As proteínas MDM2, P53, P21 e pAKT estão entre as muitas já identificadas que visam, por meio do equilíbrio direto e indireto entre si, manter o balanço entre morte e proliferação celular. Existe um leque aberto de hipóteses sobre a via de atuação dessas proteínas na tumorigênese de glândulas salivares, porém não há dados conclusivos. O objetivo deste estudo foi avaliar a expressão das proteínas MDM2, P53, P21 e pAkt em carcinoma adenóide cístico e adenocarcinoma não-específico de glândula salivar, através das técnicas de imunoistoquímica, em casos fixados em parafina; e imunofluorescência e western-blotting, em linhagens celulares provenientes dessas lesões. Os estudos imunoistoquímicos mostraram expressão de MDM2 e pAKT na maioria dos carcinomas adenóides císticos (CAC) avaliados e nos 3 casos de adenocarcinoma não-específicos (ANE). As linhagens Cac2 (proveniente de carcinoma adenóide cístico) e HSG (derivada de adenocarcinoma não-específico) também exibiram expressão das proteínas MDM2 e pAKT tanto no núcleo quanto no citoplasma celular. A proteína P53 mostrou expressão variável entre os diferentes CAC analisados e os 3 casos de ANE estudados mostraram marcação positiva para a proteína P53. Com relação às linhagens celulares, a Cac2 não expressou a proteína P53, enquanto a HSG apresentou expressão nuclear e citoplasmática dessa proteína. As glândulas salivares normais não exibiram marcação imunoistoquímica para as proteínas MDM2, P53, P21 e pAKT. Os resultados deste estudo sugerem que as proteínas pAKT e MDM2 estão envolvidas na tumorigênese e/ou progressão tumoral de carcinoma adenóide cístico e adenocarcinoma não especifico. / MDM2, P53, P21 and pAKT are proteins co-related to the balance between cell death and survival. There are many hypothesis on the role of these proteins in salivary gland tumorigenesis, however, no conclusive data have been published. Theaim of this study was to evaluate the expression of MDM2, P53, P21 and pAKT proteins on adenoid cystic carcinomas (ACC) and adenocarcinoma not otherwise specified (ANOS) through immunohistochemistry, immunofluorescence and westernblotting techniques. The immunostaining studies showed MDM2 and pAKT expression in most cases of adenoid cystic carcinoma and in all adenocarcinoma not-otherwise specified. The cell lines CAC2 (derived from adenoid cystic carcinoma) and HSG (derived from adenocarcinoma not otherwise specified) also showed nuclear and cytoplasmic expression of MDM2 and pAKT. The expression of P53 was variable in the different ACCs analyzed and was absent on CAC2 cells. However, P53 was strongly positive in all ANOS, and HSG cells also showed nuclear and cytoplasmic staining. No MDM2, P53, P21 and pAKT expression was found in normal salivary glands. Therefore, MDM2 and pAKT may participate in the tumorigenesis and/or progression of adenoid cystic carcinoma and adenocarcinoma not otherwise specified.

