BRCA1 germline mutations predispose women to early onset, familial breast and ovarian cancer. BRCA1 has been recently implicated in the cellular response to agents that disrupt the mitotic spindle. In this report, we studied BRCA1 contribution to paclitaxel response in MCF-7 breast cancer cells. We show that MCF-7 cells transfected with BRCA1 siRNA display a significant increase in resistance to paclitaxel compared with the control cells. We next demonstrate that downregulation of BRCA1 reduces the mitotic index and triggers premature cyclin B1 degradation and decrease in Cdk1 activity following paclitaxel treatment, suggesting that BRCA1 downregulation results in precocious inactivation of the spindle checkpoint. These findings were confirmed by showing that BRCA1 downregulation induces premature sister-chromatids separation in MCF-7 cells following spindle damage. Furthermore, we show that BRCA1 up-regulates the expression of the protein kinase BubR1, essential component of the functional spindle checkpoint, whose downregulation is known to result in paclitaxel resistance in MCF-7 cells. Altogether, our findings support the notion that downregulation of BRCA1 expression mediates paclitaxel resistance through premature inactivation of spindle checkpoint in MCF-7 breast cancer cells. They link BRCA1 to the mitotic checkpoint that plays an essential role in the maintenance of chromosomal stability.
Our results reveal a network of new potential Bcl-2 partners identified through the Bcl-2 immunocapture and mass spectrometry approach and analyzed by gene ontology mining. Importantly, we report for the first time the identification of galectin-7, a member of a family of β-galactoside-binding lectins, as a new mitochondrial Bcl-2 interacting partner.
Centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive α-satellite sequences, which are actively transcribed throughout the cell cycle. Centromeres play an essential role in chromosome inheritance and genome stability through coordinating kinetochores assembly during mitosis. Structural and functional alterations of the centromeres cause aneuploidy and chromosome aberrations which can induce cell death. In human cells, the tumor suppressor BRCA1 associates with centromeric chromatin in the absence of exogenous damage. While we previously reported that BRCA1 contributes to proper centromere homeostasis, the mechanism underlying its centromeric function and recruitment was not fully understood. Here, we show that BRCA1 association with centromeric chromatin depends on the presence of R-loops, which are non-canonical three-stranded structures harboring a DNA:RNA hybrid and are frequently formed during transcription. Subsequently, BRCA1 counteracts the accumulation of R-loops at centromeric α-satellite repeats. Strikingly, BRCA1-deficient cells show impaired localization of CENP-A, higher transcription of centromeric RNA, increased breakage at centromeres and formation of acentric micronuclei, all these features being R-loop-dependent. Finally, BRCA1 depletion reveals a Rad52-dependent hyper-recombination process between centromeric satellite repeats, associated with centromere instability and missegregation. Altogether, our findings provide molecular insights into the key function of BRCA1 in maintaining centromere stability and identity.
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