Le locus CDKN2A/B contient le gène codant le suppresseur de tumeur p16INK4a. Des études d’association de gènes ont identifiées que le locus CDN2A/B est associé à un risque de développement de maladies inflammatoires, dans lesquelles les macrophages jouent un rôle important. Les macrophages constituent une population cellulaire hétérogène dont la forme d’activation et les fonctions sont déterminées par les signaux environnants. Les cytokines Th1, tel que l’interféron gamma, et les antigènes bactériens, tel que le lipopolysaccharide, induisent une activation classique ou M1 des macrophages alors que les cytokines Th2, telles que les interleukines 4 et 13, induisent une activation alternative ou M2 des macrophages. Cependant, les mécanismes moléculaires impliqués dans l’acquisition de ces phénotypes sont encore mal connus. Cette étude montre que l’absence d’expression de p16INK4a (p16-/-) inhibe la réponse inflammatoire et favorise l’activation alternative des macrophages primaires murins in vitro. Afin de déterminer la relevance de ces observations in vivo, la fonction de p16INK4a dans les macrophages a été étudiée chez la souris lors d’une infection parasitaire, dans laquelle le rôle des macrophages alternatifs est connu, puis au cours du développement de l’athérosclérose dans lequel le rôle des macrophages alternatifs reste à déterminer, en utilisant la transplantation de moelle osseuse. Les souris transplantées avec de la moelle osseuse p16-/- présentent une induction de l’expression génique des marqueurs de macrophages alternatifs dans le foie après infection parasitaire, démontrant que p16INK4a influence l’activation des macrophages in vivo. Cependant, l’absence de p16INK4a dans les cellules hématopoïétiques n’influence pas le développement de l’athérosclérose dans nos conditions expérimentales. Enfin, l’invalidation de l’expression de p16INK4a par ARN interférence dans les macrophages primaires humains et la mesure d’expression de p16INK4a dans les macrophages isolés du tissu adipeux de patient obèses montrent que l’absence de p16INK4a favorise un phénotype alternatif des macrophages proche de celui des macrophages du tissu adipeux. Ces travaux de recherche identifient p16INK4a comme modulateur de l’activation des macrophages murins et humains. Mots clés : CDKI-activation alternative-inflammation-infection parasitaire-athérosclérose-obésité. / The CDKN2A/B locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Activation state and functions of macrophages are profoundly affected by environmental cytokines and microbial products. Indeed, Th1 cytokines, such as interferon gamma, and lipopolysaccharide induce a classical activation phenotype whereas Th2 cytokines, such as inteleukins 4 and 13, induce an alternative activation phenotype. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. In this study, we show that p16INK4a-deficiency (p16-/-) skews murine macrophages towards an alternative activation in vitro. The influence of p16INK4a-deficiency on macrophage activation in vivo was investigated using bone marrow transplantation, first in a parasite infectious model in which the role of alternatively activated macrophages is well characterized, then during atherosclerosis development, in which the role of alternatively activated macrophages is undefined. While mice transplanted with p16-/- bone marrow displayed higher hepatic marker expression levels of alternative activation upon parasite infection, no effect was observed on atherosclerosis development in our experimental conditions. Finally, confirming the data obtained in p16-/- macrophages, silencing of p16INK4a with siRNA in human blood-monocyte-derived macrophages also resulted in the induction of alternative activation. In addition, analysis of human adipose tissue macrophages from obese patients, which are typically alternatively activated, showed that p16ink4a expression levels were lower in ATM than in monocyte-derived macrophages of the same subjects. These findings identify a novel role for the tumor suppressor p16INK4a as modulator of murine and human macrophage activation. Keywords: CDKI-alternative activation-inflammation-parasite infectious-atherosclerosis-obesity.

