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Müer, Annika [Verfasser]. "Funktionelle Charakterisierung des p14ARF-Tumorsuppressorsignalwegs / Annika Müer." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2012. http://d-nb.info/1029792585/34.
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Abraham, Aswin George. "Nucleophosmin and p14ARF mediated regulation of p53." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:06e101f9-f0e3-42b9-ada9-7a1b1a1f4a87.
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Tumour initiation and progression occur due to oncogenic mutations that also contribute to therapeutic resistance in many human tumours. Mutations activating the "PI3K/AKT" signalling pathway and inactivating the "TP53" tumour suppressor gene are common mechanisms that cancer cells require to proliferate and escape pre-programmed cell death. p53 mutant (p53mut) tumours not only fail to respond to DNA damaging therapy, but are also described to promote therapeutic resistance by dominant negative suppression of p53 dependent promoter activity. Our work identifies the crucial interaction between the PI3K/AKT pathway and p53 mutations that regulate treatment sensitivity in tumours. Using a combination of in vitro and in vivo techniques we demonstrate that AKT inhibition promotes reduced cellular levels of p53mut via a novel Nucleophosmin 1 (NPM) mediated regulation of the tumour suppressor p14ARF and promotes re-engagement of cell cycle arrest, senescence and increased sensitivity to ionising radiation in both in vivo and in vitro systems. We show that the PI3K/AKT pathway plays an important role in the regulation of p53mut and inhibitors of this pathway can re-sensitise treatment resistant tumours. This has helped us to simultaneously highlight the cohort of patients where the greatest efficacy may be achieved in clinical practise. We further show that the AKT mediated regulation of NPM that we describe in solid tumours is relevant in Acute Myeloid Leukaemia (AML) with mutated NPM, albeit showing physiologically different effects. This further highlights the necessity for rational treatment planning with the newer targeted agents that inhibit specific signalling pathways in AML patients.
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Lindström, Mikael. "Functional characterization of the alternative reading frame protein p14ARF /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-917-x.
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Carr-Wilkinson, Jane. "Inactivation of the p53/MDM2/p14ARF pathway in neuroblastoma." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515033.
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Mistry, Sushilaben Harkisandas. "The role of p14ARF in familial and sporadic melanoma." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413193.
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Dayde, Delphine. "Liens fonctionnels entre l'EGFR et P14ARF : contribution à la carcinogenèse pulmonaire." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV071/document.
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L'EGFR est un récepteur transmembranaire à activité tyrosine kinase (TK) qui transduit des signaux de prolifération et de survie cellulaire. Dans les cancers du poumon, son activité est fréquemment dérégulée par surexpression et/ou par mutation au niveau de son domaine TK. Ces mutations sont principalement de deux types (EGFR-L858R et EGFR-Del19) et sont dites activatrices car elles induisent une activation constitutive des signalisations oncogéniques de l'EGFR. Elles sont aussi un facteur prédictif de réponse aux EGFR-TKIs qui inhibent spécifiquement ce récepteur. P14ARF est un suppresseur de tumeur qui restreint la prolifération cellulaire et maintient la stabilité génomique. Nous avons décrit son inactivation dans les cancers du poumon et démontré que son expression freine leur développement. Nos résultats récents montrent que l'expression de p14ARF est inhibée dans une très grande majorité d'adénocarcinomes pulmonaires présentant une mutation activatrice de l'EGFR. Sur la base de ces résultats nous avons émis l'hypothèse que l'inhibition de l'expression de p14ARF contribuerait à l'expansion clonale des tumeurs porteuses d'un EGFR muté. P14ARF pourrait ainsi être un frein à l'activité oncogénique de l'EGFR.Dans différents modèles d'adénocarcinomes pulmonaires exprimant un mutant EGFR-L858R nous montrons que l'expression transitoire de p14ARF active une signalisation pro-apoptotique dépendante de STAT3 et de Bcl2. En retour, l'EGFR inhibe l'expression de p14ARF et bloque ses fonctions pro-apoptotiques. Nous montrons aussi que l'activation de l'EGFR (sauvage ou muté) inhibe l'expression de p14ARF à un niveau transcriptionnel. Ceci implique une translocation nucléaire de l'EGFR contrôlée par les PI3Ks de classe III (Vps34) et la fixation de l'EGFR sur le promoteur de ARF. L'ensemble de ces travaux identifie pour la première fois un lien fonctionnel entre les voies de signalisation de l'EGFR et de p14ARF. Ils mettent en évidence un nouveau mécanisme de progression tumorale par lequel une signalisation nucléaire de l'EGFR inactive le suppresseur de tumeur p14ARF afin de permettre la croissance tumoraleEGFR is a transmembrane tyrosine kinase (TK) receptor which activates proliferative and survival signals. In lung cancer, its activity is frequently deregulated by overexpression and/or mutation in its TK domain. These mutations are mainly of two types (L858R and Del19) and are called « driver mutations » because they induce constitutive activation of EGFR oncogenic signaling. They also represent a predictive responsive factor to EGFR TKIs that specifically inhibit this receptor. P14ARF is a tumor suppressor that restricts cellular proliferation and maintains genomic stability. We described its inactivation in lung cancer and demonstrated that its expression inhibits their development. Our recent results show that the expression of p14ARF is inhibited in a majority of lung adenocarcinomas expressing an activating EGFR mutation. Based on these results we hypothesized that inhibition of p14ARF expression contributes to clonal expansion of mutated EGFR-bearing tumor. P14ARF could be a break to EGFR oncogenic activity.In different models of lung adenocarcinoma expressing a L858R EGFR mutant we show that transient expression of p14ARF activates a pro-apoptotic STAT3/Bcl-2-dependent signaling pathway. In turn, EGFR inhibits the expression of p14ARF and blocks its pro-apoptotic function. We also show that EGFR (wild type or mutated) activation inhibits the expression of p14ARF at the transcriptional level. This implies a nuclear translocation of EGFR controlled by Class III PI3K (Vps34) and its fixation to the ARF promoter. This work identifies for the first time a functional link between EGFR and p14ARF signaling pathways. They highlight a new mechanism of tumor progression by which a nuclear EGFR signaling inactivates the tumor suppressor p14ARF to allow tumor growth
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Gallagher, Stuart John. "The Role of the p14ARF Tumour Suppressor in Promoting Apoptosis." Thesis, The University of Sydney, 2008. http://hdl.handle.net/2123/3597.
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The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.
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Gallagher, Stuart John. "The Role of the p14ARF Tumour Suppressor in Promoting Apoptosis." University of Sydney, 2008. http://hdl.handle.net/2123/3597.
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Doctor of Philosophy (PhD)The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.
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Andrique, Laetitia. "Nouveaux partenaires du suppresseur de tumeurs p14ARF : découverte de nouvelles fonctions indépendantes de p53." Poitiers, 2007. http://www.theses.fr/2007POIT1403.
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Straza, Michael W. "A Tale of Two ARFs: Tumor Suppressor and Anti-viral Functions of p14ARF: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/472.
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Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer.
The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy.
In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies.
ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.
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Gamble, Laura Dawn. "MYCN and the p53-MDM2/MDMX-p14ARF network in neuroblastoma amd response to MDM2-p53 antagonists." Thesis, University of Newcastle upon Tyne, 2012. http://hdl.handle.net/10443/1350.
