Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.

Previously, we found that 3,4-(methylenedioxy)-1-(2′,3′- epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells by activating caspase-3, -8, and -9. In this study, we further investigated which upstream pathways were activated by safrole oxide during the apoptosis. Immunofluorescence assay combined with laser scanning confocal microscopy revealed that both Fas and Fas ligand (FasL) were up-regulated by the small molecule. In addition, Fas protein distribution was altered, showing a clustering distribution instead of a homogeneous one. Subsequently, Western blot analysis confirmed the up-regulations of Fas and its membrane-binding form of FasL (m-FasL), as well as P53 protein. Conversely, safrole oxide hardly affected integrin β4 subunit expression or distribution, which was reflected from the data obtained by immunofluorescence assay combined with laser scanning confocal microscopy. The results suggested that Fas/FasL pathway might be involved in safrole oxide-induced apoptosis of A549 cells, while integrin β4 might be irrelevant to the apoptosis. Nevertheless, we first found the strong expression of integrin β4 in A549 cells. The study first suggested that safrole oxide might be used as a small molecular promoter of Fas/FasL pathway to elicit apoptosis in A549 cells, which would lay the foundation for us to insight into the new strategies for lung cancer therapy.

A novel small molecule, 1-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (EOD), was synthesized in our laboratory. Previously, we reported pharmacological properties of EOD, triggering apoptosis in Human umbilical vein endothelial cells (HUVECs). Here, we further investigated the effects of EOD on the growth of A549 human lung cancer cells. EOD treatment induced apoptosis in A549 cells via up-regulating the expression of P53 protein, blocking cell cycle partly at G1 phase, and ultimately activating caspase-3. In contrast, caspase-8 might be irrelevant to EOD-triggered apoptosis. This study indicated that EOD might be a potential chemopreventive agent for lung cancer. The work would encourage us to add more novel compounds to our 'library' of small molecules derived through modern synthetic organic chemistry, and would drive us to determine the proteins that the compounds target.

