The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle.

LIM Kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, has been shown to be involved in cell cycle progression. In this study we examine the role of LIMK1 in G1 phase and mitosis. We found ectopic expression of LIMK1 resulted in altered expression of p27Kip1, the G1 phase Cyclin D1/Cdk4 inhibitor. Overexpression of LIMK1 resulted in lower levels of p27Kip1 and p27Kip1-pY88 (inactive p27Kip1). Knockdown of LIMK1 resulted in elevated levels of p27Kip1 and p27Kip1-pY88. Together, these results suggest LIMK1 regulates progression of G1 phase through modulation of p27Kip1 expression. LIMK1 is involved in the mitotic process through inactivating phosphorylation of Cofilin. Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with ?-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one other's function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation. The substrates of LIMK1, Aur-A and Cofilin, are also involved in the mitotic process. Aur-A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through regulation of the actin cytoskeleton. Aur-A phosphorylates Cofilin at multiple sites including S3 resulting in inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during progression of prophase to metaphase. Inhibition of Aur-A activity altered subcellular localization of Cofilin and induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the early mitotic stage through regulation of actin cytoskeleton reorganization. ?

Breast cancer tops the list of new cancer cases and is predicted to be the second leading cause of cancer deaths in women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth and metastasis of triple-negative and basal-like breast cancers. To study increased growth and invasive behavior in triple-negative and basal-like breast cancers we utilize both an interesting and relevant cell culture model and examination of human tissue. In this study, we use the Tamoxifen-selected, MCF-7 derivative, TMX2-28 breast cancer cell line. TMX2-28 cells are triple-negative in that they lack expression of the estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). They also have acquired a mixed basal/luminal cytokeratin profile, suggestive of a more basal-like phenotype. TMX2-28 cells are highly proliferative and invasive. In addition to our cell culture model, we also examine human tissue. Thirty frozen breast carcinoma samples were evaluated for mRNA expression. Additionally, I analyzed protein expression, using immunohistochemistry (IHC), of 50 benign reduction mammoplasty and 188 breast tumors (formalin-fixed paraffin embedded). Of the 188 breast tumors, 93 were ERα-positive and 95 were ERα-negative. Of the 95 ERα-negative samples, 24 were further classified as non-triple negative (either PR or HER2 positive), 49 were classified as triple-negative, and 22 were not further classified due to unavailability of HER2 status and were used only in analyses of ERα-negative tumors. Thirty-seven of the 188 tumor samples were ductal carcinoma in situ, 138 were invasive ductal carcinomas, and 13 were classified as other. Lastly, 23 of the 188 tumors were grade 1, 48 were grade 2, 105 were grade 3, and 12 did not have grade data available. S-phase kinase-associated protein 2 (SKP2) plays an important role in cell cycle regulation by targeting p27 for degradation. The cyclin-dependent kinase (CDK) inhibitor p27 regulates G1/S transition by binding cyclin/CDK complexes and abrogating its activity. By targeting p27 for degradation, SKP2 frees the complexes needed to progress into the S phase of the cell cycle. Evaluation of SKP2 expression in TMX2-28 revealed significantly higher levels than in other breast cancer cell lines. Despite the high levels of SKP2 expression, p27 protein was not reduced. However, levels of the Serine 10 phosphorylated form of p27 (pSer10p27), which has been associated with increased proliferation rates, was found to be increased. Furthermore, suppression of SKP2 completely eliminated the pSer10p27 and slowed cycle progression confirming