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Background: MYCN-amplification is a major negative prognostic marker, occurring in 25-30% of neuroblastomas. MYCN plays contradictory roles in promoting cell growth and sensitizing cells to apoptosis, and we have recently shown that p53 is a direct transcriptional target of MYCN, and may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14ARF pathway is inactivated in 35% of cases through MDM2-amplification or p14ARF inactivation. Neuroblastoma is therefore an ideal target for p53 reactivation using MDM2-p53 antagonists. MDMX, a homologue of MDM2, is another negative regulator of p53 which is often overexpressed in cancers and has been shown to compromise the effects of MDM2-p53 antagonists in various cancer types. MDMX expression and the effect on MDM2-p53 antagonists has not been investigated in neuroblastoma. Hypotheses 1) Reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis preferentially in MYCN-amplified cells 2) MDMX knockdown increases and p14ARF knockdown decreases the sensitivity of neuroblastoma cell lines to MDM2-p53 antagonists. Methods: The effect of MYCN, MDM2, MDMX and p14ARF was investigated on the response to MDM2-p53 antagonists using siRNA in a panel of 21 neuroblastoma cell lines. Sensitivity was measured by growth inhibition, apoptosis assays including caspase activity and fluorescent activated cell sorting, and the effect on the p53 response measured by Western blotting. Results: Using the SHEP Tet21N MYCN regulatable system, MYCN(-) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared to MYCN(+) cells and siRNA mediated knockdown of MYCN in 4 MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of cell lines treated with Nutlin-3 and MI-63, the sub-set amplified for MYCN had a significantly lower mean GI50 value and increased caspase 3/7 activity compared to the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. Knockdown of MDM2 did not alter the apoptotic response to Nutlin-3 or MI-63 but surprisingly, knockdown of MDMX resulted in decreased levels of apoptosis. MDMX expression varied amongst the neuroblastoma cell lines and positively correlated with caspase 3/7 activity following MDM2-p53 antagonist treatment. p14ARF impaired cell lines underwent less apoptosis following MDM2-p53 antagonist treatment and following Nutlin-3 treatment, 3 of 4 p14ARF impaired cell lines underwent a pronounced G1 arrest. p14ARF knockdown alone resulted in decreased caspase 3/7 activity, and following MDM2-p53 antagonist treatment there was decreased caspase 3 cleavage and activity, and decreased PARP cleavage. Conclusions: Amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wildtype p53 to MDM2-p53 antagonists and these compounds may therefore be particularly effective in treating high risk MYCN-amplified disease. This data also suggests that neuroblastomas with high MDMX expression may be more susceptible to MDM2-p53 antagonist treatment, but that cells with inactivated p14ARF predominantly undergo a G1 arrest which may protect them from apoptosis. MDMX and p14ARF status may therefore be important in addition to MYCN in determining the outcome of neuroblastomas treated with MDM2-p53 antagonists.
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Milojkovic, Ana [Verfasser]. "The tumour suppressor protein p14ARF regulates cell cycle checkpoint control and induces cell death / Ana Milojkovic." Berlin : Freie Universität Berlin, 2010. http://d-nb.info/1024744140/34.
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Junqueira, Mara de Souza. "Caracterização das vias de transformação maligna de uma nova linhagem estabelecida de melanoma murino." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-20092010-183837/.
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Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo.A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin.
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Mendonça, Samir Andrade. "Desenvolvimento e investigação da transferência gênica de p14ARF e interferon-beta em linhagens celulares de melanoma humano." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-07022019-093142/.
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O melanoma é um dos tipos de câncer de pele cuja frequência tem crescido nos últimos anos e apresentado elevada taxa de mortalidade, apesar de ter reduzida prevalência. Mesmo havendo um considerável avanço nas propostas terapêuticas nos últimos anos, ainda se vê necessário o desenvolvimento de novas abordagens, sendo a terapia gênica uma promissora possibilidade para tal. Utilizando vetores adenovirais com promotor responsivo à p53 (PGTx beta) para a transferência gênica de p19Arf (proteína supressora de tumor) e interferon-beta (citocina imunomodulatória) em células de melanoma murino com o gene Trp53 selvagem, o nosso grupo demonstrou previamente que a combinação dos dois genes, mas não o tratamento individual, promove efeito citotóxico sinérgico com a liberação de marcadores de morte imunogênica, in vitro; e significativa redução da progressão tumoral acompanhada de uma forte resposta imunológica de linfócitos T CD4+ e CD8+, células NK e neutrófilos contra desafios tumorais, in vivo. Porém, como a translação para modelos de melanomas humanos ainda estava em estágio inicial, ainda não haviam sido confirmamos se esses benefícios também seriam recapitulados. Observações inicias sugeriam que apenas a transferência gênica de interferon-beta seja suficiente para induzir morte celular em linhagens humanas portadoras de TP53 selvagem, sem ainda terem sido identificado o efeito da transferência de p14ARF e nem a necessidade de p53 endógeno para a resposta. Dessa forma, o presente projeto buscou avaliar os efeitos antitumorais provocados pela terapia gênica combinada de p14ARF e interferon-beta em modelos de melanoma humano utilizando linhagens com e sem a via da p53 integra. Para isso, foram utilizadas diferentes linhagens celulares com TP53 selvagem ou com distintas mutações e também foram construídos vetores adenovirais com o promotor constitutivo CMV, tornando assim possível a expressão dos transgenes de maneira independente do status do TP53 endógeno. O presente trabalho revelou que a transferência combinada do interferon-beta e p14ARF revelou vantagem quanto ao estímulo citotóxico e regulação negativa na dinâmica da população em ambas as linhagens UACC-62 e SK-Mel-29, independentemente do estado da via da p53. Na avaliação dos mecanismos de morte foi observado que ambas a linhagens apresentaram marcação positiva para marcadores da via da apoptose, porém com possível participação de outras modalidades de morte-celular, como a necrose, para a linhagem com o TP53 mutado (SK-Mel-29). Além disso, mostramos que os tratamentos potencialmente induzem vias de morte com caráter imunogênico pela secreção de ATP e exposição da calreticulina, sendo este último marcador mais significantemente observado mediante o tratamento combinado. Assim, recapitulamos o benefício observado em modelo murino para a transferência gênica do interferon-beta e p14ARF em modelo de melanoma humano, e investigamos marcadores importantes à translação da proposta terapêutica para o melanomaMelanoma is one of the types of skin cancer whose frequency has grown in the last years and presents a high mortality rate, despite its low prevalence. Although there has been considerable progress in therapeutic proposals in recent years, it is still necessary to develop new approaches, being gene therapy a promising possibility for this. With the use of adenoviral vectors with a p53 responsive promoter (PGTx beta) for the gene transfer of p19Arf (tumor suppressor protein) and interferon-beta (immunomodulatory cytokine) in murine melanoma cells bearing wild-type Trp53 gene, our group previously demonstrated that the combination of the two genes, but not individual treatment, promotes a synergistic cytotoxic effect with the release of immunogenic death markers in vitro; and significant reduction of tumor progression with a strong immune response mediated by CD4+ and CD8+ T lymphocytes, NK cells and neutrophils in tumor challenges in vivo. However, as the translation for human melanoma models was still at an early stage, it still was not possible to confirm whether these benefits would also be recapitulated in a human model. Initial observations suggested that interferon-beta gene transfer is sufficient to induce cell death in wild-type TP53-bearing human melanoma cell lines, with the effect of p14ARF gene transfer and the role for endogenous p53 in this response yet to be investigated. Thus, the present work aimed to evaluate the antitumor effects induced upon the combined gene transfer of p14ARF and interferon-beta in human melanoma cell lines with and without a functional p53 pathway. For this, different cell lines bearing wild-type TP53 or with different mutations were used and adenoviral vectors with the constitutive CMV promoter were also constructed, making possible the expression of the transgenes independently of the endogenous TP53 status. The present work showed that the combined transfer of interferon-beta and p14ARF was advantageous in cytotoxic stimulation and negative regulation in population dynamics for both cell lines UACC-62 and SK-Mel-29, regardless of p53 pathway status. In the evaluation of the triggered cell death mechanisms it was observed that both cell lines presented positive markers of the apoptosis pathway, but with possible participation of other cell death mechanism, such as necrosis, for the mutated TP53 cell line SK-Mel-29. In addition, we showed that the treatments potentially induced cell death pathways with immunogenic features including the secretion of ATP and calreticulin exposure, being the latter marker more significantly presented after the combined treatment. Thus, we recapitulated the benefit observed in murine model for the gene transfer of interferon-beta and p14ARF in the model of human melanoma, and investigated important markers for the translation of the melanoma therapeutic proposal
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Muniz, Viviane Palhares. "The role of Parf, a novel partner of ARF, in pancreatic ductal adenocarcinoma and in ARF signaling." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/3503.
