Abstract
Tetraploidy, a common feature in cancer, results in the presence of extra centrosomes, which has been associated with chromosome instability (CIN) and aneuploidy. Deregulation in the number of centrosomes triggers tumorigenesis. However, how supernumerary centrosomes evolve during the emergence of tetraploid cells remains yet to be elucidated. Here, generating tetraploid isogenic clones in colorectal cancer and in non-transformed cells, we show that near-tetraploid clones exhibit a significant increase in the number of centrosomes. Moreover, we find that centrosome area in near-tetraploids is twice as large as in near-diploids. To evaluate whether centrosome clustering was occurring, we next analysed the number of centrioles revealing centriole amplification. Notwithstanding, more than half of the near-tetraploids maintained in culture do not present centrosome aberrations. To test whether cells progressively lost centrioles after becoming near-tetraploid, we transiently transfected diploid cells with siRNA against ESPL1/Separase, a protease responsible for triggering anaphase, to generate newly near-tetraploid cells. Finally, using this model, we assessed the number of centrioles at different time-points after tetraploidization finding that near-tetraploids rapidly lose centrosomes over time. Taken together, these data demonstrate that although most cells reduce supernumerary centrosomes after tetraploidization, a small fraction retains extra centrioles, potentially resulting in CIN.
Highlights
Tetraploidy, a common feature in cancer, results in the presence of extra centrosomes, which has been associated with chromosome instability (CIN) and aneuploidy
To establish a model to investigate the relationship between tetraploidy and the number and structure of centrosomes, we used 2N and 4N isogenic cells derived from the colorectal cancer cell lines DLD-1, RKO and SW837, and the non-transformed RPE1
In agreement with previous data generated in our laboratory[16], we confirmed increased levels of CIN by fluorescence in situ hybridization (FISH) analysis in 4N compared to 2N cells (Fig. 1a)
Summary
Tetraploidy, a common feature in cancer, results in the presence of extra centrosomes, which has been associated with chromosome instability (CIN) and aneuploidy. To test whether cells progressively lost centrioles after becoming neartetraploid, we transiently transfected diploid cells with siRNA against ESPL1/Separase, a protease responsible for triggering anaphase, to generate newly near-tetraploid cells. Using this model, we assessed the number of centrioles at different time-points after tetraploidization finding that neartetraploids rapidly lose centrosomes over time. Centriole duplication is controlled through a centrosome-intrinsic blockade, in which duplication of the parent centriole is prevented as long as the parent and the newly-synthetized procentriole remain in a tight orthogonal association[3] The dissolution of this linkage, known as centriole disengagement, requires the activity of the kinase PLK1 and the protease separase, which permits the reduplication of the parent centriole in the cell cycle[4]. The generation of 4N cells, either by silencing ESPL1/Separase or dihydrocytochalasin B (DCB) treatment, reveals that most newly-generated 4N cells rapidly reduce extra centrosomes over time, a small fraction retains supernumerary centrosomes, resulting in CIN
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