Telomeres are specialized structures found at the ends of eukaryotic chromosomes serving as guardians of genome stability. In normal cells telomeres shorten with each cell division, but immortal cells undergoing multiple divisions constantly have to maintain telomere lengths above a critical level. This is accomplished either through expression of telomerase or the alternative recombination pathway (ALT). In the present study, we analyzed telomere dynamics of the telomerase positive human pancreatic tumor cell line MIAPaCa-2. The cells demonstrated genomic instability with a high frequency of chromosomal aberrations resulting in differences between individual karyotypes within the same cell population. The telomeres were short when compared with normal human fibroblasts, and about 39% of the chromosome ends did not have detectable telomere repeats as demonstrated by PNA-FISH. In many cases telomere signals were missing even when sister chromatids were strongly labeled. In addition, we used an internal PNA probe specific for the X chromosome, present in a single copy in these cells, in order to follow telomere dynamics on individual chromatids. High heterogeneity in telomere signals among individual X chromosomes as well as between their sister chromatids suggested sudden and stochastic loss or gain of telomere repeats. Such constant genomic instability often results in apoptosis and death of a fraction of cells present in the culture at all times. We discuss possible molecular mechanisms that may explain this observed telomere heterogeneity and possible adaptive repair mechanisms by which these cells maintain their chromosomes in order to survive such extreme and permanent genomic instability.

1.
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, et al: Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 89:10114–10118 (1992).
2.
Blackburn EH: Structure and function of telomeres. Nature 350:569–573 (1991).
3.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, et al: Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352 (1998).
4.
Bolzan AD, Bianchi MS: Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations. Mutat Res 612:189–214 (2006).
5.
Cesare AJ, Griffith JD: Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Mol Cell Biol 24:9948–9957 (2004).
6.
Chen C, Wu B, Wei T, Egholm M, Strauss WM: Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers. Mamm Genome 11:384–391 (2000).
7.
Counter CM, Hirte HW, Bacchetti S, Harley CB: Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci USA 91:2900–2904 (1994).
8.
de Lange T: Protection of mammalian telomeres. Oncogene 21:532–540 (2002).
9.
Dimri GP, Lee X, Basile G, Acosta M, Scott G, et al: A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92:9363–9367 (1995).
10.
Dunham MA, Neumann AA, Fasching CL, Reddell RR: Telomere maintenance by recombination in human cells. Nat Genet 26:447–450 (2000).
11.
Ferenac M, Polancec D, Huzak M, Pereira-Smith OM, Rubelj I: Early-senescing human skin fibroblasts do not demonstrate accelerated telomere shortening. J Gerontol A Biol Sci Med Sci 60:820–829 (2005).
12.
Fouché N, Ozgur S, Roy D, Griffith JD: Replication fork regression in repetitive DNAs. Nucleic Acids Res 34:6044–6050 (2006).
13.
Greider CW: Mammalian telomere dynamics: healing, fragmentation shortening and stabilization. Curr Opin Genet Dev 4:203–211 (1994).
14.
Greider CW, Blackburn EH: Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43:405–413 (1985).
15.
Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, et al: Mammalian telomeres end in a large duplex loop. Cell 97:503–514 (1999).
16.
Harley CB: Telomere loss: mitotic clock or genetic time bomb? Mutat Res 256:271–282 (1991).
17.
Harley CB, Futcher AB, Greider CW: Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 (1990).
18.
Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC: Telomere reduction in human colorectal carcinoma and with ageing. Nature 346:866–868 (1990).
19.
Herrmann M, Lorenz H-M, Voll R, Grunke M, Woith W, Kalden JR: A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 22:5506–5507 (1994).
20.
ISCN 2005: An International System for Human Cytogenetic Nomenclature; Shaffer LG, Tommerup N (eds) (S. Karger, Basel 2005).
21.
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, et al: Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015 (1994).
22.
Lansdorp PM: Major cutbacks at chromosome ends. Trends Biochem Sci 30:388–395 (2005).
23.
Lansdorp PM, Verwoerd NP, van de Rijke FM, Dragowska V, Little MT, et al: Heterogeneity in telomere length of human chromosomes. Hum Mol Genet 5:685–691 (1996).
24.
Lee JJ, Warburton D, Robertson EJ: Cytogenetic methods for the mouse: Preparation of chromosomes, karyotyping, and in situ hybridization. Anal Biochem 189:1–17 (1990).
25.
