Apesar do excelente prognóstico dos tumores de Wilms (TW) localizados (estádios I e II) e de histologia favorável (HF), 10% deles recidivam. Em 122 pacientes com TW com essas características, diagnosticados de 1976 e 2001, analisamos alguns fatores clínicos, como a idade por ocasião do diagnóstico e peso do tumor, em todos os pacientes; fatores biológicos, como o TP53 e a glicoproteína-p, em 40 deles; e variáveis histológicas de microestadiamento (invasão de seio renal, cápsula tumoral, vasos intra-renais e pseudocápsula inflamatória) em 28 com TW em estádio I. Correlacionando todos esses fatores com a presença de recidiva, observamos que a chance maior de recidiva estatisticamente significativa somente foi verificada em pacientes com duas ou mais variáveis de microestadiamento e/ou peso tumoral maior que 550 g / In spite of the excellent prognosis of localized favorable histology (FH) of Wilms' tumor (WT), 10% of them will relapse. In 122 TW patients with these characteristics, diagnosed between 1976 and 2001, some clinical factors have been analyzed, such as age at diagnosis and tumor weight in all patients; biological factors, like TP53 and p-glycoprotein, in 40 of them; and microsubstaging histological variables (invasion of renal sinus, tumor capsule, intrarenal vessels, and inflammatory pseudocapsule). Correlating all of those factors with relapse, we have observed that only patients with the association of two or more microsubstaging variables and/or tumor weight over 550 g showed a statistically significant higher chance of relapse

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 131 pages. Includes vita. Includes bibliographical references.

The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms. The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme Cyp2a5/CYP2A6. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific cis binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis. The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine Cyp2a5 gene are linked via hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a trans factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, CYP2A6, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, via changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR.

The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation. / In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein. / Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

The proper maintenance of the pathways governing cell growth is critical to ensure cell survival and DNA fidelity. Much of our understanding of how the cell cycle is regulated comes from studies examining the relationship between DNA viruses and the mechanisms of cell proliferation control. There are numerous examples demonstrating that viruses can alter the host cell environment to their advantage. In particular, the small DNA tumor viruses, which include adenovirus, simian-virus 40 (SV-40), and human papillomavirus (HPV), can modulate the host cell cycle to facilitate viral DNA replication. Due to the fact that these viruses infect quiescent, non-cycling cells and lack the necessary enzymes and resources to replicate their DNA (e.g. DNA polymerase), the small DNA tumor viruses must activate the host cell replication machinery in order to expedite viral DNA replication. The capacity of these viruses to perturb normal cell proliferation control is dependent upon their oncogene products, which target p53 and members of the Retinoblastoma (RB) family of proteins and inactivate their respective functions. By targeting these key cell cycle regulatory proteins, the small DNA tumor viruses induce the infected host cells to enter S-phase and activate the components involved with host cell DNA synthesis thereby generating an environment that is conducive to viral DNA replication. In contrast, the larger, nuclear-replicating DNA viruses such as those from the family Herpesviridae, do not share the same stringent requirement as the small DNA viruses to induce the infected host cell to enter S-phase. The herpesviruses encode many of the components to stimulate nucleotide biosynthesis and the necessary factors to facilitate virus DNA replication including a viral DNA polymerase and other accessory factors. Additionally, many herpesviruses encode gene products that arrest the host cell cycle, in most instances, prior to the G1/S transition point. Inducing cells to growth arrest appears to be a prerequisite for the replication of most herpesviruses. However, in addition to encoding factors that inhibit the cell cycle, many herpesviruses encode proteins that can promote cell cycle progression in a manner similar to the small DNA tumor virus oncoproteins. By targeting members of the RB family and p53 protein, the herpesvirus proteins induce S-phase and activate S-phase associated factors that playa role in DNA replication. In this manner, the herpesviruses may promote an environment that is favorable for DNA replication. Consistent with the other herpesviruses, human cytomegalovirus (HCMV)induces human fibroblasts to growth arrest. However, in other cell types, virus infection causes cells to enter S-phase. In addition, HCMV replication requires several cellular factors that are present only during S-phase. Furthermore, HCMV induces the activation of S-phase-associated events as well as the increased expression of numerous S-phase genes following infection. HCMV encodes two immediate early (IE) gene products, IE1-72 and IE2-86, which can interact with members of the RB family of proteins. Additionally, the IE2-86 protein can bind to and inhibit p53 protein function. Given the functional resemblance between the HCMV IE proteins and the oncoproteins of the small DNA tumor viruses, we hypothesized that expression of the HCMV IE proteins could modulate cell cycle control. Specifically, we determined that expression of either IE1-72 or IE2-86 can induce quiescent cells to enter S-phase and delay cell cycle exit following serum withdrawal. Moreover, IE2-86 mediates this effect in the presence or absence of p53, whereas IE1-72 fails to do so in p53-expressing cells. Furthermore, both IE1-72 and IE2-86 induce p53 protein accumulation that is nuclear localized. Because IE1-72 fails to promote S-phase entry in cells expressing p53 and induces p53 protein levels, the mechanism by which IE1-72 alters p53 levels was examined. IE1-72 elevates p53 protein levels by inducing both p19ARF protein and an ATM-dependent phosphorylation of p53 at Ser15. IE1-72 also promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. As consequence of this IE1-72-mediated increase in p53 levels, p21 protein is induced leading to a p21-dependent growth arrest in cells expressing IE1-72. These findings demonstrate that the HCMV IE proteins can alter cell proliferation control and provide further support to the notion that HCMV, through the expression of its IE proteins, induces S-phase and factors associated with S-phase while blocking cell DNA synthesis, to possibly generate an environment that is suitable for viral DNA replication.

The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage. This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393. This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes. The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues.