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Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic cancer resistant to current treatments. A better understanding of the genetic basis of PDAC progression is urgently needed to improve treatment options. The ARF tumor suppressor is inactivated in ~45% of PDAC. My thesis lab identified a new, uncharacterized ARF binding protein, Partner of ARF isoform 1A (Parf-1A). This thesis explores the hypothesis that Parf-1A plays an important role in PDAC and ARF tumor suppressor signaling
Initial studies sought to develop a novel mouse xenograft model of PDAC metastasis that would expedite testing of putative PDAC genes. Human PDAC cell lines stably expressing luciferase were generated and introduced by intracardiac injection into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored non-invasively by bioluminescence imaging and found to recapitulate PDAC tumor formation and metastatic distribution. The model was validated by the ability of ARF to suppress PDAC cancer cell migration in vitro and reduce tumor cell colonization in vivo; establishing a new bioluminescent mouse model for rapidly assessing the significance of suspected PDAC genes.
Using human PDAC cell lines and tumor specimens, we investigated the role and significance of Parf-1A to PDAC. RNAi analyses demonstrated Parf-1A is required for PDAC cell survival, proliferation and resistance to the PDAC therapeutic, oxaliplatin. PDAC cells are ARF-null; therefore these tumor promoting activities of Parf-1A were independent of ARF. Notably, immunohistochemical analyses of Parf-1A in human PDAC tumors showed Parf-1A expression is a prognostic marker of poor survival in PDAC patients. These data suggest Parf-1A is a novel biomarker of PDAC and potential target for anticancer therapy.
Other studies tested how Parf-1A influenced ARF signaling. Parf-1A depletion and overexpression showed it inhibits ARF anti-proliferative activity by mobilizing ARF from the nucleus (where it is functional) into the cytoplasm. These data show Parf-1A is a new inhibitor of ARF. Considered with findings that Parf-1A can act independent of ARF to promote PDAC tumorigenesis, such results suggest Parf-1A is a novel oncoprotein that acts through multiple pathways to facilitate tumorigenesis. Thus, Parf-1A may have broad relevance to many types of human cancers.
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VENEZIANO, Lorena. "MECHANISMS OF CHROMOSOMAL INSTABILITY: RELATIONSHIP BETWEEN TUMOR SUPPRESSORS AND SAC GENES." Doctoral thesis, Università degli Studi di Palermo, 2017. http://hdl.handle.net/10447/219878.
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Caratteristica comune di molti tumori solidi è l’aneuploidia, conseguenza dell’instabilità cromosomica (CIN). Tuttavia non sono ancora chiari i meccanismi alla base dell’aneuploidia e i percorsi che permettono la sua tolleranza. Lo Spindle Assembly Checkpoint (SAC) è un meccanismo di sorveglianza cellulare che controlla la stabilità genomica durante la mitosi. Alterazioni nei membri del SAC generano aneuploidia, ma non è ancora chiaro se questi difetti sono sufficienti per promuovere la tumori-genesi. In questo processo, infatti, il contesto genetico della cellula gioca un ruolo importante. È risaputo che i difetti di p53 aiutano le cellule a proliferare velocemente tollerando la CIN. Al contrario, l’attivazione di una p53-wt contrasta l’aneuploidia. Poco si sa, invece, circa il ruolo del tumor suppressor p14ARF nel contrastare l’aneuploidia.
In questa tesi ho voluto valutare la relazione tra alcuni geni del SAC, la cui deplezione induce aneuploidia, e i geni tumor suppressor. Inizialmente, per indagare sul ruolo di p14ARF nel contrastare l’aneuploidia, l’ho espresso ectopicamente in cellule HCT116 (quasi diploidi) dopo aver ridotto, in contemporanea, l’espressione del gene MAD2, elemento cruciale del SAC. Il silenziamento post-trascrizionale di MAD2 ha indotto alti livelli di aneuploidia e di mitosi anomale, che sono diminuiti con l’espressione contemporanea di p14ARF. Inoltre, l’espressione ectopica di p14ARF in cellule MAD2-deplete ha attivato un percorso apoptotico, associato a un incremento della proteina p53. Questa risposta non è stata rilevata in cellule HCT116 p53KO, suggerendo che p14ARF contrasta l’aneuploidia attivando un’apoptosi p53-dipendente. Poi, ho voluto approfondire la relazione tra la proteina motrice Cenp-E, che funziona solo nell’attivazione del SAC, e l’aneuploidia in cellule umane. A tal fine, ho utilizzato due diversi tipi cellulari, fibroblasti primari umani IMR90 e cellule HCT116 che non esprimono la proteina p14ARF, analizzando gli effetti della deplezione di Cenp-E fino a quattro settimane. I due tipi cellulari hanno risposto in maniera differente poiché l’aneuploidia è più tollerata dalle cellule che non esprimono p14ARF piuttosto che dai fibroblasti primari. Inoltre, l’osservazione che la riduzione dell’aneuploidia nelle IMR90 coincide con l’incremento di p14ARF e che la sua espressione ectopica riduce l’aneuploidia nelle HCT116 conferma l’abilità del gene di contrastare l’aneuploidia. Infine, per approfondire questi risultati, ho generato cellule HCT116 che esprimono una proteina p14ARF funzionale, in cui ho valutato gli effetti della deplezione di Cenp-E. In conclusione, questi risultati suggeriscono che p14ARF potrebbe avere un ruolo importante nel contrastare l’aneuploidia attivando un’apoptosi p53-dipendente e che, in maniera più generale, è coinvolto nell’ostacolare l’aneuploidia indotta da differenti stimoli.The majority of solid tumors are characterized by aneuploidy that is believed to be the consequence of chromosomal instability (CIN). The mechanisms leading to aneuploidy and the pathway (s) that allows its tolerance are not completely understood. The Spindle Assembly Checkpoint (SAC) is a cellular surveillance mechanism that works to maintain the genomic balance in mitosis. Alterations of SAC components can induce aneuploidy but it is not clear if these defects are sufficient for tumorigenesis. In this process the genetic background of the cell plays an important role. It is known that p53 defects allow cells to quickly proliferate tolerating CIN. On the contrary, activation of wt-p53 counteracts aneuploidy. Less is known about the role of the p14ARF tumor suppressor to counteract aneuploidy. In this thesis, I investigate the relationship between some of the SAC genes that if depleted induce aneuploidy and tumor suppressor genes. First, to investigate the role of p14ARF to counteract aneuploidy it was ectopically expressed in HCT116 cells (near diploid) after MAD2 depletion a crucial component of the SAC. MAD2 posttranscriptional silencing induced high levels of aneuploid cells and aberrant mitosis that decreased when p14ARF was simultaneously expressed. In addition, p14ARF ectopic expression in MAD2-depleted cells induced apoptosis associated with increased p53 protein levels. This response was not detected in HCT116 p53KO cells suggesting that p14ARF counteracts aneuploidy activating apoptosis p53-dependent. Second, I wanted to probe the relationship between the motor protein CENP-E, which works only in the SAC signaling, and aneuploidy in human cells. To this aim I used two types of cells, human primary fibroblasts (IMR90) and near diploid cells (HCT116) lacking p14ARF, and analyzed the effects of CENP-E depletion up to four weeks. These experiments showed a different response for the two cell types. Aneuploidy was tolerated for longer times in cells lacking p14ARF expression rather than in primary cells. In addition the observations that the reduction of aneuploidy in IMR90 cells was proportional to the increase of p14ARF gene expression, and that its ectopic expression in HCT116 cells reduced aneuploidy confirm the ability of p14ARF to counteract aneuploidy. Third, to improve these results I generated HCT116 cells expressing a functional p14ARF to assess the effects of CENP-E depletion. Collectively, these results suggest that the tumor suppressor p14ARF may have an important role to contrast aneuploidy activating a p53-dependent apoptosis pathway and that it is generally involved to counterbalance aneuploidy induced by different stimuli.
