Submitted by Israel Vieira Neto (israel.vieiraneto@ufpe.br) on 2015-03-05T16:35:59Z No. of bitstreams: 2 Dissertação Thaisa Feliciano de Souza.pdf: 1528321 bytes, checksum: a027e8dccdd6f0e007998541819c3f48 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T16:35:59Z (GMT). No. of bitstreams: 2 Dissertação Thaisa Feliciano de Souza.pdf: 1528321 bytes, checksum: a027e8dccdd6f0e007998541819c3f48 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-05-03 / CNPq / Nos estágios iniciais do câncer de laringe a radioterapia é uma das principais opções de tratamento e cura da doença. Entretanto, esta modalidade terapêutica resulta em reações adversas do tecido sadio adjacente ao tumor. Embora submetidos ao mesmo protocolo, os pacientes apresentam reações com intensidades diferentes, fenômeno atribuido à radiossensibilidade individual que é acarretada por falhas no rearo de danos celulares. Em termos moleculares, os mecanismos de reparação celular estão intimamente ligados aos níveis de expressão da proteína p53. Neste contexto, esta pesquisa teve como objetivo principal investigar possíveis correlações entre os níveis de expressão da proteína p53 e as principais reações adversas resultantes da radioterapia de pacientes com câncer de laringe. Para tanto, foi coletado sangue periférico de 10 pacientes com câncer de laringe, em estágio T1 e T2, submetidos ao mesmo protocolo de radioterapia. Uma parte das amostras de sangue foram irradiadas com dose de 2 Gy, enquanto outra foi mantida sem irradiar (controle). Em seguida, linfócitos foram separados e marcados com anticorpo monoclonal anti-p53 e posteriormente analisados por citometria de fluxo. Avaliou-se as reações adversas de hiperemia, rouquidão, disfagia e odinofagia nos pacientes após a última sessão de radioterapia. Observou-se um aumento estatisticamente significante da expressão da p53 após irradiação das amostras de sangue, mas com grande variação interindividual. Relacionando os graus de reações adversas de pele com a expressão basal (células não irradiadas) e expressão da p53 nas células irradiadas com 2 Gy, não foi observada correlação entre os dados. As reações adversas de rouquidão, disfagia e odinofagia tiveram correlação com a expressão basal da p53 dos pacientes, onde quanto maior a expressão basal da p53, menores foram os graus de tais reações após a radioterapia. Relacionando essas reações adversas com a porcentagem da expressão da p53 após irradiação das células, não foi observado correlação. Os resultados desta pesquisa sugerem a análise dos níveis basais da proteína p53 de linfócitos do sangue periférico, por citometria de fluxo, para prognóstico das principais reações adversas (rouquidão, odinofagia e disfagia) em pacientes com câncer de laringe nos estágios iniciais, encaminhados para radioterapia.

Made available in DSpace on 2014-06-12T23:02:31Z (GMT). No. of bitstreams: 2 arquivo4298_1.pdf: 893623 bytes, checksum: 0bcd91f818d9d6a5db4eae01d84b07ed (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os métodos imunohistoquímicos associados aos sistemas computadorizados de análise de imagens tem sido de extrema importância, como ferramentas auxiliares, no estudo da expressão de proteínas relacionadas a neoplasias malignas em pacientes portadores de doenças inflamatórias intestinais. Neste estudo, obtive-se o perfil imunohistoquímico das proteínas p53 e Ki-67 do tecido intestinal de pacientes com colite ulcerativa (n=30) um grupo tratado clinicamente e outro cirurgicamente de ambos os sexos e idade média de 38,5 anos. Os tecidos foram fixados em formalina a 10%, submetidos à rotina histológica e montados em parafina. Fragmentos de tecido (4 μm) foram submetidos à técnica de imunohistoquímica para as proteínas Ki-67 e p53. Os perfis de marcação tecidual foram quantificados através de uma estação de análise de imagem contendo um microscópio óptico equipado com uma câmera digital ambos acoplados a um computador contendo o software OPTIMAS®. Os resultados obtidos demonstram diferenças significativas nos padrões de marcação no tecido inflamado em pacientes tratados clinicamente quando comparados aos tecidos de pacientes tratados cirurgicamente. As proteínas Ki-67 e P-53 indicam marcadores imunohistoquímicos úteis para diferenciação de tecidos de colite ulcerativa que trazem características pré-neoplásicas sugestivas de malignidade