For 30 years, the tumor suppressor p53 has been a subject of intense research in nearly every discipline of scientific inquiry. While numerous surprising roles for p53 in health and disease are uncovered each year, the central role of its activation in preventing neoplastic transformation has been and will remain at the forefront of p53 research as investigators work to address an unexpectedly complex question—precisely how does p53 integrate upstream stress signals to coordinate activation of its target genes in response to stress? One manner in which to address this question is at the level of transcription initiation—after upstream signals converge on p53 and produce a number of pools of post-transcriptionally modified p53, how exactly are specific target promoters activated in such a sensitive, context-specific manner? The work presented herein aims to address the role of histone acetylation at the p21 promoter—a critical mediator of G1/S arrest—by the P/CAF acetyltransferase in response to a variety of p53-activating stresses. We show that depletion of P/CAF strongly inhibits p21 expression in response to a variety of stresses, despite normal stabilization of p53 and recruitment to target promoters. This defect in p21 expression correlates closely with abrogation of stress-induced cell-cycle arrest. Strikingly, a p53 allele lacking putative P/CAF acetylation sites was still able to direct p21 expression, which was still dependent upon P/CAF. We show further that histone acetylation at H3K14 at the p21 promoter following stress is dependent upon P/CAF. Rescue of p21 expression with wild-type P/CAF or a ∆HAT point mutant indicates that P/CAF requires an intact HAT domain, suggesting that histone acetylation at H3K14 is catalyzed by P/CAF HAT activity, not the molecular bridging of a heterologous HAT by P/CAF. Furthermore, RNA polymerase II (RNAP II) was present at the p21 proximal promoter under all basal and stress conditions, but elongation of RNAP II after stress required the presence of P/CAF. These data indicate that H3K14 acetylation by P/CAF closely correlates with the activation status of the p21 promoter, and may be necessary for activation of a larger subset of p53-responsive promoters. In addition to its critical role in p21 expression, we noted that p53 stabilization and cell-cycle arrest in response to p14ARF, but not other p53-stabilizing stresses, were also dependent on P/CAF. Cell-cycle arrest induced by p16INK4A was intact after P/CAF ablation, indicating a role for P/CAF in cell-cycle arrest specific to p14ARF-p53 signaling. Basal MDM2 levels were unaffected by P/CAF knockdown, as were p53- MDM2 and ARF-MDM2 complexes. A preliminary analysis of MDM2 localization was inconclusive, due to vastly different quantities of MDM2 in different conditions making analysis of subcellular localization difficult; however, the role of P/CAF in the relocalization of MDM2 to the nucleolus by p14ARF could potentially explain the defect in p53 stabilization, and should be explored further. These observations, underscored by recent reports that P/CAF undergoes loss of heterozygosity in several tumor types, suggest that P/CAF plays a critical role in p53-mediated cell-cycle arrest through multiple, independent mechanisms. Further study should clarify whether P/CAF is lost in tumors maintaining wild-type p53, and whether its reintroduction into these tumors confers any potential therapeutic benefit.

No description available.

O adenocarcinoma pancreático é a neoplasia maligna mais comum do pâncreas. A alta taxa de mortalidade deve-se ao diagnóstico tardio e a alta agressividade do tumor. Freqüentemente observam-se indivíduos com neoplasias de mesmo estadio apresentarem sobrevivência diferente. Isso demonstra a necessidade de incluir mais variáveis na caracterização da doença. O processo de angiogênese é essencial para o crescimento tanto do tumor primário, quanto para o metastático. A medida da densidade intratumoral da microvasculatura (DMV) por imunoistoquímica é o método mais confiável para medir a atividade angiogênica tumoral. A perda da função do gene p53 influencia a resposta à quimio e à radioterapia além de regular a angiogênese. A sobrevivência está inversamente relacionada à positividade do p53 e à DMV em neoplasias de mama, pulmão, ovários, estômago, cólon, laringe e bexiga. No adenocarcinoma pancreático os resultados são controversos. Idealizou-se essa pesquisa retrospectiva analisando-se dados clínicos e os resultados de estudos imunoistoquímicos obtidos de adenocarcinomas de pâncreas ressecados com intenção curativa. Analisou-se dados clínicos, patológicos, re-estadiamento e resultados da DMV e da expressão do gene p53 em 49 pacientes. A densidade média de microvasos foi de 46,2 vasos/mm2 sendo que esse valor foi utilizado para dividir os pacientes em grupos de baixa ou alta densidade de vasos. A coloração para p53 nuclear foi considerada positiva em 20 de 49 pacientes (40,8%). A DMV foi significativamente maior nos pacientes com tumores maiores que 3,0 cm e nos pacientes com ressecções incompletas. A expressão do gene p53 e a DMV, não foram fatores preditivos da sobrevivência pós-operatória. Não foi possível verificar relação entre a expressão do gene p53 e a densidade da microvasculatura tumoral / The prognostic significance of microvessel density and the p53 expression was evaluated. Between 1993 and 2006, 49 patients with pancreatic adenocarcinoma were ressected with curative intention. Specimens were stained immunohistochemically with antibodies anti- p53 anti-CD34. Microvessel density (MVD) was assessed scanning ten areas of the tumoral section and counted at a high power in an adequate area. The MVD ranged from 21,2 to 54,2 vessels/mm2 (mean 46,2 vessels/mm2). Specific nuclear staining for p53 was determined positive in 20 patients (40,8%). The overall median survival was 24,1 months after resection and there was no difference in survival rates according to the MVD and p53 positivity. There was also no relation between the MVD and p53 expression. MVD and p53 expression could not predict survival in these patients with pancreatic adenocarcinoma. There was no correlation with p53 expression and intratumoral microvessel density. High MVD was associated with tumor size grater than 3,0 cm and positive margins