Introdução: A doença renal crônica continua sendo um desafio no campo da pesquisa médica. Atualmente um interesse crescente tem surgido no intuito de avaliar o potencial de células tronco em retardar o avanço de doenças crônicas progressivas. Material e Métodos: Para determinar o efeito dessas células em um modelo de progressão de doença renal crônica foram usadas células linhagem negativa (Lin ) separadas magneticamente e injetadas em ratos submetidos à injúria renal. Ratos singênicos Fischer 344 foram submetidos à nefrectomia 5/6 (Nx) e divididos em 3 grupos: Nx (não tratados); NxSC1 (submetidos à infusão de 2 106 células Lin no 15º dia de pós-operatório); e NxSC3 (submetidos à infusão de 2 106 células Lin no 15º, 30º e 45º dias de pós-operatório). No 60º dia de pós-operatório clearance de inulina, imunohistoquímica e immunoblotting foram realizados. Resultados: Os animais submetidos à nefrectomia apresentaram redução do clearance de inulina (0,33 ± 0,02 ml/min/100g peso corpóreo), proteinúria (12 ± 0,5 mg/24hs) , anemia e hipertensão (145 ± 7,7 mmHg) compatíveis com doença renal crônica. A infusão de células Lin- resultou em atenuação da proteinúria (p<0,05) com relação aos animais não tratados a despeito de não ter havido diferença nos níveis de pressão arterial e aldosterona plasmática. Esses achados foram similares entre os grupos tratados com uma ou com três infusões de células. Adicionalmente a infusão de células resultou em redução do índice de glomeruloesclerose e da área intersticial relativa (p<0,05), menor infiltração do tecido renal por macrófagos e linfócitos e menor proliferação celular. A expressão tecidual do p21 e de VEGF já foi associada à aceleração da progressão da lesão renal crônica. No nosso modelo ambas as proteínas tiveram sua expressão reduzida. A redução da expressão tecidual de eNOS tem sido implicada na progressão da doença renal. Em nosso modelo houve aumento dessa expressão após infusão das células Conclusões: A infusão de células Linatenuou todos os marcadores de injúria renal em um modelo de doença precoce possivelmente através de um mecanismo imunomodulador. / Progressive renal failure continues to be a challenge. The use of bone marrowderived stem cells (SCs) represents a means of meeting that challenge. We used lineage-negative (Lin-) SCs to test the hypothesis that Lin- cell infusion decreases renal injury. Syngeneic Fischer 344 rats were submitted to 5/6 nephrectomy and divided into 3 groups: Nx (untreated); NxSC1 (receiving 2 × 106 Lin- cells on postnephrectomy day 15); and NxSC3 (receiving 2 × 106 Lin- cells on postnephrectomy days 15, 30 and 45). Controls were unoperated/untreated. On postnephrectomy day 60, clearance studies, immunohistochemistry and immunoblotting were performed. Lin- cell infusion effectively reduced postnephrectomy proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells and monocyte chemoattractant protein-1 protein expression, as well as decreasing the interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with controls, Nx rats presented greater cell proliferation, whereas NxSC1 rats and NxSC3 rats presented less cell proliferation than did Nx rats. Protein expression of p21 and VEGF increased after nephrectomy and decreased after Lin- cell infusion. Protein expression of eNOS reduced after nephrectomy and increased after cell infusion. These data suggest that SC treatment ameliorates progressive end-stage renal disease.

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles. / Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.

