Structural and functional analysis of telomeres is very important for understanding basic biological functions such as genome stability, cell growth control, senescence and aging. Recently, serious concerns have been raised regarding the reliability of current telomere measurement methods such as Southern blot and quantitative polymerase chain reaction. Since telomere length is associated with age related pathologies, including cardiovascular disease and cancer, both at the individual and population level, accurate interpretation of measured results is a necessity. The telomere Q-PNA-FISH technique has been widely used in these studies as well as in commercial analysis for the general population. A hallmark of telomere Q-PNA-FISH is the wide variation among telomere signals which has a major impact on obtained results. In the present study we introduce a specific mathematical and statistical analysis of sister telomere signals during cell culture senescence which enabled us to identify high regularity in their variations. This phenomenon explains the reproducibility of results observed in numerous telomere studies when the Q-PNA-FISH technique is used. In addition, we discuss the molecular mechanisms which probably underlie the observed telomere behavior.
References
[1]
Lange T de (2005) Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19: 2100–2110 doi:10.1101/gad.1346005.
[2]
Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345: 458–460 doi:10.1038/345458a0.
[3]
Hemann MT, Strong MA, Hao L-Y, Greider CW (2001) The Shortest Telomere, Not Average Telomere Length, Is Critical for Cell Viability and Chromosome Stability. Cell 107: 67–77 doi:10.1016/S0092-8674(01)00504-9.
[4]
Karlseder J, Smogorzewska A, Lange T de (2002) Senescence Induced by Altered Telomere State, Not Telomere Loss. Science 295: 2446–2449 doi:10.1126/science.1069523.
[5]
Cawthon RM, Smith KR, O'Brien E, Sivatchenko A, Kerber RA (2003) Association between telomere length in blood and mortality in people aged 60 years or older. The Lancet 361: 393–395 doi:10.1016/S0140-6736(03)12384-7.
[6]
Epel ES, Blackburn EH, Lin J, Dhabhar FS, Adler NE, et al. (2004) Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci U S A 101: 17312–17315 doi:10.1073/pnas.0407162101.
[7]
Canela A, Vera E, Klatt P, Blasco MA (2007) High-throughput telomere length quantification by FISH and its application to human population studies. Proc Natl Acad Sci U S A 104: 5300–5305 doi:10.1073/pnas.0609367104.
[8]
Aviv A, Hunt SC, Lin J, Cao X, Kimura M, et al. (2011) Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR. Nucleic Acids Res 39: e134 doi:10.1093/nar/gkr634.
[9]
Steenstrup T, Hjelmborg J v B, Kark JD, Christensen K, Aviv A (2013) The telomere lengthening conundrum—artifact or biology? Nucleic Acids Res 41: e131–e131 doi:10.1093/nar/gkt370.
[10]
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, et al. (1992) Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A 89: 10114–10118. doi: 10.1073/pnas.89.21.10114
[11]
Harley CB, Liu W, Blasco M, Vera E, Andrews WH, et al. (2011) A Natural Product Telomerase Activator As Part of a Health Maintenance Program. Rejuvenation Res 14: 45–56 doi:10.1089/rej.2010.1085.
[12]
Vera E, Blasco MA (2012) Beyond average: potential for measurement of short telomeres. Aging 4: 379–392.
[13]
Martens UM, Zijlmans JMJM, Poon SSS, Dragowska W, Yui J, et al. (1998) Short telomeres on human chromosome 17p. Nat Genet 18: 76–80 doi:10.1038/ng0198-76.
[14]
Baird DM, Rowson J, Wynford-Thomas D, Kipling D (2003) Extensive allelic variation and ultrashort telomeres in senescent human cells. Nat Genet 33: 203–207 doi:10.1038/ng1084.
[15]
Huffman KE, Levene SD, Tesmer VM, Shay JW, Wright WE (2000) Telomere Shortening Is Proportional to the Size of the G-rich Telomeric 3′-Overhang. J Biol Chem 275: 19719–19722 doi:10.1074/jbc.M002843200.
[16]
Lansdorp PM, Verwoerd NP, Rijke FM van de, Dragowska V, Little M-T, et al. (1996) Heterogeneity in Telomere Length of Human Chromosomes. Hum Mol Genet 5: 685–691 doi:10.1093/hmg/5.5.685.
