Abstract
In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. 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 though 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.
Highlights
Alternative Lengthening of Telomeres (ALT) is a mechanism of telomere maintenance that is utilized by a portion of cancers, in tissues of mesenchymal origin
ALT cells expressing Ku70/80 shRNAs grow poorly when compared to either the same cells cultured in the absence of DOX or ALT cells expressing shRNAs for GFP treated with DOX, and the culture undergo proliferative arrest within 12 days after induction
The growth defects triggered by depletion of Ku70/80 in ALT cells resembles that caused by downregulation of the MRE11/RAD50/Nijmegen breakage syndrome 1 (NBS1) (MRN) complex using shRNAs targeting MRE11 and NBS1
Summary
ALT is a mechanism of telomere maintenance that is utilized by a portion of cancers, in tissues of mesenchymal origin. Telomeres in ALT cells are heterogeneous in length due to rapid deletions and elongations, which are thought to occur through high rates of interchromosomal recombination including a process termed telomere sister chromatid exchange (TSCE). There is no increase in rates of general homologous recombination in these cells, suggesting that the mechanism of hyper-recombination is telomere-specific. MUS81, an endonuclease implicated in the regulation of telomere recombination, does not influence t-circle formation in ALT cells [3], suggesting that the precise contribution of the recombination machinery and the possible involvement of other pathways to the formation and maintenance of t-circles in human ALT cells remain to be defined
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