The transcription factor p53 is an important master regulator of the cellular response to stress. Mdm2 is an E3 ubiquitin ligase that is the primary negative regulator of p53. Mdm2 downregulates p53 activity through three mechanisms: proteasome-mediated degradation, exportation from the nucleus, and direct inhibition through binding. Though the roles of the Mdm2-p53 axis in cancer have been well characterized, the relationship between p53 and other diseases remain elusive. Recently, three novel Mdm2 mutations were identified in patients with premature aging. One mutation leads to the abolishment of the Mdm2 stop codon, thereby extending the Mdm2 C-terminus by five additional amino acids. The other mutation leads to alternative splicing of Mdm2, resulting in two isoforms: a full length Mdm2 protein with a point mutation in the p53 binding domain and a truncated Mdm2 protein that has a 25 amino acid deletion in the p53 binding domain. Our results indicate that the causative Mdm2 variants are hyper-stable and lead to increased p53 protein stabilization. The anti-terminating mutant Mdm2 is defective as an E3 ligase, but retains its ability to bind and dampen p53 activity. However, p53 can be hyper-activated upon induction. Analysis of patient fibroblasts, patient lymphoblastoid cell lines, and genome-edited cells that express mutant Mdm2 confirmed the aberrant regulation of p53. MdmX may also potentially play a compensatory role in this axis. Altogether, our results demonstrate that defective Mdm2 can lead to constitutive dysfunctional regulation of p53 and contribute to accelerated aging phenotypes.

p53 is a key component of the protein network that regulates cell cycle progression and prevents cancer. Under non-stressed conditions, its activity is controlled by an autoregulatory feedback loop with MDM2 that maintains low levels of the p53 protein. However, in response to stress signals, p53 is triggered to become active. MDM2 has been reported to regulate p53 by a combination of mechanisms: ubiquitination using its E3-ligase capability, chaperone activity in an ATP-dependent manner and directly transrepressing p53. Because of MDM2's central role in the control of p53, it has been the target of intense drug development efforts. A family of small molecules, the Nutlins, can bind to an MDM2 pocket modulating the p53: MDM2 complex. This leads to p53 activation and growth inhibitory effects. The aim of our study was to analyse the interactome of endogenous MDM2 and to determine whether anti-cancer drugs, such as Nutlin-3, could stabilise or disrupt sets of MDM2 interactions in order to better understand the p53- dependent and independent functions of MDM2 as a signalling hub, as well as the p53-independent activity of Nutlin-3. Results show a remarkable difference in the sets of proteins found in MDM2 complexes in control and Nutlin-3 treated cells. Two proteins, TRIM25 and OTUB2, were selected from the output list for validation based on their known functions in the ubiquitin signalling network. Binding has been studied in detail and confirmed using both in cell and in vitro techniques. The data highlight potentially novel functions for MDM2 and provides insight into the on-target p53-independent activities of Nutlin-3. Additionally, and with the aim of blocking p53 ubiquitination by MDM2, I have developed probes that are able to inhibit the ubiquitylation of p53 in vitro.

The traditional roles of the tumor suppressor protein, p53, in transcriptional regulation are mostly defined in cancer or stressed cells and are centered on control of the cell cycle, DNA repair or senescence. In this thesis, data is presented demonstrating that the p53-regulated transcriptome is highly context-dependent as illustrated using the developing kidney as a model system. To this end, we utilized whole genome transcriptome and ChIP-Seq (chromatin immunoprecipitation-high throughput sequencing) analyses in p53+/+ and p53-/- mice to identify p53 regulated pathways in the embryonic kidney. This integrated approach allowed identification of novel genes that are direct p53 targets in the developing nephron. This approach was further refined using RNA-Seq analysis of lineage-tagged FACS-isolated nephron progenitors. We find that the p53-regulated transcriptome in the embryonic kidney is mostly involved in development, morphogenesis, and metabolic pathways. Interestingly, traditional targets of p53, such as DNA repair, cell cycle and apoptosis, accounted for < 5% of differentially expressed genes. The majority of 7,893 p53-binding genomic regions contain consensus p53 binding sites. Unlike a cancer cell line in which 7% of p53 binding sites lie within proximal promoters, 78% of p53 peaks in the developing kidney