Li D, Zhu J, Firozi PF, Abbruzzese JL, Evans DB, et al: Overexpression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin Cancer Res 9:991–997 (2003).
26.
Makarov VL, Hirose Y, 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).
27.
Martens UM, Zijlmans JMJM, Poon SSS, Dragowska W, Yui J, et al: Short telomeres on human chromosome 17p. Nat Genet 18:76–80 (1998).
28.
Neumann AA, Reddel RR: Telomere maintenance and cancer – look, no telomerase. Nat Rev Cancer 2:879–884 (2002).
29.
Nilsson P, Mehle C, Remes K, Roos G: Telomerase activity in vivo in human malignant hematopoietic cells. Oncogene 9:3043–3048 (1994).
30.
Okubo M, Tsurukubo Y, Higaki T, Kawabe T, Goto M, et al: Clonal chromosomal aberrations accompanied by strong telomerase activity in immortalization of human B-lymphoblastoid cell lines transformed by Epstein-Barr virus. Cancer Genet Cytogenet 129:30–34 (2001).
31.
Olovnikov AM: Principle of marginotomy in template synthesis of polynucleotides. Dokl Akad Nauk SSSR 201:1496–1499 (1971).
32.
Poon SS, Martens UM, Ward RK, Lansdorp PM: Telomere length measurements using digital fluorescence microscopy. Cytometry 36:267–278 (1999).
33.
Rha SY, Izbicka E, Lawrence R, Davidson K, Sun D, et al: Effect of telomere and telomerase interactive agents on human tumor and normal cell lines. Clin Cancer Res 6:987–993 (2000).
34.
Riethman H, Ambrosini A, Paul S: Human subtelomere structure and variation. Chromosome Res 13:505–515 (2005).
35.
Rojanala S, Han H, Munoz RM, Vankayalapati H, Mahadevan D, et al: Aurora kinase-2 a potential molecular therapeutic target for pancreatic cancers. Proc Am Assoc Cancer Res 43:665 (2002).
36.
Rubelj I, Vondracek Z: Stochastic mechanism of cellular aging – abrupt telomere shortening as a model for stochastic nature of cellular aging. J Theor Biol 197:425–438 (1999).
37.
Sato N, Mizumoto K, Nishio S, Maehara N, Urashima T, et al: Up-regulation of telomerase activity in human pancreatic cancer cells after exposure to etoposide. Br J Cancer 82:1819–1826 (2000).
38.
Seshadri T, Campisi J: Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science 247:205–209 (1990).
39.
Shay JW, Bacchetti S: A survey of telomerase activity in human cancer. Eur J Cancer 33:787–791 (1997).
40.
Slijepcevic P, Al-Wahiby S: Telomere biology: integrating chromosomal end protection with DNA damage response. Chromosoma 114:275–285 (2005).
41.
Slijepcevic P, Bryant PE: Absence of terminal telomeric FISH signals in chromosomes from immortal Chinese hamster cells. Cytogenet Cell Genet 69:87–89 (1995).
42.
Sprung CN, Afshar G, Chavez EA, Lansdorp P, Sabatier L, Murnane JP: Telomere instability in a human cancer cell line. Mutat Res 429:209–223 (1999).
43.
Vaziri H, Schachter F, Uchida I, Wei L, Zhu X, et al: Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet 52:661–667 (1993).
44.
Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM: Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci USA 91:9857–9860 (1994).
45.
West SC: Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem 61:603–640 (1992).
46.
Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW: Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 18:173–179 (1996).
47.
Yajima T, Yagihashi A, Kameshima H, Kobayasji D, Hirata K, Watanabe N: Telomerase reverse transcriptase and telomeric-repeat binding factor protein 1 as regulators of telomerase activity in pancreatic cancer cells. Br J Cancer 85:752–757 (2001).
48.
Yoshimura SH, Maruyama H, Ishikawa F, Ohki R, Takeyasu K: Molecular mechanisms of DNA end-loop formation by TRF2. Genes Cells 9:205–218 (2004).
49.
Yunis AA, Arimura GK, Russin DJ: Human pancreatic carcinoma (MIAPaCa-2) in continuous culture: sensitivity to asparaginase. Int J Cancer 19:128–135 (1977).
50.
Yunis JJ: High resolution of human chromosomes. Science 191:1268–1270 (1976).
51.
Zahradka K, Slade D, Bailone A, Sommer S, Averbeck D, et al: Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature 443:569–573 (2006).
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.