The objective of the project is to use the RICS analysis technique in complement with confocal microscopy to determine the diffusion coefficient of the selectively labeled green fluorescent protein (GFP), GFP-EGFR and GFP-p53 in cervical cancer cells. This is a collaboration work with MD Anderson Cancer Center. The application of the study is to lay the foundation for further study in understanding the cell metabolism, subcellular morphologic and dynamic biochemical processes to aid in the diagnosis and to potentially screen cancers. Fluorescence microscopy techniques have been developed for the study of cellular processes and molecular signal pathway. However, the spatial resolution to distinguish and resolve the interactions of single molecular complexes or molecule in cells is limited by the wavelength. Hence, indirect image correlation methods complementary to the imaging techniques were developed to obtain the dynamic information within the cell. RICS is one such mathematical image processing method to determine the dynamics of the cell. HeLa cells are transfected with GFP to highlight the protein of interest. These samples were imaged with confocal microscope, Olympus FV1000 with a 60 x 1.2 NA water objective in the pseudo photon counting mode with an excitation of 488 nm argon ion laser. About 100 frames of scan area 256x256 pixels were collected from each sample at scan speed of 12.5 seconds per pixel. The stacks of images were processed with SimFCS software. The images were subjected to immobile subtraction algorithm to remove the immobile features. Consequently, each frame in the stack is subjected to 2D-correlation; then, the average 2D-spatial correlation is calculated. This 2D-spatial correlated data constitutes as RICS data which is then displayed and analyzed by fitting it to specific models. This generates a spatial temporal map of the molecular dynamics of fluorescently labeled probes within the cell. In summary, we apply RICS techniques based on correlation spectroscopy to the image data and quantify diffusion coefficient of protein of interest in cancerous cells with different treatments. This is expected to better understand cellular dynamics of cancerous cells and build better diagnostic biosensor devices for early screening.

Proefschrift--Universiteit Leiden, 2003. / Notes bibliogr.

Thèse de doctorat : Biochimie. Biologie cellulaire et moléculaire : Paris 12 : 2005. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 383 réf.

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The tumor suppressor p53 acts as a master transcription factor that controls hundreds of effecter genes in response to various cellular stresses. The flexibility of p53 to regulate its target genes with distinct functions (growth arrest, DNA repair, apoptosis etc.) is largely conferred by extensive and dynamic posttranslational modifications of the protein, including phosphorylation, acetylation, methylation, ubiquitination, sumoylation, neddylation and ADP-ribosylation. Recent evidence suggests that acetylation is indispensable for p53 activation. A major regulator of p53 acetylation, and hence p53 function, is a group of Class III histone deacetylases known as Sirtuins (SIRTs), that utilize nicotinamide adenine dinucleotide (NAD+) as substrate to catalyze the removal of acetyl groups from p53, resulting in the “silencing” of p53 activity. In an effort to determine whether a feedback loop exists whereby p53 is involved in the regulation of NAD+ metabolism, nicotinamide adenylyltransferase 2 (Nmnat2), a key NAD+ synthetase, was identified to be a novel target gene of p53, from which two transcript variants are expressed in human (TV1 and TV2). Two putative p53 response elements within the first intronic region of human Nmnat2 gene were also identified that can actively drive the expression of luciferase reporter gene in a p53-dependent manner. Most importantly, data suggests that Nmnat2, like SIRTs, is involved in the regulation of p53-mediated apoptosis and protein acetylation upon DNA damage. Furthermore, Nmnat2 isoforms exert opposite functions on SIRT-mediated deacetylation of p53. Specifically, ectopic expression of Nmnat2 TV2 isoform promotes p53 acetylation after DNA damage, whereas ectopic expression of Nmnat2-TV1 isoform suppresses it. Manipulation of SIRT activities by either RNA interference or specific inhibitors modifies p53 acetylation status the same way Nmnat2-TV2 isoform does. Collectively, the results suggest the existence of a p53-Nmnat2 feedback loop, whereby p53 can regulate its own activity positively or negatively, depending on the nature and extent of DNA damage.

Mammalian reovirus is a naturally benign virus that preferentially replicates in cancer cells (reovirus oncolysis) and has been tested as a potential cancer therapy in vitro, in vivo and in clinical trials for treating cancers from a wide range of origins. Reovirus-induced apoptosis has been shown to be important for reovirus oncolysis. The tumor suppressor protein p53 plays vital roles in mediating host apoptosis but its effect on reovirus oncolysis has not been fully understood and hence investigated. Data here show that p53 does not affect reovirus replication or reovirus-induced apoptosis in human cancer cells. However, significant enhancement of the reovirus-mediated apoptosis is induced by addition of p53 accumulators/activators such as a MDM2 antagonist Nutlin-3a or sub-lethal concentrations of chemotherapy drugs. This enhanced cell death is p53-dependent, requires NF-kappaB activation and p53 target genes p21 and bax. Furthermore, a combination of reovirus and p53 accumulators/activators directly results in significantly higher level of reovirus dissemination and spread. One of the hurdles for current chemotherapy in patients is the side effects caused by high concentrations of cytotoxic drugs. Hence, a therapeutic regime using the combination of reovirus and sub-lethal (lower concentrations of) chemotherapeutics that induce p53 activation/accumulation potentially can both enhance tumor regression and reduce the side effects in patients. Ras mutation, one of the most prevalent mutations in human cancer, has been implicated to determine the susceptibility of cancer cells to reovirus infection. However, the underlying mechanism of reovirus preferential replication needs to be further delineated. Quantitative analysis was used to compare individual steps of reovirus replication between non-transformed and Ras-transformed NIH 3T3 cells. Contrary to previous reports, reoviral protein synthesis is shown to be comparable between non-Ras and Ras-transformed cells. Meanwhile, although reovirus binding and internalization is not affected by Ras-transformation, reovirus uncoating is enhanced in Ras-transformed cells. Ras-transformation also enables reovirus to better spread to neighboring cells through apoptosis. Furthermore, reovirus infection of Ras effector mutant cells that activate specific Ras effector pathways indicate that sub-Ras pathways play different roles in enhancing reovirus preferential replication in Ras-transformed cells and therefore provide additional targets for cancer therapy.

