Abstract 2993: Homologous recombination between telomeres is present in ALT and telomerase-positive immortal cells

Abstract

Abstract Telomere homeostasis is a hallmark of malignancy and occurs via two different mechanisms. The first mechanism is telomere length control via telomerase activity, the second mechanism is characterized by the lack of any detectable telomerase activity and is known as the alternative lengthening of telomeres (ALT) pathway. There is good evidence that homologous recombination between telomeric DNA is responsible for telomere elongation in these cells and therefore enables infinite growth. Telomeres resemble fragile DNA sites, and their repetitive sequence poses a challenge for the DNA replication machinery. Replication fork stallments have been shown to occur in mammalian telomeres, giving rise to DNA lesions and genetic instability. DNA replication can resume via a homologous recombination process, which could potentially cause telomere elongation. Although telomerase-positive human cells are believed to not recombine their telomeres, we were challenged by the question whether telomeric recombination is a rare event that is missed with an insensitive detection technique. In order to address this issue we designed a novel, sensitive and specific recombination reporter system that demonstrates recombination events that have become detectable by gaining antibiotic resistance. For this purpose cells were first tagged with a subtelomeric and subsequently with a telomeric reporter cassette, after which individual clones were obtained. Once telomeres recombined, both cassettes co-localize on the same telomere. Alternative splicing of intervening sequences between two portions of a resistance gene results in antibiotic resistance, hence allowing the specific and sensitive detection of a telomeric recombination event occurring upstream of the telomeric tag. With this reporter system we show that telomeric recombination is present in ALT and telomerase-positive human fibroblasts. The frequency of recombination does not differ between these two cell lines. DNA damage caused by UV irradiation, change in heterochromatin formation from HDAC inhibition or by demethylating DNA with azacitidine did not change the frequency of homologous recombination in either ALT or telomerase-positive clones. In contrast, treatment of cells with aphidicolin, which renders telomeres fragile, significantly increased the frequency of telomeric recombination in ALT, but not in telomerase-positive cells. The introduction of telomerase into these ALT cell clones abolished the effect of the aphidicolin-induced homologous recombination. Our data show that in telomerase-positive cells and ALT cells homologous recombination between telomeres occurs with nearly the same frequency, but that the increase in telomeric recombination via fragile telomeres is dependent on the absence of telomerase activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2993. doi:10.1158/1538-7445.AM2011-2993