ࡱ> l[mOh+'0`   ( 4@HPXssAdministratorodmiNormaltAdministratoro31iMicrosoft Word 8.0@26@6% @C ՜.+,D՜.+,, hp|  IRB   Title 6> _PID_GUIDAN{F26767A9-6156-40C3-BD5C-BBB81B08F1EA}Ivica Rubelj Department of Molecular Biology, Ruer Boakovi Institute, Bijeni ka 54, 10 000 Zagreb, Croatia Abrupt Telomere Shortening in Cell Aging and cancer A strong stochastic component has been described for the appearance of senescent cells in cultures that have not completed their in vitro lifespan. The proliferative potential of individual clones show a bimodal distribution. Additionally, two cells arising from a single mitotic event can exhibit large differences in their doubling capacities. Molecular model and a computer simulation of the model has been presented that explains the observed stochastic phenomena. The model is based on a combination of both, gradual telomere shortening (GTS) and abrupt telomere shortening (ATS) (10). GTS occurs as a consequence of the inability of DNA polymerase to replicate the very end of chromosomal DNA as well as exonuclease degradation of the 5' strand at telomere ends (23). GTS could be responsible for the gradual decline in proliferative potential of a cell culture, but does not explain the stochastic aspects of cellular aging. ATS is predicted to occur through strand invasion of the 3 overhang into the telomeric/subtelomeric border region followed by formation of a t-loop structure which has later been found in normal and immortal human and mouse cells (20). ATS is mediated by Holiday structure formation which results in a deletion of distal telomeric repeats through circularisation. Our model explains the gradual increase of senescent cells in the culture by proposing that long telomeres have a stable conformation and a low probability of undergoing abrupt shortening but, as telomere shortening progresses the probability of conformation changes to an unstable form increases almost exponentially. Abrupt shortening of one or more telomeres in the cell causes cell cycle arrest within one cell division. Since it was published, all of the predictions of our model have been examined both structurally (20) and functionally (30) including the presence of ultrashort, single telomeres in senescent human cells (24), the identification of circular telomeric DNA in various mammalian cell lines (29), generation of t-loop-sized deletions at human telomeres by homologous recombination in ALT cells (30) and along with the observations of Hemann et al (31) and Smith & Whitney (8) strongly suggest catastrophic telomere deletion as the mechanism that induces the stochastic p21/pRB mediated permanent cell-cycle arrest observed in normal human cell cultures. CDEE@FF6GHHHHHH HPPjQQRSS@TT6U^^^l_$dhdh$F00\2235V67$9:J<L<N<P<R<T<V<BBjCCdh$dh$$Fe observed if abrupt shortening of a single telomere trigers the onset of cell senescence.FH%.~K 5 H v  $&*,00262\2222223344646r7789V:d:;H<T<V<>BB$CBCjCCCC^DfDDDzEEFFFFGGGGHH HLݽ0J56CJ CJhnH  0J5CJ566CJ5CJCJ 6B* CJB* CJ ;B* CJ 5B* CJB* CJ 5B* CJJLPP$QBQQQ^RfRRRzSSTTTTUUUUVZ^^&_D___``h```~aab bbbc ccd*dhnno.oooJpRppphqqq rrrr ssstB* CJ 0J5CJ0J56CJ6CJ5CJCJAesult if abrupt shortening of a single telomere triggers the onset of cell senescence.s in their doubling capacities. We present a mter simulation of the model -1-2-3rease of senescent cells in a -3-4-5-6-4-7-8 This concept is further supported by our latest experiments in which we compared the telomere lengths of a subpopulation of senescent human skin fibroblasts, present in pre-senescent cultures, with those of young cells (9). