Different expression of alternative lengthening of telomere (ALT)-associated proteins/mRNAs in osteosarcoma cell lines

Zhang,Yi; Cai,Lin; Wei,Ren-Xiong; Hu,Hao; Jin,Wei; Zhu,Xiao-Bin

doi:10.3892/ol.2011.403

Different expression of alternative lengthening of telomere (ALT)-associated proteins/mRNAs in osteosarcoma cell lines

Authors:
- Yi Zhang
- Lin Cai
- Ren-Xiong Wei
- Hao Hu
- Wei Jin
- Xiao-Bin Zhu
View Affiliations

Affiliations: Department of Orthopedics, Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, P.R. China
Published online on: September 2, 2011 https://doi.org/10.3892/ol.2011.403
Pages: 1327-1332

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Tumors, including osteosarcoma (OS), are capable of evading senescence and cell death, which is caused by telomere loss with cell division. Alternative lengthening of telomeres (ALT) is considered as the main telomere maintenance mechanism in OS. In this study, we investigated the expression of ALT-associated proteins and mRNAs in human OS cell lines. Western blotting was used to detect the protein expression in OS cell lines, while the expression of mRNA was determined by reverse‑transcriptase PCR and quantitative real-time PCR analysis. Whole-genome expression arrays were used to analyze the expression of all the mRNAs involved in telomere maintenance mechanisms (TMMs) including human telomerase reverse transcriptase, promyelocytic leukemia proteins and other related proteins. OS and normal cell lines do not express telomerase reverse transcriptase (hTERT) as a key subunit of telomerase, although they show varying levels of ALT-associated proteins and mRNAs such as PML, Rad52, MRE11 and FEN1 by Western blotting and quantitative real-time PCR analysis. A number of mRNAs that play essential roles in ALT are expressed more in OS cell lines than in the osteoblast cell line, as shown by whole-genome expression arrays. In conclusion, OS cell lines maintain their telomere length primarily through the ALT mechanism. There are numerous other proteins that regulate this process in OS; therefore, anti‑ALT therapy may be a more effective method to treat OS than anti-telomerase therapy.

