Telomere length and expression in astrocytic brain tumors

Telomere befinden sich an den Enden der linearen Chromosomen um das Genom zu schützen und zu stabilisieren. Ein Telomere besteht aus TTAGGG-Wiederholungen von DNA Sequenzen. Erst kürzlich wurde gezeigt, dass Telomere in TERRA (Telomere repeat containing RNA) umgeschrieben werden. Diese nicht-kodierenden RNAs spielen eine Rolle für Telomere erhaltenden Mechanismen (TMM) und für die Organisation von Chromatin. Die Transkription von TERRA startet im Bereich der Subtelomere und hängt vom epigenetischen Zustand der Telomere ab. Astrozytome verwenden zwei TMM, einerseits Telomeraseaktivität (TA) und andererseits einen alternativen Mechanismus (ALT = alternative lengthening of telomeres).Der ALT-Mechanismus führt in der Regel zu längeren Telomeren als der TA-Mechanismus. Eine Korrelation zwischen TMM und dem Überleben von Patienten wurde für hochgradige Astrozytome gefunden, wie für Glioblastoma multiforme (GBM). Diese Erkenntnis und die schlechte Prognose für Patienten mit GBM führen zur Notwendigkeit der weiteren Charakterisierung von TERRA in diesen Tumoren. In dieser Studie wurden Tumor-Gewebeproben (ts) von 46 Patienten mit Diagnosen Astrozytome WHO-Grad II bis IV untersucht: 12 diffuse Astrozytome (DA) Grad II, 6 anaplastische Astrozytome (AA) Grad III und 28 GBM Grad IV. Ergänzend wurden Tumor-Zelllinien untersucht, 12 etabliert aus Astrozytome und eine ALT-Zelllinie aus einem primären Osteosarkom. RNA und DNA wurden isoliert. Um die Relative Menge (RQ) der Transkriptionslevels zu messen, wurde die RNA in DNA umgeschrieben und eine Real Time PCR durchgeführt. Expressionslevel wurden mit jenen der housekeeping Gene Beta-Aktin und 36B4 verglichen. Die Telomerlänge wurde mit TRF Analyse und Realtime PCR bestimmt. Relative Mengen wurden zwischen den untersuchten Gruppen mit dem Mann- Whitney-Test verglichen. Nach logarithmischer Transfromierung der RQ-Werte wurde eine Pearson Korrelation durchgeführt. Von 12 Tumorproben wurde der epigenetische Status der Subtelomere für das Chromosom 2p durch Bisulfit-Sequenzierung bestimmt. Die Expression aller TERRA Moleküle war um das 14 und 31-fache bei GBM niedriger verglichen mit jeweils AA-und DA (p <0,05). Zelllinien der Astrozytome zeigen TERRA Expression vergleichbar wie bei GBM, im Gegensatz dazu zeigt die ALT-Zelllinie ähnliche Expression wie bei DA. Chromosom 2p-und 18p-TERRA Expression ändert sich nicht signifikant mit dem Tumorgrad. Die durchschnittliche Telomerlänge war bei niedriggradigen Tumoren DA (22 ± 15 kbp) und AA (17 ± 5 kbp) deutlich erhöht, verglichen mit GBM (9 ± 4 kbp). Die gesamte TERRA Expression korreliert mit der Telomerlänge (Pearson-Koeffizient P = 0,38, p = 0,01). Tumor Proben zeigen Unterschiede der Durchschnittswerte der CpG-Methylierung im 2pTERRA-Promotor von 89% und 83% bei jeweils niedrigen und hohen 2p-TERRA Expressionswerten. Erste Analysen zeigen einen signifikanten Zusammenhang zwischen dem Überleben der Patienten und der gefundenen TERRA Expression. Niedrige Expression von TERRA korreliert mit einer schlechten Prognose. Die relative Expression von TERRA korrelliert in astrozytären Tumoren signifikant mit der Telomerelänge, mit dem Tumorgrad und dem Methylierungsstatus des Promotors. Die relative TERRA Expression könnte ein vielversprechender Kandidat als diagnostischer und prognostischer Marker bei astrozytären Tumoren sein.Telomeres are localized at chromosomal ends to protect and stabilize the whole genome from deterioration. A telomere consists of TTAGGG repeat DNA sequences. It has been shown recently, that telomeres are transcribed into telomeric repeat-containing RNA (TERRA) involved in telomere maintenance mechanisms (TMM) and chromatin organization. TERRA transcription originates in the subtelomere and depends on the epigenetic state of the telomere. Astrocytomas utilize two TMM based on telomerase activity (TA) and on alternative lengthening of telomeres (ALT). ALT results telomeres longer than TA mechanism. Correlation between TMM and patient survival was demonstrated for high grade astrocytomas, especially glioblastoma multiforme (GBM). This finding and dismal prognosis of patients suffering from GBM emphasizes the need for detailed characterization of telomere length and TERRA in these tumors. A panel of tumor tissue samples (ts) were analyzed which originated from 46 patients with diagnoses astrocytomas WHO grade II to IV: 12 diffuse astrocytomas (DA) grade II, 6 anaplastic astrocytomas (AA) grade III and 28 GBM grade IV. Further, human tumor cell lines were analyzed, 12 originated from astrocytomas and one ALT cell line from an osteosarcoma. RNA and DNA were isolated. Relative quantities (RQ) of transcript expression levels for overall and chromosome-specific TERRA were measured by reverse transcription following real-time PCR and compared to beta actin and 36B4 levels. Telomere length was determined by TRF Southern blotting and real-time PCR. RQs were compared between groups by Mann-Whitney test and log-transformed for Pearson correlation calculation. Epigenetic state of subtelomeric region for chromosome 2p was determined by bisulphite allelic sequencing of 12 ts. Overall TERRA expression was decreased 14 and 31-fold in GBM compared to AA and DA (p<0.05), respectively. Astrocytoma cell lines contained TERRA levels found as in GBM, the ALT cell line as in DA. Chromosome 2p- and 18p-TERRA expression was not significantly altered by grade. Mean telomere length was significantly increased in low grade tumors DA (22±15 kbp) and AA (17±5 kbp) as compared to GBM (9±4 kbp). Overall TERRA expression correlates with telomere length (Pearson’s coefficient P=0.38; p=0.01). Tumor samples with low and high 2p-TERRA expression levels demonstrate different average CpG methylation at 2p-TERRA promoter of 89% and 83%, respectively. Primary analysis of expression and survival data indicates a significant connection of overall survival and expression of TERRA. Low TERRA levels correlate with a poor prognosis. Resulting data demonstrate that TERRA levels are in astrocytomas significantly related to telomere length, tumor grade and to promoter methylation status. Expression levels of TERRA can be considered promising candidates as diagnostic and potentially prognostic markers in astrocytomas