the role of SKP2 in the aggressive growth of TMX2-28 cells. Assessment of mRNA from 30 frozen human breast cancers demonstrated that SKP2 is more highly expressed in ERα-negative and basal-like breast cancers. Immunohistochemical analysis of 188 breast cancers and 50 benign reduction mammoplasty tissues confirmed that SKP2 is more highly expressed in ERα-negative breast cancers and for the first time demonstrated that triple-negative breast cancers are more likely to overexpress SKP2 than are non-triple-negative, but still ERα-negative, tumors. In contrast to some previous reports, we did not observe an inverse relationship between SKP2 and p27 expression. Only 11% of tumors expressed high SKP2 and low p27, while 32% of tumors had high SKP2 and high p27. Although no significant relationship between SKP2 and p27 expression was observed in human breast cancers, a significant positive relationship was discovered between SKP2 and pSer10p27. Furthermore, high levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple negative breast tumors than in ERα-positive breast cancers. Based on these results and those of the cell culture experiments showing complete elimination of pSer10p27 after suppression of SKP2 it appears that levels of pSer10p27 may be a better indicator of SKP2-dependent p27 degradation than are levels of p27. Therefore, that inhibiting SKP2 in triple-negative breast cancers expressing high levels of both SKP2 and pSer10p27 regardless of p27 levels may be a valid therapeutic approach. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk to patients diagnosed with triple-negative and basal-like breast cancers. Two distinct morphological/functional mechanisms are known for single cell migration in tissues: mesenchymal and amoeboid invasion. Mesenchymal movement involves the use of proteases that cause cellular lysis in tissues, thereby creating a path through which cells can invade. Amoeboid movement is protease-independent; cells find paths through the ECM by pushing and squeezing through regions of adequate size. Despite their invasive phenotype, TMX2-28 retains morphology similar to non-aggressive MCF-7 cells, suggesting that their invasion may be proteolytic-independent. We determined that TMX2-28 lack MMP-1 mRNA, and MMP-2/MMP-9 protein expression; each of which is important in protease-dependent invasion. Furthermore, TMX2-28 cells have low expression of other genes key to protease-dependent invasion including Slug, Zeb 1, Zeb 2, Vimentin, Fibronectin and N-cadherin. RhoA is a member of the Rho superfamily of GTPases that acts as a molecular switch to control signal transduction and is critical to the amoeboid invasion mechanism. TMX2-28 cells have high expression of protease-independent invasion genes such as RhoA, ROCK 1, ROCK 2, and E-cadherin. Finally, treating TMX2-28 cells with a RhoA pathway inhibitor or an shRNA targeting RhoA significantly reduces their invasiveness. These data suggest that TMX2-28 cells use a RhoA-dependent, proteolytic-independent invasion mechanism. Collectively, the data presented here demonstrate the roles of SKP2 and RhoA in triple-negative and basal-like breast cancers, making both genes, as well as their pathways, desirable therapeutic targets.

No description available.

All trans retinoic acid (atRA) has been shown to inhibit the growth of CAOV3 ovarian carcinoma cells. This results from arrest of the cell cycle during the G1 phase. G1 checkpoint is regulated by a multitude of molecules, including the retinoblastoma family of proteins, cyclin dependent kinases (Cdks), and cyclin dependent kinase inhibitors. P27, a cyclin dependent kinase inhibitor regulating G1 checkpoint has been shown to have elevated levels in response to atRA treatment. In these studies we investigated the role of p27 phosphorylation in mediating atRA induced growth inhibition. Our results show that atRA treatment of atRA sensitive CAOV3 cells leads to an increase in the levels of S10 phosphorylation of p27 in both nuclear and cytoplasmic cell compartments. This increase was accompanied by a decrease in