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture, this is the expected result if abrupt shortening of a single telomere triggers the onset of cell senescence. References: 1. Rubelj I. and Vondra ek Z. Stochastic mechanism of cellular aging - Abrupt telomere shortening as a model for stochastic nature of cellular aging. J. theor. Biol. 1999;197:425-438. 2. Makarov, V. L., Hirose, Y., Langmore, J. P., (1997) Cell 88, 657-666 3. Griffith J.D., Comeau L., Rosenfield S. et al. Mammalian telomeres end in a large duplex loop. Cell 1999;97:503-514. 4. Wang R. C., Smogorzewska A. and de Lange T., Homologous recombination generates t-loop-sized deletions at human telomeres. Cell 2004;119:355-368. 5. Baird D., Rowson J., Wynford-Thomas D. and Kipling D. Extensive allelic variation and ultrashort telomeres in senescent hs in their doubling capacities. We present a mter simulation of the model rease of senescent cells in a This concept is further supported by our latest experiments in which we compared the telomere lengths of a subpopulation of senescent human skin fibroblasts, present in pre-senescent cultures, with those of young cells (--). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture, this is the expected rs A: Biological Sciences (in press). uman cells. Nature Genet. 2003;33:203 207. 6. Regev A., Cohen S., Cohen E., Bar-Am I. and Lavi S. Telomeric repeats on small polydisperse circular DNA (spcDNA) and genomic instability. Oncogene 1998;17:3455-3461. 7. Hemann M. T., Strong M. A., Hao L.Y. and Greider C. W., The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 2001;107:67-77. 8. Smith J. R. and Whitney R. G. Intraclonal variation in proliferative potential of human diploid fibroblasts: Stochastic mechanism for cellular aging. Science 1980;207:82-84. 9. Ferenac M., Polan ec D., Huzak M., Pereira-Smith O. M., Rubelj I. Early-senescing human skin fibroblasts do not demonstrate accelerated telomere shortening, Journals of Gerontology Series in their doubling capacities. We present a mter simulation of the model -1-2-3rease of senescent cells in a -3-4-5-6-4-7-8 This concept is further supported by our latest experiments in which we compared the telomere lengths of a subpopulation of senesces in their doubling capacities. We present a mter simulation of the model -1-2-3rease of senescent cells in a -3-4-5-6-4-7-8 This concept is further supported by our latest experiments in which we compared the telomere lengths of a subpopulation of senescent human skin fibroblasts, present in pre-senescent cultures, with those of young cells (9). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture, this is the expected result if abrupt shortening of a single telomere triggers the onset of cell senescence. References: 1. Rubelj I., Vondra ek Z., J. theor. Biol. 1999; 197:425-438. 2. Makarov, V. L., Hirose, Y., Langmore, J. P., Cell 1997; 88:657-666. 3. Griffith J.D., Comeau L., Rosenfield S. et al., Cell 1999; 97:503-514. 4. Wang R. C., Smogorzewska A., de Lange T., Cell 2004;119:355-368. 5. Baird D., Rowson J., Wynford-Thomas D. et al., Nature Genet. 2003;33:203 207. 6. Regev A., Cohen S., Cohen E., et al. Oncogene 1998;17:3455-3461. 7. Hemann M. T., Strong M. A., Hao L.Y. et al. Cell 2001;107:67-77. 8. Smith J. R., Whitney R. G., Science 1980;207:82-84. 9. Ferenac M., Polan ec D., Huzak M., et al. Journals of Gerontology Series A: Biological Sciences (in press). LPP$QBQQQ^RfRRRzSSTTTTUUUUVZ^^&_D___``h```~aab bbbc ccd*dhnno.oooJpRppphqqq rrrr ssst>vBv\v;CJB* CJ 0J5CJ0J56CJ6CJ5CJCJD4o4CM2 1$dCJOJQJnH 4o4bibref@CJH*OJQJkH:o": bodyindent$dCJ   * Unknown Administrator Ivica RubeljMiljenko Huzak,Laboratorij za eksperimentalnu kancerologiju Olivia Smith-Laboratorij za molekularnu genetiku eukariotaNPG olivia smithLMGE james smithowillikctrcHerzig Zoran Vondracek AdministratorE:\RUBELJ\CSH Conference.doc@~ ~ ȧK~ ` mn   'C % & - K g } U ` ~ PQ @Q&Q @Q&Q@&QV&QX&Q @Q\&Q@Q&Q@Q&Q&Q&Q'QZ'Q\'Qj'Q'Q'Q (Q(Q(Q(Q(Q)Q)Q *Q<*Q@*Q`*Qh*Qj*Q*P@GzTimes New Roman5Symbol3& zArial3zTimes"1hCFKfR !0 l__`aaJbbDcnnnVoozpqq4rr.s$s in their doubling capacities. We present a mter simulation of the model pt telomere shortening (ATS) (1e 5' strand at telomere ends (2mortal human and mouse cells (3rease of senescent cells in a n examined both structurally (3) and functionally (4res in senescent human cells (5various mammalian cell lines (6s recombination in ALT cells (4rvations of Hemann et al (7 This concept is further supported by our latest experiments in which we compared the telomere lengths of a subpopulation of senescent human skin fibroblasts, present in pre-senescent cultures, with those of young cells (9). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture, this is the expected result if abrupt shortening of a single telomere triggers the onset of cell senescence. References: 1. Rubelj I., Vondra ek Z., J. theor. Biol. 1999; 197:425-438. 2. Makarov, V. L., Hirose, Y., Langmore, J. P., Cell 1997; 88:657-666. 3. Griffith J.D., Comeau L., Rosenfield S. et al., Cell 1999; 97:503-514. 4. Wang R. C., Smogorzewska A., de Lange T., Cell 2004; 119:355-368. 5. Baird D., Rowson J., Wynford-Thomas D. et al., Nature Genet. 2003; 33:203-207. 6. Regev A., Cohen S., Cohen E., et al., Onc. A!"#$%s in their doubling capacities. We present a mter simulation of the model rease of senescent cells in a This concept is supported by our latest experiments in which we compared the telomere lengths of the subpopulation of senescent human skin fibroblasts, present in pre-senescent cultures, with those of young cells (--). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture which should bogene 1998; 17:3455-3461. 7. Hemann M. T., Strong M. A., Hao L.Y. et al. Cell 2001; 107:67-77. 8. Smith J. R., Whitney R. G., Science 1980; 207:82-84. 9. Ferenac M., Polan ec D., Huzak M., et al. Journals of Gerontology Series A: Biological Sciences 2005 (in press). [$@$NormalmH <A@<Default Paragraph Font,J`,SubtitleCJmH4o4CM2 1$dCJOJQJnH 4o4bibref@CJH*OJQJkH:o": bodyindent$dCJ*B`2* Body TextCJ W`A Strong5q  no  X : s in their doubling capacities. We present a mter simulation of the model -1-2-3rease of senescent cells in a -3-4-5-6-4-7-8 This concept is further supported by our latest experiments in which we compared the telomere lengths of a subpopulation of senescent human skin fibroblasts, present in pre-senescent cultures, with those of young cells (9). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture, this is the expected result if abrupt shortening of a single telomere triggers the onset of cell senescence. References: 1. Rubelj I., Vondra ek Z., J. theor. Biol. 1999; 197:425-438. 2. Makarov, V. L., Hirose, Y., Langmore, J. P., Cell 1997; 88:657-666. 3. nt human skin fibroblasts, present in pre-senescent cultures, with those of young cells (9). Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture, this is the expected result if abrupt shortening of a single telomere triggers the onset of cell senescence. References: 1. Rubelj I., Vondra ek Z., J. theor. Biol. 1999; 197:425-438. 2. Makarov, V. L., Hirose, Y., Langmore, J. P., Cell 1997; 88:657-666. 3. Griffith J.D., Comeau L., Rosenfield S. et al., Cell 1999; 97:503-514. 4. Wang R. C., Smogorzewska A., de Lange T., Cell 2004;119:355-368. 5. Baird D., Rowson J., Wynford-Thomas D. et al., Nature Genet. 2003;33:203 207. 6. Regev A., Cohen S., Cohen E., et al. Oncogene 1998;17:3455-3461. 7. Hemann M. T., Strong M. A., Hao L.Y. et al. Cell 2001;107:67-77. 8. Smith J. R., Whitney R. G., Science 1980;207:82-84. 9. Ferenac M., Polan ec D., Huzak M., et al. 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