Proteomic technologies verified Anterior Gradient 2, AGR-2, as a protein over-expressed in human cancers, including breast, prostate and oesophagus cancers, with the ability to inhibit the tumour suppressor protein p53. AGR-2 gene is a hormone responsive gene with an unexpected induction by the anti-cancer drug tamoxifen highlighting the proto-oncogenic role of this protein. Anterior Gradient-2 encodes one protein that gives rise to two forms· the full length and the mature one. Full length bears a leader sequence that leads the protein to secretion. Localization studies of both forms of AGR-2 were performed using fluorescence microscopy and subcellular fractionation, in order to determine in which compartment the protein functions. Localization mutants of the mature and full length protein determined the exact sequence required for certain localization patterns. Once localization was confirmed, the mechanism of how Anterior Gradient-2 localization within the cell can inhibit p53 was initiated. Furthermore, novel peptide aptamers that bound to the protein were cloned into GFP vectors and their effect on AGR-2 was investigated. AGR-3, another member of the family, was also examined in terms of localization and function in MCF-7 cells. Yeast two hybrid analysis has identified potential nuclear and cytoplasmic binding partners for AGR-2, essential for the upstream or downstream regulation of the AGR-2 pathway. In conclusion, we present data showing models of how the Anterior Gradient protein family might function as drug-resistance survival factor in cancer as well as a p53 inhibitor, suggesting a multi-potent role of its members when it comes to trafficking, cellular localization and activation or inhibition pathways in cancer.

P53 is a tetrameric transcription factor which exhibits DNA binding activity through its core domain which encompasses the conserved domains (known as Box II, III, IV and V). The N-terminal domain of p53 provides a scaffold for binding of components of the transcriptional machinery. Phosphorylation at residues within the N-terminal transactivation domain of p53 such as Serine 20 is a crucial event in the activation of p53. It stabilises the binding of the co-activator p300, reduces the binding of the inhibitory partner Mdm2 and enhances activation of p53 target genes. The identification of enzymes that phosphorylate p53 transactivation domain is an important development in the ongoing mapping of signaling pathways that control p53-dependent transcription and resultant tumour suppression. Environmental and physiological stresses activate p53 which has led to the creation of several hypothetical models in which tumour suppressor kinases mediate p53 activation by phosphorylation at Serine 20. Although much researched the identity of the main Serine 20 kinase in cells remains undefined. In this study we have identified Calcium Calmodulin kinase superfamily (CAMK) members as potent Serine 20 kinases in cells and show that the co-transfection of p53 peptides derived from the conserved domains can modify this response. Moreover, we show that the multi-protein docking site, p53 Box V domain, is required for Serine 20 phosphorylation and ubiquitination of p53. To further define the domains required for the interaction of p53 with CAMK superfamily members, mutagenesis of p53 was performed. Using transcriptional and binding based assays we were able to establish that p53 does indeed form an interaction with Chk1 and DAPK1. Development of cell models and gene expression studies demonstrated that depletion of Chk1 and DAPK1 results in activation of the p53 signalling pathway. There may therefore be a role for kinases as negative regulators of p53 and a potential for the development of kinases as drug targets for reactivation of the p53 pathway.

Polyglutamine expansion disorders are progressive neurodegenerative diseasesthat are caused by the pathological expansion of polyglutamine repeats. Huntington'sdisease (HD) is a polyglutamine disorder caused by the expansion of an existingpolyglutamine tract in a novel protein, Huntingtin (Htt). Oxidative stress has beenimplicated in the neural dysfunction observed in multiple neurodegenerative conditionsincluding HD. The tumor suppressor p53 is a multifunctional protein that has roles inthe cell cycle, apoptosis and neurodevelopment. The role of p53 in HD-associatedneurodegeneration has been studied but not fully elucidated, nor has the role of p53 inoxidative stress toxicity been fully elucidated.Here I present work that demonstrates polyglutamine expansion inducedalterations to p53 stability, localization, and activity. The transcriptional activity of p53was found to have a role in oxidative stress mediated as well as polyglutaminemediated neurotoxicity in vitro. The expression of p53 was also altered in vivo in amouse model of HD as well as in HD brain.Taken together, these data demonstrate a role for p53 in polyglutamine and oxidativestress toxicity.

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. 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.

