Gain or loss of chromosomes (aneuploidy) could generate in a single step multiplechanges required for tumor initiation and progression. At least two possible cause ofaneuploidy, not mutually exclusive, could be suggested: mutations in genes encoding mitoticcheckpoint proteins and defects in genes controlling centrosome homeostasis. Numerousstudies have shown that centrosome amplification is a frequent event in solid tumors andseveral findings seem to link deregulation of cell cycle progression and alteration incentrosome numbers with aneuploidy, where Rb dysfunction could affect also mitotic genes.Recently, we demonstrated that both human and murine pRB deficient cells toleratedinduced aneuploidy. In addition, these cells amplified centrosomes after hydroxyureatreatment and in turn became aneuploid. On the contrary strains of HCT116 tumor cells didnot become aneuploid despite the presence of supernumerary centrosomes. Interestingly, wefound in IMR90 cells that transient loss of pRB was associated with down-regulation ofMAD2 and with over-expression of Aurora-A and PLK1 genes.From these findings we hypothesize that loss of pRb triggers centrosomeamplification by altering expression of genes encoding protein for centrosome homeostasis(i.e. cyclin E,Aurora-A, PLK1, Nek2) and inducing defects in components of the spindlecheckpoint (MAD2) that in turn gives rise to aneuploidy.Our proposal is aimed to investigate whether and what checkpoints need to bedisrupted in order for centrosome amplification give rise to aneuploidy in human cells
We propose to determine in IMR90 human fibroblasts transiently depleted of pRbwhat genes will show altered gene expression among those partecipating in cell cyclecheckpoints, chromosomal and centrosome dynamics whose differential gene expressioncould be relevant for CIN. To perform these experiments we’ll use the RT2 Profiler PCRarrays, a PCR based Pathway-focused gene expession profiling. In particular we’ll focus ongenes identified in our Microarray analysis following acute loss of pRB in MEFs. In additionwe’ll investigate if overexpression of Aurora-A and Plk1 affects at the same extentcentrosome amplification and aneuploidy in pRb depleted cells. We will investigate if theobserved MAD2 down regulation is caused by DNA methylation of the promoter region CpGislands and then to determine whether aneuploidy does not occur upon MAD2 re-expressionin pRb depleted cells. showing centrosome amplification.We will use HCT116 tumor cells as a model system to define a pathway leading toaneuploidy. In the attempt to dissect further the relationship between centrosomes andaneuploidy, we’ll silence Nucleophosmin (NPM/B23) by RNAi. Wether followingnucleophosmin RNAi centrosome amplification will occur in cells in which pRB isfunctioning, we’ll modulate the expression of genes (Aurora-A, PLK1, MAD2, BRCA1etc)we think contribute to aneuploidy. This strategy is aimed to dissect the functional relationshipbetween genes controlling centrosome and chromosomal dynamics to generate aneuploidy.
|Data di inizio/fine effettiva||1/1/07 → …|