overlies the promoter. Moreover, 25% of the differentially expressed p53-bound genes belong to nephron progenitors and nascent nephrons, including key transcriptional regulators, components of Fgf, Wnt, Bmp, and Notch pathways, and ciliogenesis genes. RNA-Seq of nephron progenitors from wild-type and mutant p53 mice demonstrated repression of the nephron stem cell marker, Cited1, but not Six2. Moreover, cytoskeleton, cell adhesion, and energy metabolism genes were downregulated in mutant progenitors consistent with the loosely organized cap mesenchyme and disrupted mesenchyme-to-epithelium transition of p53-/- progenitors. In conclusion, our studies demonstrate that p53 genomic occupancy and regulated transcriptome are distinctly different in development and cancer. p53 is a key regulator of transcriptional programs that maintain nephron niche integrity, nephron progenitor cell renewal and differentiation. / acase@tulane.edu

The intracellular pathogen Chlamydia is the causative agent of millions of new infections per year transmitting diseases like trachoma, pelvic inflammatory disease or lymphogranuloma venereum. Undetected or recurrent infections caused by chlamydial persistence are especially likely to provoke severe pathologies. To ensure host cell survival and to facilitate long term infections Chlamydia induces anti-apoptotic pathways, mainly at the level of mitochondria, and restrains activity of pro-apoptotic proteins. Additionally, the pathogen seizes host energy, carbohydrates, amino acids, lipids and nucleotides to facilitate propagation of bacterial progeny and growth of the chlamydial inclusion. At the beginning of this study, Chlamydia-mediated apoptosis resistance to DNA damage induced by the topoisomerase inhibitor etoposide was investigated. In the course of this, a central cellular protein crucial for etoposide-mediated apoptosis, the tumour suppressor p53, was found to be downregulated during Chlamydia infections. Subsequently, different chlamydial strains and serovars were examined and p53 downregulation was ascertained to be a general feature during Chlamydia infections of human cells. Reduction of p53 protein level was established to be mediated by the PI3K-Akt signalling pathway, activation of the E3-ubiquitin ligase HDM2 and final degradation by the proteasome. Additionally, an intriguing discrepancy between infections of human and mouse cells was detected. Both activation of the PI3K-Akt pathway as well as degradation of p53 could not be observed in Chlamydia-infected mouse cells. Recently, production of reactive oxygen species (ROS) and damage to host cell DNA was reported to occur during Chlamydia infection. Thus, degradation of p53 strongly contributes to the anti-apoptotic environment crucial for chlamydial infection. To verify the importance of p53 degradation for chlamydial growth and development, p53 was stabilised and activated by the HDM2-inhibiting drug nutlin-3 and the DNA damage-inducing compound etoposide. Unexpectedly, chlamydial development was severely impaired and inclusion formation was defective. Completion of the chlamydial developmental cycle was prevented resulting in loss of infectivity. Intriguingly, removal of the p53 activating stimulus allowed formation of the bacterial inclusion and recovery of infectivity. A similar observation of growth recovery was made in infected cell lines deficient for p53. As bacterial growth and inclusion formation was strongly delayed in the presence of activated p53, p53-mediated inhibitory regulation of cellular metabolism was suspected to contribute to chlamydial growth defects. To verify this, glycolytic and pentose phosphate pathways were analysed revealing the importance of a functioning PPP for chlamydial growth. In addition, increased expression of glucose-6-phosphate dehydrogenase rescued chlamydial growth inhibition induced by activated p53. The rescuing effect was even more pronounced in p53-deficient cells treated with etoposide or nutlin-3 revealing additional p53-independent aspects of Chlamydia inhibition. Removal of ROS by anti-oxidant compounds was not sufficient to rescue chlamydial infectivity. Apparently, not only the anti-oxidant capacities of the PPP but also provision of precursors for nucleotide synthesis as well as contribution to DNA repair are important for successful chlamydial growth. Modulation of host cell signalling was previously reported for a number of pathogens. As formation of ROS and DNA damage are likely to occur during infections of intracellular bacteria, several strategies to manipulate the host and to inhibit induction of apoptosis were invented. Downregulation of the tumour suppressor p53 is a crucial point during development of Chlamydia, ensuring both host