In normal cells, the p53 tumour suppressor induces cell cycle arrest or apoptosis in response to a variety of stresses, including DNA damage and ectopic oncogene expression. However, cellular pathways controlled by p53 are compromised in virtually all cancers. Defining the mechanisms regulating p53 activity in normal and tumour cells has therefore been a major priority in cell biology and cancer research. / In this study, we characterized two important regulatory mechanims of p53 activity: (i) Human papillomavirus (HPV) E6 interaction and (ii) alternative splicing. Recognized as the major etiological agents for cervical cancer, the oncogenic potential of HPVs correlates with their ability to target p53 for degradation. This study demonstrates that both p53 and HPV-18 E6 are exported from the nucleus when co-expressed, via a process that involves the C-terminal nuclear export signal (NES) of p53. However, neither nuclear export nor the p53 C-terminal NES is required for HPV-18 E6-mediated ubiquitination or degradation of p53. / This study also demonstrates that both low- and high-risk HPV E6 proteins are degraded by the ubiquitin-proteasome pathway, and thus provides an explanation for the low levels of E6 detected in cervical cancer cells. / Also reported in this study is a novel mechanism of p53 regulation arising through alternative splicing. This novel mRNA encodes a N-terminal deleted isoform of p53, termed p47. As demonstrated within, p47 does not supress cell viability but impairs both p53-mediated transcriptional activity and growth suppression. Interestingly, p47 increases both p53 monoubiquitination and nuclear export. We propose that p47 induces nuclear export of p53 by a mechanism involving monoubiquitination, as supported by recent findings from Li and colleagues (2003). The p47 protein also protects p53 from both Mdm2- and HPV-18 E6-mediated degradation. A number of cancers display abnormal localization of wildtype p53, and it will be important to examine the role of p47 in these tumours. / Taken together, the regulation of p53 activity by both HPV E6 and the alternative splice variant p47 involves alterations in p53 ubiquitination status, protein stability, and cell localization. Insight gained into these negative regulatory mechanisms may aid in the design of therapeutic strategies for reactivating wild-type p53 in HPV-associated and non-associated cancers.

Les maladies à prion sont un cas unique de pathologie où l’agent infectieux, le prion, est une protéine. La protéine prion possède plusieurs caractéristiques qui la rendent particulière vis-à-vis d’autres protéines cellulaires, telles que sa capacité à agréger et à transmettre sa conformation agrégée à la protéine soluble ainsi que la transmission des agrégats de la protéine d’une cellule à l’autre et d’un organisme à un autre. De plus en plus, on associe l’agrégation de protéines à différentes maladies humaines, comme les maladies d’Alzheimer, de Parkinson et le diabète de type 2. Certaines protéines impliquées dans ces pathologies font partie des prionoïdes, une catégorie réservée aux protéines aux propriétés agrégatives qui démontrent certaines des caractéristiques associées aux prions. Récemment, la protéine p53, un facteur de transcription fortement impliqué dans le cancer, a été montrée comme étant capable d’agréger in vitro. Une accumulation de la protéine a également été observée dans des cellules tumorales, laissant croire que l’agrégation de p53 se produit également in vivo, et pourrait avoir un rôle dans le développement du cancer. Ces observations portent à croire que la protéine p53 pourrait elle aussi faire partie des prionoïdes. L’objectif de cette étude est donc de montrer que la protéine p53 possède certaines des caractéristiques des prions. Pour ce faire, la protéine p53 recombinante a été produite pour former des agrégats de p53 et ces agrégats ont été utilisés pour déterminer si la protéine démontre des caractères prionoïdes. Les résultats obtenus montrent une agrégation in vitro de p53WT pleine longueur ainsi que de sa forme tronquée, p53C. De plus, des cellules en culture sont capables d’internaliser ces agrégats, qui co-agrègent ensuite avec la protéine p53 endogène de ces cellules. Enfin, nos résultats montrent clairement que l’internalisation des agrégats par les cellules se fait par la macropinocytose. Nous avons donc réussi à prouver que la protéine p53 agit comme un prion puisqu’elle s’agrège spontanément, ses agrégats sont internalisés par des cellules en culture et sont capables de co-agréger avec la protéine soluble.