Alternative Splicing of Fibroblast Growth Factor Receptor IgIII Loops in Cancer

Alternative splicing of the IgIII loop of fibroblast growth factor receptors (FGFRs) 1–3 produces b- and c-variants of the receptors with distinctly different biological impact based on their distinct ligand-binding spectrum. Tissue-specific expression of these splice variants regulates interactions in embryonic development, tissue maintenance and repair, and cancer. Alterations in FGFR2 splicing are involved in epithelial mesenchymal transition that produces invasive, metastatic features during tumor progression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1–3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application

The Combined Use of Known Antiviral Reverse Transcriptase Inhibitors AZT and DDI Induce Anticancer Effects at Low Concentrations

A hallmark of tumor cell survival is the maintenance of elongated telomeres. It is known that antiviral reverse transcriptase inhibitors (RTIs) such as azidothymidine (AZT) and didanosine (ddI) lead to telomere shortening at high, potentially toxic concentrations. We hypothesized that those drugs might have synergistic effects enabling successful therapy with low, nontoxic concentrations. Biologic effects of AZT and ddI were analyzed at concentrations that correspond to minimal plasma levels achieved during human immunodeficiency virus therapy. Long-term coapplication of low-dose AZT and ddI induced a significant shortening of telomeres in the tumor cell lines HCT-116, SkMel-28, MelJuso, and Jurkat. Treatment of cells with both RTI, but not with single RTI, led to a significant accumulation of γH2AX, to p53 phosphorylation, and to cell apoptosis in all cell lines. Oral low-dose dual RTI application but not low-dose single RTI application was associated with a significantly reduced tumor growth of HCT-116 cells in mice. This antiproliferative activity of the combined use of AZT and ddI at low, clinically applicable concentrations warrants clinical testing in human solid cancer

The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity