Thesis (Ph.D.)--Ohio University, August, 2009. / Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 89-107)

Thesis (Ph.D.)--Ohio University, August, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 89-107)

Thesis (Ph. D.)--University of Massachusetts Amherst, 2009. / On title page, the 'a' in ERa is symbolized by the Greek symbol for alpha. Includes bibliographical references (p. 108-124). Print copy also available.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is an important player in xenobiotic and hypoxic responses. In addition to this, my mentor has shown that ARNT is an integral cofactor of NF-kB signaling. However, these initial observations of ARNT-mediated NF-kB modulation were based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and therefore precluded the isolated examination of each isoform’s function. We show here that lymphoid malignancies exhibit higher levels of ARNT isoform 1 compared to ARNT isoform 3. However, normal T and B lymphocytes are seen to harbor equal levels of ARNT isoform 1 and 3. We hypothesize that the increase in ARNT isoform 1 is necessary for the growth of these cancer cells as suppression of isoform 1 resulted in S-phase cell cycle arrest. These findings reveal that ARNT isoform 1 potentiates cell growth by antagonizing a p53 cell cycle inhibitory mechanism and this further suggests that ARNT targeted therapies would benefit chemotherapy regimens. / text

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide expansion in the SCA7 gene resulting in progressive ataxia and retinal dystrophy. SCA7 belongs to a group of neurodegenerative disorders called polyglutamine (polyQ) diseases, that share the common feature of glutamine tract expansions within otherwise unrelated proteins. Common suggested mechanisms by which polyQ expanded proteins induce toxicity include aggregation and induction of oxidative stress.  In this work we examined the connection between oxidative stress, aggregation and toxicity in SCA7 disease. We show that expression of the SCA7 disease protein, ataxin-7 (ATXN7), results in elevated levels of ROS and oxidative stress which in turn lead to toxicity. Our results also revealed that the oxidative stress further contributes to mutant ATXN7 aggregation. Moreover, we show, for the first time, that the major source of the elevated ROS in mutant ATXN7 cells is the increased activation of NOX1 enzymes. Interestingly, our results further revealed that the increased level of NOX1 activity together with altered p53 function leads to a metabolic shift in mutant ATXN7 expressing cells. Treatments with antioxidants, a NOX1 specific inhibitor or NOX1 knock-down, all decreased the ROS level, restored the metabolic shift and ameliorated the mutant ATXN7 induced toxicity. Taken together, we conclude that mutant ATXN7 activate NOX1 enzymes which results in oxidative stress, increased mutant ATXN7 aggregation, metabolic dysfunction and toxicity. NOX1 specific inhibition could thus be a potential therapeutic strategy for SCA7. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Breast cancer is the most frequently diagnosed form of cancer in women and the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease consisting of many types of tissue neoplasia, and there appears to be no model of how a particular lesion develops into an aggressive, malignant, invasive carcinoma. Genetic mutation and aberrant epigenetic regulation are among the most common events that lead to neoplasia. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Therefore, this dissertation focuses on the mechanisms and consequences of p53 mutation during breast tumorigenesis. Genome-wide analysis of gene expression and epigenetic modifications in a panel of breast cancer cell lines suggested that p53 mutation and aberrant epigenetic silencing were cooperating mechanisms in the silencing of wild-type p53 target genes during cancer progression. Therefore, models of p53 inactivation were created in non-malignant human mammary epithelial cells to determine the role of p53 mutation on the epigenetic status of its target genes and the acquisition of malignant phenotypes. Comparisons of each model demonstrated that differing modes of p53 inactivation produced different functional consequences. Loss of wild-type p53 function alone ablated the normal cellular response to external stress stimuli, but had no affect on the expression of genes or epigenetic status in untreated cells. Introduction of missense mutant p53 protein caused very few changes when the protein was expressed at low levels. However, accumulation of mutant p53 caused a variety of gene expression changes and interfered with endogenous wild-type p53. The accumulation of mutant p53 also caused an increase in migration and invasion of the cells that expressed it. Interestingly, epigenetic aberrations were not detected in response to any of the p53 manipulations. These data suggest that accumulation of missense mutation is particularly dangerous to normal cells. They also suggest that p53 mutation and epigenetic aberration are two distinct mechanisms, which overlap and cooperate during tumorigenesis. These data suggest that treatment strategies for human breast cancer should include modalities to target both defects for increased efficacy.