A digoxigenin-labeled nonradioactive detection system was used to screen a zebrafish cDNA library for p53-like and ras-like genes. One clone was isolated and identified as an incomplete p53-like gene. The insert size of this clone is 1777 bp, which encodes part of evolutionarily conserved region II and all of regions III, IV, and V. A magnetically enriched whole zebrafish cDNA library was constructed to enhance possible recovery of ras-like genes in zebrafish. One clone, termed Zras-Bl, carried an insert of 2592 bp with an open reading frame encoding a 188 amino acid residue ras p21 protein. Based on total protein sequence, this expressed zebrafish ras p21 is most closely related to human N-ras (91% homology), with lesser homology to Ha-ras (84%) and Ki-ras (85%). Preliminary partial sequence data obtained by genomic and reverase transcriptasepolymerase chain reaction (RT-PCR) screening indicate the presence of at least one additional expressed ras gene in zebrafish. The tumorigenicity and Ki-ras mutational properties of dietary 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DBP) were compared in rainbow trout. Both chemicals elicited predominantly 12(1)G->A and 12(2)G->T mutations in trout liver tumors. Two {12(1)G->T and 12(2)G->T} and one {12(1)G->A and 12(2)G->T} double mutation were also observed in DBP livers tumors, but not in DMBA liver tumors. Some stomach tumors from both chemicals exhibited so much DNA degradation that routine PCR amplification was not possible. Among sixteen DMBA stomach tumors with intact DNA, no Ki-ras mutations were found. Of sixteen DBP stomach tumors examined, one had 12(1)G->A and two had 13(1)G->C mutations. The observed G->T transversions are compatible with apurinic mutagenesis driven by unstable DNA adducts arising from one-electron oxidation, but this is not true for the major G->A transitions or G->C transversions and rare double mutations found in this study. The low sensitivity of direct sequencing may limit the frequency of ras mutant detection in this study. / Graduation date: 1996

No description available.

Osteosarcoma (OS) is the most common primary malignant bone tumour in humans and dogs. Although medicine has made dramatic progress in treating osteosarcoma by surgery, with chemotherapy given before and after surgery, drug resistance and highly metastatic spread are often responsible for the failure of current therapies. Thus, more effective therapeutic approaches for treating osteosarcoma are needed. Previous results from our laboratory and others had shown that the basic-leucine zipper (bLZip) containing transcription factor, Zhangfei/CREBZF is a potent inhibitor of a variety of other transcription factors and has a dramatic effect on the growth of several cancer cell lines, including dog OS and human medulloblastoma cells. The objective of the studies described in this thesis was to determine the molecular mechanisms by which Zhangfei exerts its effect on dog and human OS cells. Several stressors in the microenvironment of cancer cells directly or indirectly perturb the endoplasmic reticulum (ER), which then activates the Unfolded Protein Response (UPR). The UPR modulates the effects of stress and allows tumours to survive, develop, metastasize and escape therapy. The UPR is regulated by three bLZip transcription factors—ATF6, ATF4 and Xbp1s. Since Zhangfei inhibits Luman/CREB3, a bLZip structurally similar to and closely related to ATF6 and ATF4, I initially focused my efforts on this pathway. I hypothesized that Zhangfei interacts with UPR-related bLZip transcription factors and inhibits their ability to activate the UPR signaling pathways, thereby suppressing the growth of cancer cells and increasing their susceptibility to ER stressors. To test this hypothesis, we monitored cell growth as well as levels of UPR gene transcripts and proteins in several dog and human osteosarcoma cell lines infected with adenovirus vectors expressing Zhangfei, and studied the interactions between Zhangfei and the UPR-mediator, Xbp1s. The results showed that the ectopic expression of Zhangfei in cell lines derived from dog osteosarcomas potently suppressed cell growth and inhibited their ability to activate the UPR. Further studies demonstrated that Zhangfei inhibited the UPR, at least partially, by binding to Xbp1s and suppressing its ability to activate transcription from a promoter containing unfolded protein response elements (UPRE). The leucine zipper of Zhangfei was required for this interaction, which led to the subsequent proteasomal degradation of Xbp1s. However, we also found that the effects of Zhangfei were not universal. While Zhangfei had a profound effect on the growth and UPR in some OS cell lines, it either had only a partial effect, or no effect on others. This suggested that susceptibility (or resistance) to Zhangfei may be an inherent property of OS cell lines. Since the suppressive effects of Zhangfei were not universal, and it had no obvious effects on untransformed cells and some cancer cell lines, I proposed that Zhangfei mediates its effect on cell growth and the UPR through an intermediary that is either not induced or is defective in cells that are unaffected by Zhangfei. I found that this intermediary was the tumour suppressor protein p53. The inhibitory effects of Zhangfei were only observed in the wild-type p53 expressing OS cell line U2OS while Zhangfei had no effect on the p53-null OS cell line MG63. In cells with functional p53, the ectopic expression of Zhangfei caused it to displace the ubiquitin ligase mdm2 and stabilize p53. Suppression of p53 by siRNA partially inhibited the effects of Zhangfei on the UPR and cell growth. In contrast, OS cells lacking functional p53 could be made to respond to Zhangfei if they were transfected to express wild-type p53. These results explain why Zhangfei has a profound effect on some cancer cells while having no obvious effect on others. I also characterized the interaction of Zhangfei and p53 by mapping the interacting domains on both proteins, showing that the bLZip domain of Zhangfei and the N-terminal transactivation domain (NTD) of p53 were required for their interactions. My findings reveal the profoundly inhibitory effects of Zhangfei on OS growth and the UPR, a stress-response known to promote tumour survival. I also show how Zhangfei may exert its effects. My work suggests an alternative modality for the therapy of certain types of OS, and perhaps other tumours with functional p53.

