Abstract
Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t-circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction-induced foci. These data indicate that human cells possess a mechanism to negatively regulate telomere length by trimming telomeric DNA from the chromosome ends, most likely by t-loop resolution to form t-circles. Additionally, these results indicate that some phenotypic characteristics attributed to alternative lengthening of telomeres (ALT) result from increased mean telomere length, rather than from the ALT mechanism itself.
Highlights
In human cells, telomeric DNA is composed of doublestranded arrays of a (TTAGGG)n repeat unit, terminating in a single-stranded G-rich 30 overhang (Moyzis et al, 1988; de Lange et al, 1990; Makarov et al, 1997; Wright et al, Received: 19 November 2008; accepted: 23 January 2009; published online: 12 February 2009& 2009 European Molecular Biology Organization1997)
Increased telomerase activity resulted in telomere elongation and telomere length heterogeneity in telomerase-positive cells HT1080 and HeLa telomerase-positive cells were transduced with pBABEpuro containing hTR driven by the U3 promoter, or with an empty vector control
The frequency of telomere exchange events was unaltered in telomerase-positive cells with elongated telomeres Long, heterogeneous telomeres, the presence of extrachromosomal telomeric repeat (ECTR) DNA and the colocalisation of telomeric DNA with promyelocytic leukemia (PML) protein partly phenocopied the telomeres of cells that use the alternative lengthening of telomeres (ALT) mechanism, so we investigated whether the hTR-transduced HeLa and HT1080 cells had other features of ALT, including elevated telomeric exchange events, usually referred to as telomere sister chromatid exchanges (t-SCEs) (Bailey et al, 2004; Bechter et al, 2004; Londono-Vallejo et al, 2004; Muntoni and Reddel, 2005)
Summary
Telomeric DNA is composed of doublestranded (ds) arrays of a (TTAGGG)n repeat unit, terminating in a single-stranded (ss) G-rich 30 overhang (Moyzis et al, 1988; de Lange et al, 1990; Makarov et al, 1997; Wright et al, Received: 19 November 2008; accepted: 23 January 2009; published online: 12 February 2009& 2009 European Molecular Biology Organization1997). Levels of hTR and hTERT are both limiting for telomerase activity and telomere length in immortalised cells, and telomerase-positive cancer cell lines typically have a very small number of telomerase molecules and stably short telomeres (Cristofari and Lingner, 2006; Cao et al, 2008). In such situations, telomerase is preferentially recruited to the shorter telomeres (Bianchi and Shore, 2008)
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