As proteínas MDM2, P53, P21 e pAKT estão entre as muitas já identificadas que visam, por meio do equilíbrio direto e indireto entre si, manter o balanço entre morte e proliferação celular. Existe um leque aberto de hipóteses sobre a via de atuação dessas proteínas na tumorigênese de glândulas salivares, porém não há dados conclusivos. O objetivo deste estudo foi avaliar a expressão das proteínas MDM2, P53, P21 e pAkt em carcinoma adenóide cístico e adenocarcinoma não-específico de glândula salivar, através das técnicas de imunoistoquímica, em casos fixados em parafina; e imunofluorescência e western-blotting, em linhagens celulares provenientes dessas lesões. Os estudos imunoistoquímicos mostraram expressão de MDM2 e pAKT na maioria dos carcinomas adenóides císticos (CAC) avaliados e nos 3 casos de adenocarcinoma não-específicos (ANE). As linhagens Cac2 (proveniente de carcinoma adenóide cístico) e HSG (derivada de adenocarcinoma não-específico) também exibiram expressão das proteínas MDM2 e pAKT tanto no núcleo quanto no citoplasma celular. A proteína P53 mostrou expressão variável entre os diferentes CAC analisados e os 3 casos de ANE estudados mostraram marcação positiva para a proteína P53. Com relação às linhagens celulares, a Cac2 não expressou a proteína P53, enquanto a HSG apresentou expressão nuclear e citoplasmática dessa proteína. As glândulas salivares normais não exibiram marcação imunoistoquímica para as proteínas MDM2, P53, P21 e pAKT. Os resultados deste estudo sugerem que as proteínas pAKT e MDM2 estão envolvidas na tumorigênese e/ou progressão tumoral de carcinoma adenóide cístico e adenocarcinoma não especifico. / MDM2, P53, P21 and pAKT are proteins co-related to the balance between cell death and survival. There are many hypothesis on the role of these proteins in salivary gland tumorigenesis, however, no conclusive data have been published. Theaim of this study was to evaluate the expression of MDM2, P53, P21 and pAKT proteins on adenoid cystic carcinomas (ACC) and adenocarcinoma not otherwise specified (ANOS) through immunohistochemistry, immunofluorescence and westernblotting techniques. The immunostaining studies showed MDM2 and pAKT expression in most cases of adenoid cystic carcinoma and in all adenocarcinoma not-otherwise specified. The cell lines CAC2 (derived from adenoid cystic carcinoma) and HSG (derived from adenocarcinoma not otherwise specified) also showed nuclear and cytoplasmic expression of MDM2 and pAKT. The expression of P53 was variable in the different ACCs analyzed and was absent on CAC2 cells. However, P53 was strongly positive in all ANOS, and HSG cells also showed nuclear and cytoplasmic staining. No MDM2, P53, P21 and pAKT expression was found in normal salivary glands. Therefore, MDM2 and pAKT may participate in the tumorigenesis and/or progression of adenoid cystic carcinoma and adenocarcinoma not otherwise specified.

<p>TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes.</p><p>The cell cycle inhibitor p21^Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect.</p><p>The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal <i>p21</i> promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as <i>Id2</i> that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene <i>Meox2</i>, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur.</p><p>This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.</p>

TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes. The cell cycle inhibitor p21Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect. The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal p21 promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as Id2 that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene Meox2, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur. This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.

