Evidence suggests that loss of the RB pathway affects mitosis, so Van Harn and colleagues arrested mouse embryonic fibroblasts in which the three Rb family members were ablated (TKO-MEFs) in G2 by serum starvation. This induced the cyclin-dependent kinase inhibitor p21, produced double-strand DNA breaks and activated the homologous recombination repair pathway. Inhibiting DNA damage response (DDR) kinases accelerated cell cycle re-entry and progression into mitosis after release from serum starvation and downregulated p21 expression. These data indicate that serum-starved RB pathway-deficient cells accumulate DNA damage during progression through the preceding S phase and thereby activate DDR pathways. Moreover, metaphase spreads of TKO-MEFs showed defects in centromeric sister chromatid cohesion and more chromatid breaks than in controls, suggesting damage persists in mitosis. Consistent with this, the authors showed that serum-starved TKO-MEFs that re-entered the cell cycle after serum addition had increased copy number alterations. Sister chromatid cohesion is initiated during S phase by the cohesin complex, and although the authors did not find evidence of improper loading of the cohesin complex they suggested that this process might be defective when the RB pathway is inactivated.
Manning and colleagues used RNA interference to downregulate RB1 expression in TERT-immortalized RPE1 cells (which are diploid epithelial cells) and found that the loss of RB1 induces aneuploidy and chromosome missegregation. Knock down of RB1 did not affect proliferation but increased the mitotic index by twofold. In particular, the authors found a high proportion of cells in prometaphase, indicating that loss of RB1 delayed mitotic progression. On further analysis, the authors observed increased inter-centromeric distance between sister chromatids, defects in chromosome congression, sister kinetochore orientation and alignment on the metaphase plate. The authors proposed that these mitotic defects might originate from a defect in the centromere structure, which is regulated by the cohesin and condensin complexes. They found that chromatin association of RAD21 — a subunit of the cohesin complex — was reduced in mitotic cells lacking RB1, suggesting that its loss impairs the loading or maintenance of the cohesin complex at the centromere.
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