the levels of skp2 protein, protein that plays a critical role in the degradation of p27. Similar results were not observed in SKOV3 cells which are not growth inhibited by atRA treatment. Finally, we demonstrated that overexpression of a mutant of p27 that cannot be phosphorylated on S10 induces a dominant negative effect on the endogenous p27 activity. This dominant negative effect reverses the atRA effect on p27 binding to CDKs, on inhibition of CDK activity, on the expression of S phase genes and ultimately on the inhibition of growth of ovarian carcinoma cells. These results suggest that hypophosphorylation of the serine10 locus of p27 might be one of the mechanisms that lead to atRA resistance of ovarian carcinoma cell. It is possible that atRA resistant ovarian tumors constitute an environment that hinders S10 phosphorylation and that by modulating the activity of the kinase(s) responsible for this event the atRA resistance can be overcome. / Microbiology and Immunology

Os tumores de glândula salivar compreendem em torno de 2 a 6,5% dos tumores de cabeça e pescoço. Entre os tumores de glândula salivar, o adenoma pleomórfico é benigno e o mais comum. O carcinoma adenoide cístico e adenocarcinoma polimorfo de baixo grau encontram-se entre os mais frequentes malignos. Jab1 é uma de muitas proteínas que afetam diversos estágios da tumorigênese sendo importante na regulação variadas vias de sinalização e/ou proteínas como p27 e AP-1, a última composta por c-jun e c-fos, que são principalmente relacionadas com o ciclo celular e proliferação celular. O objetivo desse trabalho foi avaliar a expressão imuno-histoquímica das proteínas Jab1, p27, c-jun e c-fos no adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico das glândulas salivares. Foi realizada análise imuno-histoquímica semi-quantitativa das células marcadas nos tumores de glândula salivar e glândula salivar normal de acordo com o escore 0 (células sem expressão), 1(> 0 <= 5% de células marcadas), 2 (> 5 <= 50%) and 3 (> 50%). Para Jab1, c-jun e c-fos foi considerado apenas marcação nuclear e para p27, nuclear e citoplasmática, separadamente. Os resultados foram analisados utilizando-se os testes de Kruskal-Wallis, de Mann-Whitney, do Qui-quadrado e o teste de correlação de Spearman, cujo nível de significância foi de p<0,05 e processados com o auxílio do software GraphPad Prisma 5.0. A análise estatística revelou que a expressão de Jab1 foi significante no adenoma pleomórfico e no carcinoma adenoide cístico em relação aos ductos e no adenocarcinoma polimorfo de baixo grau em relação ao carcinoma adenoide cístico (p=0,0136, 0,0001 e 0,0344, respectivamente); a expressão de p27 nuclear foi significante no adenoma pleomórfico e no adenocarcinoma polimorfo de baixo grau quando comparados ao carcinoma adenoide cístico (p=0,0074 e 0,0004, respectivamente) e a expressão citoplasmática em todos os grupos quando comparados aos ácinos; c-fos, a expressão foi significativa nos ductos ao compará-los ao adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico (p=0,0002, 0,0048 e 0,0352, respectivamente). O teste de correlação de Spearman de Jab1, p27, c-jun e c-fos em cada lesão separadamente revelou que no adenoma pleomórfico houve correlação significativa entre Jab1 e p27 (r=0,371; p=0,020) e entre c-jun e c-fos (r=0,452; p=0,004). No adenocarcinoma polimorfo de baixo grau, houve correlação entre Jab1 e p27 (r=0,494; p=0,044) e no carcinoma adenoide cístico, entre p27 e c-fos (r=0,513; p=0,035). Foi concluído que a tumorigênese do adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico parece estar associada à expressão de Jab1 e p27. / Salivary gland tumors comprise about 2 to 6.5% of the head and neck tumors. Among the salivary gland tumors, pleomorphic adenoma is the most common and benign tumor. Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are the most frequent malignant tumors. Jab1 is one of many proteins which affects many stages of the tumorigenesis and regulates positively and negatively several pathways and/or proteins such as p27 and AP-1, the latter composed by c-jun and c-fos, which are mostly related to cell cycle and cell proliferation. The aim of this study was to evaluate the immunoexpression of the proteins Jab1, p27, c-jun and c-fos in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma of the salivary glands. The semi-quantitative immunohistochemical analysis was performed in salivary gland tumors and in normal salivary gland according to the score 0 (no stained cells), 1 (> 0 <= 5% of stained cells), 2 (> 5 <= 50%) and 3 (> 50%). Nuclear immunostaining alone was considered for Jab1, c-jun and c-fos proteins and cytoplasmic and nuclear staining for p27. Results were analyzed in GraphPad Prisma 5.0 software using Kruskal-Wallis, Mann-Whitney and Chi-square tests and Spearman correlation test in which significancy level was p<0,05. Statistical analysis revealed that Jab1 expression was significant in pleomorphic adenoma and adenoid cystic carcinoma in relation to ducts and in polymorphous low-grade adenocarcinoma in relation to adenoid cystic carcinoma (p=0,0136, 0,0001 e 0,0344, respectively); the p27 nuclear expression was significant in pleomorphic adenoma and in polymorphous low-grade adenocarcinoma when compared to adenoid cystic carcinoma (p=0,0074 e 0,0004, respectively) and cytoplasmic immunostaining was significant in all groups when compared to acini; c-fos expression was significant in ducts if compared to pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (p=0,0002, 0,0048 e 0,0352, respectively). Spearman correlation test to Jab1, p27, c-jun and c-fos in each lesion separately revealed significant correlation between Jab1 and p27 (r=0,371; p=0,020) and c-jun and c-fos (r=0,452; p=0,004) in pleomorphic adenoma. There was correlation between Jab1 and p27 (r=0,494; p=0,044) in polymorphous low-grade adenocarcinoma and between p27 and c-fos (r=0,513; p=0,035) in adenoid cystic carcinoma. In conclusion, tumorigenesis in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma seems to be associated to expression of Jab1 and p27.

Os tumores de glândula salivar compreendem em torno de 2 a 6,5% dos tumores de cabeça e pescoço. Entre os tumores de glândula salivar, o adenoma pleomórfico é benigno e o mais comum. O carcinoma adenoide cístico e adenocarcinoma polimorfo de baixo grau encontram-se entre os mais frequentes malignos. Jab1 é uma de muitas proteínas que afetam diversos estágios da tumorigênese sendo importante na regulação variadas vias de sinalização e/ou proteínas como p27 e AP-1, a última composta por c-jun e c-fos, que são principalmente relacionadas com o ciclo celular e proliferação celular. O objetivo desse trabalho foi avaliar a expressão imuno-histoquímica das proteínas Jab1, p27, c-jun e c-fos no adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico das glândulas salivares. Foi realizada análise imuno-histoquímica semi-quantitativa das células marcadas nos tumores de glândula salivar e glândula salivar normal de acordo com o escore 0 (células sem expressão), 1(> 0 <= 5% de células marcadas), 2 (> 5 <= 50%) and 3 (> 50%). Para Jab1, c-jun e c-fos foi considerado apenas marcação nuclear e para p27, nuclear e citoplasmática, separadamente. Os resultados foram analisados utilizando-se os testes de Kruskal-Wallis, de Mann-Whitney, do Qui-quadrado e o teste de correlação de Spearman, cujo nível de significância foi de p<0,05 e processados com o auxílio do software GraphPad Prisma 5.0. A análise estatística revelou que a expressão de Jab1 foi significante no adenoma pleomórfico e no carcinoma adenoide cístico em relação aos ductos e no adenocarcinoma polimorfo de baixo grau em relação ao carcinoma adenoide cístico (p=0,0136, 0,0001 e 0,0344, respectivamente); a expressão de p27 nuclear foi significante no adenoma pleomórfico e no adenocarcinoma polimorfo de baixo grau quando comparados ao carcinoma adenoide cístico (p=0,0074 e 0,0004, respectivamente) e a expressão citoplasmática em todos os grupos quando comparados aos ácinos; c-fos, a expressão foi significativa nos ductos ao compará-los ao adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico (p=0,0002, 0,0048 e 0,0352, respectivamente). O teste de correlação de Spearman de Jab1, p27, c-jun e c-fos em cada lesão separadamente revelou que no adenoma pleomórfico houve correlação significativa entre Jab1 e p27 (r=0,371; p=0,020) e entre c-jun e c-fos (r=0,452; p=0,004). No adenocarcinoma polimorfo de baixo grau, houve correlação entre Jab1 e p27 (r=0,494; p=0,044) e no carcinoma adenoide cístico, entre p27 e c-fos (r=0,513; p=0,035). Foi concluído que a tumorigênese do adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico parece estar associada à expressão de Jab1 e p27. / Salivary gland tumors comprise about 2 to 6.5% of the head and neck tumors. Among the salivary gland tumors, pleomorphic adenoma is the most common and benign tumor. Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are the most frequent malignant tumors. Jab1 is one of many proteins which affects many stages of the tumorigenesis and regulates positively and negatively several pathways and/or proteins such as p27 and AP-1, the latter composed by c-jun and c-fos, which are mostly related to cell cycle and cell proliferation. The aim of this study was to evaluate the immunoexpression of the proteins Jab1, p27, c-jun and c-fos in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma of the salivary glands. The semi-quantitative immunohistochemical analysis was performed in salivary gland tumors and in normal salivary gland according to the score 0 (no stained cells), 1 (> 0 <= 5% of stained cells), 2 (> 5 <= 50%) and 3 (> 50%). Nuclear immunostaining alone was considered for Jab1, c-jun and c-fos proteins and cytoplasmic and nuclear staining for p27. Results were analyzed in GraphPad Prisma 5.0 software using Kruskal-Wallis, Mann-Whitney and Chi-square tests and Spearman correlation test in which significancy level was p<0,05. Statistical analysis revealed that Jab1 expression was significant in pleomorphic adenoma and adenoid cystic carcinoma in relation to ducts and in polymorphous low-grade adenocarcinoma in relation to adenoid cystic carcinoma (p=0,0136, 0,0001 e 0,0344, respectively); the p27 nuclear expression was significant in pleomorphic adenoma and in polymorphous low-grade adenocarcinoma when compared to adenoid cystic carcinoma (p=0,0074 e 0,0004, respectively) and cytoplasmic immunostaining was significant in all groups when compared to acini; c-fos expression was significant in ducts if compared to pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (p=0,0002, 0,0048 e 0,0352, respectively). Spearman correlation test to Jab1, p27, c-jun and c-fos in each lesion separately revealed significant correlation between Jab1 and p27 (r=0,371; p=0,020) and c-jun and c-fos (r=0,452; p=0,004) in pleomorphic adenoma. There was correlation between Jab1 and p27 (r=0,494; p=0,044) in polymorphous low-grade adenocarcinoma and between p27 and c-fos (r=0,513; p=0,035) in adenoid cystic carcinoma. In conclusion, tumorigenesis in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma seems to be associated to expression of Jab1 and p27.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Avaliou-se a expressÃo das proteÃnas bax, bcl-2 e P27 no carcinoma espinocelular invasivo da cavidade bucal atravÃs da tÃcnica da imunohistoquÃmica para conhecer-se o perfil apoptÃtico destes tumores. Buscou-se detectar os fatores epidemiolÃgicos e o comportamento clÃnico dos pacientes acometidos por esta neoplasia. Procurou-se empregar parÃmetros estatÃsticos diferentes do odds ratio para comparaÃÃes posteriores. Analisando-se quarenta e oito pacientes da Santa Casa da MisericÃrdia de Fortaleza, Cearà foi possÃvel conhecer os fatores sexo, escolaridade, renda familiar, tabagismo (forma e tempo de consumo,tempo de abandono, quantidade consumida), etilismo(tipo de bebida, tempo de consumo, quantidade consumida, freqÃÃncia). Com a anÃlise do laudo histopatolÃgico da peÃa cirÃrgica soube-se das informaÃÃes referentes ao tumor em si (localizaÃÃo, gradaÃÃo histolÃgica, tamanho, linfonodos envolvidos). Obteve-se fragmentos da peÃas cirÃrgicas e foram