cell survival and metabolic support conducive to chlamydial growth. / Intrazellulär lebende Chlamydien führen jährlich zu Millionen an Neuinfektionen und lösen Krankheiten wie das Trachom, eine Entzündung des Auges, sowie entzündliche Beckenerkrankungen oder Lymphogranuloma venereum, eine venerische Lymphknotenentzündung, aus. Unentdeckte oder wiederkehrende Infektionen, ausgelöst durch chronisch persistierende Chlamydien, führen häufig zu schwerwiegenden Komplikationen. Um das Überleben der Wirtszelle und dauerhafte Infektionen zu ermöglichen, induzieren Chlamydien antiapoptotische Signalwege, hauptsächlich auf Höhe der Mitochondrien, und beeinträchtigen darüber hinaus die Aktivität proapoptotischer Proteine. Energie, Kohlenhydrate, Aminosäuren, Lipide und Nukleotide bezieht der Krankheitserreger vollständig aus der Wirtszelle. Erst dadurch wird sowohl die Vermehrung der Bakterien, als auch das Wachstum der chlamydialen Inklusion ermöglicht. Zu Beginn dieser Arbeit wurde die Chlamydien-vermittelte Resistenz gegenüber induziertem Zelltod nach Schädigung der DNA durch den Topoisomerase-Inhibitor Etoposid untersucht. Im Zuge dessen wurde entdeckt, dass der Tumorsuppressor p53, ein zentrales zelluläres Protein entscheidend für die Etoposid-induzierte Apoptose, während Chlamydien-Infektionen herunterreguliert wird. Nachdem verschiedene chlamydiale Stämme und Serovare untersucht wurden, konnte festgestellt werden, dass es sich bei der Herunterregulierung von p53 um ein allgemeines Merkmal chlamydialer Infektionen von humanen Zellen handelt. Die Reduzierung der Proteinmenge von p53 wird dabei durch den PI3K-Akt Signalweg, Aktivierung der E3-Ubiquitin-Ligase HDM2 und abschließendem Abbau durch das Proteasom vermittelt. Zusätzlich wurde ein interessanter Unterschied zwischen Infektionen humaner und muriner Zellen entdeckt. Sowohl Aktivierung des PI3K-Akt Weges, als auch der Abbau von p53 konnten in Chlamydien-infizierten Mauszellen nicht beobachtet werden. Kürzlich wurde darüber berichtet, dass während chlamydialer Infektionen reaktive Sauerstoffspezies produziert werden und die DNA der Wirtszelle geschädigt wird. Demnach trägt der Abbau von p53 entscheidend dazu bei, ein für chlamydiale Infektionen maßgebliches, anti-apoptotisch geprägtes Umfeld zu generieren. Um die Bedeutung des Abbaus von p53 für Wachstum und Entwicklung von Chlamydien zu ermessen, wurde p53 durch den HDM2-inhibierenden Wirkstoff Nutlin-3, sowie die DNA-Schäden induzierende Verbindung Etoposid stabilisiert bzw. aktiviert. Die Entwicklung der Chlamydien, sowie die Ausbildung der Inklusion wurden dadurch überraschenderweise stark beeinträchtigt bzw. waren fehlerhaft. Die Vollendung des chlamydialen Entwicklungszyklus wurde verhindert, was den Verlust der Infektivität nach sich zog. Interessanterweise erlaubte das Entfernen des p53-aktivierenden Stimulus die Ausbildung der bakteriellen Inklusion und die Wiedererlangung der Infektivität. Eine ähnliche Beobachtung konnte in Zelllinien mit einer p53-Defizienz gemacht werden. Da bakterielles Wachstum und Ausbildung der Inklusion durch aktiviertes p53 stark eingeschränkt war, wurde vermutet, dass p53-vermittelte Inhibierung des zellulären Metabolismus am fehlerhaften Wachstum der Chlamydien beteiligt ist. Analyse von Glykolyse und Pentosephosphatweg (PP-Weg) zeigten den Stellenwert eines funktionierenden PP-Wegs für das Wachstum der Chlamydien auf. Zusätzlich konnte durch Überexpression der Glucose-6-phosphat-Dehydrogenase das durch aktiviertes p53 gehemmte Wachstum der Chlamydien wiederhergestellt werden. Dieser Effekt war noch deutlicher in p53-defizienten Zellen, die mit Etoposid bzw. Nutlin-3 behandelt wurden. Demnach tragen auch p53-unabhängige Aspekte zur Einschränkung des chlamydialen Wachstums bei. Das Entfernen von reaktiven Sauerstoffspezies durch Antioxidationsmittel war jedoch nicht ausreichend zur Wiedererlangung der chlamydialen Infektivität. Demnach sind nicht nur die anti-oxidativen Eigenschaften des PP-Wegs sondern auch das Bereitstellen von Vorläufermolekülen für die Nukleotidsynthese, sowie dessen Beitrag zur DNA-Reparatur entscheidend für erfolgreiches Wachstum von Chlamydien. Veränderung der Signaltransduktion der Wirtszelle wurde bereits bei einigen Krankheitserregern nachgewiesen. Da reaktive Sauerstoffspezies und DNA Schäden häufig bei Infektionen intrazellulärer Bakterien auftreten, entstanden unterschiedliche Strategien, den Wirt zu manipulieren und das Einleiten des Zelltodes zu verhindern. Das Herunterregulieren des Tumorsuppressors p53 ist entscheidend während der Entwicklung von Chlamydien. Sowohl das Überleben der Wirtszelle, als auch die für chlamydiales Wachstum förderliche Unterstützung durch den Stoffwechsel werden dadurch gewährleistet.