As proteínas p53, MDM-2, c-Kit, ErbB-2, PCNA e p-glicoproteína foram estudadas em 42 amostras de osteossarcoma ao diagnóstico, através da técnica de imunohistoquímica, e foram correlacionados estes achados com o prognóstico destes pacientes. O p-53 foi positivo em 23,1% (9/39), PCNA em 71,4% (25/35), p-glicoproteína em 40,5% (15/37), MDM-2 em 34,8% (8/23), c-kit em 67,6% (25/37) e ErbB-2 em 17,9% (7/39). Na análise univariada, a presença de metástases ao diagnóstico, a positividade de p53 e ErbB-2 influenciaram o prognóstico individualmente, entretanto, na análise multivariada, a presença de metástase ao diagnóstico revelou-se o único fator de prognóstico estatisticamente significante / We study, by imunohistochemistry technique, the proteins p53, MDM-2, c-Kit, ErbB-2, PCNA and p-glycoprotein in samples of osteosarcoma tumors at diagnosis and its correlation with the prognosis of this patients. The p-53 was positive in 23,1% (9/39), PCNA in 71,4% (25/35), p-glycoprotein in 40,5% (15/37), MDM-2 in 34,8% (8/23), c-kit in 67,8%(25/37) and ErbB-2 in 17,9% (7/39) of the samples. In the univariate analysis, the presence of metastasis at diagnosis, the positivity of p-53 and ErbB-2 influenced the prognosis individually, otherwise, in the multivariate analysis, the presence of metastasis at diagnosis was the only prgnostic factor statistically significant

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest. Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment. For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues. Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS. The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility.

Les glycosides cardiotoniques sont des inhibiteurs des pompes sodium / potassium utilisés pour le traitements des insuffisances cardiaques, qui détiennent également des activités anticancéreuses et épigénétiques récemment caractérisées. Toutefois, dans l’objectif de repositionner ces médicaments comme traitement anticancéreux, les mécanismes sousjacents aux activités anticancéreuses et épigénétiques des glycosides cardiotoniques restent à être déterminés. Dans nos travaux, nous révélons que la proscillaridine A est le glycoside cardiotonique qui détient des activités anticancéreuses et épigénétiques les plus puissantes dans des lignées de cancer du côlon, de leucémies et de sarcomes pédiatrique. De plus, nous avons identifié que l’activité anticancéreuse de la proscillaridine A corrèle positivement avec le niveau d’expression protéique du proto-oncogène MYC dans un panel de 14 lignées cellulaires cancéreuses. Dans les lignées cellulaires exprimants un haut niveau de MYC telles que les lignées leucémiques, la proscillaridine A agit comme un inhibiteur de MYC et module sa stabilité protéique ainsi que la régulation transcriptionnelle et translationnelle de ces cibles. Cette inhibition est induite par la baisse significative de l’expression des enzymes épigénétiques les lysines acétyltransférases (KATs), qui contrôlent l’ajout des résidus d’acétylcoenzyme A sur les histones et sur d'autres protéines dont MYC. La baisse d’expression des KATs résultent à une baisse de l’acétylation des résidus de l’histone 3 et à une reprogrammation de l’acétylome des cellules cancéreuses surexprimant MYC. Ces changements au niveau de la chromatine induisent une reprogrammation transcriptionnelle et phénotypique des cellules surexprimant MYC, qui se traduit par une perte de la transcription des programmes oncogéniques et l’induction des programmes associés à la différenciation cellulaire. Pour finir, nous avons évalué le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec le médicament épigénétique la décitabine dans des lignées cancéreuses exprimants des niveaux différentiels de MYC. Dans une lignée résistante à la proscillaridine A et exprimant de faible niveau de MYC (lignée de cancer de côlon), la décitabine et la proscillaridine A démontrent des activités épigénétiques synergiques tandis que dans une lignée sensible à la proscillaridine A et surexprimant MYC (lignée de sarcome pédiatrique), la décitabine et la proscillaridine A démontrent des activités antiprolifératives synergiques. Dans ces travaux, nous avons donc démontré le potentiel de repositionner la proscillaridine A dans les cancers surexprimant MYC. Également, nous démontrons le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec la décitabine et nous suggérons d’étudier cette combinaison de médicaments dans les cancers plus résistants à la proscillaridine A. / Cardiac glycosides are sodium/potassium pomps’ inhibitors used for the treatment of heart failure, and whose anticancer and epigenetic activities have been recently characterized. However, in order to repurpose cardiac glycosides as anticancer drugs, mechanistic studies are required to identify the anticancer and epigenetic mechanism of actions. In our experiments, proscillaridin A exhibited the most powerful anticancer and epigenetic activities in colon cancer, leukemia, and sarcoma cell lines. Moreover, we demonstrated that in a panel of 14 cancer cell lines, proscillaridin A anticancer activities positively correlated with MYC