In addition to the canonical full-length p53 (FLp53), the TP53 gene produces twelve protein isoforms through alternative RNA splicing or initiation of transcription and translation. Two of<br>these isoforms, D133p53a and p53b, have been identified as endogenous regulators of cellular senescence. Cellular senescence is a durable cell cycle arrest that inhibits the continued replication of aged and DNA-damaged cells. This process is a critical mechanism of tumor suppression that<br>prevents initiation and malignant progression and has been leveraged to treat cancers including glioblastoma. However, removal of senescent cells by macrophages is needed to restore tissue homeostasis. This process is impacted by a variety of factors. For example, senescent cells accumulate in aged individuals and can promote chronic inflammation and disease through the senescence-associated secretory phenotype (SASP). As the global population ages, it will become more critical to understand the function of cellular senescence in disease. Targeting senescent cells, either through elimination (senolysis) or reprogramming, may have potential therapeutic value in individuals with a high senescent cell burden. Aged or DNA-damaged cells adopt a senescence-associated p53 isoform profile characterized by reduced expression of D133p53a and increased expression of p53b. Critically, restoration of D133p53a rescues cells from senescence and enhances DNA repair. Targeting p53<br>isoforms may represent a mechanism by which cells can be reprogrammed. A thorough understanding of the contexts in which senescent cells maintain beneficial or harmful roles is<br>critical to developing senescence therapeutics in cancer and aging.

It is well known that ionizing radiation is genotoxic, and can trigger heritable mutations in the germ cells of an animal. Recently, researchers have used hypervariable expanded simple tandem repeat (ESTR) regions of DNA to explore this phenomenon. ESTRs facilitate the examination of induced genetic mutations using relatively low radiation doses and fewer mice than more traditional approaches. Numerous studies have examined the responses of ESTRs to radiation in the germ line; however the mechanism behind germ line mutations at ESTR loci is poorly understood. Current hypotheses propose that error-prone DNA repair, which allows for misalignment of DNA strands through replication slippage produces in changes in ESTR size. P53 is involved in DNA replication as well as repair of DNA damage, apoptosis and other cancer-related processes. We use p53-deficient heterozygous male mice to examine the role of p53 in germ line mutations at ESTR loci. Males were irradiated with a variety of dose combinations both prior to and post-meiosis, and were mated to unirradiated wildtype females. DNA from the adults and offspring was analyzed for mutations at ESTR loci using DNA fingerprinting. Surprisingly, the study found no significant differences in germ line mutation rate between any treatment groups, including the 0Gy and 1Gy treatments. I discuss the possibility that these results are due to the p53 deficiency of the males, and that p53 homozygosity is necessary for radiation-induced germ line mutations at ESTR loci to occur. I conclude that further studies need to be done, including a control study using wildtype males of the same background strain as that of the p53 deficient line in order to verify our results. / Thesis / Master of Science (MS)

Tumorigenesis is the result of uncontrolled cell growth due to the deregulation of cell cycle checkpoints 1. Period 2 (Per2) is a tumor suppressor that oscillate in expression in a 24-hour cycle 2, 3. Here, we show that Per2 interacts with the tumor suppressor protein p53. Both G1 and G2 checkpoint pathways involve a p53 dependent pathway which can trigger the cell to go through cell arrest or programmed cell death4. Understanding all the mitigating factors involved in regulating cell cycle progression under DNA damage can offer a better idea in how cells become immortal. Initially discovered through screening of a human liver cDNA library, the novel interaction between p53-Per2 was further documented using co-precipitation. Interestingly, under genotoxic stress conditions, p53 and Per2 were not found to bind which leads us to suspect that Per2 does not affect active p53 which may possibly be due to post translational modifications of its active state. Furthermore we investigated p53's ability to act as a transcription factor in the presence of Per2, showing that the Per2-p53 complex prevents p53 from binding to DNA. This implies that the tetramerization of p53 may also be another factor in Per2's ability to bind to p53. A truncated p53 lacking the last 30 amino acids that theoretically increase p53's ability to form a tetramer showed a drastic reduction in binding to Per2 5, 6. On the other hand, p53 lacking the tetramerization domain showed binding similar to wildtype. Consequently we speculate that the ability of Per2 to modulate p53 and act as a tumor suppressor protein may be dependent on either the post translational modifications of p53 or its oligomeric state. / Master of Science