Existeixen moltes evidències que indiquen que les diferents funcions descrites de p21(Cip1) es deuen, en certa part, a la seva localització intracel·lular. Mentre que la p21(Cip1) nuclear inhibeix la proliferació cel·lular, la p21(Cip1) citoplasmàtica és capaç de regular la supervivència i la mobilitat cel·lular. El fet que les funcions de p21(Cip1) en cada un dels compartiments cel·lulars tinguin papers oposats fa que sigui d'un gran interès l'estudi dels canvis de localització de p21(Cip1) i els mecanismes que regulen aquesta translocació, així com les possibles vies que p21(Cip1) pugui fer servir per a localitzar-se en els diferents compartiments cel·lulars.En aquesta tesi s'han realitzat dos estudis diferenciats, però amb un objectiu comú ja que tots dos es centren en l'anàlisi dels mecanismes reguladors de la localització cel·lular de p21(Cip1). En la primera part d'aquesta tesi, observem la importància de la fosforilació de p21(Cip1) per part de la proteïna cinasa C (PKC). Aquesta fosforilació té lloc en el residu Ser153, molt proper a la senyal de localització nuclear (NLS) de la p21(Cip1) i es capaç de regular la localització cel·lular de p21(Cip1). Aquests resultats, juntament amb altres treballs, demostren que la fosforilació de p21(Cip1) afavoreix la seva localització en el citoplasma. Diferents aproximacions experimentals ens van permetre observar com aquesta fosforilació inhibeix la unió entre p21(Cip1) i CaM. D'aquest primer treball se'n deriva un model de regulació de la localització cel·lular de p21(Cip1) en el qual la unió a CaM i la fosforilació per PKC tenen papers oposats. D'una banda, la unió de p21(Cip1) a CaM inhibeix la seva fosforilació per part de PKC i afavoreix la localització de p21(Cip1) en el nucli. D'altra banda, la fosforilació de p21(Cip1) en el residu Ser153 indueix una localització citoplasmàtica de p21(Cip1). Treballs posteriors ens van permetre observar com la sortida de p21(Cip1) del nucli cap al citoplasma també està regulada. Després del dany al DNA, els nivells cel·lulars de p21(Cip1) incrementen, especialment en el nucli, per tal d'assegurar una aturada del cicle cel·lular. Observem com en resposta al dany al DNA, p21(Cip1) s'acumula no només en el nucleoplasma de les cèl·lules sinó que també s'acumula en el nuclèol. En les cèl·lules danyades els components nucleolars es troben desorganitzats i el nuclèol perd els contactes amb l'embolcall nuclear i presenta unes estructures esfèriques en el seu interior. La p21(Cip1) present en les estructures esfèriques del nuclèol està en un equilibri dinàmic amb la p21(Cip1) del nucleoplasma i la presència de p21(Cip1) en aquestes estructures correlaciona amb una inhibició de l'export p21(Cip1) cap al citoplasma. Aquests resultats donen suport a l'existència d'una via d'export de proteïnes nuclears a través del nuclèol, semblant a la que intervé en l'export de ribosomes, la qual es veuria afectada amb la desestructuració dels nuclèols en resposta al dany cel·lular. A més, amb els resultats obtinguts no descartem la possibilitat que p21(Cip1) pugui ser modificada en el nuclèol ja que en resposta al dany al DNA també s'hi localitzen altres proteïnes implicades en diferents vies de modificació post-traduccional. Així doncs, l'acumulació nuclear de p21(Cip1) en resposta al dany al DNA no es deu únicament a un increment de la seva transcripció sinó que aquesta acumulació també es deguda a la inhibició de la sortida de p21(Cip1) cap al citoplasma a través del nuclèol.En conclusió, tant l'entrada com la sortida de la p21(Cip1) del nucli és un mecanisme altament regulat que farà que la localització de p21(Cip1) pugui variar en diferents situacions fisiològiques de la cèl·lula. Aquest fet és de gran importància per al correcte funcionament cel·lular ja que com hem descrit anteriorment, p21(Cip1) és una proteïna amb funcions oposades depenent de la seva localització intracel·lular: oncogènica al citoplasma i supressora de tumors al nucli. / It is well known that p21(Cip1) is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. The importance of p21(Cip1) cellular localization indicates that it has to be precisely regulated.On one side we observed the importance of p21(Cip1) phosphorylation by PKC inducing its cytoplasmic localization. PKC phosphorylates p21(Cip1) at Ser 153 and when phosphorylated, p21(Cip1) can not bind to CaM. From this study we conclude that CaM and PKC have an opposite role in the regulation of p21(Cip1) localization: CaM binding to p21(Cip1) prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization; while when phosphorylated at Ser153, p21(Cip1) is located at the cytoplasm.On the other side the export of p21(Cip1) from the nucleus to the cytoplasm is also regulated. After DNA damage, p21(Cip1) increases and accumulates in the nucleus to ensure cell cycle arrest. We observed that after DNA damage p21(Cip1) accumulates not only in the nucleoplasm but also in the disrupted nucleoli. In damaged cells the nucleolar components are disorganized and nucleoli have lost their contacts with the nuclear envelope and appear with spherical structures inside. The nucleolar p21(Cip1) forms a dynamic equilibrium between the nucleolus and the nucleoplasm and correlates with the inhibition of p21(Cip1) nuclear export. This result proves the existence of a nucleolar export route to the cytoplasm for p21(Cip1) similar to the one described for the ribosome export. Moreover, the results obtained suggested that p21(Cip1) could be modified in the nucleolus in response to DNA damage as different proteins involved in post-translation modifications also localize in the nucleoli after the damage. Thus, after DNA damage, p21(Cip1) accumulates in the nucleus due to an increase in its transcription and due to an inhibition of its export to the cytoplasm.All this results together indicate the importance of p21(Cip1) localization depending on the cellular context and that its localization is precisely regulated by different pathways.

As we age, our ability to cope with a variety of stresses significantly decreases. One of the features of an ageing organism is the dramatic increase in the number of cells arrested in the G1 phase, a process known as senescence. It is well established that the senescence phenotype leads to a change in the way cells respond to stress. However, the molecular mechanisms by which these cells cope and/or respond to a variety of environmental challenges remain unknown. In general, cells respond to stress by engaging a variety of mechanisms; one of them is the assembly of cytoplasmic foci known as stress granules (SGs). These entities are considered as part of the survival pathways that are activated at the beginning of any stress to protect key cellular elements which allow a quick recovery if the stress is rapidly removed. However, we do not know whether SGs formation is activated during senescence. In this study, we investigated the formation and the role of SGs in senescent cells exposed to various stresses. We demonstrated that while SGs can assemble in response to oxidative stress (OS) during all the steps leading to senescence activation, their number significantly increases at late stage of senescence. This increase correlates with a rapid decrease in the expression of the cyclin kinase inhibitor p21, one of the main players in the activation of the senescence phenotype. Although the OS-induced recruitment of p21 mRNA to SGs correlates with a significant increase in its half-life, this translocation interferes with p21 translation only at late senescence. This translation inhibition could be explained by the co-recruitment of CUGBP1, a known translation activator during senescence of p21, and p21 mRNA to SGs. Therefore, our data suggest that SGs formation and the reduction in p21 protein levels represent two main events through which senescent cells respond to stress conditions.

No description available.

Antisense oligodeoxynucleotides (asODNs) show strong binding and high selectivity and can be constructed to recognize specific cellular targets such as gene regulated mRNA. Radiolabelled asODNs have the potential to image gene expression through mRNA targeting and could be a valuable tool in the early assessment of outcome to cancer treatment. We have explored the potential of in vivo imaging of p21 gene expression, using fluorine-18 labelled asODNs ([18F]asODNs) and in vitro techniques, recognizing the relationship between the expression of this gene and resistance of cancer cells to radiation therapy. Radiolabelling of fully phosphorothioated, 20-mer ODNs was performed using the [18F]-labelled prosthetic group, 4-N-[18F]fluorobenzyl-2-bromoacetamide ([18F]FBBA). [18F]FBBA was first synthesized in an automated synthesis unit, resulting in a modest radiochemical yield. Methods to improve the yield were investigated using a metal catalyst-assisted borohydride exchange resin. Alkylation of [18F]FBBA to ODN resulted in radiochemical yields of 40%. Cellular uptake and retention studies were performed in human carcinoma cells expressing p21+/+ (HCT116) and the p21 knock-out cell line, 80S4, using both [18F]-labelled antisense and random sequence ODNs. Nonradioactive FBBA-labelled ODNs were used to evaluate the antisense effectiveness and distribution of the FBBA-modified ODNs. In vitro studies demonstrated that FBBA did not interfere with the antisense effect of ODNs against p21 mRNA; however, the probes required a transfection agent to observe an antisense effect. Cell fractionation studies with [18F]ODNs revealed increasing accumulation of liposome-transfected [18F]asODN in the cytoplasm of HCT116 cells over time. A biocompatible spermine-grafted block copolymer (SP) was subsequently evaluated as a potential vector to improve the delivery of [18F]asODN into cells. SP was shown to direct [F]-labelled ODNs to the cytoplasm, whereas naked [F]ODNs remained sequestered in vesicles, and liposome-transfected [F]ODNs localized mostly in the nucleus. Selective uptake and retention of [18F]asODN was observed in p21+/+ cells only when the probe was transfected with SP. Based on these studies, it can be concluded that [18F]asODNs have the potential to image gene expression, however the focus may need to be directed to find an appropriate vector which can rapidly deliver [18F]-labelled asODNs to the target tissue in vivo.

With the dawn of metazoans, the ability of cells to communicate with each other became of paramount importance in maintaining tissue homeostasis. The transforming growth factor β (TGF-β) signaling pathway, which plays important roles during embryogenesis and in the adult organism, signals via a heterodimeric receptor complex consisting of two type II and two type I receptors. After receptor activation through ligand binding, Smads mediate the signal from the receptor complex to the nucleus, where they orchestrate transcription. Depending on the context of activation, TGF-β can mediate a plethora of cellular responses, including proliferation, growth arrest, apoptosis and differentiation. In cancer, TGF-β can act as both as a tumor suppressor and promoter. During early stages of tumorigenesis, TGF-β prevents proliferation. However, TGF-β is also known to promote tumor progression during later stages of the disease, where it can induce differentiation of cancer cells towards a migratory phenotype. The aim of this thesis was to investigate how cells can differentiate their response upon TGF-β pathway activation. The first paper describes the role of Notch signaling in TGF-β induced growth arrest, demonstrating that TGF-β promotes Notch activity and that Notch signaling is required for prolonged TGF-β induced cell cycle arrest. In the second and third paper, we investigate the role of SIK, a member of the AMPK family of kinases, mediating signaling strength of TGF-β through degradation of the TGF-β type I receptor ALK5. While the second paper focuses on the effect of SIK on ALK5 stability and subsequent alterations in TGF-β signaling, the third paper emphasizes cooperation between SIK, Smad7 and the E3 ligase Smurf in degradation of ALK5. Finally, the fourth paper explores a novel role of SIK during TGF-β induced epithelial to mesenchymal transition (EMT). SIK binds to and degrades the polarity protein Par3, leading to enhanced EMT.

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/12/09). Includes bibliographical references.