protooncogene expression level. In high MYC expressing cell lines such as leukemia, proscillaridin A inhibited MYC expression through protein destabilization and through transcriptomic and translational regulation of MYC targets. Theses inhibitions are induced by the loss of lysine acetylatransferase (KAT) expressions, which are epigenetic enzymes controlling the addition of acetyl-coenzyme A on histones and other proteins such as MYC. KAT inhibitions are responsible for the global loss of histone 3 acetylation and acetylome reprogrammation in high MYC expressing cancer cells. These chromatin changes induced transcriptomic and phenotypic reprogrammation, defined by a loss of the transcription of oncogenic programs and the induction of cell differentiation. To finish, we evaluated the anticancer and epigenetic synergic potential of proscillaridin A in combination with the epigenetic drug the decitabine in cancer cell lines expressing different MYC levels. In a cancer cell line resistant to proscillaridin A treatments and expressing low MYC level (colon cancer cell line), the combination of decitabine and proscillaridin A demonstrated synergistic epigenetic activity although, in a cell line sensitive to proscillaridin A treatments and expressing high MYC level (sarcoma cell line), the combination of decitabine and proscillaridin A exhibited synergistic anti-proliferative activity. To conclude, we highlighted the potential of repurposing proscillaridin A as an anticancer treatment in high MYC expressing cells. Furthermore, we demonstrated the anticancer and epigenetic synergistic potential of proscillaridin A in combination with decitabine and we propose to study the drug combination in cancers that are resistant to proscillaridin A treatment.

No / Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1beta-mediated inflammatory signaling. Resveratrol suppressed IL-1beta-induced activation of NF-kappaB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1beta-induced NF-kappaB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IkappaB kinase, IkappaBalpha phosphorylation, and inhibition of nuclear translocation of NF-kappaB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-kappaB activation. Inhibition of PI3K by wortmannin attenuated IL-1beta-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1beta-induced activation of NF-kappaB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-kappaB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-kappaB.

A neoplasia endócrina múltipla tipo 2 (NEM-2) é uma síndrome tumoral herdada por mutações germinativas no proto-oncogene RET (RET) e transmitida por herança autossômica dominante. Atualmente, a indicação de tireoidectomia total preventiva é recomendada a indivíduos portadores de mutações no RET. Analisamos a aplicação do método Eletroforese em Gel Sensível à Conformação (CSGE) no rastreamento de mutações hot-spots do RET. Sete famílias com NEM-2 foram rastreadas pelo CSGE, seqüenciamento gênico e análise do Polimorfismo Conformacional de Cadeia Simples (SSCP). Usando o CSGE e SSCP, identificamos cinco das seis (83,3%) mutações verificadas pelo seqüenciamento: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile e Met918Thr. Foram analisados 128 amplicons englobando mutações hot-spots do RET e 116 dentre 128 (90.6%) concordaram com o seqüenciamento genético. Os polimorfismos 691 e 769 também foram documentados pelo CSGE e SSCP. Os dados obtidos por CSGE e SSCP foram totalmente (100%) concordantes. O CSGE revelou ser metodologia sensível, rápida, fácil de ser executada e de baixo custo na detecção de mutações nos códons 620, 634, 648, e 918, as quais constituem grande maioria (~95%) dos pacientes com NEM-2. Quanto à mutação Val804Met (prevalência na população inferior a 3%), o método necessita ser otimizado. Concluímos que o CSGE é uma metodologia efetiva para o rastreamento de mutações que mais freqüentemente ocorrem no RET como causadora de NEM-2. / Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by activating germline mutations in RET proto-oncogene (RET). Presently, the prophylactic total thyroidectomy is recommended to all RET mutations carriers. Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for the RET hot-spot mutations. Seven MEN2 families were studied by CSGE, as well as by Single Strand Conformational Polymorphism (SSCP) and direct sequencing analysis. Using CSGE and SSCP, we were able to detect five out of the six (83.3%) RET mutations verified by direct sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile and Met918Thr. RET polymorphisms 691 and 769 were verified by CSGE and SSCP. In our sample, data obtained using CSGE were fully concordant (100%) with SSCP findings. Thus, CSGE showed to be a sensitive, fast, low-cost, and ease procedure to detect RET mutations in codons 620, 634, 648, and 918 which are reported as the most prevalent RET variants (~95%) in large MEN2 series. As to the Val804Met mutation (prevalence in the population lower than 3%), this method still needs to be optimized. We concluded that CSGE is an effective screening method for the most frequent RET hot-spot disease-causing mutations.

