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DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity


Alternative lengthening of telomeres (ALT)1 is likely to be an important target for anticancer treatment as 10% of cancers depend on this telomere maintenance mechanism for continued growth2, and inhibition of ALT can cause cellular senescence3. However, no ALT inhibitors have been developed for therapeutic use because of the lack of a suitable ALT activity assay and of known ALT-specific target molecules. Here we show that partially single-stranded telomeric (CCCTAA)n DNA circles (C-circles) are ALT specific. We provide an assay that is rapidly and linearly responsive to ALT activity and that is suitable for screening for ALT inhibitors. We detect C-circles in blood from ALT+ osteosarcoma patients, suggesting that the C-circle assay (CC assay) may have clinical utility for diagnosis and management of ALT+ tumors.

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Figure 1: Detection of C-circles in ALT+ cells by C-circle (CC) assay.
Figure 2: CC assay is specific for ALT.
Figure 3: CC assay is rapidly responsive to ALT activity changes.
Figure 4: C-circles are increased in blood from ALT+ osteosarcoma patients.


  1. Bryan, T.M., Englezou, A., Gupta, J., Bacchetti, S. & Reddel, R.R. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J. 14, 4240–4248 (1995).

    Article  CAS  Google Scholar 

  2. Henson, J.D. et al. A robust assay for alternative lengthening of telomeres (ALT) in tumors demonstrates the significance of ALT in sarcomas and astrocytomas. Clin. Cancer Res. 11, 217–225 (2005).

    CAS  PubMed  Google Scholar 

  3. Perrem, K. et al. Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids. Oncogene 18, 3383–3390 (1999).

    Article  CAS  Google Scholar 

  4. McEachern, M.J., Krauskopf, A. & Blackburn, E.H. Telomeres and their control. Annu. Rev. Genet. 34, 331–358 (2000).

    Article  CAS  Google Scholar 

  5. Dunham, M.A., Neumann, A.A., Fasching, C.L. & Reddel, R.R. Telomere maintenance by recombination in human cells. Nat. Genet. 26, 447–450 (2000).

    Article  CAS  Google Scholar 

  6. Jiang, W.Q. et al. Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of MRE11/RAD50/NBS1 complex. Mol. Cell. Biol. 25, 2708–2721 (2005).

    Article  CAS  Google Scholar 

  7. Potts, P.R. & Yu, H. The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins. Nat. Struct. Mol. Biol. 14, 581–590 (2007).

    Article  CAS  Google Scholar 

  8. Murnane, J.P., Sabatier, L., Marder, B.A. & Morgan, W.F. Telomere dynamics in an immortal human cell line. EMBO J. 13, 4953–4962 (1994).

    Article  CAS  Google Scholar 

  9. Perrem, K., Colgin, L.M., Neumann, A.A., Yeager, T.R. & Reddel, R.R. Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells. Mol. Cell. Biol. 21, 3862–3875 (2001).

    Article  CAS  Google Scholar 

  10. Yeager, T.R. et al. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res. 59, 4175–4179 (1999).

    CAS  Google Scholar 

  11. Henson, J.D. . The Role of Alternative Lengthening of Telomeres in Human Cancer. PhD thesis. Univ. Sydney (2006).

    Google Scholar 

  12. Grudic, A. et al. Replication protein A prevents accumulation of single-stranded telomeric DNA in cells that use alternative lengthening of telomeres. Nucleic Acids Res. 35, 7267–7278 (2007).

    Article  CAS  Google Scholar 

  13. Nabetani, A. & Ishikawa, F. Unusual telomeric DNAs in human telomerase-negative immortalized cells. Mol. Cell. Biol. 29, 703–713 (2009).

    Article  CAS  Google Scholar 

  14. Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Article  CAS  Google Scholar 

  15. Zellinger, B., Akimcheva, S., Puizina, J., Schirato, M. & Riha, K. Ku suppresses formation of telomeric circles and alternative telomere lengthening in Arabidopsis. Mol. Cell 27, 163–169 (2007).

    Article  CAS  Google Scholar 

  16. Zhao, Y., Hoshiyama, H., Shay, J.W. & Wright, W.E. Quantitative telomeric overhang determination using a double-strand specific nuclease. Nucleic Acids Res. 36, e14 (2008).

    Article  Google Scholar 

  17. Fasching, C.L., Bower, K. & Reddel, R.R. Telomerase-independent telomere length maintenance in the absence of ALT-associated PML bodies. Cancer Res. 65, 2722–2729 (2005).

    Article  CAS  Google Scholar 

  18. Varley, H., Pickett, H.A., Foxon, J.L., Reddel, R.R. & Royle, N.J. Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells. Nat. Genet. 30, 301–305 (2002).

    Article  Google Scholar 

  19. Cerone, M.A., Autexier, C., Londono-Vallejo, J.A. & Bacchetti, S. A human cell line that maintains telomeres in the absence of telomerase and of key markers of ALT. Oncogene 24, 7893–7901 (2005).

    Article  CAS  Google Scholar 

  20. Cesare, A.J. & Griffith, J.D. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Mol. Cell. Biol. 24, 9948–9957 (2004).

    Article  CAS  Google Scholar 

  21. Wang, R.C., Smogorzewska, A. & de Lange, T. Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355–368 (2004).

    Article  CAS  Google Scholar 

  22. Pickett, H.A., Cesare, A.J., Johnstone, R.L., Neumann, A.A. & Reddel, R.R. Control of telomere length by a trimming mechanism that involves generation of t-circles. EMBO J. 28, 799–809 (2009).

    Article  CAS  Google Scholar 

  23. Ziegler, A., Zangemeister-Wittke, U. & Stahel, R.A. Circulating DNA: a new diagnostic gold mine? Cancer Treat. Rev. 28, 255–271 (2002).

    Article  CAS  Google Scholar 

  24. Tsang, J.C. & Lo, Y.M. Circulating nucleic acids in plasma/serum. Pathology 39, 197–207 (2007).

    Article  CAS  Google Scholar 

  25. Bryan, T.M., Englezou, A., Dalla-Pozza, L., Dunham, M.A. & Reddel, R.R. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat. Med. 3, 1271–1274 (1997).

    Article  CAS  Google Scholar 

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We thank Tumour Bank (Children's Hospital Westmead, Australia) for patient blood specimens, kConFab (Australia) for LFS-05 skin sample, P.R. Potts (University of Texas Southwestern) for MMS21 antibody, M.A. Cerone (Breakthrough Breast Cancer Research Centre Institute of Cancer Research) for C3-c16 cells, P. Crowe and R. Das Gupta (Prince of Wales Hospital) for DOS16 cells, W.-Q. Jiang and K. Perrem (Children's Medical Research Institute) for IIICF/c-10, 11, 16 and 17 cells and GM847/hTERT-3 and GM847/hTERT-6 cells, respectively, D. Spector (Cold Spring Harbor Laboratory) for EYFP-SP100 construct and L. Naldini (San Raffaele Telethon Institute for Gene Therapy) for pMD2-VSVG envelope vector, and A. Muntoni for critical review of this manuscript.

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J.D.H. conceived and developed the assay and wrote the manuscript. L.I.H. established the LFS-05F-24 cell line. Experiments were performed by J.D.H., Y.C., L.I.H., A.C.C., A.Y.M.A. and H.A.P. R.R.R. was responsible for all aspects of the project and edited the manuscript.

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Correspondence to Roger R Reddel.

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Henson, J., Cao, Y., Huschtscha, L. et al. DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity. Nat Biotechnol 27, 1181–1185 (2009).

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