[17]
Krejci K, Koch J (1998) Improved detection and comparative sizing of human chromosomal telomeres in situ. Chromosoma 107: 198–203 doi:10.1007/s004120050297.
[18]
Bekaert S, Koll S, Thas O, Van Oostveldt P (2002) Comparing telomere length of sister chromatids in human lymphocytes using three-dimensional confocal microscopy. Cytometry 48: 34–44 doi:10.1002/cyto.10105.
[19]
Vida？ek N？, ？uku？i？ A, Ivankovi？ M, Fulgosi H, Huzak M, et al. (2010) Abrupt telomere shortening in normal human fibroblasts. Exp Gerontol 45: 235–242 doi:10.1016/j.exger.2010.01.009.
[20]
Londono-Vallejo JA, DerSarkissian H, Cazes L, Thomas G (2001) Differences in telomere length between homologous chromosomes in humans. Nucleic Acids Res 29: 3164–3171. doi: 10.1093/nar/29.15.3164
[21]
Graakjaer J, Pascoe L, Der-Sarkissian H, Thomas G, Kolvraa S, et al. (2004) The relative lengths of individual telomeres are defined in the zygote and strictly maintained during life. Aging Cell 3: 97–102 doi:10.1111/j.1474-9728.2004.00093.x.
[22]
Graakjaer J, Londono-Vallejo J a, Christensen K, K？lvraa S (2006) The Pattern of Chromosome-Specific Variations in Telomere Length in Humans Shows Signs of Heritability and Is Maintained through Life. Ann N Y Acad Sci 1067: 311–316 doi:10.1196/annals.1354.042.
[23]
Njajou OT, Cawthon RM, Damcott CM, Wu S-H, Ott S, et al. (2007) Telomere length is paternally inherited and is associated with parental lifespan. Proc Natl Acad Sci U S A 104: 12135–12139 doi:10.1073/pnas.0702703104.
[24]
Gilson E, Londo？o-Vallejo A (2007) Telomere length profiles in humans: all ends are not equal. Cell Cycle Georget Tex 6: 2486–2494. doi: 10.4161/cc.6.20.4798
[25]
Martens UM, Chavez EA, Poon SSS, Schmoor C, Lansdorp PM (2000) Accumulation of Short Telomeres in Human Fibroblasts Prior to Replicative Senescence. Exp Cell Res 256: 291–299 doi:10.1006/excr.2000.4823.
[26]
Rubelj I, Vondracek Z (1999) Stochastic Mechanism of Cellular Aging—Abrupt Telomere Shortening as a Model for Stochastic Nature of Cellular Aging. J Theor Biol 197: 425–438 doi:10.1006/jtbi.1998.0886.
[27]
Rubelj I, Huzak M, Brdar B (2000) Sudden senescence syndrome plays a major role in cell culture proliferation. Mech Ageing Dev 112: 233–241 doi:10.1016/S0047-6374(99)00090-1.
[28]
Petersen S, Saretzki G, Zglinicki T von (1998) Preferential Accumulation of Single-Stranded Regions in Telomeres of Human Fibroblasts. Exp Cell Res 239: 152–160 doi:10.1006/excr.1997.3893.
[29]
Von Zglinicki T, Pilger R, Sitte N (2000) Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic Biol Med 28: 64–74 doi:10.1016/S0891-5849(99)00207-5.
[30]
Wang Y, Patel DJ (1993) Solution structure of the human telomeric repeat d[AG3(T2AG3)3] G-tetraplex. Structure 1: 263–282 doi:10.1016/0969-2126(93)90015-9.
[31]
Biffi G, Tannahill D, McCafferty J, Balasubramanian S (2013) Quantitative visualization of DNA G-quadruplex structures in human cells. Nat Chem 5: 182–186 doi:10.1038/nchem.1548.
[32]
Gorbunova V, Seluanov A, Pereira-Smith OM (2002) Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis. J Biol Chem 277: 38540–38549 doi:10.1074/jbc.M202671200.
[33]
Young JI, Smith JR (2001) DNA Methyltransferase Inhibition in Normal Human Fibroblasts Induces a p21-dependent Cell Cycle Withdrawal. J Biol Chem 276: 19610–19616 doi:10.1074/jbc.M009470200.