confeccionadas lÃminas para a verificaÃÃo da invasividade atravÃs da coloraÃÃo em hematoxilina e eosina. Em seguida foi realizada a reaÃÃo de imunohistoquÃmica foi pelo mÃtodo da estrepto-avidina-biotina-peroxidase objetivando-se avaliar a expressÃo das proteÃnas supracitadas. As lÃminas com marcaÃÃo positiva foram submetidas a contagem de no mÃnimo mil cÃlulas e este resultado foi empregado nos mÃtodos LI (Labelling Index) e HS(HScore). A avaliaÃÃo dos resultados foi atravÃs de mÃtodos descritivos, sendo os testes realizados o qui-quadrado e o teste exato de Fisher com nÃvel de significÃncia a dez por cento.Os resultados apontaram uma mÃdia de idade de cinqÃenta e sete anos e o maior nÃmero de indivÃduos do sexo masculino, analfabetos, com renda familiar de um salÃrio-mÃnimo na sua maioria. A forma de consumo de tabaco, em maior nÃmero encontrada, foi o cigarro industrializado com vinte anos de consumo. A ingestÃo de bebidas destiladas superou a de fermentadas com mÃdia de vinte anos. O soalho bucal foi a localizaÃÃo com maior nÃmero de casos, com uma mÃdia de tamanho de trÃs e meio centÃmetros, o estadiamento patolÃgico predominante foi o pT2, com gradaÃÃo histolÃgica moderadamente diferenciada em maior quantidade. Dentre os cruzamentos realizados, houve correlaÃÃo estatÃstica entre o tamanho de tumores com as faixas etÃrias(P=0,084 para &#945;=10%); tamanho dos tumores com envolvimento de linfonodos(P=0,085 para &#945;=10%); sexo dos pacientes com envolvimento dos linfonodos (P=0,03 para &#945;=10%). Os resultados das reaÃÃes de imunohistoquÃmica mostraram um maior percentual de casos positivos para bax (77,1%) seguido de P27(45,9%) e bcl-2 (16,6%). As mÃdias dos LI encontradas apontaram bax com 67, 766; seguida de bcl-2 com 10,804; e P27 com 7,989. Cruzando-se os H-scores dos marcadores entre si encontrou-se correlaÃÃo positiva entre bax e P27 (0,245 na correlaÃÃo de Pearson). Os parÃmetros estatÃsticos avaliados foram: mÃdia e seu erro padrÃo; mediana; moda; desvio padrÃo; curtose e seu erro padrÃo; mÃnimo; mÃximo e quartis. Os resultados mostram uma tendÃncia a apoptose nos carcinomas espinocelulares bucais / The evaluation of the expression of bax, bcl-2 and p27 proteins at invasive squamous cell carcinoma of oral cavity was done using the immunohistochemistry technique to know about apoptotic profile of these neoplasms. The epidemiological factors and the clinical behavior of the patients were detected, too. Statistical parameters different from odds ratio was employed for future comparisons. Forty-eight patients from Santa Casa da MisericÃrdia de Fortaleza, CearÃ, Brazil was analyzed and with this analysis was possible to know the prognostic factors: sex of patients, instruction grade, familiar gains, tobacco consumption (form of consumption, duration of consumption, period of forsaking and quantity consumed), alcohol consumption (nature of drinking, duration of consumption, period of forsaking, quantity consumed, periodicity). In a posterior moment, with the histopathological report from the surgical specimen was possible to know about localization of neoplasm, size of neoplasm, histological grade, nodes involvement and invasiveness. Pieces of the neoplasm were achieved from surgical specimen and glass slides were done to analyze the invasiveness by hematoxilin-eosin coloration. After the immunohistochemistry reaction by streptoavidin biotin technique was done to evaluate the expression of proteins above. The glass slides with positive reaction were submitted under a counting and, at least, one thousand cells were counted. The results from counting were submitted under the LI(Labelling Index) and HS(H Score) methods. The evaluation of the results was made using descriptive methods and the statistical tests were qui-square and Fisherâs exact test with 10% significance. The results showed the mean age was 57 years old, more males than females, analphabets and one minimum wage the mean familiar gain. The main form of tobacco consumption was industrialized cigarette with 20 years of consumption. The swallow of distillers