Gain or loss of chromosomes (aneuploidy) could generate in a single step multiple changes required for tumor initiation and progression. At least two possible cause of aneuploidy, not mutually exclusive, could be suggested: mutations in genes encoding mitotic checkpoint proteins and defects in genes controlling centrosome homeostasis. Numerous studies have shown that centrosome amplification is a frequent event in solid tumors and several findings seem to link deregulation of cell cycle progression and alteration in centrosome numbers with aneuploidy, where Rb dysfunction could affect also mitotic genes.Recently, we demonstrated that both human and murine pRB deficient cells tolerated induced aneuploidy. In addition, these cells amplified centrosomes after hydroxyurea treatment and in turn became aneuploid. On the contrary strains of HCT116 tumor cells did not become aneuploid despite the presence of supernumerary centrosomes. Interestingly, we found in IMR90 cells that transient loss of pRB was associated with down-regulation of MAD2 and with over-expression of Aurora-A and PLK1 genes.From these findings we hypothesize that loss of pRb triggers centrosome amplification 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 spindle checkpoint (MAD2) that in turn gives rise to aneuploidy.
Our proposal is aimed to investigate whether and what checkpoints need to be disrupted in order for centrosome amplification give rise to aneuploidy in human cells.We propose to determine in IMR90 human fibroblasts transiently depleted of pRb what genes will show altered gene expression among those partecipating in cell cycle checkpoints, chromosomal and centrosome dynamics whose differential gene expression could be relevant for CIN. To perform these experiments we’ll use the RT2 Profiler PCR arrays, a PCR based Pathway-focused gene expession profiling. In particular we’ll focus on genes identified in our Microarray analysis following acute loss of pRB in MEFs. In addition we’ll investigate if overexpression of Aurora-A and Plk1 affects at the same extent centrosome amplification and aneuploidy in pRb depleted cells. We will investigate if the observed MAD2 down regulation is caused by DNA methylation of the promoter region CpG islands and then to determine whether aneuploidy does not occur upon MAD2 re-expression in pRb depleted cells. showing centrosome amplification.We will use HCT116 tumor cells as a model system to define a pathway leading to aneuploidy. In the attempt to dissect further the relationship between centrosomes and aneuploidy, we’ll silence Nucleophosmin (NPM/B23) by RNAi. Wether following nucleophosmin RNAi centrosome amplification will occur in cells in which pRB is functioning, 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 relationship between genes controlling centrosome and chromosomal dynamics to generate aneuploidy.
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