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Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaf 147-169).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 177-202). Also available in electronic version.

The DNA damage response is an important barrier to tumorigenesis. Impairment of p53 function is crucial to tumorigenesis by allowing evasion of p53 dependent responses. The mechanisms involve either (i) missense mutations, (ii) partial abrogation of signaling pathways or effector molecules that regulate p53, (iii) epigenetic deregulation. The tyrosine to cysteine mutation, Y220C, in p53 is found in around 100,000 new cancer cases per annum. This mutation destabilizes the core domain by 4 kcal mol-1 and destabilizes p53 under physiological conditions. The large to small mutation results in the fusing of two shallow pockets to create an extended surface cleft that a number of different fragments bind. The small molecule PK083, 1-(-ethyl-9H-carbazol-3-yl)-N-methanamine, binds the mutant-specific crevice with a KD = 150 μM and raised the protein mutant's half-life to over 15 minutes vs. 4 minutes in the absence of the ligand. This presents an ideal starting point towards the design of a p53 rescuing drug. A library of carbazoles was designed and synthesized, guided by SAR studies, crystallographic information and computational chemistry, with the aim of optimizing the structure toward a more potent PK083 analogue. Affinity gains were achieved by exploitation of direct fluorine-protein interactions between PK9255 (N-methyl-1-(9- (2,2,2-trifluoroethyl)-9H-carbazol-3-yl)methanamine), and the backbone carbonyls of Leu145 and Trp146 and the thiol of Cys220, resulting in a Kd = 28 μM. Further affinity gains were achieved through SAR studies targeting the proline-rich subsite II. Chemistry was optimized to allow a diversity-oriented synthesis toward 2,6,9- substituted carbazoles. A small library of PK083 analogues, where the subsite II targeting group was a halogen, ether, ester, amide or heterocycle were synthesized, identifying the heterocyclic compounds as most potent. A scan of heterocyclic compounds was carried out to identify the most potent heterocyclic substitution.

p53 is a pivotal tumour suppressor in mammalian cells. It protects the integrity of a number of cellular pathways, preventing the malignant transformation of cells. There is however perhaps nothing more efficient at disrupting cellular pathways than a virus. Viruses infiltrate cells commandeering the normal growth and survival pathways for their narcissistic needs. While the association between viral infections and the induction of p53 has long been recognised, there is controversy surrounding the ultimate role of p53 during a virus infection. The classical model of p53 in an adenovirus infection is that p53 is a formidable obstacle which needs to be overcome. Adenoviruses overcome p53 by degrading the protein and removing its ability to transactivate its target genes. However the degradation is not immediate and there is increasing evidence which would suggest p53 is actually beneficial to an adenovirus infection. In the introductory chapter, I review what is known about p53 and virus infections. What emerges from this review is the sheer number of interactions that occur between viruses and p53, indicating its importance in an infection. Additionally it shows that adenoviruses are not the only virus shown to benefit from the presence of p53. What beneficial role p53 may be fulfilling in an adenovirus infection is unclear. The experiments reported in this thesis investigate the functions of p53 in an adenovirus infection. In Chapter Three, immunoblots on a panel of adenovirus infected cells reveal that p53 levels do not correlate with the level of the classical p53 target proteins. This indicates that p53 is disconnected from its target genes during an infection. Promoter/reporter assays carried out on infected cells show that adenovirus can directly regulate p53 target genes independently of p53. In Chapter Five, I show this regulation is dependent on E1a, with transient transfection of E1a resulting in the marked activation of p53 target promoters. E1a mediated transactivation appears to be reliant on the largest splice variant of E1a (E1a-289R) and the presence of pRB. Electrophoresis mobility shift assays reveal that the transcription factor Sp1 is involved. In Chapter Four, p53 transcription in an adenovirus infection was directly assayed by using an artificial p53 consensus response element. The results show that p53 is unable to activate its consensus response element during an infection. However, I show that p53 is transcriptionally competent in an infection, and is able to transactivate a mutant derivative of the p53 consensus sequence. This shows that p53 is not only transcriptional competent but has a gain-of-function in an infection. This gain-of-function requires E1a, and appears not to be due to a change in the DNA binding affinity of p53. The data in this thesis show that adenoviruses not only appear to inhibit and control the normal transcriptional profile of p53 but appear to modify p53, giving it a new transcriptional profile. This provides a possible mechanism by which p53 could aid an adenovirus infection.

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

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?

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