A neoplasia endócrina múltipla tipo 2 (NEM-2) é uma síndrome tumoral herdada por mutações germinativas no proto-oncogene RET (RET) e transmitida por herança autossômica dominante. Atualmente, a indicação de tireoidectomia total preventiva é recomendada a indivíduos portadores de mutações no RET. Analisamos a aplicação do método Eletroforese em Gel Sensível à Conformação (CSGE) no rastreamento de mutações hot-spots do RET. Sete famílias com NEM-2 foram rastreadas pelo CSGE, seqüenciamento gênico e análise do Polimorfismo Conformacional de Cadeia Simples (SSCP). Usando o CSGE e SSCP, identificamos cinco das seis (83,3%) mutações verificadas pelo seqüenciamento: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile e Met918Thr. Foram analisados 128 amplicons englobando mutações hot-spots do RET e 116 dentre 128 (90.6%) concordaram com o seqüenciamento genético. Os polimorfismos 691 e 769 também foram documentados pelo CSGE e SSCP. Os dados obtidos por CSGE e SSCP foram totalmente (100%) concordantes. O CSGE revelou ser metodologia sensível, rápida, fácil de ser executada e de baixo custo na detecção de mutações nos códons 620, 634, 648, e 918, as quais constituem grande maioria (~95%) dos pacientes com NEM-2. Quanto à mutação Val804Met (prevalência na população inferior a 3%), o método necessita ser otimizado. Concluímos que o CSGE é uma metodologia efetiva para o rastreamento de mutações que mais freqüentemente ocorrem no RET como causadora de NEM-2. / Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by activating germline mutations in RET proto-oncogene (RET). Presently, the prophylactic total thyroidectomy is recommended to all RET mutations carriers. Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for the RET hot-spot mutations. Seven MEN2 families were studied by CSGE, as well as by Single Strand Conformational Polymorphism (SSCP) and direct sequencing analysis. Using CSGE and SSCP, we were able to detect five out of the six (83.3%) RET mutations verified by direct sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile and Met918Thr. RET polymorphisms 691 and 769 were verified by CSGE and SSCP. In our sample, data obtained using CSGE were fully concordant (100%) with SSCP findings. Thus, CSGE showed to be a sensitive, fast, low-cost, and ease procedure to detect RET mutations in codons 620, 634, 648, and 918 which are reported as the most prevalent RET variants (~95%) in large MEN2 series. As to the Val804Met mutation (prevalence in the population lower than 3%), this method still needs to be optimized. We concluded that CSGE is an effective screening method for the most frequent RET hot-spot disease-causing mutations.

