RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts

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Abstract
Incorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings could be the result of targeting cellular proteins in addition to pRb by HPV16-E7 oncoprotein. To get a more detailed picture on the role of pRb in chromosomal instability and centrosome amplification, we analyzed the effects of the acute loss of retinoblastoma gene function in primary conditional Rb deficient mouse embryonic fibroblasts (MEFs). Moreover, since pRb is a transcriptional repressor, microarray analysis was done on pRb-competent and pRb-deficient MEFs to evaluate changes in expression of genes for centrosome homeostasis and for correct mitosis. Acute loss of pRb induces centrosome amplification and aneuploidy in the vast majority of cells analyzed. A time course analysis shows a decrease of cells with amplified centrosomes after 40 days from the adenoviral infection. At this time only 12% of cells still show amplified centrosomes. Interestingly, cells with pRb constitutive loss show a similar percentage of cells with amplified centrosomes. DNA-Chip analyses in MEFs wt (mock infected) and pRb depleted (Ad-Cre infected) cells reveal differential expression of genes controlling both centrosome duplication and mitotic progression. Our findings suggest a direct link between pRb status, centrosome amplification and chromosomal instability, and define specific mitotic genes as targets whose gene expression has to be altered to achieve or maintain aneuploidy.