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Detection of telomeric DNA circles in human ALT cells using rolling circle amplification Tkac, Jan


Telomeres, the nucleoprotein structures at the ends of linear chromosomes, maintain genomic stability by protecting chromosome ends from fusion, degradation, and processing by the DNA double-strand break repair machinery. Telomere shortening, which occurs naturally in somatic cells during aging, leads to cellular senescence or apoptosis. In contrast, germline cells and cancer cells acquire unlimited replicative potential by activating a telomere lengthening mechanism, generally via reactivating the enzyme telomerase reverse transcriptase. To date, drug development targeting cellular immortalization in cancer has focused on telomerase inhibition. However, in a subset of tumours and in vitro-immortalized cell lines, telomeres are maintained by homologous recombination-mediated pathways, termed alternative lengthening of telomeres (ALT). ALT tumours are expected to be refractory to anti-telomerase therapies, so the ability to rapidly and reliably screen for ALT status in tumour-derived cells is essential for guiding therapeutic strategies that target cellular immortalization. One characteristic of ALT-mediated telomere maintenance is the presence of extrachromosomal telomeric repeat-containing DNA circles (t-circles), which provide an attractive target for detection in screening applications. Current methods oft-circle detection require considerable amounts of cells, making them unsuitable for analysis of limited clinical samples. We optimized a screen for ALT status based on a novel technique of rolling circle amplification (RCA) oft-circles from extrachromosomal DNA extracts of human ALT cells. We demonstrate that RCA requires a much lower number of cells than previously established t-circle detection methods, and screening many samples can be performed in parallel, making RCA suitable for analyzing clinical samples. T-circles were reproducibly detected in human immortalized ALT cell lines, but not in telomerase-utilizing cell lines. In addition, ectopic over-expression of telomerase in an ALT cell line does not appear to affect t-circle formation. This suggests that presence of active telomerase within a cell does not inhibit all telomeric recombination reactions. The potential for RCA as a tool to screen tumour samples for ALT activity and the link between telomerase and ALT-based telomere lengthening mechanisms are discussed.

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