As an Adenovirus infects a host cell a multitude of molecular interactions occur, some driven by the virus and some driven by the cell it is infecting. Many of these areas of Adenovirus biology have been intensely studied over the last half century, however, many questions remain unanswered. The aim of this study was to investigate, more closely, a long studied molecular interaction, namely the role of the tumour suppressor p53 in the Adenovirus life cycle, and also to investigate the related, but much less studied, interaction between Adenoviruses and the host cell DNA repair machinery. Controversy surrounds the role of p53 in the Adenovirus life cycle, with current dogma favouring the view that p53 is inactivated, as it presumably presents an obstacle to a productive infection. In Chapter 3, a standardised infection protocol was developed to examine this area of Adenovirus biology more closely. This was followed with an array of cell viability and western blotting analyses that not only showed p53 was not an antagonist of the Adenovirus life cycle, but in some cases p53 acted as a protagonist. Isogenic cell lines were used to reinforce this point. Following this, data were provided that virus DNA replication was linked to the ability of an Adenovirus to kill cells. Furthermore, p53 was shown by immunofluorescence to be present in infected cells at a time that corresponded with virus DNA replication, albeit at low levels. By adding p53 back into cells, it was shown that the number of Adenovirus progeny could be stimulated to levels produced in genetically wild type TP53 cells. A selection of promoter/reporter assays and infection/transfection assays then showed how p53 might be aiding the virus life cycle. These data showed that low levels of p53 cooperated with the Adenovirus transactivator, E1A, to promote late gene expression, and this translated into a modest increase in virus late antigens in infected cells. Taken together these data show that, contrary to current dogma, p53 generally aids an Adenovirus infection and it may do this through promoting virus late gene expression. Recent data have emerged suggesting Adenoviruses must disable the host DNA double-strand break machinery to achieve a productive infection. As this area of Adenovirus biology is in its infancy, and as p53 has recently been identified as an integral component of these DNA repair processes, the contributions of the host cell repair machinery to Adenovirus biology were examined in Chapters 4 and 5. In Chapter 4, western blotting showed that upon Adenovirus infection, a key component of the homologous recombination repair machinery, Rad51, was markedly up-regulated. This up-regulation occurred independently of other key repair proteins, and was found to be a generalised feature of an Adenovirus infection. Surprisingly, p53 did not appear to be involved in this up-regulation, and neither were several other nodal host regulatory proteins. The up-regulation was then linked to Adenovirus DNA replication using a temperature-sensitive mutant Adenovirus, ts125. In Chapter 5, functional analysis of this up-regulated protein showed that Rad51 colocalised with Adenovirus replication centres. This colocalisation coincided with a time when virus DNA replication was occurring. Furthermore, transient over-expression of Rad51 drastically increased the amount of virus progeny produced. This effect was reproduced in two very different cell types and with a selection of attenuated mutant viruses. Finally, several models were proposed that might account for this newfound effect of Rad51 on the Adenovirus life cycle. The data presented in this thesis shows that Adenovirus not only interacts with key molecular machinery within the host cell, but also manipulates this machinery to its own end. These data add additional layers of complexity to current knowledge of the virus/host cell relationship, and thus reveal new avenues of research for future work.

Bibliography: leaves 170-196. / xix, 199 leaves, [84] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The results presented in this thesis establish the growth-suppressive activity of the human GAS1 gene and provide the first direct evidence that GAS1 can inhibit the growth of tumours. In addition, this study demonstrates that the antiproliferative effect of GAS1 are mediated by a p53 dependent pathway and that functional inactivation of p53 by either mutation and/or overexpression of the MDM2 oncogene product inhibits the GAS1 mediated growth-suppression. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1997?