The long-range goal of this project is to investigate the protective effects of conjugated linoleic acid (CLA) against carcinogenesis. In this dissertation, we demonstrate the mechanisms of CLA action on cell cycle progression and repression of polycyclic aromatic hydrocarbon (PAH)-induced Cyclooxygenase-2 (COX-2) in breast and colon cancer cells. CLA reduced the expression of factors required for G1 to S-phase transition including cyclins D1 and E, and hyperphoshorylated retinoblastoma Rb protein. In contrast, the over-expression of mutant p53 (175Arg to His) in MCF-7 cells prevented the CLA-dependent accumulation of p21 and the reduction of cyclin E levels suggesting that the expression of wild-type p53 is required for CLA-mediated activation of the G1 restriction point. We also report, CLA reduced the expression of COX-2 promoter activity induced by the aromatic hydrocarbon receptor (AhR)-ligand benzo[a]pyrene (B[a]P). Mutagenesis or deletion of potential xenobiotic responsive elements (XREs) within the COX-2 promoter abrogated its ability to be induced by the high affinity AhR-ligand TCDD. In addition, promoter studies using a XRE-dependent CYP1A1 plasmid revealed CLA can inhibit PAH-induced AhR/XRE-driven genes. In both studies, the t10,c12-CLA isomer was more effective than c9,t11-CLA in inhibiting cell proliferation and AhR/XRE-dependent genes. Taken together, these data suggest that the anti-cancerous properties of CLA appear to be a function, at least in part, of the relative content of specific isomers and their 1) ability to elicit a p53 response that leads to the accumulation of pRb and cell growth arrest, and 2) ability to inhibit PAH-induced cyclooxygenase-2 promoter activity through an AhR-dependent mechanism.

Head and neck cancer is diagnosed in more than 50,000 Americans each year, resulting in roughly 11,000 deaths. For this disease, a typical therapeutic regimen involves cisplatin, a radiosensitizer, given alongside targeted irradiation. While technological advances such as IMRT have been useful in sparing normal tissues from radiotherapy, the salivary glands occupy much of the head and neck and surround several lymph nodes, and thus, non-diseased salivary glands are often damaged. This causes reduced salivary output, damaged oral mucosa, dysphagia, malnutrition and tooth decay. Often, these side-effects are so severe that patients discontinue treatment, however, in many cases, salivary gland damage is permanent, and treatment options are palliative. Specifically, muscarinic-cholinergic agonists are used to enhance secretion from remaining salivary cells, although due to non-specific action, these drugs have a number of ill-effects. It is clear that therapies are needed to prevent radiation-induced salivary gland damage, as well as to restore glandular function in patients who are already suffering.Previous work from our group has shown that salivary gland dysfunction results from loss of acinar cells to radiation-induced apoptosis. Importantly, a single intravenous dose of IGF1 can prevent apoptosis and preserve salivary output when given immediately prior to irradiation. Because of its broad effects, however, IGF1 may never be a viable clinical option. Instead, our goal is to identify signaling events that mediate the radioprotective effects of IGF1 downstream of Akt. Because radiation-induced apoptosis in salivary glands is p53-dependent, we assessed the contributions of the p53 homologs p63 and p73 to the DNA damage response. Here, we show that IGF1 enhances cell cycle arrest following irradiation by reducing inhibitory binding of deltaNp63 to the p21 promoter. We hypothesize that IGF1-induced cell cycle arrest may allow time for DNA repair, thus preventing apoptosis and maintaining salivary function. In addition, we indicate chronic signaling events downstream of p63 that may contribute to permanent loss of salivary function by blocking differentiation of salivary progenitor cells. Together, these results indicate that p63 may be a valid therapeutic target for both prevention of damage and restoration of function in irradiated salivary glands.

In biomolecules, the relationship among the sequence, molecular structure, and biological function are of very importance in the development of nanotechnology such as drug discovery. Previous studies involving multiple sequence alignment of biomolecules have demonstrated that interdependent or associated sites are indicative of the structural and functional characteristics of biomolecules, as an extension to methods such as consensus sequences analysis. In this thesis, a new method to detect associated sites in aligned sequence ensembles is proposed. It involves the use of multiple sub-tables (or levels) of two-dimensional contingency table analysis. The idea is to incorporate analysis by following an approach known as granular computing, which represents information at different levels of granularity or resolution. When associations of multiple sites in the sequence alignment converge, they reflect points of interrelatedness among the sites in the biomolecules. The study involves two different phases of analysis. The first phase includes labeling of the molecular sites in the p53 protein multiple sequence alignment according to the detected patterns. The sites are consequently labeled into three different types based on their site characteristics - conserved sites, associated sites, and hypervariate sites. To identify and label the associated sites, the proposed method is employed. In the second phase, the significance of the extracted site patterns is evaluated with respect to some of the structural and functional characteristics of the p53 protein. The results indicate that the extracted site patterns in combination with conserved sites are significantly associated with some of the known functionalities of p53 such as post translational modifications and the mutation frequency of the sites, hence establishing the link between these identified sites and the defined functionality. Furthermore, when these sites are aligned with p63 and p73, the homologs of p53, based on the common domains, the sites significantly discriminate between the human sequences of the p53 family. Therefore, the study confirms the importance of these detected sites that could indicate their differences in cancer suppressing property.