- Letter to the Editor
- Open Access
Identification of patient-derived glioblastoma stem cell (GSC) lines with the alternative lengthening of telomeres phenotype
© The Author(s). 2019
- Received: 13 March 2019
- Accepted: 3 May 2019
- Published: 16 May 2019
Glioblastoma multiforme (GBM) is an aggressive brain tumor with a poor overall prognosis. Current standard of care involves surgical resection followed by adjuvant treatment with radiation (RT), temozolomide, and tumor treating fields (TTF) . Despite this aggressive treatment modality, median overall survival is approximately 15 months. Telomeres are terminal DNA elements found at eukaryotic chromosomal ends consisting of hexagonal repeats of (TTAGGG)n which are essential for maintaining genomic stability . To maintain telomere length and circumvent the end-replication problem, most cancer cells express telomerase . Telomerase is composed of two subunits: a catalytic component with reverse-transcriptase activity encoded by the gene TERT, and an 11 base-pair RNA template encoded by the gene TERC . Mutations in the promoter region for TERT occur in approximately 60–80% of GBM, leading to increased telomerase activity and enabling replicative immortality . A defining feature of anaplastic astrocytomas and a small fraction of secondary GBM, is activation of a telomerase-independent alternative lengthening of telomeres (ALT) mechanism, driven by homologous recombination (HR) machinery . ALT tumors can readily be detected by assaying for the presence of extrachromosomal telomeric DNA C-Circles (CCs) via qPCR or ALT-associated telomere foci by FISH on pathological specimens . ALT+ high grade glioma (HGG) are enriched in tumors with loss of function mutations in ATRX (alpha-thalassemia/mental retardation X-linked) and less commonly, SMARCAL1. When these chromatin remodeling genes are inactivated, the resultant replication stress and aberrant HR at telomeres is hypothesized to lead to ALT . Mutations in both ATRX and SMARCAL1 are mutually exclusive with TERT promoter mutations suggesting functional redundancy between these two mechanistic pathways [3, 4].
Here, we sought to identify and characterize ALT+ GBM by screening through a panel of 24 patient-derived GBM stem cell lines (GSCs). We tested for ALT using a novel qPCR method that measures both telomere content (TC), which is indicative of overall telomere length, and DNA C-Circles (CCs), which are specific and quantifiable markers for ALT activity . Telomerase expression was assessed by quantifying mRNA levels of TERT using whole transcriptome sequencing. ATRX protein expression was measured by immunoblotting.
To date, only 2 ALT+ glioma cell lines have been documented (TG-20 and JHH-GBM14) [5, 12], however in these prior studies ALT was assayed for by immunofluorescent detection of telomere/PML body foci and lack of telomerase activity via the telomerase repeat amplification protocol (TRAP) assay. We report here that detection of DNA CCs via qPCR and mRNA quantification of TERT are also usable biomarkers that can reliably detect ALT and may be more applicable in a clinical setting as both assays require minute amounts of DNA and RNA. In conclusion, identification of these ALT+ GSCs will enable future explorations of the mechanisms and biology of the ALT phenotype, and will serve as pre-clinical models to test novel chemotherapeutic agents in an effort to improve outcomes in a subset of high-grade gliomas and secondary GBM.
This study was supported in part by the Radiological Society of North America (RSNA) Prince Research Resident Grant (2018–2019).
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
AF conceived the experiments and took lead in writing the manuscript. JY, VS, RE, CD, CA, SD, DI all aided in the experimental design and analysis of data. JH and EPS developed the theoretical framework, supervised the project and experiments, and helped revise the manuscript.All authors read and approved the final manuscript.
Ethics approval and consent to participate
Yes, reviewed by the IRB. Acquisition of these human cell lines was covered under an institutional protocol “LAB04–0001,” with full informed consent obtained from each person.
Consent for publication
The authors declare that they have no competing interests.
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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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