistical significance (p = 0.0292). These results suggest that the association between methylation of the P16INK4a gene and TIMP-2 can promote genomic instability, which can act as a marker in the evolution of the disease. Future studies on the molecular alterations of the extracellular matrix, which precede morphological changes, maybe they can function as a signal, separating the patients with higher risk of evolution of vulvar lichen sclerosus to differentiated VIN and / or cancer of the vulva

No description available.

Le radon est un carcinogène pulmonaire avéré chez l'homme à fortes doses mais l'existence d'un risque de cancer à faibles doses n'est pas établie car les approches épidémiologiques se heurtent, à ce niveau de doses, à de nombreux facteurs confondants. Ces données soulignent la nécessité de disposer d'un modèle d'étude des tumeurs pulmonaires chez l'animal. <br />Le but de ce travail de thèse est de caractériser, sur le plan moléculaire, une série de tumeurs pulmonaires radon-induites chez le rat qui comprend des adénocarcinomes (AC), des carcinomes épidermoïdes (Sq) et des carcinomes adénosquameux (ASq) formés des composantes cellulaires AC et Sq. Une analyse cytogénétique globale de ces tumeurs par la technique de CGH a permis de définir des déséquilibres récurrents de régions chromosomiques, sur lesquelles ont été ciblés des gènes candidats potentiellement impliqués dans le développement des tumeurs, comme p16Ink4a, p19Arf, Rb1, K-Ras ou c-Myc. Une analyse plus précise des voies p16Ink4a/Cdk4/Rb1 et p19Arf/Mdm2/Tp53 a été menée et a permis de mettre en évidence une inactivation fréquente de la voie Rb1, par la perte d'expression de la protéine p16Ink4a, indiquant que cette voie joue un rôle majeur dans le développement de ces tumeurs pulmonaires. Enfin, une analyse transcriptomique comparative des trois types de tumeurs induites a révélé que les tumeurs complexes ASq ont un profil d'expression génique qui reflète leur nature composite mais qu'elles présentent également des spécificités. <br />L'ensemble de ce travail à mis à jour des caractéristiques moléculaires communes aux tumeurs pulmonaires murines et humaines qui encouragent à poursuivre l'étude des spécificités des tumeurs pulmonaires radio-induites chez le rat pour aider à une meilleure caractérisation de ces tumeurs chez l'homme.

No description available.

The tumour suppressor protein p16INK4a (p16) is a cyclin-dependent kinase (CDK) inhibitor that plays a key role in the regulation of the cell cycle by controlling the progression of cells through the G1 to S phase transition. Dysregulation of the protein through deletion, silencing or mutation of the gene encoding p16 is implicated in a range of different cancers including melanoma, cervical and oesophageal to name a few. p16 is composed of four ankyrin repeats and it has a very low thermodynamic and kinetic stability and rapidly unfolds even in the absence of denaturants. This low stability means that the protein is highly vulnerable to point mutations, which can result in functional inactivation through a range of different mechanisms such as deletion of key binding contacts, disruption of secondary or tertiary structure and consequent destabilisation leading to unfolding or aggregation. Heavy-chain antibodies are a unique form of antibody devoid of light chains found in the serum of the Camelid family (camels and llamas). Despite the absence of light chains, heavy-chain antibodies have evolved to complement traditional antibodies and retain the full binding capacity seen in canonical IgG antibodies. The single variable domain, known as a nanobody, is, at 15 kDa, the smallest antigen binding fragment, a tenth the size of a standard IgG antibody. The small size and relative ease of production, coupled with an unusually high stability, makes nanobodies useful tools as biological reagents, crystallography chaperones and therapeutics. The research contained within this PhD looks at the development of nanobodies to target p16. By leveraging the high stability of selected nanobodies, the aim was to obtain binders that could stabilise and reactivate a range of unstable cancer-associated mutants. The initial stages of the project focused on generating and optimising the expression and purification of p16 constructs prior to immunisation of animals to raise nanobodies. A high-throughput approach was taken to generate forty-five different p16 constructs with a range of different solubility and purification tags. These constructs were assessed in a multi-factorial expression screen, which resulted in the identification of a p16 construct with a ten-fold improvement in soluble expression levels compared with previous studies. A range of biophysical techniques, including circular dichroism and chemical denaturation, were performed to characterise this protein fully prior to immunisation. The second part of this project utilised a phage display library of two immune nanobody libraries generated against p16 and a p16 variant stabilised by previously published second-site mutations. This process yielded a large number of diverse nanobodies. Biophysical characterisation of these nanobodies was first performed, and they were found to have a range of chemical and thermal stabilities. Assays were then developed to test the ability of the nanobodies to stabilise p16. Two nanobodies were found to dramatically stabilise wild-type p16, with an increase in stability of approximately 44 % and 60 %, respectively. Furthermore, these nanobodies were also able to stabilise a subset of cancer-associated point mutants. Although there are NMR structures of p16, as well as a crystal structure of p16 bound to CDK6, the resolution of is very low, most likely due to the high backbone flexibility of p16. The last part of the project aimed to obtain a higher-resolution structure of p16 by using the two stabilising nanobodies as crystallisation chaperones. The more stabilising of the two nanobodies resulted in crystals that diffracted to a resolution of less than 2 $\AA$, a significant improvement compared with the previously published structure. In conclusion, a number of nanobodies were generated against tumour-associated p16 and shown to be capable of stabilising p16, allowing structure determination to high resolution and restoration of the stability of cancer-associated mutants to wild-type levels. In the project, a range of different approaches for nanobody production were explored, and these will be important for future applications. Moreover, the crystal structure of the p16-nanobody complex showed that the nanobody binds on the opposite face of p16, to the face involved in binding to CDKs; thus, this nanobody could potentially be exploited as a pharmacological chaperone to stabilise and restore the activity of cancer-associated mutant p16 in the cell.

