pRb loss and chromosomal instability in human cells.

Abstract

pRb loss and chromosomal instability in human cells. Recent studies suggest that Retinoblastoma tumor suppressor (RB) plays important roles in the prevention of chromosomal instability by regulating genes that control cell cycle progression and mitotic events. We investigated the effects of stable post-transcriptional silencing of RB in primary human fibroblasts (IMR90) and in near-diploid colon cancer cells (HCT116) focusing on chromosome missegregation mechanisms. Stable depletion of pRb was achieved by infection with the retroviral vector MSCV-LMP670 encoding a microRNA (miR670) targeting RB transcript. Cytogenetic, immunofluorescence microscopy and time-lapse video-microscopy analyses showed that pRb loss caused aneuploidy, micronuclei formation and multipolar spindles. By qRT-PCR and western blot analyses we detected changes in the expression of genes involved in centrosomes duplication, centromere assembly and mitotic checkpoint regulation. Moreover, depletion of pRB induced dysregulation of genes involved in epigenetics and it seems to be associated to global DNA hypomethylation in IMR90 cells. Finally, pRB loss conferred to HCT116 cells a proliferative advantage, instead human primary fibroblasts showed premature senescence features including increased levels of p53, p21, p14 and p16 genes expression. Our results demonstrate that inactivation of RB may promote chromosomal instability by compromising several pathways. In normal cells aneuploidy alone does not seem sufficient to cause malignant trasformation, but in a permissive cellular context could enhance proliferative capacity.