VILNIAUS UNIVERSITETAS GAMTOS MOKSLŲ FAKULTETAS MIKROBIOLOGIJPOS IR BIOTECHNOLOGIJOS KATEDRA Sandra Armalytė Ląstelių žymens p16ink4A raiškos tyrimai kiekybinės PGR metodu Santrauka Ankstyvai gimdos kaklelio vėžio diagnostikai šiuo metu plačiausiai taikomas citologinis tyrimas (PAP testas). PAP testo jautrumas ir specifiškumas ankstyvojoje gimdos kaklelio vėžio diagnostikoje yra gana žemas. Diagnozuojant priešvėžinius ar vėžinius pakitimus, gaunama 15-50% klaidingai neigiamų rezultatų ir iki 30% klaidingai teigiamų rezultatų. Todėl yra aktualu sukurti naujus metodus, kurie leistų citologiniame mėginyje tiksliau identifikuoti dėl ŽPV infekcijos transformuotas, pakitusias ląsteles. Dėl šių priežasčių šiuo metu nemažas dėmesys skiriamas ląstelių žymens p16INK4A nustatymui. Ląstelių žymuo p16INK4A yra specifiškai indukuojamas, įvykus ŽPV sukeltai ląstelių onkogeninei transformacijai. Taigi, šis žymuo gali padėti aptikti mažus priešvėžinių ar vėžinių pokyčių židinius. Tačiau reikalingi tolesni išsamūs tyrimai, patvirtinantys šio žymens diagnostinę vertę. Šio tyrimo tikslas, naudojant atvirkštinės transkripcijos kiekybinės PGR metodą, ištirti ląstelių žymens p16ink4A RNR raišką, esant intraepiteliniams gimdos kaklelio ląstelių pakitimams arba gimdos kaklelio vėžiui ir įvertinti šio žymens sąsajas su priešvėžinių ar vėžinių pokyčių išsivystymo laipsniu. Šiame darbe šis žymuo buvo tiriamas RNR lygmenyje ir nustatyta jo diagnostinė vertė. Naudojant kiekybinį atvirkštinės... [toliau žr. visą tekstą] / VILNIUS UNIVERSITY FACULTY OF NATURAL SCIENCES DEPARTAMENT OF MICROBIOLOGY AND BIOTECHNOLOGY Sandra Armalytė Studies on the expression of cellular marker p16INK4A by quantitative PCR Summary For the diagnostics of cervical cancer cytological test (Pap smear) is commonly used. The Papanicolaou (Pap) test is a cytological staining technique, which allows the identification of asymptomatic women who have preneoplastic lesions or early cancer of the uterine cervix. The success of the Pap smear test is limited with respect to sensitivity and specificity. False negative rates for cervical premalignant lesions and cervical cancer lie between 15% and 50% and false positive rates of approximately 30% have been reported. The failure of the Pap test to eradicate this potentially preventable disease outlines the limitations of current screening programmes and emphasises the need for the identification of specific biomarkers for dysplastic epithelial cells of the cervix to aid in primary screening and lesion diagnosis. Therefore, attempts are made to detect the cellular marker p16INK4A. Protein p16INK4A, an indirect marker of cell cycle dysregulation, is commonly expressed in cervical dysplasias and carcinomas. This biomarker can help to diagnose cervical premalignant lesions or cervical cancer. More experiments are needed to confirm the diagnostic value of this marker. The aim of the current study was to investigate the expression of p16INK4A RNA by quantitative reverse transcriptase PCR... [to full text]

Tumor Susceptibility Gene 101, TSG101, exhibits multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the signals involve in the regulation of TSG101 gene functions are unclear. In this present study, we observed congruous TSG101 up-regulation and the differentiation status of keratinocyte in both human foreskin tissue and reconstructed organotypic skin culture. In addition, we found an essential and downstream role of TSG101 in calcium-induced early keratinocyte differentiation since TSG101 siRNA inhibits this process. Our results also indicate a PKC-dependent mechanism is involved based on the following findings. First, a PKC agonist, TPA up-regulates TSG101 and keratin 10 under low calcium condition. Second, co-treatment of keratinocytes with GF 109203X, a PKC inhibitor, blocks TPA-induced TSG101 and keratin 10 up-regulation. Previous report indicates TSG101 gene exhibits a TATA-less and Sp1-containing promoter. Our analysis further shows that both calcium