Tumor suppressor p53, the guardian of the genome, has been intensely studied molecule owing to its central role in maintaining cellular integrity. While the level of p53 protein is maintained low in unstressed conditions, there is a rapid increase in the functional p53 protein levels during stress conditions. It is now well documented in literature that p53 protein accumulates in the cells following DNA damage by posttranslational modifications leading to increased stability and half life of protein. Additionally, recent studies have also highlighted the significance of increased p53 translation during stress conditions. Interestingly, an alternative initiation codon has been shown to be present within the coding region of p53 mRNA. Translation initiation from this internal AUG results in an N-terminally truncated p53 isoform, described as ΔN-p53. However, the mechanisms underlying co-translational regulation of p53 and ΔN-p53 are still poorly understood. Studies have suggested that synthesis of both p53 and its ΔN-p53 isoform is regulated during cell cycle and also stress and cell-type specific manner. Interestingly, reports also demonstrate continued synthesis of both p53 isoforms during stress conditions. In contrast, global rates of cap-dependent translation initiation are shown to be reduced during stress conditions. This translation attenuation is observed mainly due to restricted availability of critical initiation factors. Interestingly, preferential synthesis of a vital pool of survival factors persists even during these circumstances. Studies have suggested that this selective translation is mediated via alternative mechanisms of translation initiation. One of the important mechanisms used for protein synthesis during these conditions is internal initiation. In this mechanism, the ribosomes are recruited to a complex RNA structural element known as ‘Internal Ribosome Entry Site (IRES)’, generally present in the 5’ untranslated region (UTR) of mRNA. Therefore, it is possible that the translation of p53 and ΔN-p53 could also be regulated by IRES mediated translation, especially during stress conditions. In this thesis the role of internal initiation in translational control of p53 and ΔN-p53 has been investigated. Additionally, the putative secondary structure of p53 IRES RNA has been determined. Further, it has been shown that polypyrimidine tract binding (PTB) protein acts as an important regulator of p53 IRES activities. The probable mechanism of action of PTB protein has also been investigated. The results suggest that interaction with PTB alters the p53 IRES conformation which could facilitate translation initiation. Finally, the possible physiological significance of existence of p53 IRES elements has been addressed. In the first part of the thesis, the presence of internal ribosome entry site within p53 mRNA has been investigated. As a first step, the 5’UTRs mediating the translation of both p53 and ΔN-p53 were cloned in the intercistronic regions of bicistronic constructs. Results of in vivo transfection of these bicistronic constructs suggested the presence of two IRES elements within p53 mRNA, with activities comparable to known viral and cellular IRESs. The IRES directing the translation of p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of ΔN-p53 extends further into the protein-coding region. To further validate, stringent assays were performed to rule out the possibility of any cryptic promoter activity, re-initiation/scanning or alternative splicing in the p53 mRNA. Transfection of in vitro synthesized bicistronic RNAs confirmed the presence of IRES elements within p53 mRNA. Incidentally, this constitutes the first report on translational control of p53 by internal initiation. In the second part of the thesis, the secondary structure of p53 IRES RNA has been investigated. Structural analysis of p53 RNA was performed using structure-specific nucleases and modifying chemicals. The results obtained from chemical modification and nuclease probing experiments were used to constrain Mfold predicted structures. Based on this, a putative secondary structure model for p53 IRES RNA has been derived. Sequence alignment suggested that the p53 IRES RNA showed significant sequence conservation across mammalian species. To study the effect of mutations on the IRES structure, mutant p53 IRESs were used that harbor silent mutations at critical locations within the p53 IRES element. Incidentally, one of the mutant constructs used in the study was observed to be a naturally occurring mutation in a chronic lymphocyte leukemia patient. RNA structure analyses of these two mutant p53 IRES RNAs were performed. The nuclease mapping data suggested conformational alteration in these mutant RNAs with respect to wild type. Consistently, a comparative Circular-Dichroism spectroscopy of the Wt and mutant RNAs also validated the conformational alteration of the mutant RNAs. This also suggested that the presence of mutations in p53 IRES might result in decreased induction of p53 protein following DNA damage due to altered RNA structure. This might constitute as one of the mechanisms leading to tumor development in some types of cancers. In the third part of the thesis, the role of important cellular proteins that might modulate p53 IRES mediated translation has been studied. These cellular proteins act as IRES interacting trans-acting factors (ITAFs). Polypyrimidine tract binding (PTB) protein is an important ITAF implicated in regulating IRES mediated gene expression during apoptosis. It was observed that PTB protein specifically interacts with both the IRES elements within p53 mRNA. Interestingly, the affinity of interaction of PTB protein with both p53 IRES RNAs was observed to be significantly different. In order to determine the contact points of PTB on p53 IRES, a foot-printing assay using structure specific nuclease and recombinant-PTB protein was performed on p53 RNA. The data from foot-printing as well as primer extension inhibition assay (toe-printing analysis) suggested the presence of multiple PTB binding sites on p53 IRES RNA. Based on these results, a deletion mutant was generated that showed reduced PTB binding and also reduced IRES activity as compared to wild type. Further, to study the role of PTB in mediating p53 translation, the expression of PTB gene was partially silenced by using PTB specific siRNA. Partial depletion of endogenous PTB protein showed a significant decrease in the p53 IRES activities. These results suggest that PTB protein is essential for the p53 IRES activities. To understand the probable mechanism by which PTB regulates p53 IRES mediated translation, CD spectroscopy analysis of p53 IRES RNA was performed in the absence and presence of PTB protein. Interestingly, CD spectra analysis of the p53 RNA in the presence of PTB suggested a specific conformational change in p53 IRES, which might probably facilitate ribosome loading during internal initiation. This also suggests that abnormal expression of p53 ITAFs might lead to reduced p53 induction following DNA damage conditions. It could also be another event leading to malignant transformation of cells bearing wild type p53. It is highly tempting to speculate that the levels of p53 ITAFs could also be used as tumor biomarkers. In the fourth part of the thesis, the physiological relevance of existence of IRES elements within p53 mRNA has been investigated. The levels of p53 and ΔN-p53 proteins are known to be regulated in a cell cycle phase-dependent manner. The IRES activities of both p53 IRES elements were investigated at different phases of cell cycle. The activity of the IRES responsible for translation of p53 protein was found to be highest at G2-M transition and the maximum IRES activity corresponding to ΔN-p53 synthesis was observed at G1-S transition. These results suggested that the p53 IRES activities are regulated in a cell-cycle phase-dependent manner. Next, the regulation of p53 IRES mediated translation during stress conditions was studied. Human lung carcinoma cell line, A549 cells (that endogenously express both the p53 isoforms), were exposed to DNA damaging drug, doxorubicin. The level of p53 protein was observed to increase in a time-dependent manner. Interestingly, PTB protein, which is predominantly nuclear, was found to translocate to the cytoplasm during stress condition in a time-dependent manner. Under similar conditions, p53 protein was observed to reverse translocate from the cytoplasm to nucleus, probably to function as a transcription factor. Next, the influence of partial PTB silencing on p53 isoforms in the presence of cell stress (mediated by doxorubicin) was investigated. The data indicated reduced levels of both p53 and ΔN-p53 when PTB gene expression was partially silenced. These observations constitute “the proof of concept” that relative abundance of an ITAF, such as PTB protein, might contribute to regulating the coordinated expression of the p53 isoforms. The thesis reveals the presence as well as the physiological relevance of existence of IRES elements within p53 mRNA. The novel discovery of p53 IRES elements may provide new insights into the underlying mechanism of translational regulation. The modulation of the p53 IRES activities by PTB protein suggests that the regulated expression of p53 isoforms depends on the integrity of IRES elements and availability of cellular proteins that can serve as p53 ITAFs. Thus, studies pertaining to the identification of mutations within p53 IRES region as well as abnormal expression of p53 ITAFs such as PTB in cancer cells may have far reaching implications. These studies might lead to further advances in the field of cancer detection, prognosis and design of novel therapeutic strategies.

