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Abstract
Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised.
Highlights
Chromosomal instability (CIN) is a hallmark of almost all human cancer cells, and reflects ongoing changes of chromosome structure and number over time
For the evaluation of ploidy levels in post-stasis Variant human mammary epithelial cells (vHMEC) lines (830 and 440212) throughout the cell culture, a combination of β-tubulin immunofluorescence with fluorescent in situ hybridization (FISH) was performed. This immunoFISH protocol enabled the different nucleus inside the same cytoplasm to be visualized, allowing the ploidy of mononucleated (MN) and binucleated (BN) cells to be recorded. vHMEC were analyzed at an early culture stage (PD19 and PD21, for 830 and 440212, respectively) just after a period of selection when clones with p16INK4a inactivation (Figure S1A) acquire proliferation capacity due to promoter hypermethylation [20, 22]
There is a significant correlation between centrosome aberrations and advancing disease [4]
Summary
Chromosomal instability (CIN) is a hallmark of almost all human cancer cells, and reflects ongoing changes of chromosome structure and number over time. Potential mechanisms for the generation of centrosome aberrations in cancer cells include alterations in proteins controlling the centrosome duplication cycle, which could initiate multiple cycles of centriole replication -overduplication -within a single cell cycle [5]; or accumulation of extra centrosomes when cell division does not occur because of mitotic slippage, endoreduplication, aborted cytokinesis or cell fusion [6, 7]. Another possible scenario for increasing numbers of centrosomes involves de novo formation of centrioles during interphase [8]. In mouse models, supports the notion that loss of telomere repeats contributes to carcinogenesis [13]
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