View Figures

View References

1	Arndt CA and Crist WM: Common musculoskeletal tumors of childhood and adolescence. N Engl J Med. 341:342–352. 1999. View Article : Google Scholar : PubMed/NCBI
2	Endo-Munoz L, Cumming A, Sommerville S, Dickinson I and Saunders NA: Osteosarcoma is characterised by reduced expression of markers of osteoclastogenesis and antigen presentation compared with normal bone. Br J Cancer. 103:73–81. 2010. View Article : Google Scholar : PubMed/NCBI
3	Tabori U and Dome JS: Telomere biology of pediatric cancer. Cancer Invest. 25:197–208. 2007. View Article : Google Scholar
4	Brachner A, Sasgary S, Pirker C, et al: Telomerase- and alternative telomere lengthening-independent telomere stabilization in a metastasis-derived human non-small cell lung cancer cell line: effect of ectopic hTERT. Cancer Res. 66:3584–3592. 2006. View Article : Google Scholar
5	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
6	Smith CC, Aylott MC, Fisher KJ, Lynch AM and Gooderham NJ: DNA damage responses after exposure to DNA-based products. J Gene Med. 8:175–85. 2006. View Article : Google Scholar : PubMed/NCBI
7	Smogorzewska A, Karlseder J, Holtgreve-Grez H, Jauch A and de Lange T: DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2. Curr Biol. 12:1635–1644. 2002. View Article : Google Scholar : PubMed/NCBI
8	D’Adda di Fagagna F, Reaper PM, Clay-Farrace L, et al: A DNA damage checkpoint response in telomere-initiated senescence. Nature. 426:194–198. 2003.PubMed/NCBI
9	Bakkenist CJ, Drissi R, Wu J, Kastan MB and Dome JS: Disappearance of the telomere dysfunction-induced stress response in fully senescent cells. Cancer Res. 64:3748–3752. 2004. View Article : Google Scholar : PubMed/NCBI
10	Shay JW and Wright WE: Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol. 1:72–76. 2000. View Article : Google Scholar : PubMed/NCBI
11	Makarov VL, Hirose Y and Langmore JP: Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell. 88:657–666. 1997. View Article : Google Scholar : PubMed/NCBI
12	Palm W and de Lange T: How shelterin protects mammalian telomeres. Annu Rev Genet. 42:301–334. 2008. View Article : Google Scholar : PubMed/NCBI
13	Artandi SE and DePinho RA: Telomeres and telomerase in cancer. Carcinogenesis. 31:9–18. 2010. View Article : Google Scholar
14	Weinrich SL, Pruzan R, Ma L, et al: Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet. 17:498–502. 1997. View Article : Google Scholar : PubMed/NCBI
15	Henson JD, Neumann AA, Yeager TR and Reddel RR: Alternative lengthening of telomeres in mammalian cells. Oncogene. 21:598–610. 2002. View Article : Google Scholar : PubMed/NCBI
16	Xu ZX, Zou WX, Lin P and Chang KS: A role for PML3 in centrosome duplication and genome stability. Mol Cell. 17:721–732. 2005. View Article : Google Scholar : PubMed/NCBI
17	Nakayama J, Tahara H, Tahara E, et al: Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet. 18:65–68. 1998. View Article : Google Scholar : PubMed/NCBI
18	Blasco MA, Funk W, Villeponteau B and Greider CW: Functional characterization and developmental regulation of mouse telomerase RNA. Science. 269:1267–1270. 1995. View Article : Google Scholar : PubMed/NCBI
19	Avilion AA, Piatyszek MA, Gupta J, Shay JW, Bacchetti S and Greider CW: Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res. 56:645–650. 1996.PubMed/NCBI
20	Beattie TL, Zhou W, Robinson MO and Harrington L: Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding. Mol Biol Cell. 11:3329–3340. 2000. View Article : Google Scholar : PubMed/NCBI
21	Kickhoefer VA, Stephen AG, Harrington L, Robinson MO and Rome LH: Vaults and telomerase share a common subunit, TEP1. J Biol Chem. 274:32712–32717. 1999. View Article : Google Scholar : PubMed/NCBI
22	Kickhoefer VA, Liu Y, Kong LB, et al: The telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle. J Cell Biol. 152:157–164. 2001. View Article : Google Scholar : PubMed/NCBI
23	Liu Y, Snow BE, Hande MP, et al: Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo. Mol Cell Biol. 20:8178–8184. 2000. View Article : Google Scholar : PubMed/NCBI
24	Ulaner GA, Huang HY, Otero J, et al: Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma. Cancer Res. 63:1759–1763. 2003.PubMed/NCBI
25	Terasaki T, Kyo S and Takakura M: Analysis of telomerase activity and telomere length in bone and soft tissue tumors. Oncol Rep. 11:1307–1311. 2004.PubMed/NCBI
26	Fujiwara-Akita H, Maesawa C, Honda T, Kobayashi S and Masuda T: Expression of human telomerase reverse transcriptase splice variants is well correlated with low telomerase activity in osteosarcoma cell lines. Int J Oncol. 26:1009–1016. 2005.PubMed/NCBI
27	Sotillo-Piñeiro E, Sierrasesúmaga L and Patiñno-García A: Telomerase activity and telomere length in primary and metastatic tumors from pediatric bone cancer patients. Pediatr Res. 55:231–235. 2004.PubMed/NCBI
28	Yu ST, Chen L, Wang HJ, Tang XD, Fang DC and Yang SM: hTERT promotes the invasion of telomerase-negative tumor cells in vitro. Int J Oncol. 35:329–336. 2009.PubMed/NCBI
29	Jegou T, Chung I, Heuvelman G, et al: Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line. Mol Biol Cell. 20:2070–2082. 2009. View Article : Google Scholar : PubMed/NCBI
30	Sanders RP, Drissi R, Billups CA, Daw NC, Valentine MB and Dome JS: Telomerase expression predicts unfavorable outcome in osteosarcoma. J Clin Oncol. 22:3790–3797. 2004. View Article : Google Scholar : PubMed/NCBI
31	Herbert BS, Gellert GC, Hochreiter A, et al: Lipid modification of GRN163, an N3′→P5′ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Oncogene. 24:5262–5268. 2005.PubMed/NCBI
32	Pascolo E, Wenz C, Lingner J, et al: Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol Chem. 277:15566–15572. 2002. View Article : Google Scholar : PubMed/NCBI
33	Perrem K, Colgin LM, Neumann AA, Yeager TR and Reddel RR: Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells. Mol Cell Biol. 21:3862–3875. 2001. View Article : Google Scholar : PubMed/NCBI
34	Costa A, Daidone MG, Daprai L, et al: Telomere maintenance mechanisms in liposarcomas: association with histologic subtypes and disease progression. Cancer Res. 66:8918–8924. 2006. View Article : Google Scholar : PubMed/NCBI
35	Bhattacharyya S, Sandy A and Groden J: Unwinding protein complexes in ALTernative telomere maintenance. J Cell Biochem. 109:7–15. 2010.PubMed/NCBI
36	Mankouri HW and Hickson ID: The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific ‘dissolvasome’? Trends Biochem Sci. 32:538–546. 2007.PubMed/NCBI
37	Raynard S, Zhao W, Bussen W, et al: Functional role of BLAP75 in BLM-topoisomerase IIIalpha-dependent holliday junction processing. J Biol Chem. 283:15701–15708. 2008. View Article : Google Scholar : PubMed/NCBI
38	Jiang WQ, Zhong ZH, Henson JD, Neumann AA, Chang AC and Reddel RR: Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex. Mol Cell Biol. 25:2708–2721. 2005. View Article : Google Scholar : PubMed/NCBI
39	Sugawara N, Wang X and Haber JE: In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol Cell. 2:209–219. 2003. View Article : Google Scholar : PubMed/NCBI
40	Miyazaki T, Bressan DA, Shinohara M, Haber JE and Shinohara A: In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J. 3:939–949. 2004. View Article : Google Scholar : PubMed/NCBI
41	Saharia A and Stewart SA: FEN1 contributes to telomere stability in ALT-positive tumor cells. Oncogene. 28:1162–1167. 2009. View Article : Google Scholar : PubMed/NCBI
42	Zeng S, Xiang T, Pandita TK, et al: Telomere recombination requires the MUS81 endonuclease. Nat Cell Biol. 11:616–623. 2009. View Article : Google Scholar : PubMed/NCBI
43	Fan Q, Zhang F, Barrett B, Ren K and Andreassen PR: A role for monoubiquitinated FANCD2 at telomeres in ALT cells. Nucleic Acids Res. 37:1740–1754. 2009. View Article : Google Scholar : PubMed/NCBI
44	Wu G, Jiang X, Lee WH and Chen PL: Assembly of functional ALT-associated promyelocytic leukemia bodies requires Nijmegen Breakage Syndrome 1. Cancer Res. 63:2589–2595. 2003.PubMed/NCBI
45	Henson JD, Cao Y, Huschtscha LI, et al: DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity. Nat Biotechnol. 27:1181–1185. 2009. View Article : Google Scholar : PubMed/NCBI