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. In the first part of the thesis, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by crossing the conditional Mdm4 and Mdm2 alleles to an erythroid-specific-cre (EpoRGFP-Cre ) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, interestingly, Mdm4 only contributes to p53 regulation at a specific phase of the differientation program.<p><p>Moreover, a large body of evidence indicates that aberrant expression of either MDM2 or MDM4 impairs p53 tumor suppression function and consequently favors tumor formation. Overexpression of MDM2 was observed in 10% of 8000 human cancers from various sites, including lung or stomach, and MDM4 was found amplified and/or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach and breast cancers. Remarkably, selective MDM4 amplification occurs in about 65% of human retinoblastomas. In contrast, MDM2 amplifications are relatively rare (about 5%) in retinoblastomas, indicating that depending on the tumor context (cell type, initiating oncogene, …), MDM4, rather than MDM2, overexpression might be selected for as a more efficient mean of suppression of p53 function. As part of a large effort to better understand why different cell types require distinct combinations of mutations to form tumours, we will examine the molecular basis for selective up-regulation of Mdm4 in retinoblastomas. In this context, we have successfully generated 2 conditional transgenic mouse lines expressing either mycMdm2 or mycMdm4 driven by the PCAGGs promoters in the ROSA26 locus. Since a cassette containing a floxed transcriptional stop element is inserted upstream of the transgenes, we can achieve tissue-specific expression and spatio-temporal regulation of the transgenes by using different Cre and CreER. By the use of N-terminal myc-tag fused with the transgenes, we are able to compare the expression levels of the transgenes. Finally, due to C-terminal IRES-GFP element, we can easily identify transgene expressing cells. One of our aims is to use this Mdm4 conditional transgenic mouse line as the first, non-chimeric, mouse model of retinoblastoma that can be used as an appropriate preclinical model to improve treatment of this disease.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

<p>In Sweden approximately 2800 new lung cancer patients are diagnosed every year. Radiotherapy is used with curative intention in certain groups of patients. The aim of this thesis is to study the basis of differences in radioresistance and the possibility to predict response to radiotherapy.</p><p>In the first study we investigated, using the comet assay, four lung cancer cell lines with different sensitivity towards radiation. A clear dose-response relationship for radiation-induced DNA single strand and double strand breaks were found. All cell lines showed a remarkably efficient repair of both the DNA single strand and double strand breaks one hour after irradiation. However, further studies in one radioresistant and one radiosensitive cell line demonstrated that repair during the first 15 min had the best accordance with radiosensitivity measured as surviving fraction.</p><p>In the second and third study, sequencing studies of the p53 gene were performed on cell lines as well as on tumour material. Cell lines that were expressing a mutation in exon 7 were associated with increased radiosensitivity compared with tumor cell lines with mutations in other exons. In the clinical study, 10 patients were found to be mutated in the p53 gene whereas the other 10 patients were not. No correlation to clinical parameters could be drawn.</p><p>In the fourth study, serum from 67 patients with a confirmed diagnosis of non-small cell lung cancer was investigated for the presence of p53 antibodies. P53 antibodies in sera, taken prior to radiation treatment, were associated with increased survival.</p><p>The summary of this thesis indicates that the p53 gene has an impact on the effect of radiotherapy in lung cancer. The presence of p53 antibodies might be of clinical interest for predicting survival after radiotherapy. Further studies on the importance of the p53 gene on early repair are of interest. </p>