INTRODUÇÃO: Na Neoplasia Endócrina Múltipla tipo 2 (NEM2), o desenvolvimento do Carcinoma Medular de Tireoide (CMT), Feocromocitoma (FEO) e Hiperparatireoidismo primário (HPT) está associado à mutações germinativas ativadoras no proto-oncogene RET. Casos de CMT esporádico podem apresentar mutações somáticas no RET (~40%). A variabilidade fenotípica observada em casos de CMT e FEO familiais associados à NEM2 indica o envolvimento de eventos genéticos adicionais que seriam responsáveis pelas diferenças clínicas observadas nos indivíduos afetados (idade de desenvolvimento, progressão e agressividade do tumor). Outras alterações genéticas no RET como duplas mutações, SNPs e haplótipos específicos podem influenciar na susceptibilidade, agressividade e modulação do fenótipo NEM2. Entretanto, os estudos de outros genes envolvidos no processo da tumorigênese NEM2 ainda estão em andamento. Recentemente foi mostrado que RET ativado controla a expressão de proteínas inibidoras do ciclo celular (p18 e p27). Mutações germinativas no gene p27 foram recentemente associadas à susceptibilidade de tumores neuroendócrinos e estão associadas à síndrome NEM4 (Neoplasia endócrina múltipla tipo 4). Mutações somáticas, inativadoras de p27, são raramente encontradas em vários tipos de tumores. Entretanto, diversos estudos documentaram que a redução na expressão e a sublocalização citoplamática de p27 são controladas por alterações pós-transducionais e/ou epigenéticas. OBJETIVOS: o estudo teve como objetivos avaliar a participação de genes, recentemente associados ao RET ativado, em tumores de pacientes com NEM2 e também verificar se polimorfismos no gene p27 estariam atuando como moduladores de fenótipo em uma grande família com NEM2. CASUÍTICA: foram analisadas 66 amostras tumorais advindas de 36 pacientes com diagnóstico clínico e genético de NEM2 e 28 indivíduos pertencentes a uma grande família com NEM2A-CMTF e mutação C620R no gene RET. MÉTODOS: As análises somáticas do p27 e também de p15, p18 e RET foram realizadas por PCR e sequenciamento direto de DNA e análise de microssatélites para p27 foi realizada por PCR e eletroforese capilar. Análises de expressão e localização da proteína p27 celular foram realizadas por Western blot e imunohistoquímica. A análise da modulação de fenótipo na família com NEM2A foi realizada por meio da amplificação do éxon 1 do gene p27 na amostra de sangue total. RESULTADOS: Não foram encontradas mutações somáticas no gene p27 e também nos genes p15 e p18. Entretanto, verificamos baixa expressão proteica de p27 em tumores CMT e FEO, a qual se encontrava relacionada com o tipo e agressividade do códon mutado no RET, principalmente em tumores que apresentavam mutação RET no códon 634 (controle x 634 p=0,05; controle x 634/791 p= 0,032; 620 x 634 p=0,045; 620 x 634/791 p= 0,002; 620 x 634 + 634/791 p=0,036). Notou-se também correlação positiva entre os níveis de expressão de p27 na localização nuclear, analisada por imunohistoquímica, e o genótipo TT do SNP p27 p.V109G (p=0,03). CONCLUSÕES: Alterações moleculares somáticas no gene p27 nos tumores NEM2 não são frequentes. Entretanto, a redução na expressão e a localização citoplasmática de p27 provavelmente estão associadas a alterações somáticas em outros genes que controlam os processos de fosforilação da proteína p27 (eventos pós-transducionais) / INTRODUCTION: In Multiple Endocrine Neoplasia type 2 (MEN2) the development of medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) are associated with activating germline mutations in RET proto-oncogene. Cases of sporadic MTC may have somatic RET mutations (~ 40%). The phenotypic variability observed in cases with familial MTC/MEN2 and PHEO/MEN2 indicates the probable involvement of additional genetic events that could be responsible for the clinical differences observed in the affected individuals (age development, progression and aggressiveness of the tumor). Other genetic alterations such as RET double mutations, SNPs and specific haplotypes may influence susceptibility, aggressiveness and MEN2 phenotype modulation. However, studies of other genes involved in the tumorigenesis of MEN2 are still in progress. Recently, it was shown that the activated RET controls the expression of cell cycle inhibitory proteins (p18 and p27). Germline mutations in the p27 gene have