Telomere dysfunction incriminates cells that can’t divide [with video]
Cells that arrest cell division during mitosis eventually die or stop the cell cycle in the following G1 phase. Both of these outcomes are proposed to be tumor suppressive mechanisms, since escape from prolonged mitotic arrest may render cells aneuploid. Despite its importance in tumor suppression, the molecular pathway that predisposes arrested mitotic cells to die or stop the cell cycle in the subsequent G1 phase had not been determined.Here, we found that the chromosome ends (i.e. telomeres), which are normally protected from activating a DNA damage response, become de-protected during prolonged mitotic arrest, activating the DNA damage checkpoint machinery. We propose that this programmed telomere de-protection protects cells that escape prolonged mitotic arrest from aneuploidy and serves as a molecular switch that reacts to improper mitosis progression.
HFSP Long-Term Fellow Makoto Hayashi and colleaguesauthored on Mon, 14 May 2012
Telomeres are the nucleoprotein complexes that protect eukaryotic chromosome ends from being recognized as damaged DNA. Vertebrates’ telomeres are composed of double stranded 5’-TTAGGG-3’ repeats that are bound by the telomere-specific “shelterin” protein complex. The shelterin components TRF2 and POT1 are predominantly involved in chromosome end protection by preventing ATM- and ATR-dependent DNA damage checkpoint activation. Disruption of TRF2 or POT1 activates DNA damage response signaling, resulting in phosphorylation of histone H2AX (gamma-H2AX) within the telomeric and subtelomeric chromatin and the induction of a p53-dependent cell cycle arrest or apoptosis. Replication-dependent telomere shortening also causes telomere dysfunction in human somatic cells after the exhaustion of telomeric repeats, which induces a permanent state of cell cycle arrest termed replicative senescence. In addition to these known pathways of telomere dysfunction, we recently found that prolonged mitotic arrest also induces telomere de-protection in primary and transformed human cell lines.
Figure: Immunofluorescent image of human diploid primary fibroblast exposed to a microtubule drug, Vinblastine, for 24 hours. Blue: Chromosome, Green: Telomere, Red: Histone H2AX phosphorylation on Ser139 (gammga-H2AX).
When cells enter mitosis, the spindle assembly checkpoint is activated, which delays mitosis progression until all chromosomes are aligned properly and ready to separate. If cells encounter a problem and are arrested in mitosis, they eventually die during mitotic arrest, or skip cytokinesis and progress into the subsequent G1 phase of the cell cycle, a process termed mitotic slippage. Cells that escape from prolonged mitotic arrest or progress through mitotic slippage succumb to various fates in the following cell cycle, such as apoptosis or p53-dependent cell cycle arrest. Cell death during mitotic arrest, as well as apoptosis and cell cycle arrest after escape from prolonged mitotic arrest have been proposed to be tumor suppressive mechanisms, since a failure to induce these pathways may allow cells to keep dividing with an abnormal number of chromosomes, leading to aneuploidy and driving tumor progression. However, the molecular mechanisms that trigger growth arrest or cell death pathways during mitotic arrest have previously not been identified.
In the course of experiments designed to explore the putative roles of cohesin in telomere maintenance, we discovered that prolonged mitotic arrest induces gamma-H2AX focus formation specifically at the telomeres of mitotic chromosomes. Prolonged mitotic arrest also caused loss of the telomeric 3’-overhang and ATM activation, which is consistent with the idea that chromosome ends were rendered de-protected and exposed to the DNA damage checkpoint machinery during mitotic arrest. Cells that possess mutations in the ATM kinase, but not ATR, failed to accumulate telomeric gamma-H2AX during mitotic arrest, indicating that ATM has a predominant role in the mitotic telomere de-protection pathway. We further found that TRF2 was dissociated from telomeres during prolonged mitotic arrest and that over expression of TRF2 suppressed the damage phenotype, indicating loss of TRF2 as the molecular basis for 3’-overhang loss and ATM activation. Primary cells suffering from mitotic telomere dysfunction underwent a p53-dependent cell cycle arrest in the subsequent G1 phase after release from mitotic arrest, whereas cells with incompetent p53 function continued their cell cycle progression with an abnormal number of chromosomes. Our results support the hypothesis that mitotic telomere de-protection leads to an activation of a p53-dependent DNA damage checkpoint pathway in the following cell cycle, eventually causing cell cycle arrest or apoptosis. Finally, we discovered that Aurora B kinase, which is upregulated in mitosis, is involved in regulating the telomeric gamma-H2AX foci formation, suggesting that mitotic telomere de-protection is actively induced during mitotic arrest.
From those results, we propose that programmed exposure of chromosome ends during prolonged mitotic arrest induces DNA damage checkpoint signaling and therefore serves as a mitotic-duration checkpoint that is responsible for elimination of cells that have failed to progress through mitosis properly. Our results also finally shed light on the mechanism of action of extensively used chemotherapeutic drugs such as Taxol, Vinblastine and Velcade, which all inhibit mitotic progression and cause cell death.
A Telomere Dependent DNA Damage Checkpoint Induced by Prolonged Mitotic Arrest. Makoto T. Hayashi, Anthony J. Cesare, James A. J. Fitzpatrick, Eros Lazzerini-Denchi and Jan Karlseder. Nature Structural & Molecular Biology, 2012 Mar 11; 19(4): 387-394.