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Gretton, Svetlana. "Investigation of the functional role of the transcription factor, CTCF, in the regulation of human Bax and p14ARF genes." Thesis, University of Essex, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589445.
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CTCF (CCCTC-binding factor) is a ubiquitously expressed, multifunctional and conserved ll-Zinc finger transcription factor. CTCF is involved in the regulation of various genes, including those responsible for proliferation and apoptosis, using different mechanisms. One such mechanisms is based on reversible poly(ADP-ribosyl)ation (PARylation) of CTCF at the CTCF target sites (CTSs) and requires cooperation between CTCF and the PARylation enzyme, PARP-l. The main aim of this study was to investigate the role ofCTCF, PARP-l and PARylation in the epigenetic regulation of a pro-apoptotic gene, Bax and tumour suppressor gene, p 14ARF. We found that in all cells, breast and non-breast, the levels of Bax mRNA and protein were similar, with chromatin in the active state. However, CTCF binding was enhanced at the Bax promoter in breast cancer cells and tumours. We propose that in breast cancer cells and tumours, possibly in cooperation with P ARP-l; CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis. Four breast cell lines with the wild type p14ARF gene but different levels of p14ARF mRNA expression were used as models to study p14ARF regulation by CTCF. The relationship was observed between the levels of p14ARF expression, active chromatin marks and CTCF and PARP-l binding. The presence of both proteins at the p14ARF CTS was associated with the active state of p14ARF, whereas the presence of CTCF but not P ARP-l was associated with the silent state of p14ARF. Additionally, in this study a novel matrix Bio Vyon™IProtein A was optimised, evaluated and used for chromatin immunoprecipitation (ChIP) assays with tissue samples. The studies regarding the role of CTCF PARylation in cell proliferation were initiated and preliminary results obtained indicating that importance of CTCF PARylation for its normal functions.
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Chen, Jingyu. "Nucleolar stress stimulates the NF-kappaB pathway : mechanism underlying the proapoptotic effects of aspirin." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28901.
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The nucleolus is a multifunctional organelle that, in addition to its primary role in ribosome biogenesis, has emerged as a critical stress sensor and coordinator of stress response. However, the molecular nature of how nucleoli sense stress and coordinate downstream cellular consequence remains poorly understood. NF-κB signalling is a critical regulator of stress response. Many cellular stresses that disrupt nucleolar function also stimulate the NF-κB pathway. However, the role of NF-κB as a downstream effector of nucleolar stress has not yet been examined. Aspirin, a known chemopreventative agent, stimulates the NF-κB pathway to mediate apoptosis but the upstream mechanisms are unclear. In this thesis, I identified a novel nucleolar stress response pathway that culminates in activation of NF-κB signalling, and demonstrated the significance of this nucleolar pathway in the anti-tumour effects of aspirin. Using multiple approaches, I made the novel observations that disruption of the Pol I complex activates the cytoplasmic NF-κB signalling pathway. I show that multiple stress stimuli of NF-κB pathway induce degradation of the crucial Pol I complex component, rDNA transcription initiation factor IA (TIF-IA). I identified the tumour suppressor, p14ARF and the Pol I complex component, upstream binding factor (UBF) as mediators of this degradation. I revealed that inhibition of CDK4 activity lies upstream of UBF/p14ARF-facilitated TIF-IA degradation. Furthermore, using different approaches I show that blocking aspirin/CDK4i-mediated degradation of TIF-IA blocks the effects of these agents on nucleolar morphology and NF-κB signalling. Finally, I show this nucleolar stress response pathway, containing a UBF/p14ARF/TIF-IA axis, is utilized by aspirin to kill colon cancer cells. Taken together, this data presented in this thesis advances understanding of nucleolar stress response, and has therapeutic implications with regard to the anti-tumour effects of aspirin.
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Ozenne, Peggy. "Contrôle de la signalisation oncogénique du mutant L858R de l'EGFR par la protéine suppresseur de tumeur p14ARF dans les adénocarcinomes pulmonaires." Phd thesis, Université de Grenoble, 2011. http://tel.archives-ouvertes.fr/tel-00767340.
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Contrôle de la signalisation oncogénique du mutant L858R de l'EGFR par la protéine suppresseur de tumeur p14ARF dans les adénocarcinomes pulmonaires. Le récepteur à l'EGF (EGFR) est un oncogène puissant impliqué dans le développement des cancers du poumon. Dans ces cancers, la présence de mutations activatrices de l'EGFR (majoritairement L858R et Del19) est un facteur prédictif de réponse aux agents pharmacologiques qui ciblent spécifiquement ce récepteur (EGFR-TKI). Cependant, l'association entre réponse thérapeutique et mutation est plus complexe que prévue, soulignant la nécessité d'approfondir la compréhension des mécanismes moléculaires impliqués dans développement de ces cancers. Nous avons précédemment montré que la quasi-totalité des tumeurs pulmonaires avec des mutations activatrices de l'EGFR présente une expression faible ou indétectable de la protéine suppressive de tumeur p14ARF. Ces résultats nous ont conduites à émettre l'hypothèse que l'expression de p14ARF était un frein essentiel à l'expansion clonale de ces cellules. Nous décrivons pour la première fois une relation fonctionnelle entre p14ARF et du mutant L858R de l'EGFR dans laquelle p14ARF inhibe la croissance de cellules exprimant ce mutant en induisant leur apoptose. Les effets suppresseurs de tumeur de p14ARF impliquent une fonction pro-apoptotique originale de STAT3 qui conduit à l'inhibition de l'expression de la protéine anti-apoptotique Bcl-2. De plus, nous montrons que les cellules EGFR-L858R maintiennent leur avantage de croissance en inhibant l'expression de p14ARF et la signalisation pro-apoptotique STAT3/Bcl-2 qui en découle. Nos résultats identifient également p14ARF comme une nouvelle cible transcriptionnelle de STAT3, mettant ainsi en évidence une boucle de rétrocontrôle positif entre ces deux protéines qui pourrait entretenir la signalisation pro-apoptotique médiée par STAT3. Sur la base de ces résultats, nous suggérons que la réactivation de la voie p14ARF/STAT3/Bcl-2 pourrait être une nouvelle stratégie thérapeutique dans le traitement de ces cancers.
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Leduc, Camille. "Fonctions anti-prolifératives de la protéine suppressive de tumeur p14ARF dans les cancers broncho-pulmonaires : contribution de la protéine du rétinoblastome Rb." Université Joseph Fourier (Grenoble), 2005. http://www.theses.fr/2005GRE10078.
