Inactivation of 14-3-3σ Influences Telomere Behavior and Ionizing Radiation-Induced Chromosomal Instability

Abstract

Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G₂ and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3ς gene has been reported to be a checkpoint control gene, since it promotes G₂ arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3ς^−/− cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G₁ checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G₂ after irradiation with gamma rays, corroborating the role of the 14-3-3ς protein in G₂/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G₂ checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3ς^−/− cells are defective in maintaining G₂ arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G₂/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.

ACKNOWLEDGMENTS

This investigation was supported by NS34746 (T.K.P.). R.J.W. is a chercheur boursier sénior of the Fonds de la Recherche en Santé du Québec (FRSQ) and acknowledges support by the Canadian NCI (grant #010049).

Thanks are due to Bert Vogelstein for providing reagents and Raj Pandita for her advice and help.