Abstract
Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human cancer.
Highlights
Telomeres consist of multiple kilobases of repeated TTAGGG sequence at the ends of chromosomes and are protected by a sequence-specific protein cap [1]
Consistent with previous observations in other cell line models [24, 25, 27,28,29], MOG-G-UVW and SF188 ATRXKO clones did not display ultrabright telomeric foci (Fig 3A). These characteristic markers of alternative lengthening of telomeres (ALT) were detectable in ATRXKO clones derived from U-251 and UW479 (Fig 3B), but not in empty vector control clones when assessed one month after clonal expansion (S2 Fig)
We assessed the effect of ATRX loss on this ALT characteristic in ATRXKO clones derived from U-251 and UW479, as well
Summary
Telomeres consist of multiple kilobases of repeated TTAGGG sequence at the ends of chromosomes and are protected by a sequence-specific protein cap [1]. Due to the limitations of cellular replication machinery, in the absence of a telomere length maintenance mechanism, telomeres will shorten with each cell division. In proliferating normal somatic cells, one or more telomeres will shorten to a critical length, causing cells to undergo senescence. Cell-line specific effects of ATRX loss in glioma openhandsoverflowinghearts.org). Core facilities were funded through a National Cancer Institute (https://www.cancer.gov) Cancer Center Support grant (P30 CA006973). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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