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Il est clairement établi que p14ARF fait 'partie d'un réseau linéaire p14ARF/MDM2Ip53 capable d'inhiber la croissance cellulaire en réponse aux activations oncogéniques. Cependant, son inactivation est fréquemment retrouvée dans les cancers broncho-pulmonaires exprimant une protéine p53 mutée ce qui nous a conduit à émettre l'hypothèse que p14ARF pouvait présenter des fonctions biologiques indépendantes de p53. Dans cette étude, nous avons montré que p14ARF est capable d'inhiber la croissance de lignées tumorales pulmonaires humaines nulles pour p53 en induisant un arrêt du cycle cellulaire en phase G2 précédant la survenue d'une apoptose, et conduisant à la régression de tumeurs « in vivo» chez la souris nude. Différents travaux nous ayant conduit à envisager la protéine du rétinoblastome (Rb) comme un médiateur de l'activité anti-proliférative de p14ARF indépendante de p53, nous avons recherché l'existence de relations directes entre ces deux protéines. Dans ce cadre, nous avons montré que l'acétylation de Rb médiée par l'histone acétyltransférase TipOO conduit à sa dégradation par le protéasome et avons mis en évidence qu'en inhibant ce processus, p14ARF stabilise la protéine Rb dans les cellules. Enfin, nous avons montré que le traitement de cellules tumorales bronchiques humaines avec des agents alkylants induit l'accumulation de Rb sous une forme hypoacétylée de façon dépendante de p14ARF. L'ensemble de ces travaux situe donc p14ARF comme un régulateur majeur de la croissance cellulaire quelque soit le statut de p53, et identifie donc un nouveau mécanisme par lequel p14ARF contrôle la voie Rb pour médier son activité anti-proliferativeLit is clearly established that the p14ARF tumour-suppressor protein belongs to a linear p14ARF /MDM2/p53 pathway that inhibits cell growth in response to oncogenic stimuli. However, our previous demonstration of a frequent co-inactivation of p14ARF and p53 proteins in human lung tumors led us to investigate the possibility that p14ARF could display p53-independent functions. Ln this study, we showed that p14ARF inhibits the growth of p53-null human tumoral pulmonary ceillines by inducing a G2 arrest that precedes apoptosis, and leading to the regression of lung tumours established into nude mice. Several studies have led us to consider the retinoblastoma protein (Rb) as a potential mediator of the p53-independent anti-proliferative activity of p14ARF. To test this hypothesis, we investigated the existence of direct relationships belween these Iwo proteins. Ln this setting, we showed that the Tip60-mediated Rb acetylation stimulates its proteasomal degradation and provided evidencethat p14ARF stabilizes Rb by preventing its Tip60-dependent acetylation. Ln addition, we present data indicating that hypoacetylated Rb accumulates in a p14ARF-dependent manner following a cell treatment with alkylating agents. Overall, our results situate p14ARF as a critical regulator of cell growth whatever the p53 status, and identify a new mechanism by which p 14ARF controls the Rb pathway to trigger its anti-proliferative activity
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CILLUFFO, Danilo. "Identification of molecular markers involved in cellular response to aneuploidy in normal and tumor cells." Doctoral thesis, Università degli Studi di Palermo, 2020. http://hdl.handle.net/10447/395230.
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Fauvin, Dominique. "Caractérisation de nouvelles fonctions du suppresseur de tumeurs p14ARF à travers deux exemples, le récepteur à activité tyrosine kinase ErbB3 et le facteur de transcription UBF1." Poitiers, 2009. http://www.theses.fr/2009POIT1406.
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Hashemi, Jamileh. "Germline CDKN2A/ARF alterations in human melanoma /." Stockholm : Karolinska institutet, 2002. http://diss.kib.ki.se/2002/91-7349-148-9.
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Holzer, Kerstin [Verfasser], and Peter J. [Akademischer Betreuer] Goebell. "Auswertung von Häufigkeitsverteilungen und Korrelationen der Proteine Hdm2, P53, P63, P14ARF und P16INK4a im invasiven Harnblasenkarzinom an digitalisierten Tissue Mikroarrays / Kerstin Anna Holzer. Betreuer: Peter J. Goebell." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2013. http://d-nb.info/1033030031/34.
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Di, Tommaso Anne. "Etude du suppresseur de tumeurs p14ARF chez les primates : identification des variations en N-terminal, et leurs impacts, sur la localisation de ARF et la régulation du cycle cellulaire." Poitiers, 2006. http://www.theses.fr/2006POIT2312.
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Les protéines ARF primates présentent de très fortes similitudes entre elles, aussi bien au niveau de la taille que de la séquence en acides aminés. Toutefois quatre différentes ARF ont pu être détectées. Les variations moléculaires en N-terminal bien que n’ayant pas entraîné des modifications dans la localisation nucléolaire des protéines ARF, ont cependant engendré des effets distincts dans la voie classiquement admise de la régulation du cycle cellulaire par le suppresseur de tumeurs p14[exp. ]ARF. Ainsi, il existerait une association forte entre Mdm2 et p14[exp. ]ARFA[ind. ]31H[ind. ]60 indépendamment de p53 et tout aussi efficace dans la régulation du cycle cellulaire. D’autre part, la thréonine en position 31 chez l’homme s'inscrit dans un rôle tout à fait nouveau quant à la stabilisation et l'activation de la protéine p21 selon un mécanisme post-transcriptionnel, fondamental dans la fonction de la protéine p14[exp. ]ARF. Bien que les variations étudiées ne perturbent pas le rôle de suppresseur de tumeurs de la protéine ARF primate, elles ont néanmoins pour conséquence d’entraîner les différentes protéines ARF primates dans des voies de régulation encore peu connuesThe Alternative Reading Frame (ARF) tumor suppressor primarily inhibits cell proliferation by activating p53-dependent cell cycle arrest and/or apoptosis. To gain more insight into ARF function, we recently examined the first exon coding sequences of ARF from 14 different species of primates, including human, monkeys and apes. The sequences were nearly identical except for several discrete amino acid variations that distinguished human ARF from other primate forms of ARF. The impact of those changes on the biological activities of ARF was tested in this study. We found that conversion of Ala31 to Thr31, which is specific to human, enhanced ARF’s ability to upregulate p21Cip1 expression. By comparison, combined alteration of ARF amino acids Thr31 and Leu60 (T31L60) to Ala31 and His60 (A31H60) increased ARF-Mdm2 association. This work shows that some p14ARF residues impart specific effects on its biological activities that may influence the efficacy of ARF-mediated tumor suppression
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Overkamp, Tim. "Bedeutung des p53-Signalwegs für Apoptoseaktivierung und Zellzyklusarrestregulation durch das p14 ARF Tumorsuppressorgen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16619.
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BH3-only Proteine, eine pro-apoptotische Untergruppe der Bcl-2 Proteinfamilie, sind zentrale Mediatoren von apoptotischen Signalen durch die Regulierung intrinsischer Apoptose-signalwege. Unsere Arbeitsgruppe hat vor kurzem gezeigt, dass Apoptose, die durch den p14ARF Tumorsuppressor induziert wird über die p53-abhängige Aktivierung des BH3-only Proteins Puma/Bbc3 vermittelt wird. Interessanterweise induziert p14ARF aber auch in p53 defizienten Zellen Zellzyklusarrest und Apoptose. Die dahinterliegenden Signalwege sind jedoch nicht bekannt. In dieser Arbeit berichten wir, dass das BH3-only Protein Bmf (Bcl-2 modifying factor) beim p14ARF-induzierten Zelltod in p53 defizienten Zellen eine wichtige Rolle spielt. Expression von p14ARF führt zu einer Induktion der PERK Kinase, daran anschließender Phosphorylierung von eIF2α sowie Aktivierung der stromabwärts liegenden Transkriptionsfaktoren ATF4 und CHOP. Diese Signalkaskade ist normalerweise Teil einer zellulären Antwort auf fehl- oder ungefaltete Proteine im Endoplasmatischen Retikulum (ER), der sogenannten ‘unfolded protein response’ (UPR), die zum einen durch verminderte Translationsinitiation und Hochregulierung von Chaperonen die Menge der fehlgefalteten Proteine reduzieren soll. Allerdings induziert p14ARF keinen ER Stress, sondern den PERK‒CHOP Signalweg. Die Transkriptionsfaktoren ATF4 und CHOP binden direkt in der Promotorregion von bmf und sind für dessen transkriptionelle Regulation verantwortlich. Unsere Daten zeigen, dass der PERK‒eIF2α‒ATF4‒CHOP Signalweg eine wesentliche Rolle bei der Induktion von Apoptose durch p14ARF spielt. Dieser Weg könnte ein Sicherungsmechanismus sein, der es den Zellen auch nach Verlust von p53 erlaubt Apoptose einzuleiten, nachdem p14ARF durch Onkogene hochreguliert wurde.BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family of proteins, are central mediators of apoptosis signals by regulating the intrinsic apoptosis pathway. We have recently shown, that apoptosis triggered by the p14ARF tumour suppressor protein is mediated by the p53-dependent activation of the BH3-only protein Puma/Bbc3. Nevertheless, expression of p14ARF in p53-family deficient cells is capable of inducing both cell cycle arrest and apoptosis, but the signalling pathways initiated remain elusive. Here, we report that the BH3-only protein Bmf (Bcl-2 modifying factor) is involved in cell death in p53-deficient cells triggered by p14ARF. Expression of p14ARF leads to the induction of the PERK kinase, subsequent phosphorylation of eIF2α and activation of transcription factors ATF4 and CHOP. This signalling cascade is usually part of the ‘unfolded protein response’ (UPR), which is activated upon ER stress to reduce the amount of misfolded proteins by reduction of global protein translation initiation and upregulation of chaperones. Of note, p14ARF does not induce ER stress but activates the PERK‒CHOP pathway. ATF4 and CHOP transcription factors directly bind to the promotor region of bmf and induce its transcription. These data suggest that the PERK‒eIF2α‒ATF4‒CHOP signalling pathway may play a substantial role in mediating p14ARF-triggered apoptosis. This pathway could play the role of a ‘fail-safe’ mechanism that allows cells, even after loss of p53, to undergo apoptosis induced by upregulation of p14ARF by oncogenes.