recently been associated with the susceptibility to neuroendocrine tumors and are associated with the MEN4 syndrome (Multiple endocrine neoplasia type 4). Somatic inactivating mutations p27 are rarely found in many types of tumors. However, several studies have documented that reduced expression and subcellular location of p27 is controlled by post-transductional changes and/or epigenetic factors. OBJECTIVES: This study aimed to evaluate the role of genes recently associated with RET activated in tumors from MEN2 patients and also check whether polymorphisms in the p27 gene would be acting as modulators of phenotype in a large MEN2 family. PATIENTS: We analyzed 66 tumor samples from 36 patients with clinical and genetic diagnosis of MEN2 and from 28 individuals belonging to a large family with FMTC/MEN2A and RET C620R mutation. METHODS: The analyses of somatic p27, p15, p18 and RET were performed by PCR and direct sequencing of DNA and microsatellite analysis was performed for p27 by PCR and capillary electrophoresis. Expression analysis and subcellular localization of p27 protein were performed by Western blot and immunohistochemistry. The analysis of phenotype modulation in MEN2A families was performed by the amplification of exon 1 of the p27 gene in a whole blood sample. RESULTS: There were no somatic mutations in the p27 gene and also in the p15 and p18 genes. However, we verified a low p27 protein expression in MTC/MEN2 and PHEO/MEN2 that showed a definite correlation with the type and aggressiveness of the mutated RET codon, mainly in those tumors from cases with germline RET codon 634 mutations (control vs 634, p=0,05; control vs 634/791, p= 0,032; 620 vs 634, p=0,045; 620 vs 634/791, p= 0,002; 620 vs 634 + 634/791, p=0,036). It was also verified a positive correlation between the immunohistochemistry expression of nuclear p27 subcellular location and the p27 p.V109G TT genotype (p=0,03). CONCLUSIONS: The reduction in the expression of p27 and its subcellular localization are likely to be associated with somatic changes in other genes that control the processes of phosphorylation of p27 protein through post-transductional events

INTRODUÇÃO: Na Neoplasia Endócrina Múltipla tipo 2 (NEM2), o desenvolvimento do Carcinoma Medular de Tireoide (CMT), Feocromocitoma (FEO) e Hiperparatireoidismo primário (HPT) está associado à mutações germinativas ativadoras no proto-oncogene RET. Casos de CMT esporádico podem apresentar mutações somáticas no RET (~40%). A variabilidade fenotípica observada em casos de CMT e FEO familiais associados à NEM2 indica o envolvimento de eventos genéticos adicionais que seriam responsáveis pelas diferenças clínicas observadas nos indivíduos afetados (idade de desenvolvimento, progressão e agressividade do tumor). Outras alterações genéticas no RET como duplas mutações, SNPs e haplótipos específicos podem influenciar na susceptibilidade, agressividade e modulação do fenótipo NEM2. Entretanto, os estudos de outros genes envolvidos no processo da tumorigênese NEM2 ainda estão em andamento. Recentemente foi mostrado que RET ativado controla a expressão de proteínas inibidoras do ciclo celular (p18 e p27). Mutações germinativas no gene p27 foram recentemente associadas à susceptibilidade de tumores neuroendócrinos e estão associadas à síndrome NEM4 (Neoplasia endócrina múltipla tipo 4). Mutações somáticas, inativadoras de p27, são raramente encontradas em vários tipos de tumores. Entretanto, diversos estudos documentaram que a redução na expressão e a sublocalização citoplamática de p27 são controladas por alterações pós-transducionais e/ou epigenéticas. OBJETIVOS: o estudo teve como objetivos avaliar a participação de genes, recentemente associados ao RET ativado, em tumores de pacientes com NEM2 e também verificar se polimorfismos no gene p27 estariam atuando como moduladores de fenótipo em uma grande família com NEM2. CASUÍTICA: foram analisadas 66 amostras tumorais advindas de 36 pacientes com diagnóstico clínico e genético de NEM2 e 28 indivíduos pertencentes a uma grande família com NEM2A-CMTF e mutação C620R no gene RET. MÉTODOS: As análises somáticas do p27 e também de p15, p18 e RET foram realizadas por PCR e sequenciamento direto de DNA e análise de microssatélites para p27 foi realizada por PCR e eletroforese capilar. Análises de expressão