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Redox Regulation of Human P53 Tumor Suppressor Gene Activity: Identification of Redox Genes That Play Role in Human P53 Reporter Gene Activity
(Izmir Institute of Technology, 2008) Ataç, Beren; Koç, Ahmet
The occurence of p53 gene mutations in many human tumors shows that p53 protein plays an important role in preventing cancers. One of the most important function of p53 protein is its ability to stimulate transcription of other genes that inhibit cell cycle progression and DNA repair mechanisms or apoptosis pathways. p53 gene activity is controlled by a series of mechanisms among which redox regulation has taken little attention. Because of its importance as a tumor suppressor, its role as a cell cycle control protein and transcription factor, we decided to focus on p53 transcriptional activity. Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription of reporter genes. Several studies have shown that the p53 protein is prone to oxidative inactivation. Although yeast does not contain a p53 gene, the similarity of cell cycle control mechanisms and oxidative stress response pathways prompted us to ask whether human p53 was active in yeast cells lacking individual antioxidant genes. In this study, using yeast deletion mutants, p53 reporter gene activity was assayed in different antioxidant mutants that were identified by REDOX-Cys-Search bioinformatic tool by previous studies. Seven antioxidant genes were found to be important in regulating p53 activity. These genes played role in phosphatidylinositol pathway, protein dephosphorylation, cellular iron metabolism, DNA mismatch repair, and three other unknown biochemical pathways.Identification of these new proteins that regulate p53 activity may have broad implications in understanding the complex behaviour of p53 and tumor formation in humans.
Deciphering 5-Fluorouracil Mediated Molecular Mechanisms Required for Cell Death
(Izmir Institute of Technology, 2011) Can, Geylani; Baran, Yusuf
The chemotherapy agent 5-Fluorouracil (5-FU) is an antimetabolite that has been in use to treat several cancers for decades. In cells, it is converted into three distinct fluoro-based nucleotide analogues which interfere with DNA-synthesis and repair leading to impairment of the genome and, eventually apoptotic cell death. Current knowledge also state that 5-FU induced damage is signaling through a p53-dependent induction of death inducing complex (DISC) formation and further caspase-8 activation in certain cell types and members of the TNF-receptor family has been proposes to be required for the process. Here, we introduce calcium (Ca2+) as a messenger for p53 activation in the cellular response triggered by 5-FU. Using a combination of pharmacological and genetic approaches, we show that treatment of cultured colon carcinoma cells stimulates entry of extracellular Ca2+ through L-type plasma membrane channels and that this event direct posttranslational phosphorylation of at least two specific p53 serine residues (ser15 and ser33) by means of Calmodulin (CaM) activity. Obstructing this pathway by the Ca2+-chelator BAPTA or by two different inhibitors of CaM efficiently blocks 5-FU-induced cell death. The fact that a widely used therapeutic drug, such as 5-FU, is signaling by these means could provide new therapeutic intervention points, or specify new combinatorial treatment regimes.
Regulation of Human P53 Tumor Suppressor Gene Activity by Thiol-Depentdet Oxidoreductases
(Izmir Institute of Technology, 2011) Bekki, Gözde; Koç, Ahmet
Background: Occurrence of p53 mutations in more than half of human tumors indicates the importance of p53 gene in cancer prevention. Nevertheless, oxidation of cysteine-SH groups in p53 protein can inactivate the protein under oxidative conditions. The importance of p53 as a tumor suppressor and insufficient studies about redox regulation of p53 gene lead us to study redox regulation of p53 protein. Methods: In this study, yeast (Saccharomyces cerevisiae) was used as an in vivo model. All potential thiol-dependent antioxidant genes in yeast were identified based on specific characters in their structure, via REDOXCysSearch bioinformatics tool. To study human p53 gene activity in yeast cells, p53 gene and p53 RE Lac-Z reporter that is inducible by p53, were transformed into yeast. Antioxidant gene mutants were analyzed for LacZ reporter gene activity. Results: We identified several yeast mutants (Î”sac1, Î”hnt3 and Î”map1) with lower p53 activity with respect to wild-type yeast. Conclusion: Due to conserved mechanisms of cell cycle regulation and oxidative stress tolerance between yeast and mammals, we believe that results from yeast studies may help us to understand the redox regulation of p53 in human cells. Thus, a new perspective may appear in the redox regulation of p53 gene.

Master Degree / Yüksek Lisans Tezleri

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