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy in Western countries. A better understanding of the etiology and risk factors associated with this disease is crucial for the development of early detection protocols as well as more effective therapies. Epidemiological data has shown that the risks of EOC are highest among peri- or post- menopause women, while increased parity or the use of oral contraceptives is preventive. These data suggest that alterations in reproductive factors are associated with ovarian cancer risks; however, the molecular mechanisms underlying such a link remain to be understood. For decades, EOC was believed to arise from the epithelium that surrounds the ovarian surface, yet this concept fails to explain the morphological resemblance of ovarian epithelial neoplasms with the epithelial cells of the Müllerian-derived female reproductive tract. Alternative ideas have argued that EOC may originate from extra- or para-ovarian tissues such as the fallopian tube and ovarii rete. Studies of the origin of EOC will provide a better understanding of the disease and advance the protocols for early diagnosis. The aims for this thesis are to establish in vivo ovarian tumor models based on the germ cell deficient Wv/Wv mice that mimicking menopausal physiology. The Wv mice harbor a point mutation in c-Kit, which reduces its tyrosine kinase activity to about 1%, resulting in a premature loss of ovarian germ cells and follicles that recapitulates the initiation of menopause in human. We have developed ovarian tumor models by deleting the tumor suppressor genes p53 or p27kip1 in Wv/Wv mice. We found that both Wv/Wv:p27+/- and Wv/Wv :p27 -/- mice developed ovarian epithelial tumors, which consist of papillary structures lined by hyperchromatic neoplastic cells. Positive Cytokeratin 8 (CK8) staining indicated the epithelial origin of these tumors. In vitro primary cultures of mouse ovarian surface epithelial (MOSE) cells from wildtype, p27+/- and p27 -/- mice further confirmed the growth advantage caused by p27 deficiency. However, neither p27 +/- nor p27 -/- MOSE cells were transformed in vitro, probably due to the compensatory increase of cyclin dependent kinase inhibitor (CKI) proteins including p21, p16, p19. When p53 was deleted unilaterally in the ovarian surface epithelial cells of Wv/Wv:p53 loxP/loxP mice by single administration of Adenovirus containing Cre activity (Ad-Cre), ovarian tumors developed after long latency. The ovarian tumors were significantly enlarged when compared with the uninfected ovary from the same mouse. However, most of the lesions in Wv:p53 conditional knockout tumors was negative for epithelial and follicular markers. In vitro deletion of p53 in MOSE cells significantly increased the proliferation and passage numbers of these cells. A compensatory increase of the CKI protein p16, as well as the cellular senescence level was also observed in p53 deleted MOSE cells, suggesting that p53 deletion alone was not sufficient to bypass p16- mediated tumor defense mechanisms in MOSE cells. Taken together, single deletion of p27 and p53 significantly amplified the phenotype of benign tubular adenomas in Wv/Wv mouse. However, neither p27 nor p53 deletion was sufficient to induce the development of malignant ovarian carcinomas in Wv/Wv mice, probably due to the up-regulation of CKI family proteins such as p21, p16 or p19.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 10

The human p63 gene discovered in 1997 encodes a series of protein isoforms that differ in their N- and/or C-terminal sequences. These isoforms have widely differing properties in promoting or repressing p53-related functions such as growth arrest and apoptosis. In addition, p63 appears to play important roles in the maintenance and differentiation of epithelial cell populations. Many studies have shown that p63, particularly Np63, is expressed in normal epithelium and also highly expressed in squamous cell carcinomas of surface epithelia. Methods. We have refined the analysis of the expression patterns of p63 isoforms by the use of a quantitative RT-PCR technique applied to micro-dissected normal oral mucosal samples. We have also studied p63 expression in squamous cell carcinoma of the head and neck (SCCHN) compared to normal oral mucosa taken from the same patient. Furthermore, tobacco-exposed buccal mucosa was compared to age and gender matched non exposed controls. RT-PCR for telomerase and immunohistochemical analysis for detection of p53 and Ki-67 proteins was further performed. We also explored the function of p63 in SCCHN cells by using small interfering RNA (siRNA) to silence the expression of different p63 isoforms in cell lines originating from SCCHN. The effect of p63 knockdown was studied using a Fluorescein Diacetate based cytotoxicity assay and immunohistochemistry looking at expression of differentiation markers. The response of the siRNA treated cells to radiation and cytostatic drug was also investigated. We have further studied normal oral wound healing using immunohistochemistry and quantitative PCR. By the use of a macro array comparing siRNA treated cells with non-treated cells a possible connection between the BRCA1 gene and p63 expression was shown and further studied with the use of cHIP and luciferase reporter assays. Results. The p63 isoforms are expressed in normal epithelium, with the highest levels consistently found in basal and parabasal layers. Extensive use of tobacco had no effect on p63. The quantitative PCR showed statistically increased levels of the ΔNp63 and p63isoforms. No correlation was found between p63-isoform expression patterns and proliferation. The exploration of the function of p63 in SCCHN cells by the use of small interfering RNA (siRNA) showed a statistically significant decreased survival for tumour cells when all p63 isoforms, the N-terminal truncated or the  isoforms were inhibited. No effect on cell proliferation or expression of epithelial differentiation markers was observed. We also demonstrated that inhibition of p63 expression sensitizes cells to the effects of ionizing radiation and cisplatin. The study of normal oral wound healing using immunohistochemistry and quantitative PCR showed significant changes in p63 isoform expression. The Np63 isoform was mainly expressed in the basal layer in the non proliferating and migrating cells while TAp63 was almost absent. The BRCA1 study showed p63 to bind to the BRCA1 promoter and activate the expression of BRCA1 protein. Summary. The p63 proteins have different functions and the balance between the isoforms and their localisation within the epithelium seems to be important for normal wound healing as well as cancer cell survival.

