Background & Significance
Telomeres are repetitive regions of DNA that functionally cap the ends of chromosomes. In normal somatic cells, telomeric DNA is eroded with each cell division, while in cancer cells, activation of a telomere maintenance mechanism is a prerequisite for replicative immortality. 10-15% of cancers utilise a homology-directed repair mechanism known as Alternative Lengthening of Telomeres (ALT) to maintain their telomeres. Cancers engaging in ALT are typically aggressive with poor prognosis. While many aspects of the ALT mechanism have been characterised, the cell cycle regulation of telomere synthesis and extension remains to be elucidated.
Methods & Results
We have used cell cycle synchronisation, followed by rolling circle DNA amplification, microscopy and DNA fibre analysis, to investigate the intricacies of telomere extension during G2 and M phases of the cell cycle in ALT cancer cells. We have identified, for the first time, two modes of telomere synthesis that are specific to ALT cells. First, non-productive telomere synthesis, which is defined as the generation of newly synthesised extrachromosomal telomeric repeat (ECTR) DNA without telomere lengthening, and second, productive telomere synthesis, which is defined as nascent telomere extension and telomere length maintenance.
We demonstrate that ALT cells arrested in G2-phase synthesise substantial amounts of telomeric DNA, but rather than the telomeres being extended, newly synthesised telomeric DNA takes the form of ECTRs that are then released into the cytoplasm during M-phase, coinciding with the inability of the cell to retain G2 nascent telomere synthesis in the subsequent G1-phase. In contrast, productive telomere synthesis occurs specifically during prometaphase.
Conclusions
Contrary to previous reports, we demonstrate that productive telomere synthesis occurs in prometaphase. This has implications for the therapeutic targeting of ALT cancers via cell-cycle disruption and telomere-targeted therapies. We are currently investigating the mechanisms that drive early mitotic telomere extension.