and TPA stimulate phosphorylation of Sp1 and the corresponding TSG101 wild type promoter activity, but not the activity of Sp1 site mutant TSG101 promoter. The co-treatment with GF 109203X blocks the above effects of calcium and TPA, implying that this is a PKC signaling-dependent process. Taken together, these data suggest a PKC-Sp1 signaling is involved in early differentiation switch of keratinocyte through up-regulation of TSG101. Functional inactivation experiment indicates that tsg101 is a tumor suppressor in mouse model. However, many studies using human tumor specimens or conditional knockout mouse give discrepant and contradictive results. Therefore, the role of TSG101 in human cancer remains illusive. Here we demonstrate an inverse correlation between TSG101 and p16INK4a or acetylated- histone H4 protein expression profiles in human head and neck squamous cell carcinomas (HNSCC) (N=98, p<0.001). Using conditioned human HEp2 cells, we confirm that TSG101 negatively modulates p16INK4a expression. Chromatin immunoprecipitation and the subsequent PCR analysis reveal that TSG101 dose-dependently decreases the amount of acetylated histone H4-associated chromatin on p16INK4a promoter. In addition, TSG101 interacts and colocalizes with HDAC1 and SUMO-1 in the nucleus. Furthermore, TSG101 confers a dose-dependent effect on promoting HDAC1 SUMOylation, hence its activity. Taken together, our data demonstrate for the first time that TSG101 can promote SUMO-1 modification of HDAC1, which impacts on down-regulation of p16INK4a gene expression, providing evidence whereby TSG101 might participate in the epigenetic silencing of p16INK4a during the development of HNSCC.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-08-21T20:57:06Z No. of bitstreams: 1 VicenilmaMartins.pdf: 7618309 bytes, checksum: 3d94fb00454be15b5872822df938424e (MD5) / Made available in DSpace on 2017-08-21T20:57:06Z (GMT). No. of bitstreams: 1 VicenilmaMartins.pdf: 7618309 bytes, checksum: 3d94fb00454be15b5872822df938424e (MD5) Previous issue date: 2017-04-28 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Infection by HPV is described in the literature in 30-50% of cases of penile cancer. The immunohistochemical of p16INK4a is used as an indication of the presence of HPV and prognostic marker for squamous carcinomas various sites. However, its role in penile carcinoma has not yet been completely elucidated. The aim of the study was to prospectively analyze whether the expression of p16INK4a is associated with the presence of HPV and histological parameters in penile cancer. A prospective study was conducted in the period 2014 to 2016 with 55 cases of patients with penile carcinoma. HPV DNA was detected by PCR in fresh tumor tissue and immunohistochemistry for analysis of p16INK4a protein in paraffin waxed tissue. The evaluation of histological parameters was performed after total inclusion of the tumor tissue. HPV DNA (lowrisk and high-risk genotypes) was found in 49 (89.1%) cases, 46 (83.6%) HR-HPV. Of the 22 p16INK4a positive cases, HR-HPV DNA was present in 21 (95.5%) of them (p = 0.032). Regarding histological parameters, p16INK4a and HR-HPV were significantly associated only with the tumor subtype (p = 0.036 and p = 0.032, respectively), being that, all carcinomas with basaloid characteristics were positive p16INK4a. Using a fresh tissue sample, our sample showed the highest incidence of HPV in the literature. Expression of the p16INK4a protein was significantly associated with the presence of high oncogenic HPV virus and may serve as a marker for the presence of this virus. The p16INK4a protein was not associated with the histological prognostic parameters, except for the tumor subtype. / A infecção pelo HPV é descrita na literatura em 30-50% dos casos de câncer de pênis. A expressão imunohistoquímica de p16INK4a é utilizada como indicativo da presença de HPV e marcador prognostico para carcinomas escamosos de vários sítios. No entanto, seu papel no carcinoma