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.

Thesis (M.S.)--Mississippi State University. College of Veterinary Medicine. / Title from title screen. Includes bibliographical references.

Submitted in the fulfillment of the requirement for the Degree of Master's in Chemistry, Durban University of Technology, 2017. / P53 is a tumour suppressor protein that is dysfunctional in most human cancer cells. Mutations in the p53 genes result in the expression of mutant proteins which accumulate to high levels in tumour cells. Several studies have shown that majority of the mutations are concentrated in the DNA-binding domain where they destabilize its conformation and eliminate the sequence- specific DNA-binding to abolish p53 transcription activities. Accordingly, this study involved an investigation of the effects of mutations associated with cancer, based on the framework of sequences and structures of p53 DNA-binding domains, analysed by SIFT, Pmut, I-mutant, MuStab, CUPSAT, EASY-MM and SDM servers. These analyses suggest that 156 mutations may be associated with cancer, and may result in protein malfunction, including the experimentally validated mutations. Thereafter, 54 mutations were further classified as disease- causing mutations and probably have a significant impact on the stability of the structure. The detailed stability analyses revealed that Val143Asp, Ala159Pro, Val197Pro, Tyr234Pro, Cys238Pro, Gly262Pro and Cys275Pro mutations caused the highest destabilization of the structure thus leading to malfunctioning of the protein. Additionally, the structural and functional consequences of the resulting highly destabilizing mutations were explored further using molecular docking and molecular dynamics simulations. Molecular docking results revealed that the p53 DNA-binding domain loses its stability and abrogates the specific DNA-binding as shown by a decrease in binding affinity characterized by the ZRANK scores. This result was confirmed by the residues Val143Asp, Ala159Pro, Val197Pro, Tyr234Pro and Cys238Pro p53-DNA mutant complexes inducing the loss of important hydrogen bonds, and introduced non-native hydrogen bonds between the two biomolecules. Furthermore, Molecular dynamics (MD) simulations of the experimental mutant forms showed that the structures of the p53 DNA-binding domains were more rigid comparing to the wild-type structure. The MD trajectories of Val134Ala, Arg213Gly and Gly245Ser DNA-binding domain mutants clearly revealed a loss of the flexibility and stability by the structures. This might affect the structural conformation and interfere with the interaction to DNA. Understanding the effects of mutations associated with cancer at a molecular level will be helpful in designing new therapeutics for cancer diseases. / M