drinking was bigger than fermentation ones with 20 years of drinking at mean. The floor of the mouth was the anatomic site with more number of cases and the mean size of neoplasm was 1.4 inches. The preponderant pathological staging detected was pT2 and the preponderant histological grade was moderately differentiated. Among the cross tabs realized, there were statistical correlation between size of tumors and age (P=0.084; &#945;=10%), between size of tumors and nodes involvement (P=0,085; &#945;=10%), between sex of patients and nodes involvement (P=0.03; &#945;=10%). The results from immunohistochemical reactions were more positive to bax (77.1% of positive cases), P27(45.9% of positive cases) and bcl-2 (16.6% of positive cases). The mean of LI showed bax at first position (67.766); second bcl-2 (10.804) and P27(7.989). The cross tabs among HS showed statistical positive correlation between bax and P27 (0.245 at Pearsonâs correlation). The statistical parameters were: mean and its standard error, median, mode, standard deviation, kurtosis and its standard error, minimum, maximum and percentiles. The conclusions showed apoptosis propensity at oral squamous cell carcinoma

Poor prognosis for high-risk neuroblastoma patients makes it necessary to find novel treatmentstrategies. This work aims to understand the cell cycle behavior of various high-riskneuroblastoma cell lines following chemotherapy treatment. Here, we mapped the expressionof cell cycle dependent proteins, p21 and p27, in seven high-risk neuroblastoma celllines. All cell lines showed an overall impaired growth following doxorubicin treatment.However, regrowth was observed in all cell lines between day 6 to 15 by forming colonies.The expression of p21 and p27 was measured in all cell lines showing an upregulationof p21 in 3 out of 5 p53 mutated cell lines while it was downregulated in the 2 cell lineswith a p53 wild type. Furthermore, inhibition assays using inhibitors of CHK1/2, p21 ,andSKP2 were performed. The results were promising as the CHK1/2 inhibitor reduced cellviability in all tested cell lines, while the p21 inhibitor had an effect in 3 out of 6 testedcell lines and the SKP2 inhibitor in 4 out of 6 tested cell lines. Confluency measurementover 15 days showed impaired growth following treatment with the CHK1/2 inhibitor for3 out of 6 tested cell lines and p21 inhibitor in 1 out of 6 tested cell lines. The obtainedresults were encouraging and might aid in finding a novel treatment strategy preventingresistance and relapse in neuroblastoma. However, further studies are needed in order tovalidate the efficacy and safety of these promising drugs in neuroblastoma patients. / Dålig prognos för högrisk neuroblastompatienter gör det nödvändigt att hitta nya behandlingsstrategier.Detta arbete syftar till att förstå cellcykelbeteendet hos olika högrisk neuroblastomcellinjerefter kemoterapibehandling. I denna studie kartlades uttrycket av cellcykelberoendeproteinerna, p21 och p27, i sju högrisk neuroblastomcellinjer. Alla cellinjervisade en total nedsatt tillväxt efter doxorubicinbehandling. Återväxt observerades emellertidmellan dag 6 och 15 genom bildandet av kolonier. Uttrycket av p21 och p27 mättesi alla cellinjer. Resultaten visade en uppreglering av p21 i 3 av 5 p53-muterade cellinjermedans det nedreglerades i de två cellinjerna med en p53-vildtyp. Vidare utfördes inhiberingsanalysermed användning av hämmare mot CHK1/2, p21 eller, SKP2. Resultatenär lovande då CHK1/2-hämmaren reducerade cellviabiliteten i alla testade cellinjer, medanp21-hämmaren hade en effekt i 3 av 6 testade cellinjer och SKP2 i 4 av 6 testadecellinjer. Konfluensmätning under 15 dagar visade nedsatt tillväxt efter behandling medCHK1/2-hämmaren för 3 av 6 testade cellinjer och p21-hämmare i 1 av 6 testade cellinjer.De erhållna resultaten är lovande och kan hjälpa till att hitta en ny behandlingsstrategi somförhindrar resistens och återfall i neuroblastom. Ytterligare studier behövs emellertid föratt validera effektiviteten och säkerheten för dessa lovande läkemedel hos neuroblastompatienter.