de pênis ainda não está totalmente elucidado. O objetivo do estudo foi analisar, de forma prospectiva, se a expressão de p16INK4a está associada a presença de HPV e parâmetros histológicos no câncer de pênis. Estudo prospectivo realizado no período de 2014 a 2016 com 55 casos de pacientes com carcinoma pênis. Foi realizada a detecção do DNA HPV por PCR em tecido tumoral a fresco e a imunohistoquímica para análise da proteína p16INK4a em tecido parafinado. A avaliação de parâmetros histológicos foi realizada após inclusão total do tecido tumoral. O DNA HPV (genótipos de baixo risco e alto risco) foi encontrado em 49 (89,1%) casos, sendo 46 (83,6%) HRHPV. Dos 22 casos p16INK4a positivo, o DNA HR-HPV esteve presente em 21 (95,5%) deles (p = 0,032). Em relação a parâmetros histológicos, a p16INK4a e o HR-HPV foram significativamente associados apenas ao subtipo do tumor (p = 0,036 e p=0,032, respectivamente), sendo que todos os carcinomas com características basalóides foram p16INK4a positivo. Utilizando amostra de tecido a fresco, a nossa casuística mostrou a mais alta incidência de HPV da literatura. A expressão da proteína p16INK4a foi significativamente associada à presença do vírus HPV de alto risco oncogênico e pode servir como marcador da presença desse vírus. A proteína p16INK4a não foi associada aos parâmetros prognósticos histológicos, exceto ao subtipo do tumor.

Replicative exhaustion of endothelial cells (ECs) contributes to the pathogenesis of age-related vascular disorders, including atherosclerosis and impaired wound healing. Conversely, abnormal proliferation of ECs underlies the development of EC-derived malignancies, such as haemangioblastoma and angiosarcoma. The central objective of this thesis was to delineate mechanisms that regulate the replicative lifespan of human ECs and molecular alterations that occur during immortalisation of ECs. The gradual shortening of telomeres (chromosome-end structures) is one mechanism that restricts the replicative lifespan of human ECs. Telomere shortening initiates an irreversible growth arrest or senescence through activation of a TP53-mediated DNA damage response. Expression of the cyclin-dependent kinase inhibitor, p16INK4a, is also increased and reinforces senescence via the retinoblastoma pathway. Overexpression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity and extends the lifespan of human ECs, but is not sufficient for immortalisation. The current study demonstrated that p16INK4a repression by promoter methylation was a frequent event during immortalisation of hTERT-transduced bone marrow ECs (BMECs), occurring in 5 of 12 clones. Repression of p16INK4a concurred with the development of recurring chromosomal aberrations, which appeared to be a consequence of telomere dysfunction and chromosome fusions. Loss of p16INK4a and the development of a complex karyotype were associated with a more transformed phenotype in hTERT-immortalised BMECs. The investigations described in this thesis were the first to associate loss of p16INK4a expression with the accumulation of chromosome aberrations. Repression of p16INK4a in only a subset of immortal BMECs provided impetus for investigating whether there was a functionally analogous defect in the hTERT-immortalised BMECs that retained p16INK4a expression. In normal human cells, oncogenic Ras upregulates p16INK4a and induces senescence independently of telomere shortening. This thesis demonstrates that the immortal BMECs that retained p16INK4a expression had a defective response to oncogenic Ras, which may have contributed to the immortalisation of these cells. Whole genome and proteome analyses identified additional alterations in gene copy number and protein expression specific to p16INK4a-positive or -negative immortal BMECs. Overall, these investigations provide new insight to the potential consequences of p16INK4a repression during carcinogenesis and describe novel molecular alterations that occur during immortalisation of human ECs.