<p>Healthy cells require input from growth factor signaling pathways to maintain cell metabolism and survival. Growth factor deprivation induces a loss of glucose metabolism that contributes to cell death in this context, and we have previously shown that maintenance of glycolysis after growth factor deprivation suppresses the activation of p53 and the induction of the pro-apoptotic protein Puma to prevent cell death. However, it has remained unclear how cell metabolism regulates p53 activation and whether this increased glycolysis promotes cell survival in the face of additional types of cell stress. To examine these questions, we have utilized a system in which stable overexpression of the glucose transporter Glut1 and hexokinase 1 in hematopoietic cells drives growth-factor independent glycolysis. This system allows us to examine the effects of glucose metabolism in the absence of other signaling events activated downstream of growth factor receptors. Here, we demonstrate that elevated glucose metabolism, characteristic of cancer cells, can suppress PKC&#948;-dependent p53 activation to maintain cell survival after growth factor withdrawal. In contrast, DNA damage-induced p53 activation was PKC&#948;-independent and was not metabolically sensitive. Both stresses required p53 serine 18 phosphorylation for maximal activity but led to unique patterns of p53 target gene expression, demonstrating distinct activation and response pathways for p53 that were differentially regulated by metabolism.</p><p>Unlike the growth factor-dependence of normal cells, cancer cells can maintain growth factor-independent glycolysis and survival and often demonstrate dramatically increased rates of glucose uptake and glycolysis, in part to meet the metabolic demands associated with cell proliferation. Given the ability of elevated glucose metabolism to suppress p53 activity in the context of metabolic stress, we examined the effect of increased glucose uptake on leukemogenesis using a mAkt-driven model of leukemia and adoptive transfer experiments. We show here that elevated glucose uptake promoted leukemogenesis in vivo, perhaps through suppression of p53 transcriptional activity. During the process of leukemogenesis, cancerous cells can acquire growth factor independent control over metabolism and survival through expression of oncogenic kinases, such as BCR-Abl. While targeted kinase inhibition can promote cancer cell death, therapeutic resistance develops frequently and further mechanistic understanding regarding these therapies is needed. Kinase inhibition targets the necessary survival signals within cancerous cells and may activate similar cell death pathways to those initiated by growth factor deprivation. As we have demonstrated that loss of metabolism promotes cell death after growth factor withdrawal, we investigated whether cell metabolism played a role in the induction of apoptosis after treatment of BCR-Abl-expressing cells with the tyrosine kinase inhibitor imatinib. Consistent with oncogenic kinases acting to replace growth factors, treatment of BCR-Abl-expressing cells with imatinib led to reduced metabolism and p53- and Puma-dependent cell death. Accordingly, maintenance of glucose uptake inhibited p53 activation and promoted imatinib resistance, while inhibition of glycolysis enhanced imatinib sensitivity in BCR-Abl-expressing cells with wild type p53 but had little effect on p53 null cells. Together, these data demonstrate that distinct pathways regulate p53 after DNA damage and metabolic stress and that inhibition of glucose metabolism may enhance the efficacy of and overcome resistance to targeted molecular cancer therapies.</p> / Dissertation