Identification and characterisation of new genes important for p53- dependent, stress-induced apoptosis.

Polato, Federica (2006). Identification and characterisation of new genes important for p53- dependent, stress-induced apoptosis. PhD thesis The Open University.



The present thesis is aimed at the characterisation of an unknown gene named DRAGO (DRugs Activated Gene Overexpressed), isolated in the Laboratory of Molecular Pharmacology at the Mario Negri Institute as a p53-inducible gene by differential expression analysis in cells following genotoxic insults. The structure of the human DRAGO has been molecularly characterised. The coding region contains exons 2, 3, 4, 5 and the first 26 bases of exon 6. The remaining bases of this exon (4 Kb) constitute the 3’ untranslated region (3’UTR). Interestingly, the gene presents two large (45-50 Kb) introns. Both the presence of such introns, and of the large 3’UTR suggest a strong gene regulation at transcriptional and post-transcriptional levels. This observation is also supported by the presence of several AU and CU rich regions, which are known to be involved in degradation/stability of mRNA. A fragment upstream the 5’ of the coding region of DRAGO was isolated and demonstrated to have promoter activity. In transfection experiments the promoter activity of the isolated region was two-fold increased by human p53. An even stronger promoter activity in response to p73 and its isoforms was observed. Moreover, DNp73 was able to induce DRAGO expression co-operating with p73 and its isoforms.

The presence of DNA mutations in the gene has been investigated by using single strand conformation polymorphism (SSCP) analysis and DNA sequencing. For these studies, 12 human cancer cell lines of different origin and four cell sublines made resistant to anticancer drugs were analysed but no mutations were detected. The investigation was also extended to human tumor biopsies and again no mutations could be detected. The functional role of the DRAGO gene has been studied by ectopically overexpressing it in eukaryotic cells. An inhibitory effect on cell growth and the formation of a large number of vacuoles leading to the disruption of the plasmatic membrane could be observed upon its overexpression. The phenotypic changes induced by DRAGO overexpression were therefore evaluated using specific deletion mutants and it was demonstrated that cells can tolerate the expression of DRAGO mutant with deletion of a few aminoacids in the C-terminus. The computational analysis of the protein revealed that DRAGO protein is likely a transmembrane protein. The theoretical prediction was supported by immunofluorescence experiments using deletion mutants transiently expressed in cells and by the analysis of GFP (green fluorescent protein) -derived deletion mutants clones, showing that the fluorescence was localised in the membrane. DRAGO-null mice have been generated: they are viable and do not present any macroscopical alterations. Mouse embryo fibroblasts (MEF) were isolated from homozygously deleted mice. DRAGO -/- MEFs were more resistant to drug treatment than wild-type MEF due to the inability of the cells to activate apoptosis. This newly isolated gene can be therefore considered as a novel potential target for the development of new therapeutic anticancer agents.

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