La progression dans le cycle cellulaire est contrôlée par de vagues oscillantes de cyclines et des kinases cycline-dépendantes (Cdk). Ces kinases sont régulées positivement par l’association des sous-unités cyclines régulatrices et négativement en se liant aux inhibiteurs de Cdk. Parmi ces derniers, p27 inhibe tous les complexes cycline-Cdk quelle que soit la phase cellulaire et agit en tant que régulateur négatif principal de la prolifération cellulaire dans une variété de cellules et de tissus. Intrinsèquement, p27 phosphorylé est ubiquitiné et dégradé par le complexe SCFSkp2-Cks1. Des études génétiques de la souris, ainsi que des examens cliniques chez l’homme, ont montré que p27 est un important suppresseur de tumeur. Le gène est rarement muté. Cependant, p27 est fréquemment réprimé dans les cancers humains en raison d’une augmentation de l’expression de Skp2 et de Cks1 dans le noyau, ce qui est généralement associée à un mauvais pronostic. La localisation subcellulaire de Cks1 est donc d'une importance primordiale dans le contrôle de la prolifération cellulaire. Les résultats récents de notre laboratoire ont montré une interaction entre Cks1 et les protéines de transport nucléaire importine α1 et β3. Aussi, l’analyse de la séquence primaire de Cks1 a également révélé un signal de localisation nucléaire classique (NLS) à son extrémité C-terminale. Des mutations ont été effectuées sur le NLS suspect pour déterminer si oui ou non l'import nucléaire de Cks1 était contrôlé par cette séquence. Un inhibiteur synthétique de l’importine β a également été utilisé pour étudier l’import de Cks1 dans le noyau. Les résultats indiquent que l’extrémité C-terminale de Cks1 est en effet un NLS puisque les mutations de Cks1 et l'inhibition de l’importine β conduisent, tous deux, à l'accumulation de Cks1 dans le cytoplasme. Ces résultats ont été utiles pour mieux comprendre le mécanisme régulant la localisation de Cks1. Toutefois, des travaux futurs sont nécessaires pour mieux comprendre l'impact de la séquestration cytoplasmique de Cks1 sur le cancer et ainsi espérer aboutir à l'identification de nouvelles cibles pharmacologiques impliqués dans la prolifération cellulaire. / Progression through the cell cycle is controlled by oscillating waves of cyclins and cyclin-dependent kinases (Cdk). These kinases are regulated positively by association with cyclin regulatory subunits and negatively by binding of Cdk inhibitors. Among the latter, p27 inhibits all cyclin-Cdk complexes regardless of the cell cycle phase and acts as a primary negative regulator of cell proliferation in a variety of cell types and tissues. Intrinsically, phosphorylated p27 is ubiquitinated and degraded by the SCFSkp2-Cks1 complex. Mouse genetic studies and human clinical investigations have shown p27 as an important tumor suppressor, which gene is rarely mutated. However, p27 is frequently downregulated in human cancers due to an increased expression of nuclear Cks1 and this is usually associated with a poor prognosis. The subcellular localization of Cks1 is thus of primordial importance in the control of cell proliferation. Recent results from our laboratory have shown an interaction between Cks1 and nuclear transport proteins α1 and β3 importin. Analysis of the primary sequence of Cks1 also revealed a classic nuclear localization signal (NLS) at its C-terminal. Mutations have been done on the suspected NLS to determine whether or not Cks1’s nuclear import is regulated by this motif. A synthetic inhibitor of β importin has also been used to study the mechanism of Cks1 import. Results indicated that the C-terminal end of Cks1 is indeed a NLS since mutations of Cks1 and inhibition of β importin both lead to accumulation of Cks1 in the cytoplasm. These outcomes were helpful to better understand the mechanism regulating Cks1 localization. However, future works are required to further understand the impact of cytoplasmic sequestration of Cks1 on cancer and hopefully lead to the identification of novel pharmacological targets involved in cell proliferation.