Depletion of Ku70/80 reduces the levels of extrachromosomal telomeric circles and inhibits proliferation of ALT cells

Abstract

In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. t In contrast, cancer cells counteract the loss of telomeric DNA either by inducing the expression of telomerase or by activating the Alternative Lengthening of Telomeres (ALT) pathway. ALT cells are characterized by heterogeneous telomeres and the presence of extrachromosomal circular double-stranded DNA molecules containing telomeric repeat sequences. These telomeric circles (t-circles) are thought to be generated through a recombination process and utilized as templates for telomere elongation by rolling-circle-replication, although their precise mechanism of formation and role in telomere maintenance and cell proliferation is largely unknown. Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length. These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.

Geroconversion: irreversible step to cellular senescence

“…Noteworthy, there is a correlation with longevity in vivo [77][78][79] Replicative senescence of human cells is a variant of stress-induced senescence (Fig. 4D).…”

Alternative mechanisms of telomere lengthening: Permissive mutations, DNA repair proteins and tumorigenic progression

“…RPA knockdown induces growth arrest and accumulation of single-stranded telomeric DNA in U-2 OS and GM847 ALT cells [66], suggesting functions in telomere end processing. Knockdown of the Ku70/80 heterodimer, which binds to broken DNA ends to facilitate non-homologous end joining, impairs the growth of CCL75.1 ALT cells and reduces ECTRs, although telomere shortening does not occur [67]. Likewise, XRCC3, a component of the homologous recombination machinery that resolves Holliday junctions, is required for the maintenance of ECTRs in GM847 and WI-38 VA-13/2RA ALT cells [59], as its knock-down reduces ECTRs in these cell lines.…”

Inter-telomeric recombination is present in telomerase-positive human cells

“…52 In ALT cells telomere length is unchanged when Ku70/80 is depleted, but the levels of t-circles are drastically reduced, which is associated with a robust growth inhibition. 53 Treatment strategies which tackle one target and block both telomere length control mechanisms are clearly advantageous over a combined strategy where each individual pathway is targeted simultaneously. The increasing complexity of connected and redundant pathways of telomere Incubation of human cells with aphidicolin induced a fragile telomere phenotype in human fibroblasts expressing hTERT and Hela 1.3 cells.…”