All-trans retinoic acid (atRA) mediated growth inhibition results in the arrest of the cell cycle during the G1 phase in CAOV3 cells but not SKOV3 ovarian carcinoma cells. The G1 checkpoint is regulated by a multitude of molecules such as the retinoblastoma family of proteins, cyclins, cyclin dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CDKis). CAOV3 cells, which are atRA sensitive, have been shown to express p16INK4a (p16), a cyclin dependent kinase inhibitor regulating the G1 checkpoint. However, atRA resistant SKOV3 cells do not express p16. In these studies, we investigated the role of p16 in mediating atRA induced growth arrest. Our results show that overexpression of p16 in SKOV3 cells leads to growth inhibition following atRA treatment. However, the inhibition is short-term due to the loss of p16 expression. Nevertheless, these results show that p16 plays a role in atRA mediated growth inhibition in ovarian carcinoma cells and that modulation of p16 expression can determine the growth response to atRA. Additionally, we also examined the effect of atRA treatment on the expression of homeobox genes in the CAOV3 cells and SKOV3 cells model system. Homeobox genes comprise a family of transcription factors which function during embryonic development to control pattern formation, differentiation and proliferation. Besides their dominant role during embryogenesis, they are also expressed in adults. In human tumors, an association between the deregulation of the expression of homeobox genes and oncogenic transformation has been reported. It is known that some homeobox genes are atRA targets due to the presence of retinoic acid response element (RARE) either in their promoter region or in their 3' region. In these studies we examined the expression of 13 homeobox genes in CAOV3 cells and SKOV3 cells following ethanol or atRA treatment. The 13 homeobox genes were analyzed because previous studies done by our laboratory observed differences in expression of these homeobox genes when comparing atRA sensitive oral squamous carcinoma cells (SCC) to atRA resistant oral squamous cell carcinoma cells. Of the 13 homeobox genes analyzed in the ovarian carcinoma cell model system, we found HOXA1 and HOXB4 to be upregulated by atRA in CAOV3 cells but not in SKOV3 cells. We also found that the induction of HOXA1 and HOXB4 mRNA expression in CAOV3 cells occurred as a respond to atRA treatment and is not due to a generalized response because of overall growth reduction. Interestingly, HOXA1 has two alternatively spliced forms. The mRNA expression of the truncated form of HOXA1 is highly induced by atRA when compared to its full length form. HOXB1, which is HOXA1 target gene, was not upregulated following atRA treatment. These results suggest that: 1) expression of p16 plays a role in mediating atRA growth inhibition; 2) HOXA1 and HOXB4 also play a role in mediating growth suppression by atRA; and 3) the truncated form of HOXA1 is induced by atRA treatment and may play a role in mediating growth inhibition by atRA, perhaps by acting in a dominant negative fashion. / Microbiology and Immunology

Head and neck cancers (HNSCC) are highly heterogeneous disease, results from two major carcinogens - tobacco and/or alcohol, or HR HPV infection. This thesis was based on 60 biopsies of head and neck tumours embedded into paraffin after histological verification. HPV infection, including particular types was monitored in different HNSCC regions by multiplex qPCR. Subsequent IHC demonstrated expression of p16INK4A and p53 as a possible diagnostic biomarker. Based on the information, patients with HNSCC can benefit from antiEGFR therapy by Cetuximab, but so far without defined predictors, the analysis of point mutations of Ras gene family (Kras, Nras) and Braf gene was performed. These mutations were monitored as potential predictive biomarkers, in correlation with gender, age and other risk factors. For all statistical processing the Chi-x2 test was used. Key words: Head and neck cancers, biopsy, HPV types, PCR, p16INK4A, p53, molecular predictors, Kras, Nras, Braf

No description available.