The alternative lengthening of telomeres (ALT) is a recombination-based mechanism of telomere maintenance activated in 5–20% of human cancers. In Saccharomyces cerevisiae, survivors that arise after inactivation of telomerase can be classified as type I or type II ALT. In type I, telomeres have a tandem array structure, with each subunit consisting of a subtelomeric Y′ element and short telomere sequence. Telomeres in type II have only long telomere repeats and require Sgs1, the S. cerevisiae RecQ family helicase. We previously described the first human ALT cell line, AG11395, that has a telomere structure similar to type I ALT yeast cells. This cell line lacks the activity of the Werner syndrome protein, a human RecQ helicase. The telomeres in this cell line consist of tandem repeats containing SV40 DNA, including the origin of replication, and telomere sequence. We investigated the role of the SV40 origin of replication and the effects of Werner protein and telomerase on telomere structure and maintenance in AG11395 cells. We report that the expression of Werner protein facilitates the transition in human cells of ALT type I like telomeres to type II like telomeres in some aspects. These findings have implications for the diagnosis and treatment of cancer. 1. Introduction As progressive loss of telomere DNA is associated with senescence [1], maintenance of telomere function is essential for indefinite cell proliferation. Most cancer cells rely on expression of telomerase for suppression of telomere shortening [2]. However 5%–20% percent of cancers maintain telomeres by the alternative lengthening of telomere (ALT), a recombination-based mechanism [3]. Telomere maintenance mechanisms are a potential prognostic indicator [3] and promising target in cancer diagnosis and therapy [4–6]. Increasing evidence supports that Werner protein (WRN), a RecQ helicase and exonuclease, plays a direct role in telomere maintenance [7] and promotion of tumor cell growth [8]. WRN epigenetic silencing in human cancers leads to hypersensitivity to treatment with a number of chemotherapeutic drugs [9]. Germline mutations in the WRN gene cause an autosomal recessive disorder, Werner syndrome (WS). WS is characterized by symptoms suggestive of premature aging and by the development of mesenchymal neoplasms [10]. Strikingly, the ALT mechanism is more prevalent in tumors arising from tissues of mesenchymal origin, such as osteosarcomas, than in those of epithelial origin [11]. It has been suggested that the telomere-telomere recombination in WRN-deficient, telomere dysfunctional
References
[1]
L. Hayflick and P. S. Moorhead, “The serial cultivation of human diploid cell strains,” Experimental Cell Research, vol. 25, no. 3, pp. 585–621, 1961.
[2]
C. M. Counter, A. A. Avilion, C. E. Lefeuvre et al., “Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity,” The EMBO Journal, vol. 11, no. 5, pp. 1921–1929, 1992.
[3]
J. D. Henson, A. A. Neumann, T. R. Yeager, and R. R. Reddel, “Alternative lengthening of telomeres in mammalian cells,” Oncogene, vol. 21, no. 4, pp. 598–610, 2002.
[4]
A. Siddiqa, D. A. Cavazos, and R. A. Marciniak, “Targeting telomerase,” Rejuvenation Research, vol. 9, no. 3, pp. 378–390, 2006.
[5]
R. Villa, M. G. Daidone, R. Motta et al., “Multiple mechanisms of telomere maintenance exist and differentially affect clinical outcome in diffuse malignant peritoneal mesothelioma,” Clinical Cancer Research, vol. 14, no. 13, pp. 4134–4140, 2008.
[6]
S. Zimmermann and U. M. Martens, “Telomeres and telomerase as targets for cancer therapy,” Cellular and Molecular Life Sciences, vol. 64, no. 7-8, pp. 906–921, 2007.
[7]
A. S. Multani and S. Chang, “WRN at telomeres: implications for aging and cancer,” Journal of Cell Science, vol. 120, no. 5, pp. 713–721, 2007.
[8]
P. L. Opresko, J. P. Calvo, and C. von Kobbe, “Role for the Werner syndrome protein in the promotion of tumor cell growth,” Mechanisms of Ageing and Development, vol. 128, no. 7-8, pp. 423–436, 2007.
[9]
R. Agrelo, W. H. Cheng, F. Setien et al., “Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 23, pp. 8822–8827, 2006.
[10]
A. Ozgenc and L. A. Loeb, “Werner Syndrome, aging and cancer,” Genome Dynamics, vol. 1, pp. 206–217, 2006.
[11]
J. D. Henson, J. A. Hannay, S. W. McCarthy et al., “A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas,” Clinical Cancer Research, vol. 11, no. 1, pp. 217–225, 2005.
[12]
P. R. Laud, A. S. Multani, S. M. Bailey et al., “Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway,” Genes and Development, vol. 19, no. 21, pp. 2560–2570, 2005.
[13]
F. B. Johnson, R. A. Marciniak, M. McVey, S. A. Stewart, W. C. Hahn, and L. Guarente, “The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase,” The EMBO Journal, vol. 20, no. 4, pp. 905–913, 2001.
[14]
V. Lundblad and J. W. Szostak, “A mutant with a defect in telomere elongation leads to senescence in yeast,” Cell, vol. 57, no. 4, pp. 633–643, 1989.
[15]
V. Lundblad and E. H. Blackburn, “An alternative pathway for yeast telomere maintenance rescues est1- senescence,” Cell, vol. 73, no. 2, pp. 347–360, 1993.
[16]
S. C. Teng and V. A. Zakian, “Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae,” Molecular and Cellular Biology, vol. 19, no. 12, pp. 8083–8093, 1999.
[17]
H. Cohen and D. A. Sinclair, “Recombination-mediated lengthening of terminal telomeric repeats requires the Sgs1 DNA helicase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 6, pp. 3174–3179, 2001.
[18]
J. Y. Lee, J. L. Mogen, A. Chavez, and F. B. Johnson, “Sgs1 RecQ helicase inhibits survival of Saccharomyces cerevisiae cells lacking telomerase and homologous recombination,” Journal of Biological Chemistry, vol. 283, no. 44, pp. 29847–29858, 2008.
[19]
T. R. Yeager, A. A. Neumann, A. Englezou, L. I. Huschtscha, J. R. Noble, and R. R. Reddel, “Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body,” Cancer Research, vol. 59, no. 17, pp. 4175–4179, 1999.
[20]
C. L. Fasching, K. Bower, and R. R. Reddel, “Telomerase-independent telomere length maintenance in the absence of alternative lengthening of telomeres-associated promyelocytic leukemia bodies,” Cancer Research, vol. 65, no. 7, pp. 2722–2729, 2005.
[21]
R. A. Marciniak, D. Cavazos, R. Montellano, Q. Chen, L. Guarente, and F. B. Johnson, “A novel telomere structure in a human alternative lengthening of telomeres cell line,” Cancer Research, vol. 65, no. 7, pp. 2730–2737, 2005.
[22]
H. Saito and R. E. Moses, “Immortalization of Werner syndrome and progeria fibroblasts,” Experimental Cell Research, vol. 192, no. 2, pp. 373–379, 1991.
[23]
B. T. Kurien and R. H. Scofield, “Western blotting,” Methods, vol. 38, no. 4, pp. 283–293, 2006.
[24]
R. A. Marciniak, D. B. Lombard, F. B. Johnson, and L. Guarente, “Nucleolar localization of the Werner Syndrome protein in human cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 12, pp. 6887–6892, 1998.
[25]
S. S. Poon and P. M. Lansdorp, “Quantitative fluorescence in situ hybridization (Q-FISH),” Current Protocols in cell Biology, chapter 18, unit 18.4, 2001.
[26]
S. M. Bailey, E. H. Goodwin, and M. N. Cornforth, “Strand-specific fluorescence in situ hybridization: the CO-FISH family,” Cytogenetic and Genome Research, vol. 107, no. 1-2, pp. 14–17, 2004.
[27]
V. A. Zakian, “Structure, function, and replication of Saccharomyces cerevisiae telomeres,” Annual Review of Genetics, vol. 30, pp. 141–172, 1996.
[28]
M. A. Rehman, G. Fourel, A. Mathews et al., “Differential requirement of DNA replication factors for subtelomeric ARS consensus sequence protosilencers in Saccharomyces cerevisiae,” Genetics, vol. 174, no. 4, pp. 1801–1810, 2006.
[29]
M. A. Rehman and K. Yankulov, “The dual role of autonomously replicating sequences as origins of replication and as silencers,” Current Genetics, vol. 55, no. 4, pp. 357–363, 2009.
[30]
K. Myung, A. Datta, C. Chen, and R. D. Kolodner, “SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination,” Nature Genetics, vol. 27, no. 1, pp. 113–116, 2001.
[31]
L. Crabbe, R. E. Verdun, C. I. Haggblom, and J. Karlseder, “Defective telomere lagging strand synthesis in cells lacking WRN helicase activity,” Science, vol. 306, no. 5703, pp. 1951–1953, 2004.