O carcinoma epidermoide de lábio (CEL), que é causado pela exposição crônica e excessiva à radiação ultravioleta do sol, é precedido por uma desordem potencialmente maligna, a queilite actínica (QA). No entanto, ainda não é possível determinar quais casos de QA evoluirão para CEL. O método mais utilizado por patologistas para prever o prognóstico de desordens potencialmente malignas é a graduação histológica das displasias epiteliais. Entretanto, o sistema é subjetivo e ineficiente quanto ao seu valor preditivo. Acredita-se que o epitélio adjacente ao CEL tenha alterações genéticas semelhantes ao corpo da neoplasia. O objetivo deste estudo foi graduar as displasias epiteliais na margem do CEL e verificar a presença de proteína p53 mutada nessas áreas. Foram utilizados 40 casos de CEL com epitélio adjacente, no qual a displasia epitelial foi classificada pelo sistema proposto pela Organização Mundial da Saúde (OMS) e pelo sistema binário. Os casos foram, ainda, submetidos a reações de imuno-histoquímica com anticorpo anti-proteína p53 mutada. Entre os 40 casos estudados, 15 apresentaram displasia epitelial discreta, 18 moderada e 7 intensa. Pelo sistema binário, 36 casos foram classificados como de baixo risco e 4 como de alto risco. A marcação imuno-histoquímica para a proteína p53 mutada foi encontrada no epitélio adjacente de 32 casos (80%) dessa amostra. Considerando-se as duas graduações estudadas, a marcação foi detectada em 11/15 casos de displasia discreta, 16/18 de moderada e 5/7 de intensa pelo sistema da OMS e em 29/36 casos de baixo risco e 3/4 casos de alto risco. Concluiu-se, assim, que o epitélio adjacente ao CEL, mesmo exibindo poucas alterações morfológicas pode ter um comprometimento genético importante em genes que comandam a estabilidade genômica. / The lip squamous cell carcinoma (LSCC), which is caused by chronic and excessive exposure to solar ultraviolet radiation, is preceded by a potentially malignant disorder, the actinic cheilitis (AC). However, it is not possible to determine which AC cases will progress to LSCC. The method used by pathologists to predict the prognosis of potentially malignant disorders is the histologic grading of epithelial dysplasia. Nevertheless, this system is subjective and inefficient as to its predictive value. It is considered that the epithelium adjacent to LSCC has genetic alterations similar to the main tumor. The aim of this study was to grade the epithelial dysplasia on the LSCC border and also to verify the presence of mutated p53 protein in these areas. Forty LSCC cases with adjacent epithelium were retrieved from our files. The epithelial dysplasia was classified by the system proposed by the World Health Organization (WHO) as well as the binary system. Three m sections were submitted to the antibody against mutated p53 protein by means of immunohistochemistry. Among the 40 cases studied, 15 were classified as mild dysplasia, 18 moderate and 7 severe. When the binary system was considered, 36 cases were classified as low risk and four as high risk. Immunostaining revealed the presence of mutated p53 protein in the adjacent epithelium of 32 cases (80%). Analyzing the two grading systems separately, the staining was detected in 11/15 cases of mild dysplasia, 16/18 of moderate and 5/7 of intense; 29/36 of low-risk cases and 3/4 cases high-risk. In conclusion, even if it shows few morphological changes, the epithelium adjacent to LSCC may have a genetic involvement in important genes that control genomic stability.

The tumour suppressor p53 protein plays a central role in the DNA damage response/checkpoint pathways leading to DNA repair, cell cycle arrest, apoptosis and senescence. The activation of p53-mediated pathways is primarily facilitated by the binding of tetrameric p53 to two 'half-sites', each consisting of a decameric p53 response element (RE). Functional REs are directly adjacent or separated by a small number of 1-13 'spacer' base pairs (bp). The p53 RE is detected by exact or inexact matches to the palindromic sequence represented by the regular expression [AG][AG][AG]C[AT][TA]G[TC][TC][TC] or a position weight matrix (PWM). The use of matrix-based and regular expression pattern-matching techniques, however, leads to an overwhelming number of false positives. A more specific model, which combines multiple factors known to influence p53-dependent transcription, is required for accurate detection of the binding sites. In this thesis, we present a logistic regression based model which integrates sequence information and epigenetic information to predict human p53 binding sites. Sequence information includes the PWM score and the spacer length between the two half-sites of the observed binding site. To integrate epigenetic information, we analyzed the surrounding region of the binding site for the presence of mono- and trimethylation patterns of histone H3 lysine 4 (H3K4). Our model showed a high level of performance on both a high-resolution data set of functional p53 binding sites from the experimental literature (ChIP data) and the whole human genome. Comparing our model with a simpler sequence-only model, we demonstrated that the prediction accuracy of the sequence-only model could be improved by incorporating epigenetic information, such as the two histone modification marks H3K4me1 and H3K4me3.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.