e localização da proteína p27 celular foram realizadas por Western blot e imunohistoquímica. A análise da modulação de fenótipo na família com NEM2A foi realizada por meio da amplificação do éxon 1 do gene p27 na amostra de sangue total. RESULTADOS: Não foram encontradas mutações somáticas no gene p27 e também nos genes p15 e p18. Entretanto, verificamos baixa expressão proteica de p27 em tumores CMT e FEO, a qual se encontrava relacionada com o tipo e agressividade do códon mutado no RET, principalmente em tumores que apresentavam mutação RET no códon 634 (controle x 634 p=0,05; controle x 634/791 p= 0,032; 620 x 634 p=0,045; 620 x 634/791 p= 0,002; 620 x 634 + 634/791 p=0,036). Notou-se também correlação positiva entre os níveis de expressão de p27 na localização nuclear, analisada por imunohistoquímica, e o genótipo TT do SNP p27 p.V109G (p=0,03). CONCLUSÕES: Alterações moleculares somáticas no gene p27 nos tumores NEM2 não são frequentes. Entretanto, a redução na expressão e a localização citoplasmática de p27 provavelmente estão associadas a alterações somáticas em outros genes que controlam os processos de fosforilação da proteína p27 (eventos pós-transducionais) / INTRODUCTION: In Multiple Endocrine Neoplasia type 2 (MEN2) the development of medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) are associated with activating germline mutations in RET proto-oncogene. Cases of sporadic MTC may have somatic RET mutations (~ 40%). The phenotypic variability observed in cases with familial MTC/MEN2 and PHEO/MEN2 indicates the probable involvement of additional genetic events that could be responsible for the clinical differences observed in the affected individuals (age development, progression and aggressiveness of the tumor). Other genetic alterations such as RET double mutations, SNPs and specific haplotypes may influence susceptibility, aggressiveness and MEN2 phenotype modulation. However, studies of other genes involved in the tumorigenesis of MEN2 are still in progress. Recently, it was shown that the activated RET controls the expression of cell cycle inhibitory proteins (p18 and p27). Germline mutations in the p27 gene have recently been associated with the susceptibility to neuroendocrine tumors and are associated with the MEN4 syndrome (Multiple endocrine neoplasia type 4). Somatic inactivating mutations p27 are rarely found in many types of tumors. However, several studies have documented that reduced expression and subcellular location of p27 is controlled by post-transductional changes and/or epigenetic factors. OBJECTIVES: This study aimed to evaluate the role of genes recently associated with RET activated in tumors from MEN2 patients and also check whether polymorphisms in the p27 gene would be acting as modulators of phenotype in a large MEN2 family. PATIENTS: We analyzed 66 tumor samples from 36 patients with clinical and genetic diagnosis of MEN2 and from 28 individuals belonging to a large family with FMTC/MEN2A and RET C620R mutation. METHODS: The analyses of somatic p27, p15, p18 and RET were performed by PCR and direct sequencing of DNA and microsatellite analysis was performed for p27 by PCR and capillary electrophoresis. Expression analysis and subcellular localization of p27 protein were performed by Western blot and immunohistochemistry. The analysis of phenotype modulation in MEN2A families was performed by the amplification of exon 1 of the p27 gene in a whole blood sample. RESULTS: There were no somatic mutations in the p27 gene and also in the p15 and p18 genes. However, we verified a low p27 protein expression in MTC/MEN2 and PHEO/MEN2 that showed a definite correlation with the type and aggressiveness of the mutated RET codon, mainly in those tumors from cases with germline RET codon 634 mutations (control vs 634, p=0,05; control vs 634/791, p= 0,032; 620 vs 634, p=0,045; 620 vs 634/791, p= 0,002; 620 vs 634 + 634/791, p=0,036). It was also verified a positive correlation between the immunohistochemistry expression of nuclear p27 subcellular location and the p27 p.V109G TT genotype (p=0,03). CONCLUSIONS: The reduction in the expression of p27 and its subcellular localization are likely to be associated with somatic changes in other genes that control the processes of phosphorylation of p27 protein through post-transductional events