Loss of Wild-Type ATRX Expression in Somatic Cell Hybrids Segregates with Activation of Alternative Lengthening of Telomeres

Kylie Bower,Sara L Cole,Roger R Reddel,Elsa L Moy,Loretta M S Lau,Christine E Napier,Rebecca A Dagg,Emma L Duncan,Shawn Ahmed

doi:10.1371/journal.pone.0050062

Abstract

Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT.

Highlights

Telomeres contain repetitive DNA, (TTAGGG)n, to which specific proteins bind and act in concert to maintain chromosome integrity [1]
We previously showed that fusing MeT-5A with GM847 resulted in 18 of 18 hybrid lines ceasing proliferation after 20– 45 population doublings (PD), and that 13 eventually recommenced proliferation after 50–100 days (Figure 1; Table S1) [21]
We found that all hybrid lines which lacked ATRX expression were homozygous for the G Single Nucleotide Polymorphism (SNP), meaning that the ATRX allele from the telomerase-positive parental cell line was lost from the hybrid, whereas the ATRX allele from the ALTpositive parent was retained

Summary

Introduction

Telomeres contain repetitive DNA, (TTAGGG)n, to which specific proteins bind and act in concert to maintain chromosome integrity [1]. In normal human somatic cells, telomeres undergo progressive shortening with cell division [2], which accounts for the observation that these cells undergo a limited number of cell division cycles before permanently ceasing proliferation and becoming senescent [3]. Immortalized human cells avoid senescence via the activity of a telomere lengthening mechanism (TLM) that counteracts the normal process of telomere attrition [4]: via either the reverse transcriptase enzyme, telomerase [5] or the alternative lengthening of telomeres (ALT) mechanism [6]. ALT occurs in a minority of cancers overall, and in more than 50% of leiomyosarcomas, osteosarcomas, astrocytic tumors grades 2 and 3, and undifferentiated pleomorphic sarcomas [7,8,9]. An understanding of how this TLM is repressed in normal cells and activated in cancers may provide possibilities for new forms of treatment

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