Inactivation of p53 by mutation occurs in half of human tumours. The majority of these mutations affect the DNA-binding core domain and hence impair DNA binding and hinder transcription of p53 target genes. A wealth of data indicates that even cancers carrying wild type p53 protein, evolve mechanisms to render the p53 pathway inactive. Thus, inactivation of the p53 response, either by mutation or the alternative mechanisms, allows unpurturbed tumour growth. Recent work identified Anterior Gradient-2 (AGR2) as a protein overexpressed in wild type p53 expressing tumours and it was subsequently shown that AGR2 inhibits p53 pathway. In this study I confirmed that AGR2 protein inhibits p53 and AGR2 depletion potentiates p53-dependent DNA damage response. As there were no physiological signals known that regulate the AGR2-p53 axis, here I set out to identify pathways that activate or inhibit AGR2. I found that transforming growth factor β(TGF- β) triggers AGR2 protein reduction and this is concomitant with the stabilisation and increased activity of p53 protein. TGF-β halts AGR2 transcription in a SMAD4- dependent manner and triggers AGR2 protein degradation involving an ATM kinase. I found that SMAD nuclear interacting protein (SNIP1) mediated the ATMdependent AGR2 degradation. Interestingly, SNIP1 overexpression by itself promoted AGR2 protein degradation. I found that AGR2 protein degradation was proteasome independent and involed autophagy-lysosomal degradation pathway. As the mechanism of p53 inhibition by AGR2 is not known, I reasoned that identifying interactors of AGR2 may potentially further our understanding of the mechanism accounting for AGR2-mediated p53 inhibtion. I isolated the ATP binding protein Reptin in the yeast two-hybrid system and subsequently validated it as an AGR2 binding partner. Mutations of the two ATP binding motifs in Reptin resulted in altered oligomerization and thermostability of Reptin and affected the AGR2-Reptin complex stability. I also identified the Reptin docking site and it was mapped to a divergent octapeptide loop. I found that AGR2-Reptin complex coimmunoprecipitated with the p53 protein. Subsequently, I showed that Reptin protein can influence p53 activity, and depending on local concentration, either inhibit the transcription of p53-genes or chaperone its DNA binding activity. Interestingly, I found that Reptin formed a stable complex, independent of AGR2, with p53 R175H, p53 F270A, p53 S269D and p53 S269A, which has implication for the Reptin function in cancers bearing mutant form of p53 protein.