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Younes, Mohamad. "Etude des relations entre les mutations EGFR/KRAS et les altérations de la voie p53/p14arf et caractérisation d'une nouvelle cible thérapeutique, le complexe neurotensine et son récepteur1, dans les cancers bronchiques non à petites cellules." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00839459.
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Nous avons étudié, le statut de la voie p53/p14arf dans l'adénocarcinome bronchique muté pour EGFR ou KRAS ainsi que l'impact du complexe NTS/NTSR1 sur la progression tumorale et la survie du cancer bronchique non à petites cellules. Dans une série de 96 patients, les mutations de TP53, KRAS et EGFR ont été détectées dans respectivement 45.2%, 15.8% et 34.4% des cas. La diminution de l'expression de p14arf a été observée dans 57.1% des cas. Le déficit de la voie p53/p14arf a été observé dans 83,3% et 76.9% des tumeurs mutées pour EGFR et KRAS respectivement, ce qui suggère que les altérations de la voie p53/p14arf sont des événements communs dans ces tumeurs mais pas systématiques. L'expression du NTSR1 a été mise en évidence par immunohistochimie dans 60.4% des adénocarcinomes de stades I (n= 139); 47% de stade II (n = 74) et 50% de stade III (n = 133). A l'analyse univariée et multivariée, l'expression du NTSR1, était associée significativement à un mauvais pronostic en termes de survie globale (p = 0,0081 pour les stade I, p = 0.0087 pour les stade II-III). Par contre, l'expression de NTSR1 n'était pas associée avec une différence significative en terme de survie globale pour le carcinome épidermoïde ou le carcinome à grandes cellules. Les études, in vivo et in vitro, ont montré que le complexe NTS/NTSR1 favorise la progression tumorale en augmentant l'expression HER2 et HER3 et la transactivation d' EGFR, HER2 et HER3 par l'activation de MMP1 et la libération de HB-EGF et de NRG1. Ces résultats suggèrent que le complexe NTS/NTSR1 contribue activement à l'agressivité et à la progression tumorale bronchique
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Kovi, Ramesh C. "Defining the Role of CtBP2 in p53-Independent Tumor Suppressor Function of ARF: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/433.
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ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF’s p53-independent tumor suppressor activity is not understood.
The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF’s p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF’s ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein.
CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression.
To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF.
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Fonseca, Maria Cristina Dias Ferrão. "P53, MDM2 and P14ARF immunohistochemical expression in retinoblastoma." Master's thesis, 2010. http://hdl.handle.net/10316/18584.
Full textAbstract:
Introdução: O retinoblastoma é o tumor maligno intraocular primário mais comum
em idade pediátrica. Knudson apresentou a proposta do modelo two-hit, permitindo distinguir dois grandes grupos de tumores: hereditários e não hereditários. Estabeleceu-se um nexo de
causalidade entre a mutação no gene supressor tumoral RB1 (cromossoma 13q14) e o
desenvolvimento do retinoblastoma. Evidências actuais sugeriram que a inactivação bialélica
do gene RB1 é a lesão iniciadora, mas não é suficiente para a progressão completa do
retinoblastoma [1]. Assim, para explicar o desenvolvimento deste tumor, foi proposta a
hipótese de uma expressão alterada da via supressora tumoral p14ARF-MDM2-p53 [2].
Trabalhos anteriores demonstraram que a expressão da proteína p14ARF era
indetectável, ao contrário da Mdm2, que se apresentava sobre-expressa no retinoblastoma [3].
Objectivos: O objectivo deste trabalho foi avaliar a expressão imunohistoquímica dos
componentes da via p53 (p14ARF, Mdm2 e p53) para melhor compreender a patogenia e
diferenciação moleculares do retinoblastoma. Tentou-se também, correlacionar a expressão
destas proteínas com parâmetros clínicos dos doentes, nomeadamente o padrão de
hereditariedade do tumor, estádio de Reese-Ellsworth e prognóstico vital.
Material e métodos: Foram obtidos 24 cortes histológicos de retinoblastomas de 22
doentes enucleados, provenientes do material em arquivo no Laboratório de Patologia
Oftálmica dos HUC. Os seus registos clínicos foram consultados para recolher informação,
incluindo idade, género, padrão de hereditariedade, estádio de Reese-Ellsworth e prognóstico
vital. O estudo imunohistoquímico foi realizado em cortes histológicos de retinoblastoma
incluídos em parafina.
Resultados: Foi obtida positividade da expressão das proteínas p53, p14ARF e Mdm2
em 87,5% (21/24), 87,5% (21/24) e 95,8% (23/24) das 24 amostras de retinoblastomas,
respectivamente. Globalmente, a expressão de p53 não se correlaciona positivamente com a
P53,MDM2 AND P14ARF IMMUNOHISTOCHEMICAL EXPRESSION IN RETINOBLASTOMA
11
expressão de p14ARF (p=0,343) nem de Mdm2 (p=1,000). Adicionalmente, a expressão de
p14ARF foi demonstrada principalmente em amostras de tumores com positividade de
expressão para as proteínas p53 e Mdm2, simultaneamente. Igualmente, não foi possível
estabelecer qualquer relação entre as expressões das proteínas p53, p14ARF e Mdm2 e os
parâmetros clínicos analisados (padrão de hereditariedade, estádio de Reese-Ellsworth e
prognóstico vital).
Conclusões: Neste estudo observámos que 87,4% dos casos apresentaram marcação
nuclear e nucleolar da proteína p14ARF e, concomitantemente, documentámos a sobreexpressão
da desta proteína em metade dos casos, contrariamente a resultados de trabalhos
anteriores [3].
De acordo com os nossos resultados, obtivemos sobre-expressão da proteína Mdm2
em 79,2% das amostras de retinoblastomas, o que está de acordo com a hipótese que defende
que a sobre-expressão do MDM2 será um elemento importante na patogenia molecular do
retinoblastoma [2,4].
A pequena amostra de doentes utilizada neste estudo comprometeu os resultados
finais, nos quais não se demonstrou qualquer relação estatisticamente significativa entre os
parâmetros considerados. Futuros estudos devem ser realizados no sentido de estabelecer o verdadeiro valor prognóstico destes marcadores histológicos, recorrendo a uma amostra populacional de dimensões superiores.Introduction: Retinoblastoma is the most common primary ocular malignancy in pediatric age. Knudson proposed his two-hit model, allowing the distinction of retinoblastoma in two major classes: heritable and non-heritable. Retinoblastoma was first considered to arise from a well known mutation in the RB1 tumor-suppressor gene (chromosome 13q14). Currently, evidence supports that biallelic inactivation of RB1 gene is the initiating event, but not sufficient for fully malignant progression [1]. The hypothesis of altered expression of p14ARF-MDM2-p53 surveillance pathway components was proposed as an attempt to explain fully retinoblastoma development [2]. Previous studies proposed that p14ARF protein expression was undetectable, in contrast with Mdm2 protein overexpression in retinoblastoma [3]. Objectives: The aim of this study was to evaluate the immunohistochemical expression of p53 pathway components (p14ARF, Mdm2 and p53) in order to a better understanding of the molecular pathogenesis and differentiation of retinoblastoma. Additionally, it was attempted to correlate the expression of these proteins with retinoblastoma’s heritable pattern, Reese-Ellsworth staging and vital prognosis. Methods: A cohort of 24 retinoblastoma tissue samples from 22 enucleated cases was obtained from the registry of HUC’s Ophthalmic Pathology Laboratory. Clinical records were consulted to collect information including gender, age, heritable pattern, Reese-Ellsworth stage and prognosis. Immunohistochemistry was performed on formalin-fixed, paraffinembedded retinoblastoma tissue samples using primary antibodies against p53, p14ARF and Mdm2. Results: Positive p53, p14ARF and Mdm2 expression was obtained in 87.5% (21/24), 87.5% (21/24) and 95.8% (23/24) of the 24 samples, respectively. Overall, p53 protein expression was not positively correlated neither with p14ARF (p=0.343) nor Mdm2 expression P53,MDM2 AND P14ARF IMMUNOHISTOCHEMICAL EXPRESSION IN RETINOBLASTOMA 9 (p=1.000). In addition, p14ARF expression was mainly found in tissue samples that were positive for both p53 and Mdm2. Moreover, we did not obtain a positive relationship between p53, p14ARF and Mdm2 expression and the analyzed clinical parameters (heritable pattern, vital prognosis and Reese-Ellsworth staging). Conclusions: In our study, we obtained 87.4% of positive p14ARF nuclear and nucleolar expression and we even documented the presence of p14ARF overexpression in half of the cases, in opposition to previous reports [3]. According to our results, there was a Mdm2 overexpression in 79.2% of retinoblastoma samples, which supports the hypothesis that MDM2 overexpression may be an important element in retinoblastoma molecular pathogenesis [2,4]. The small cohort of patients involved in this study compromised the final results, which did not show any statistical significance. Further studies need to be performed in order to establish the true prognostic value of these histological markers, using a larger retinoblastoma patient’s population.
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Freedberg, D. E., S. H. Rigas, J. Russak, W. Gai, M. Kaplow, I. Osman, F. Turner, et al. "Frequent p16-independent inactivation of p14ARF in human melanoma." 2008. http://hdl.handle.net/10454/5973.
Full textAbstract:
BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16.
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Ivanchuk, Stacey M. "Identification and characterization of novel p14ARF tumour suppressor binding partners." 2005. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=232762&T=F.
Full text
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Hemmati, Philipp [Verfasser]. "Tumorbiologische Relevanz und Regulation des p14ARF Tumorsuppressorsignalweges / von Philipp Hemmati." 2010. http://d-nb.info/1010915339/34.
Full text
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Hung, Chia, and 黃嘉宏. "The association between cellular expression of MDM2 and p14ARF with Chronic Kidney Disease." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/98809748696984866714.
Full textAbstract:
碩士中山醫學大學
醫學研究所
100
Objectives According to Department of Health report of the rank of public mortality etiolgy, chronic kidney disease had been the tenth rank. Chronic kidney disease not only causes patient mortality by itself, Chronic kidney disease related morbidities also induce patient mortality(Wen, Cheng et al. 2008; Bowling, Feller et al. 2011). One of the pathogenesis of Chronic kidney disease was apoptosis, the correlation between apoptotic makers with acute kidney injury had been reported before(Kaushal, Basnakian et al. 2004; Bengatta, Arnould et al. 2009; Havasi and Borkan 2011), therefore, we studied if these apoptotic pathway makers also had the correlation to the severity of Chronic kidney disease on our kidney tissue samples. Methods We totally collect seventy Chronic kidney disease patients who received Nephroectomy due to all kinds of etiologies within three years. We retrospectively studies the correlation between Immunohistochemical stains of these kidney tissues by MDM2 and p14ARF with the clinical severity of Chronic kidney disease. Results We showed the glomerular and interstitial fibrosis scores had the significant difference between the advanced chronic kidney disease( eGFR < 45 ml/min/1.73 m2) and non- advanced chronic kidney disease( eGFR >= 45 ml/min/1.73 m2). In the area of normal tubular cytoplasm and atrophy tubular cytoplasm, p14ARF had stronger staining in the group of advanced chronic kidney disease( p vaule were 0.009 and 0.011 respectively，statistical significant difference). After further analysis, we found the patients who were over sixty five years- old, abnormal BMI( too light or over weight), had the diagnosis of Urothelial cell carcinoma (UCC), high serum Creatinine level and stronger staining p14ARF of normal tubular cytoplasm all had higher risk for advanced chronic kidney disease(Odds ratio and 95% Confidence Interval were 2.81 and 1.06－7.44； 66.00 and 6.08－716.18； 10.50 and 2.34－47.04； 3.75 and 1.08－13.07；558.00 and 48.24－6453.81；5.76 and 1.62－20.45). Conclusion We showed the Immunohistochemical stain of p14ARF over normal tubular cytoplasm area had positive correlation with the clinical severity of Chronic kidney disease.
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Wang, Yu-Chien, and 王郁茜. "Etiological association of alterations in p53-hdm2-p14ARF pathway with lung tumorigenesis in Taiwan." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/19243168942193336071.
Full textAbstract:
碩士國立臺灣師範大學
生物研究所
91
Lung cancer is the leading and second cause of cancer deaths among women and men in Taiwan, respectively. However, the molecular mechanisms involved in lung tumorigenesis in Taiwan remain poorly defined. There is increasing evidence that alterations in tumor suppressor genes and oncogenes are common in many forms of human cancer including lung cancer. We found that p53 gene mutation frequency was 17% in resected non-small cell lung cancers (NSCLC). However, p53 protein overexpression frequency was 48%. To further identify the molecular basis for this p53 immunohistochemical abnormality, we performed a genetic and epigenetic study of the p53 upstream proteins, p14ARF and hdm2, in NSCLC patients. Specimens of resected NSCLC from 113 patients were recruited in this study. Protein expression and mRNA expression of p14ARF and hdm2 were examined by immunoshitochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Methylation-based PCR assay was conducted to detect promoter methylation of the p14ARF gene. Loss of heterozygosity (LOH), homozygous deletion, and mutation of p14ARF gene were also examined. All data of p14ARF and hdm2 analyses were compared among patients with various clinicopathological parameters and with the p53 protein expression level. The data indicated that 45% and 41% lung cancer patients showed low or absent of hdm2 protein and mRNA expression, respectively. We also found that alternative splicing was the major mechanism which caused hdm2 gene alteration. In addition, we examined the frequency of protein expression of Akt kinase because Akt is known to associate with phosphorylation and nuclear localization of hdm2. The results indicated that the negative Akt kinase protein expression was correlated with negative hdm2 protein expression. With regard to p14ARF analyses, the data indicated that 34% and 31% lung cancer patients showed low or absent of p14ARF protein and mRNA expression, respectively. The frequency of p14ARF promoter hypermethylation, LOH, homozygous deletion, and mutation was 30%, 24%, 9% and 2%, respectively. We suggested that promoter hypermethylation was the major mechanism which caused p14ARF gene alteration. Note that 92% (35/38) patients with p53 overexpression showed absence or low expression of hdm2 protein and near overexpression of p14ARF protein. It indicated that the p53 overexpression was indeed induced by the dysregulation of the upstream proteins, hdm2 and p14ARF. Interestingly, most of the NSCLC patients with dysregulation of the p53-hdm2-p14ARF pathway were suffered late stage and SQ type of cancer. In addition, the patients with p53 overexpression had poor prognosis (P=0.013). The study was the first report which examines all possible alteration pathways in p53-hdm2-p14ARF gene/protein deregulation in the same series of NSCLC, and examines their relationship with the clinical data of NSCLC. In addition, it was also the first report on the alternative splicing of hdm2 mRNA in lung cancer. In conclusion, the alteration of p53-hdm2-p14ARF regulation pathway plays an important role in tumorigenesis of lung cancer in Taiwan, and could be potentially used as a molecular prognostic marker.