[32]
J. V. Grobelny, A. K. Godwin, and D. Broccoli, “ALT-associated PML bodies are present in viable cells and are enriched in cells in the G2/M phase of the cell cycle,” Journal of Cell Science, vol. 113, no. 24, pp. 4577–4585, 2000.
[33]
K. Perrem, L. M. Colgin, A. A. Neumann, T. R. Yeager, and R. R. Reddel, “Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells,” Molecular and Cellular Biology, vol. 21, no. 12, pp. 3862–3875, 2001.
[34]
C. M. Counter, W. C. Hahn, W. Wei et al., “Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 25, pp. 14723–14728, 1998.
[35]
C. M. Counter, M. Meyerson, E. N. Eaton et al., “Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase,” Oncogene, vol. 16, no. 9, pp. 1217–1222, 1998.
[36]
S. Makovets, T. L. Williams, and E. H. Blackburn, “The telotype defines the telomere state in Saccharomyces cerevisiae and is inherited as a dominant non-Mendelian characteristic in cells lacking telomerase,” Genetics, vol. 178, no. 1, pp. 245–257, 2008.
[37]
M. L. Yamamoto, R. Reliene, J. Oshima, and R. H. Schiestl, “Effects of human Werner helicase on intrachromosomal homologous recombination mediated DNA deletions in mice,” Mutation Research, vol. 644, no. 1-2, pp. 11–16, 2008.
[38]
P. H. Chen, W. B. Tseng, Y. Chu, and M. T. Hsu, “Interference of the simian virus 40 origin of replication by the cytomegalovirus immediate early gene enhancer: evidence for competition of active regulatory chromatin conformation in a single domain,” Molecular and Cellular Biology, vol. 20, no. 11, pp. 4062–4074, 2000.
[39]
S. Anant, S. A. Axenovich, S. L. Madden, F. J. Rauscher, and K. N. Subramanian, “Novel replication inhibitory function of the developmental regulator/transcription repressor protein WT1 encoded by the Wilms' tumor gene,” Oncogene, vol. 9, no. 11, pp. 3113–3126, 1994.
[40]
A. M. Castellino, P. Cantalupo, I. M. Marks, J. V. Vartikar, K. W. C. Peden, and J. M. Pipas, “Trans-dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP,” Journal of Virology, vol. 71, no. 10, pp. 7549–7559, 1997.
[41]
C. P. Baur, K. Klausing, M. Scheffner, H. Stahl, and R. Knippers, “Protein-DNA interactions at the Simian Virus 40 origin of replication,” Biochimica et Biophysica Acta, vol. 951, no. 2-3, pp. 388–395, 1988.
[42]
M. C. San Martin, C. Gruss, and J. M. Carazo, “Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel,” Journal of Molecular Biology, vol. 268, no. 1, pp. 15–20, 1997.
[43]
C. L. Fasching, A. A. Neumann, A. Muntoni, T. R. Yeager, and R. R. Reddel, “DNA damage induces alternative lengthening of telomeres (ALT)-associated promyelocytic leukemia bodies that preferentially associate with linear telomeric DNA,” Cancer Research, vol. 67, no. 15, pp. 7072–7077, 2007.
[44]
W. Q. Jiang, Z. H. Zhong, A. Nguyen et al., “Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins,” Journal of Cell Biology, vol. 185, no. 5, pp. 797–810, 2009.
[45]
G. Blandert, N. Zalle, Y. Daniely, J. Taplick, M. D. Gray, and M. Oren, “DNA damage-induced translocation of the Werner helicase is regulated by acetylation,” Journal of Biological Chemistry, vol. 277, no. 52, pp. 50934–50940, 2002.
[46]
R. Vaitiekunaite, D. Butkiewicz, M. Krze？niak et al., “Expression and localization of Werner syndrome protein is modulated by SIRT1 and PML,” Mechanisms of Ageing and Development, vol. 128, no. 11-12, pp. 650–661, 2007.
[47]
C. Von Kobbe, A. May, C. Grandori, and V. A. Bohr, “Werner syndrome cells escape hydrogen peroxide-induced cell proliferation arrest,” The FASEB Journal, vol. 18, no. 15, pp. 1970–1972, 2004.
[48]
Y. Zhao, A. J. Sfeir, Y. Zou et al., “Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells,” Cell, vol. 138, no. 3, pp. 463–475, 2009.
