Identification of mechanisms underlying chromosomal instability in human cells by DNA microarrays and siRNA

Progetto: Research project

Description

The occurrence of chromosomal alterations, both structural and numerical, scored in the majority of solid tumors, conferring significant variation between tumors and tumor cells of the same histological origin, are believed to destabilize the genome and further increase the occurrence of new changes. Thus, the underlying chromosomal instability (CIN) and consequent aneuploidy, observed in tumors and in early stage carcinomas, strongly suggest that it could be considered a prerequisite in initiation as well as in tumor progression. CIN could be a transient phenomenon consisting in a two stages process, where centrosome amplification would lead to aberrant spindles and catastrophic variations in chromosome number. The project takes advantage of results we already got both in human and murine pRB deficient cells as well as in HCT116 strains. Recently we demonstrated that both human and murine pRB deficient cells were able to tolerate induced aneuploidy. In addition, we showed that these cells amplify centrosome after hydroxyurea treatment and in turn become aneuploidy. Moreover, we got results that strains of HCT116 tumor cells, with different genetic background, did not become aneuploid despite the presence of supernumerary centrosomes. The overall goal of this proposal is to further elucidate the pathway(s) that likely mediates aneuploidy in mammalian cells. We propose two experimental approaches to address the role of Rb and genes involved in genetic stability and/or centrosome homeostasis in the attempt to draw a pathway whose alteration could result in CIN.The first approach will consist in transcriptomic studies of genes that likely are differentially expressed in human pRB deficient cells that undergo aneuploidy and centrosome amplification. Moreover, we’ll look at the immediate-early effects of Rb and p53 loss in human fibroblasts transient silencing of p53 and Rb by RNA interference.The second approach is aimed to further define whether in addition to Rb genes associated with centrosome homeostasis and/or genetic instability could be involved in a pathway that mediates aneuploidy. As a model system we will use strains of HCT116 in which centrosome amplification but not aneuploidy occurred. RNA interference strategies will be used to abolish the function of genes previously involved in centrosome control or in genetic stability. In particular we will silence the Nucleophosmin/B23 gene that is found associated with the correct centrosome dupication and the Aurora A/stk15 kinase involved in a pathway leading to centrosome maturation. Another target will be the BRCA1 gene that is often associated with genomic instability and whose product has been located at the centrosome in mitosis. RNA interference strategy will be accomplished also in primary normal cells to validate the hypothesis that alterations in the expression of some of these genes could underlie aneuploidy. Then, understanding of the molecular bases for aneuploidy could result in the medium long term in new strategies for the successful treatment of cancer.

Layman's description

In the attempt to get additional clues on a pathway for CIN in human cells, we propose two experimental approaches to address the role of Rb as well as of some genes involved in genetic stability and/or centrosome homeostasis. Firstly, since our data suggest the involvement of pRB that is known as a transcrption repressor, to prevent aneuploidy associated with centrosome amplification, we propose to investigate changes in gene expression in pRB deficient, both E7 expressing and silenced by siRNA, and pRB proficient human fibroblasts after HU treatment by using DNA microarrays. The comparison between human fibroblasts depleted of pRB by siRNA and expressing the HPV-E7 oncoprotein, that has been shown to induce centrosome amplification in cells that lack RB function (Duensing S. and Munger K. J. Virology 77, 12331-5, 2003), could help to discriminate the effects because of the presence of E7 that targets not only pRB. The use of pRB silenced human cells is suggested by the results of the immediate-early effects of acute Rb loss in conditional deficient pRB MEFs. However, because checkpoints in mouse are less stringent than in human cells, we will look first at the immediate early effects on centrosome and genetic stability in human fibroblasts. Likely, the results from the microarray analysis might suggest altered expression of genes that could be candidates to investigate in addition to those indicated above. Secondly, starting from our findings in HCT116 cells, we will investigate in these cells as well as in normal human fibroblasts, the effects of silencing Rb by siRNA and modulate expression of genes such as BRCA1, Aurora-A. In particular Aurora-A will be either over-expressed or silenced by siRNA to affect the processes, mentioned above, in which is involved. In addition we’ll silence by siRNA Nucleophosmin (NPM/B23) that is a licensing factor for centrosome duplication after its CDK2/cyclinE mediated phosphorylation (Okuda M.et al.,Cell 103, 127-140, 2000). Then, if nucleophosmin is a down stream target of pRB its loss could result also in centrosome amplification in cells in which pRB is functioning (i.e. in HU arrested cells). This strategy will be accomplished also in normal human fibroblasts (IMR90) to test the hypothesis that alterations in centrosome numbers alone in not sufficient to generate aneuploidy.
StatoAttivo
Data di inizio/fine effettiva1/1/05 → …