Made available in DSpace on 2016-08-10T10:39:12Z (GMT). No. of bitstreams: 1 NATHALIA AMARAL NOGUEIRA.pdf: 6074559 bytes, checksum: db2409d0f003801fe0f55ef172d6687e (MD5) Previous issue date: 2016-03-14 / In developing countries, uterine cervical cancer is responsible for a large percentage of deaths in middle-aged women, and presents high rates of incidence, especially due to the lack of efficient screening of precursor lesions and cancer. Biomarkers can increase the accuracy and effectiveness of screening programs, diagnosis and treatment of cervical cancer precursor lesions. Among the markers related to cell proliferation changes, p16INK4a excels. P16INK4aexpression may suggest the presence of transformed cells, in the lesions seemingly normal morphology, and it also predicts the faster development of neoplastic cells. The prognostic role of p16INK4a in cervical cancer is not clear. The effect of p16INK4a expression in the survival of cervical cancer patients has been investigated and the results are conflicting. The main objective of this study was to investigate the potential prognostic role of p16INK4a in cervical carcinomas. The series comprising 161 patients attended on Araújo Jorge Hospital - Goiânia, Goiás, Brazil - from 2006 to 2007, with histologically confirmed cervical cancer, assessed for p16INK4a expression by means of immunohistochemistry. The results were compared by Chi-square test or Fisher s exact test and survival analysis by Kaplan-Meier and Log-rank tests. Overexpression of p16INK4a was observed in 145 (90.0%) of the cases. No statistically significant association was observed between p16INK4a expression and clinical pathological aspects of cervical carcinomas. The overall survival for the group was 69.0%. When survival was evaluated in relation to p16INK4a expression, cases with overexpression showed a better survival rate (70.3%), towards those with low expression (56.2%), despite the difference between groups was not statistically significant. When compared in relation to clinical staging, cases with stage I and II showed a better survival (71.9%), compared to those with stage III and IV (54.8%) (p= 0.04), translating the best prognosis of cases diagnosed ealier. Our results do not confirm the association between the p16INK4a expression and prognosis of uterine cervical cancer. / Nos países em desenvolvimento, o câncer de colo uterino é responsável por grande porcentagem de mortes em mulheres de meia idade e apresenta altas taxas de incidência, especialmente devido à falta de prevenção eficiente de lesões precursoras ou de câncer inicial. Biomarcadores podem aumentar a acurácia e a efetividade de programas de rastreamento, diagnóstico e tratamento das lesões precursoras do câncer. Dentre os marcadores relacionados às alterações da proliferação celular, destaca-se p16INK4a, cuja expressão pode sugerir a presença de células transformadas, ainda que as lesões apresentem morfologia aparentemente normal, e traduzir a evolução mais rápida das células neoplásicas. O papel prognóstico de p16INK4a no câncer de colo uterino não é claro. O efeito da hiperexpressão da p16INK4a na sobrevida de pacientes com câncer de colo uterino tem sido investigado e os resultados são conflitantes. O principal objetivo deste estudo foi investigar o potencial papel prognóstico de p16INK4a em carcinomas de colo uterino. A casuística consistiu de 161 pacientes com câncer de colo uterino confirmados histologicamente e assistidos no Hospital Araújo Jorge, em Goiânia-GO, no período de 2006-2007. A avaliação da expressão de p16INK4a foi feita por meio de reação imuno-histoquímica. Os resultados foram comparados pelo teste do Chi-quadrado ou teste exato de Fisher e as análises de sobrevida pelos testes de Kaplan-Meier e Log-rank. Os resultados demonstraram a hiperexpressão de p16INK4a em 145 casos (90,0%), enquanto a hipoexpressão foi observada em 16 casos (10,0%). Nenhuma associação estatisticamente significativa foi observada entre a expressão de p16INK4a e os aspectos clinicopatológicos dos carcinomas de colo uterino avaliados. A sobrevida global para o grupo foi de 69,0%. Quando a sobrevida foi avaliada em relação à expressão de p16INK4a, observou-se que os casos com hiperexpressão apresentaram sobrevida de 70,3%, comparados aos de hipoexpressão que apresentaram sobrevida de 56,2%. A diferença entre os grupos não foi estatisticamente significativa. Com relação ao estadiamento clínico, observou-se que os casos com estádio I e II apresentaram melhor sobrevida (71,9%) em relação àqueles com estádio III e IV (54,8%) (p=0,04), traduzindo o melhor prognóstico dos casos diagnosticados mais precocemente. Nossos resultados não confirmaram a associação entre a expressão de p16INK4a e o prognóstico dos tumores avaliados.