In Sweden approximately 2800 new lung cancer patients are diagnosed every year. Radiotherapy is used with curative intention in certain groups of patients. The aim of this thesis is to study the basis of differences in radioresistance and the possibility to predict response to radiotherapy. In the first study we investigated, using the comet assay, four lung cancer cell lines with different sensitivity towards radiation. A clear dose-response relationship for radiation-induced DNA single strand and double strand breaks were found. All cell lines showed a remarkably efficient repair of both the DNA single strand and double strand breaks one hour after irradiation. However, further studies in one radioresistant and one radiosensitive cell line demonstrated that repair during the first 15 min had the best accordance with radiosensitivity measured as surviving fraction. In the second and third study, sequencing studies of the p53 gene were performed on cell lines as well as on tumour material. Cell lines that were expressing a mutation in exon 7 were associated with increased radiosensitivity compared with tumor cell lines with mutations in other exons. In the clinical study, 10 patients were found to be mutated in the p53 gene whereas the other 10 patients were not. No correlation to clinical parameters could be drawn. In the fourth study, serum from 67 patients with a confirmed diagnosis of non-small cell lung cancer was investigated for the presence of p53 antibodies. P53 antibodies in sera, taken prior to radiation treatment, were associated with increased survival. The summary of this thesis indicates that the p53 gene has an impact on the effect of radiotherapy in lung cancer. The presence of p53 antibodies might be of clinical interest for predicting survival after radiotherapy. Further studies on the importance of the p53 gene on early repair are of interest.

The tumour suppressor p53 is essential for genome stability and loss of its function can lead to human cancer. The functional roles of p53 are regulated by a variety of mechanisms, some of which are not well understood. However, the murine double minute 2 (MDM2) protein, a major negative regulator of p53, has been found to be overexpressed in many human cancer cell lines in which p53 was not mutated; thus establishing MDM2 as a target for cancer therapeutics. MDM2 is defined as both an oncoprotein and an E3-ubiquitin ligase; its interactions with p53 are controlled through multiple domains, providing different possible pathways to inhibit MDM2 and therefore reactivate p53 function. Previous work undertaken in the Ball laboratory has shown that the MDM2 RING domain plays a critical role in p53 ubiquitination; thus screening for modulation of its activity by small molecules could provide new leads for the inhibition of the E3 ligase activity of MDM2. The MDM2 RING domain was cloned, expressed and purified so that it could be studied using a series of in vitro experiments. The generation of a library of short (12-mer) peptides as potential inhibitors of the MDM2 RING domain was investigated using phage display against His-tagged RING protein to screen the peptide ligands. In order to study the specificity of these peptides towards MDM2 (res. 396-491 and 396-479) compared with MDM4 and BRCA1, the MDM4 RING domain (res. 395-490 and 395-478) and BRCA1 (res. 1-304) domain were expressed and purified for further characterisation. A small selection of peptides was isolated and their binding affinity and activity as MDM2 inhibitors evaluated by in vitro ELISA, affinity chromatography and ubiquitination assays. One peptide in particular, KCCYFETHMPRH, was found to bind to MDM2 and was able to inhibit MDM2-mediated ubiquitination of p53 in vitro. Preliminary optimisation of this peptide by alanine scan revealed a peptide with a 2-fold increased potency. Since peptides provide comparatively weak therapeutic leads due to a combination of poor cellular uptake and susceptibility to cleavage by proteases, cyclic peptidomimetics based upon this lead were developed using side-chain to side-chain cyclisation. These peptidomimetics were successfully generated by the synthesis and incorporation of novel N-propargylated glycine and N-azidoalkyl glycine building blocks into a peptide sequence by Solid Phase Peptide Synthesis (SPPS). Following a Copper-catalysed Azide-Alkyne Cycloaddition (CuAAC) on solid phase or in solution, these peptoid-peptide hybrids were isolated, purified and characterised.