Magíster en Ciencias de la Ingeniería, Mención Química / Ingeniero Civil en Biotecnología / Durante las últimas décadas, una rama de la investigación biotecnológica se ha enfocado en desarrollar y optimizar procesos de producción de proteínas recombinantes orientadas a aplicaciones terapéuticas. En particular, los péptidos terapéuticos ofrecen actualmente un nuevo potencial comercial para la industria farmacéutica y biotecnológica al generar estrategias efectivas en el tratamiento de diversas patologías como cáncer y alteraciones metabólicas entre otras. Estudios previos han demostrado la capacidad supresora de tumores de péptidos con blanco intracelular como p53p-Ant y PNC-27. Estos corresponden a regiones derivadas de la proteína p53, factor de regulación transcripcional que inhibe la progresión del ciclo celular e induce reparación celular o apoptosis, en caso de estrés genotóxico. Ambos péptidos contienen en su secuencia, un péptido de penetración celular, el cual les entrega la capacidad de atravesar la membrana plasmática. Así, bajo distintos mecanismos, con p53p-Ant y PNC-27 se ha reportado apoptosis o bien necrosis de manera selectiva en ciertos tipos de células tumorales. El presente trabajo tuvo por objetivo expresar en E. coli los péptidos p53p-Ant y PNC-27, y purificarlos mediante el sistema IMPACT. Éste es un mecanismo de expresión y purificación mediado por inteínas que permite purificar a través de una columna de afinidad, proteínas recombinantes con su secuencia nativa sin el uso de proteasas y minimizando pasos cromatográficos. Se logró producir ambos péptidos en forma soluble. La expresión soluble del péptido PNC-27 se consiguió a partir de los dos vectores de expresión proporcionados por el sistema IMPACT (pTXB1 y pTYB11), esta expresión resultó ser fuertemente dependiente de las condiciones de cultivo ya que se requiere de una inducción a baja temperatura (12 °C). Por su parte, la expresión soluble de p53p-Ant depende tanto del sistema de expresión como de las condiciones de cultivo. El diseño de las construcciones genéticas, en particular las que incluyen el vector de expresión pTYB11, permitió una efectiva purificación de los péptidos utilizando un único paso cromatográfico. Más aún, los niveles de rendimiento (0,4 1,2 mg L-1) superan los reportados para péptidos con el mismo sistema y los niveles de pureza obtenidos permitirían desarrollar investigación avanzada con ensayos biológicos in vivo o in vitro.

Objetivos. Determinar la influencia de los marcadores: antígeno Ki67 y del gen p53 como factores pronóstico independiente en la sobrevida de los pacientes con Glioblastoma Multiforme. Materiales y métodos. Se revisó un total de 150 casos de pacientes con diagnóstico preliminar de Glioblastoma Multiforme (GBM) atendidos en el Instituto Nacional de Enfermedades Neoplásicas, entre los años 2008 y 2013 y se seleccionaron 60 casos que cumplían con los criterios de inclusión requeridos, información clínico patológica y seguimiento adecuado. Resultados. La media de edad es de 51 años (8-73 años), conformado por 34 hombres (56.7%) y 26 mujeres (43.3%). La mediana de sobrevida global (SG) es menor en el grupo de pacientes que sobreexpresaron el antígeno Ki67 (>= 20%), frente a los pacientes que tuvieron niveles de expresión moderada del antígeno (>= 10%). (26.5 vs 40 meses). Asimismo, se evidencia que la SG es mayor en los pacientes que expresan positivamente el gen p53 (>20%), frente a los pacientes que no llegaron a expresarlo. (40 vs 30 meses). Conclusiones. Tanto la expresión del antígeno ki67 como la expresión del gen p53 se pueden determinar como factores pronóstico de la sobrevida de pacientes que hayan sido diagnosticados con GBM con el fin de mejorar la calidad de vida de estos pacientes dándoles la posibilidad de recibir un tratamiento más específico acorde a los valores de estos marcadores inmunohistoquímicos.Objectives.To evaluate the influence of markers: Ki67 antigen and p53 gene as independent prognostic factors in the survival of patients with Glioblastom Multiform. Materials and methods. A total of 150 cases of patients with a preliminary diagnosis of Glioblastom Multiform (GBM) treated at the National Institute of Neoplastic Diseases between 2008 and 2013 were reviewed and 60 cases were selected that satisfy the inclusion criteria required, clinical pathological information and adequate follow-up information. Results. The mean age is 51 years (8-73 years), made up of 34 men (56.7%) and 26 women (43.3%). The median overall survival (OS) was lower in the group of patients who overexpressed the Ki67 antigen (> = 20%), compared to the patients who had moderate levels of antigen expression (> = 10%). (26.5 vs. 40 months). As well evidenced that the OS is higher in patients who positively express the p53 gene (> 20%), compared to patients who did not express it. (40 vs. 30 months. Conclusion. Both, the expression of the ki67 antigen and the expression of the p53 gene could be determined as prognostic factors for the survival of patients who have been diagnosed with GBM in order to improve the quality of life of these patients giving them the possibility of receiving a more specific treatment according to the values of these immunohistochemically markers.