Le cancer du col utérin est un des cancers les plus prévalents dans le monde et représente une cause majeure de mortalité par cancer chez la femme. Le dépistage de ce cancer est actuellement basé sur l’examen clinique, la colposcopie, la cytologie du col utérin et l’histopathologie dont la reproductibilité inter observateur est médiocre surtout en ce qui concerne les lésions de bas grade (CIN 1). Bien qu’amélioré par de nouveaux marqueurs (p16INK4A), la recherche de nouveaux marqueurs spécifiques des lésions du col utérin nécessite l’élucidation des mécanismes de la cancérogenèse parmi lesquels les altérations épigénétiques, but de notre recherche. Nous avons évalué au cours des différentes étapes de la cancérogenèse du col utérin la méthylation globale de l'ADN par immunohistochimie et HPLC. L’hyperméthylation des îlots de CpG de trois gènes candidats impliqués dans la signalisation cellulaire (CDH13, DAPK1, TWIST1) a été étudié par une technique de PCR – méthylation spécifique (MSP) après modification de l’ADN au bisulfite de sodium. L’étude immunohistochimique à l'aide d’anticorps dirigés contre la 5-MeCyd objective une hypométhylation globale de l’ADN tardive n’apparaissant que dans les cancers invasifs. Ces résultats ont été confirmés par HPLC. Par contre, l’hyperméthylation des îlots de CpG des gènes candidats étudiés est présente dés le stade lésions précancéreuses. Ces résultats suggèrent qu’au cours de la cancérogenèse du col utérin, l’hypométhylation globale et l’hyperméthylation des îlots de CpG sont deux mécanismes indépendants. L’étude du profil de méthylation des régions promotrices des gènes CDH13, DAPK1, TWIST1 pourrait constituer un marqueur potentiel des lésions précancéreuses et cancéreuses du col de l’utérus / Background: The uterine cervix cancer is one of the main cancer among women worldwide and represents an important cause of death in women especially in developing countries. The screening of this cancer is based on clinic tests, colposcopy, cytology and histopathology which have a low inter observer reproducibility, specially concerning CIN1 lesions. Although improved by new markers (p16INK4A), the discovery of new specific markers requires the elucidation of mechanisms of carcinogenesis such as epigenetic alterations, aim of our study. Material and methods: We evaluated during uterine cervix carcinogenesis global DNA methylation by immunohistochemistry and HPLC. CpGs islands hypermethylation of three genes implied in cellular signalizing (CDH13, DAPK1, TWIST1) were evaluated using specific methylation PCR (MSP) after bisulfite DNA modification. Results: Immunohistochemistry study using anti 5-MeCyd antibodies objectives a late global DNA hypomethylation appearing only in invasive uterine cervix carcinoma. These results were confirmed by HPLC. However, CpGs islands hypermethylation of the promoter of CDH13, DAPK1 and TWIST1 genes studied were detected in precancerous lesions. Conclusions: Our results suggest that during uterine cervix carcinogenesis, global DNA hypomethylation and gene hypermethylation were two independent mechanisms. The methylation profile of the promoter regions of CDH13, DAPK1 and TWIST1 genes would represent a potential marker for the diagnosis of the precancerous and cancerous uterine cervix lesions

