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

<div><p>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.</p> </div

Detection of ALT activity by C-circle assay.

<p>A. Dot blot of C-circle assay products for each parental and hybrid line. Serial dilutions of genomic DNA from GM847 cells indicate linearity of the assay within this range. The presence of C-circles indicates the cell line utilizes the ALT mechanism. B. Quantitation of C-circle assay levels by densitometry. The results from two separate experiments are presented relative to the signal obtained by GM847 cells. Bars, average; error bars, range.</p

Growth curves of hybrid cell lines.

<p>GM847 cells were fused with J82, TE-85 or A549 telomerase-positive cell lines using polyethylene glycol. Proliferation of each hybrid clone was plotted as a function of population doubling and the number of days following fusion. Growth of MeT-5A/GM847 hybrid lines has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone.0050062-Duncan1" target="_blank">[21]</a>.</p

Correlation between ALT activity and lack of normal ATRX expression.

a<p>6/7 ALT hybrids underwent a period of growth arrest compared to 3/12 TEL hybrids (p = 0.0198, Fisher’s exact test);</p>b<p>TRAP assay: “+” indicates detectable telomerase activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g002" target="_blank">Figure 2</a>);</p>c<p>TRF: “+” indicates a terminal restriction fragment length pattern characteristic of ALT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g003" target="_blank">Figure 3</a>);</p>d<p>CC assay: “+” indicates C-circle levels above the cut-off level for ALT activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g004" target="_blank">Figure 4</a>);</p>e<p>TLM: telomere lengthening mechanism deduced from TRAP, TRF and CC assay data; ALT and TEL indicate ALT-positive and telomerase-positive, respectively;</p>f<p>ATRX protein: “+:” indicates normal pattern of immunofluorescence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g005" target="_blank">Figure 5</a>) and normal levels on Western (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g006" target="_blank">Figure 6</a>);</p>g<p>ATRX SNP: a single nucleotide polymorphism (rs3088074, c.3017C>G, p.Q929E) for which each of the parental lines is homozygous identified the parental origin(s) of the ATRX alleles in all hybrids except the TE-85/GM847 lines for which the SNP was not determined (nd), because TE-85 and GM847 both have G at this location;</p>h<p>The magnitude of the C peak was significantly smaller than that of the G peak;</p>i<p>ATRX exon 1: “+” indicates the presence of wild-type ATRX exon 1 as determined by PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g007" target="_blank">Figure 7</a>).</p

Agarose gel electrophoresis of ATRX exon 1 PCR products.

<p>ATRX exon 1 of each of the indicated samples was amplified by PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#s2" target="_blank">Materials and Methods</a>. The PCR products (expected size 418bp) were subjected by agarose gel electrophoresis and verified by Sanger sequencing. GM02063 is the parent cell strain from which GM847 cells were derived by SV40-induced immortalization.</p