| Abstract |
p21WAF1/Cip1 (p21) plays an essential role in important biological processes such as cell growth, differnentiation etc. acting as an inhibitor of the cell cycle in G1 and G2. Under conditions of p21 gene induction by extracellular factors such as cytokines, genotoxic stress or differentiation factors, p21 represses the activity of cyclin/CDK (Cyclin-Dependent Kinase) complexes and the activity of PCNA (Proliferating Cell Nuclear Antigen) which is a subunit of DNA Polymerase δ, resulting in the inhibition of DNA synthesis during S phase. In the present dissertation, we studied the molecular mechanisms that control p21 gene expression at the transcriptional level. In the first part, we have undertaken a functional characterization of the proximal p21 promoter and we found that two members of the Sp1 family of transcription factors (Sp1 and Sp3) bound, with different affinity, to six binding sites present in the proximal region between nucleotides -120 and -45 that were named Sp1 sites 1-6. Mutagenesis of these sites combined with transient transfections of SL2 cells that lack endogenous Sp1 or related activities, showed that sites 1 and 2 are important for the transactivation of the p21 promoter by Sp1 and Sp3. Utilizing a mutant Sp1 lacking the oligomerization domain D (Sp1 ΔD) we showed that the transactivation of the p21 promoter is accomplished by synergistic interactions between Sp1 proteins bound to adjacent sites on the proximal region. In the second part, we characterized the distal region of the p21 promoter by mutagenesis and by constructing artificial promoters and we found that the region between nucleotides -2,325 to -2,280, that includes a binding site for the tumor suppressor p53 protein, is a strong transcriptional enhancer of the p21 gene in human hepatome HepG2 cells. We showed that p53 cannot transactivate the p21 promoter in SL2 cells that lack endogenous Sp1 and Sp3 and that the p53-mediated transactivation of the p21 promoter is inhibited by mutations in Sp1 site 3. In parallel, we showed that none of the 6 Sp1 sites is necessary for the transactivation of the p21 promoter by Smad proteins which are mediators of the Transforming Growth Factor β (TGFβ) signaling pathway. Based on these findings, we proposed that p21 promoter is regulated by physical and functional interactions between p53 bound to the distal region and Sp1 or Sp3 bound to the proximal promoter region. Direct binding of Sp1 and Sp3 to p53 was shown both in vitro and in vivo. We found that Sp1 domains A, B and DBD interact with the C-terminal region of p53 (amino acids 293-393) whereas a mutant p53 that lacks the above region could not transactivate the p21 promoter. We also found that Sp1 and Sp3 physically and functionally interact with p73 which is homologous to p53 but is expressed in a cell specific manner. In the third part of the dissertation, we investigated the importance of Sp1 in cell processes that are mediated by p53 such as genotoxic stress induced by chemotherapeutic drugs. We found that the anticancer drug 5-Fluorouracil (5-FU) induces the transcription of the p21 gene only in cells that express a functional p53 protein. p53 activation by 5-FU is caused by the site-specific phosphorylation at serine 15. We also found that Mithramycin, a compound that is used for the treatment of Paget s disease and in certain forms of cancer, has a dual effect on cells. On one hand, it strongly activates p53 by a similar mechanism to 5-FU i.e. by phosphorylation at serine 15. On the other hand, Mithramycin, due to its property to inhibit the binding of Sp1 to GC-rich motives in the promoters of genes, represses the transcriptional induction of the p21 and PUMA (p53-Upregulated Mediator of Apoptosis) genes by 5-FU but has no effect on the basal levels of transcription of the above genes. Finally, we showed that Mithramycin is a strong apoptotic inducer in HepG2 cells presumably due to the inhibition of p21 gene induction by this drug. In summary, the results of the present dissertation show that transcriptional activation of p21 gene requires a number of protein-protein interactions between factors that bind to the distal p21 promoter region and factors that bind to the proximal p21 promoter region. Repression of these interactions inhibits cell cycle arrest by p21, resulting in uncontrolled cell proliferation. These results also support an important role of Sp1 transcription factor in the basal and the inducible activity of the p21 gene. The p21 protein, due to its cell cycle inhibitory properties, is a prime tool for the treatment of disease of uncontrolled cell proliferation. Understanding the molecular mechanisms of p21 gene regulation is of outmost importance for anticancer research.
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Μηχανισμοί μεταγραφικής ρύθμισης του γονιδίου του αναστολέα του κυτταρικού κύκλου p21 WAF1/Cip-1 του ανθρώπου
