Telomere length modulation in human astroglial brain tumors - PubMed (original) (raw)

Telomere length modulation in human astroglial brain tumors

Domenico La Torre et al. PLoS One. 2013.

Abstract

Background: Telomeres alteration during carcinogenesis and tumor progression has been described in several cancer types. Telomeres length is stabilized by telomerase (h-TERT) and controlled by several proteins that protect telomere integrity, such as the Telomere Repeat-binding Factor (TRF) 1 and 2 and the tankyrase-poli-ADP-ribose polymerase (TANKs-PARP) complex.

Objective: To investigate telomere dysfunction in astroglial brain tumors we analyzed telomeres length, telomerase activity and the expression of a panel of genes controlling the length and structure of telomeres in tissue samples obtained in vivo from astroglial brain tumors with different grade of malignancy.

Materials and methods: Eight Low Grade Astrocytomas (LGA), 11 Anaplastic Astrocytomas (AA) and 11 Glioblastoma Multiforme (GBM) samples were analyzed. Three samples of normal brain tissue (NBT) were used as controls. Telomeres length was assessed through Southern Blotting. Telomerase activity was evaluated by a telomere repeat amplification protocol (TRAP) assay. The expression levels of TRF1, TRF2, h-TERT and TANKs-PARP complex were determined through Immunoblotting and RT-PCR.

Results: LGA were featured by an up-regulation of TRF1 and 2 and by shorter telomeres. Conversely, AA and GBM were featured by a down-regulation of TRF1 and 2 and an up-regulation of both telomerase and TANKs-PARP complex.

Conclusions: In human astroglial brain tumours, up-regulation of TRF1 and TRF2 occurs in the early stages of carcinogenesis determining telomeres shortening and genomic instability. In a later stage, up-regulation of PARP-TANKs and telomerase activation may occur together with an ADP-ribosylation of TRF1, causing a reduced ability to bind telomeric DNA, telomeres elongation and tumor malignant progression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Terminal Restriction Fragment Analysis.

Representative autoradiogram of southern blot analysis. Telomere length was reduced in astroglial tumors as compared with NBT, but elongated telomere was frequently found in GBMs as compared with both AAs and LGGs (p<.001). A Bar Graph. Shows telomere length (x axis) in different grade of astrocytomas and controls (y axis). Error bars indicate standard deviation. ** indicates significance of p<.001. Abbreviations: Kbs: kilobase pair; NBT: normal brain tumor; LGA: Low grade Astrocytoma; AA: Anaplastic Astrocytoma; GBM: Glioblastoma Multiforme; LMW: Low molecular weight; HMW: High molecular weight; MW: Molecular Weight.

Figure 2

Figure 2. Telomerase activity and h-TERT mRNA expression.

Telomerase activity significantly correlates with h-TERT mRNA expression and WHO in astroglial brain tumors. The h-TERT expression levels in GBMs resulted statistically higher as compared with those in LGAs. The expressions in AAs differed significantly as compared with that in LGAs. A correlation was found between h-TERT and WHO grading (p<.001). Telomerase activity is expressed as relative telomerase activity (RTA) according to the formula showed in the methods. Error bars indicate standard deviation. * p<.05; ** p<.001. Abbreviations: NBT: normal brain tumor; LGA: Low grade Astrocytoma; AA: Anaplastic Astrocytoma; GBM: Glioblastoma Multiforme; h-TERT human telomerase reverse transcriptase.

Figure 3

Figure 3. Expression levels of Telomere-associated proteins among tumors with different grade of malignancy.

Quantitative mRNA expression levels of TRF1, TRF2, TANKs, and PARP-1 was determined by real-time RT-PCR analysis. Relative quantification for these genes was expressed as fold variation over control, and calculated by the ΔΔCt method, using control samples (actin) as calibrators. A TRF1 expression levels progressively decrease from LGAs to GBMs. The expression in LGAs was significantly higher than that in both GBMs (P = .007) and AAs (P = .009). B TRF2 expression levels in LGAs resulted statistically higher as compared with those in GBMs (P = .008), and showed a tendency toward a lower expression in AAs as compared with LGAs C Increased expression of TANKs resulted in AAs compared with LGAs and GBMs. D PARP1 mRNA expression levels resulted statistically higher in AAs as compared with those in LGAs. Moreover, PARP expression in GBMs showed a tendency toward the higher expression as compared with LGAs. Abbreviations: LGA: Low grade Astrocytoma; AA: Anaplastic Astrocytoma; GBM: Glioblastoma Multiforme; TRF1, Telomeric repeat-binding factors 1; TRF2, Telomeric repeat-binding factors 2; PARP1, Poly (ADP-ribose) polymerase 1.

Figure 4

Figure 4. Western blot analysis of TRF1, TRF2, h-TERT, TANKs, and PARP-1.

Left: representative autoradiogram; right: graphs with quantitative data. Both TRF1 and TRF2 are over expressed in LGAs as compared with AAs and GBMs. h-TERT, TANKs, and PARP1 showed a tendency toward a higher expression in malignant astrocytomas (i.e. AAs and GBMs). Abbreviations: NBT: Normal Brain Tissue; LGA: Low grade Astrocytoma; AA: Anaplastic Astrocytoma; GBM: Glioblastoma Multiforme; TRF1, Telomeric repeat-binding factors 1; TRF2, Telomeric repeat-binding factors 2; h-TERT human telomerase reverse transcriptase; PARP1, Poly (ADP-ribose) polymerase 1.

Figure 5

Figure 5. Biomolecular profiles of expression for each tumor histotype.

Relative quantification of studied genes showed that expression levels of those genes are similar along with different histological grades. In LGA, an up-regulation of TRF1 occurs when telomerase has not yet been activated or is down regulated by TRF1. In AA, an up regulation of PARP-Tankirase complex and telomerase activation occurs. The ADP-ribosylation of TRFs, mediated by PARP1, diminish its ability to bind telomeric DNA, causing TRF1 down-regulation and allowing telomerase to elongate progressively telomeres. Finally, a down-regulation of TRF1 and TRF2, increasing telomerase activity, persistent over-expression of PARP-TANKs and elongated telomere are typical features in GBM. Black arrows indicates telomere length along with different stages of carcinogenesis and tumor progression in astroglial brain tumors. Abbreviations: LGA: Low grade Astrocytoma; AA: Anaplastic Astrocytoma; GBM: Glioblastoma Multiforme; TRF1, Telomeric repeat-binding factors 1; TRF2, Telomeric repeat-binding factors 2; h-TERT human telomerase reverse transcriptase; PARP1, Poly (ADP-ribose) polymerase 1.

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This Paper was supported in part by Italian Ministry of University and Research; Grant number: prot. 2008979M8K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.

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