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Wen-Ting, Yeh, and 葉文婷. "Analysis of gene alterations of p14ARF-p53 and p16INK4a-Rb pathways in bladder cancers." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/87649100212305782273.
Full textAbstract:
碩士高雄醫學大學
醫學研究所
89
Bladder cancer is a significant health problem in the world. According to previously reports, alteration on chromosome 9 is the most common events in bladder cancer. The 9p21 gene cluster, harboring growth suppressive genes p14ARF and p16INK4a, play active roles in the p53 and Rb growth control pathways, respectively. p16INK4a is a cyclin- dependent kinase inhibitor functioning upstream Rb. p14ARF restrains cell growth by abrogating Mdm2 inhibition of the p53 activity and facilitates p53 mediated cell cycle arrest and apoptosis. Recent studies revealed that inactivation of p16INK4a/p14ARF gene might lead to cell carcinogenesis. In the present study, the mechanisms of inactivation of p16INK4a and p14ARF genes in bladder cancers were analyzed. The relative contribution of p14ARF/p53 and p16INK4a/Rb pathways in bladder cancer development was also evaluated. In this study, homozygous deletions of the p16INK4a and p14ARF genes were analyzed in 53 human bladder cancers by using Southern hybridization. The results showed that homozygous deletion of p16INK4a gene was observed in 12 (23%) samples. In contrast, a relatively higher percentage (43%, 23/53) of samples showed homozygous deletions of p14ARF. In addition, most of the p14ARF gene deletions occurred exclusively on the exon 1β region (91%, 21/23). However, only 2 samples contained deletion in exon 1β, 1α and exon 2. We also found that p14ARF gene deletion in well and morderate differentiated tumors and poor differentiated tumors were 6 (27%) and 17 (63%) respectively. PCR- SSCP and sequencing were performed to analyze the mutation status. Results showed that no mutations were detected in exon 1αand 1β in any cases. Only one mutation in exon 2 cause a missense mutation in the p16INK4a and p14ARF reading frame (2.5%；1/40) was found. This mutation was identified in p16INK4a codon 108 (GAT→CAT), resulting in a change of aspartic acid to histidine. The mutation is inside the ankyrin repeat motifs in a region that is required for the protein to bind and inhibit CDK4. The same mutation in the p14ARF open reading frame was at codon 122 (CGA→CCA), resulting in a change of arginine to proline. DNA methylation patterns in the CpG islands of the p16INK4a and p14ARF gene were determined by MSP. p16INK4a CpG island methylation was found in 24 of 40 (60%) tumors, but no methylation was detected in p14ARF in any cases. An analysis of the p16INK4a and p14ARF mRNA expression was carried out by RT-PCR. Results showed that p16INK4a mRNA expression was not detected in 27 of 49 (55%) tumors, including 12 cases harboring deletion and 15 cases containing methylation. However, expression of the p16INK4a gene was also observed in 7 cases with DNA methylation, suggesting partial methylation on one allele. Suppressed p14ARF mRNA expression was detected in 28 of 49 tumors (57%). But, 5 of the 28 samples with suppressed p14ARF mRNA expression contained no detectable alteration. Uncoupling between p14 transcript and p14ARF alteration suggest a novel mechanism of inactivation existed. In this study immunohistochemistry examined the expression of P16 and P14 together with RB and P53 status in bladder cancers. The results indicated that P14 was frequently absent in bladder cancers (27/39, 69%). Abnormal expression of P53 was also observed in 27 of 39 (69%) samples. Concomitant absent P14 expression and staining of P53 was frequently observed in bladder cancer (17/39; 44%). P16 protein was undetectable in 25 of 39 (64%) samples. Absence RB protein expression was seen in 23% (9/39), however, overexpression was frequently observed in 38% (15/39) samples. Furthermore, result indicated that RB protein unexpression was significantly correlated in invasive bladder cancer. Concomitant P16 and P53 protein abnormal expression was frequently observed also in bladder cancer (44%, 17/39). Taken together, the results showed that in cases of bladder cancer, at least one protein was altered, and alterations of tumors in both p16INK4a/Rb and p14ARF/p53 pathways was more often (77%, 30/39) than tumors with one or no alteration of these two major pathways. Our results show that in bladder cancers, p14ARF is a primary target of homozygous deletion, whereas p16INK4a is the hotspot of methylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulatory pathways during bladder cancer development.
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Liao, Chien-Huang, and 廖建皇. "Molecular Mechanisms of 1-Nitropyrene, Benezo [a] pyrene, p14ARF and Taxol mediated Telomerase Regulation in H1299 NSCLC Cell Line." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/93833661872611337847.
Full textAbstract:
碩士中山醫學大學
毒理學研究所
90
貳、英文摘要 Telomerase activity is not detectable in most somatic cell but is upregulated in 85~95% of human canner, which suggests important role of telomerase in neoplastic transformation. Telomerase revese transcriptase was the determinant rate-limiting step in telomerase regulation, and its active promoter activity was involved in telomerase reactivation. Therefore we have cloned the telomerase revese transcriptase promoter (h.TERT-p548). In addition , we co-transfected a series of constructs containing unidirectionally delected fragments： h.TERT-p-212~+50 (core promoter)、h.TERT-p -196~+50 (deleted SP1 site)、h.TERT-p-177~+50 (deleted SP1 and c-MYC site)、h.TERT-p-155~+50 (deleted SP1、c-MYC and AP-2 site), respectively, to examine whether B[a]P、1-NP、p14ARF and taxol regulated telomerase activation through those transcription factors. In the present study, we first employed TRAP and RT-PCR to elucidate whether the regulation of telomerase activity and h.TERT mRNA expression in human NSCLC cell by treatment with B[a]P、1-NP and p14ARF . The telomerase activation was not influence by treament with B[a]P、1-NP and p14ARF ；h.TERT mRNA level was increased 1~2 fold by low concentration of B[a]P and 1-NP , and that was increased 1~1.5 fold by treatment with p14ARF .h.TERT-p548 was activated by low concentration of B[a]P, and repressed by 1-NP、p14ARF and taxol when experiment with transient transfection and luciferase reporter assay . In addition , they did not modulate these transcription factor to regulate h.TERT transcription activation. Significantly, after we alone transfecting h.TERT-p-177 or h.TERT-p-155 plasmid DNA , each still expressed 50~60% transcription activation.It indicated that except for c-Myc, AP-2 and NF-E2 have the ability to regulate telomerase reactivation. Finally our flow cytometric studed show that B[a]P、1-NP and p14ARF slightly increase G0/G1 percentage , and taxol cause cell cycle arrest in G2/M phase leading apoptosis . Furthermore, B[a]P、1-NP、p14ARF and taxol can induce cytotoxicity in H1299 and casue cell death using MTT and colony formation assay . In summary, this study provides data showing that B[a]P、1-NP、p14ARF and taxol all were able to regulate h.TERT transcriptionactivation , but independently of c-Myc . Maybe they dependent on other transcription factors to regulate telomerase reactivation.