Considerando que várias mutações gênicas estão envolvidas no estabelecimento dos tumores, surge a idéia de que o alcance da melhor eficiência no tratamento do câncer é dado pela entrega de múltiplos genes. Este trabalho apresenta a construção, produção e caracterização funcional in vitro e in vivo do vetor adenoviral bicistrônico Adp16IRESp53 e dos monocistrônicos Adp16 e Adp53 em modelo de câncer de pulmão. Nossos resultados indicam uma forte indução de morte celular nas células H358 transduzidas com Adp16IRESp53 em comparação com vetores monocistrônicos Adp16, Adp53 ou o reporter AdeGFP e/ou AdLacZ. Nos ensaios in vivo, utilizando modelo xenografico onde as células H358 foram implantadas no subcutâneo de camundongos atímicos Balb/C nude, pudemos confirmar também in vivo a significativa inibição do crescimentos dos tumores tratados com Adp16IRESp53. Em conclusão, a remediação simultânea de p16INK4a e p53, mediada pelo arranjo bicistrônico, pode ser considerada como uma estratégia promissora para terapia gênica do câncer de pulmão. / This work presents the construction, production and functional evaluation in vitro and in vivo of the bicistronic adenoviral vector Ap16IRESp53 as well as the monocistronic vectors Adp16 and Adp53 in a lung cancer model. Considering that several mutation events are involved in tumorigenesis, comes the idea that a greater efficiency in cancer treatment would be reached with delivery of multiples genes. Our data demonstrate a strong cell death effect in H358 cells transduced with Adp16IRESp53 when compared with Adp16, Adp53 or the reporter AdeGFP and/or AdLacZ. For the in vivo studies, we have used H358 cells implanted subcutaneously in athymic Balb/c nude mice. Our data show significant suppression of tumors treated with the therapeutic adenoviral vector, Adp16IRESp53. In conclusion, the simultaneous replacement of p16INK4a and p53, mediated by the bicistronic vector, may prove to be a promising strategy for gene therapy of lung cancer.

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.

Considerando que várias mutações gênicas estão envolvidas no estabelecimento dos tumores, surge a idéia de que o alcance da melhor eficiência no tratamento do câncer é dado pela entrega de múltiplos genes. Este trabalho apresenta a construção, produção e caracterização funcional in vitro e in vivo do vetor adenoviral bicistrônico Adp16IRESp53 e dos monocistrônicos Adp16 e Adp53 em modelo de câncer de pulmão. Nossos resultados indicam uma forte indução de morte celular nas células H358 transduzidas com Adp16IRESp53 em comparação com vetores monocistrônicos Adp16, Adp53 ou o reporter AdeGFP e/ou AdLacZ. Nos ensaios in vivo, utilizando modelo xenografico onde as células H358 foram implantadas no subcutâneo de camundongos atímicos Balb/C nude, pudemos confirmar também in vivo a significativa inibição do crescimentos dos tumores tratados com Adp16IRESp53. Em conclusão, a remediação simultânea de p16INK4a e p53, mediada pelo arranjo bicistrônico, pode ser considerada como uma estratégia promissora para terapia gênica do câncer de pulmão. / This work presents the construction, production and functional evaluation in vitro and in vivo of the bicistronic adenoviral vector Ap16IRESp53 as well as the monocistronic vectors Adp16 and Adp53 in a lung cancer model. Considering that several mutation events are involved in tumorigenesis, comes the idea that a greater efficiency in cancer treatment would be reached with delivery of multiples genes. Our data demonstrate a strong cell death effect in H358 cells transduced with Adp16IRESp53 when compared with Adp16, Adp53 or the reporter AdeGFP and/or AdLacZ. For the in vivo studies, we have used H358 cells implanted subcutaneously in athymic Balb/c nude mice. Our data show significant suppression of tumors treated with the therapeutic adenoviral vector, Adp16IRESp53. In conclusion, the simultaneous replacement of p16INK4a and p53, mediated by the bicistronic vector, may prove to be a promising strategy for gene therapy of lung cancer.