Abstract
BackgroundIncorrect 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.ResultsAcute 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.ConclusionOur 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.
Highlights
Incorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer
Centrosome amplification after acute loss of pRb in primary murine fibroblasts To evaluate the effects of Rb acute loss on centrosomes we used Mouse Embryonic Fibroblasts (MEFs) carrying conditional Rb alleles (RbLoxP/LoxP) in which two LoxP sites are inserted into the introns surrounding exon 19 of the Rb gene [34]
Growing MEFs (MEF RbLoxP/LoxP) were infected with adenoviruses expressing the Cre recombinase (Ad-Cre) in order to generate cells with truncated pRb (Rb-/-), by excision of exon 19, that are functionally equivalent to Rb null cells
Summary
Incorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. Some studies suggest that aneuploidy in cancer cells could arise from defects in the mitotic spindle checkpoint. This mitotic checkpoint monitors that in metaphase, all chromosomes are properly aligned and attached to the mitotic spindle before progressing to anaphase. In human and murine cells mutations of some of these mitotic genes such as: hBUB1, hSecurin, hMAD2 and hBUBR1 induced aneuploidy in dividing diploid cells over several generations, they were rarely mutated in human tumors [3,4,5,6,7]). MAD2 overexpression was associated with stable inactivation of the retinoblastoma tumor suppressor and E2F-1 activation [9]
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