Transient expression of wild-type or biologically inactive telomerase allows the formation of artificial telomeres in mortal human cells.

Telomere seeding, the formation of artificial telomeres, has been routinely successful in immortalized but not normal human cells. We compared seeding efficiencies in preimmortal and immortal SV40-transformed cells using plasmid telomeres with T(2)AG(3) tracts of 1600 and 3200 bp. Seeding occurred only in immortal cells, indicating that transformed preimmortal cells behave like normal cells vis à vis formation of new telomeres and that T-antigen inhibition of cellular checkpoints is insufficient to allow seeding. Telomerase is active in immortal but not preimmortal cells, which do not express the reverse transcriptase hTERT. Upon transient expression of hTERT, seeds with 1600 bp of T(2)AG(3) formed telomeres in preimmortal cells. Comparable seeding efficiencies were obtained with wild-type hTERT or the HA-tagged protein that is catalytically active but unable to maintain endogenous telomeres. No seeding occurred with catalytically inactive hTERT. Given that telomerase expression was transient and that longer seeds did not form telomeres in the absence of the enzyme, seeding may not be elicited merely by elongation of telomeric sequences. We propose that modification of the telomeric terminus by telomerase may contribute to telomere seeding by leading to formation of a structure that impedes rejoining of this terminus with chromosomal sequences.

[1]  R. Reddel,et al.  Telomere maintenance by recombination in human cells , 2000, Nature Genetics.

[2]  B. van Steensel,et al.  Control of Human Telomere Length by TRF1 and TRF2 , 2000, Molecular and Cellular Biology.

[3]  J. Hodgkin,et al.  MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans , 2000, Nature.

[4]  B. Lehnert,et al.  DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. Bacchetti,et al.  Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres. , 1999, Nucleic acids research.

[6]  E. Blackburn,et al.  Ku is associated with the telomere in mammals. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[7]  W. Hahn,et al.  Inhibition of telomerase limits the growth of human cancer cells , 1999, Nature Medicine.

[8]  M. Robinson,et al.  Telomere shortening and apoptosis in telomerase-inhibited human tumor cells. , 1999, Genes & development.

[9]  T. de Lange,et al.  Ku Binds Telomeric DNA in Vitro * , 1999, The Journal of Biological Chemistry.

[10]  J. Griffith,et al.  Mammalian Telomeres End in a Large Duplex Loop , 1999, Cell.

[11]  E. Blackburn,et al.  Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Broccoli,et al.  p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. , 1999, Science.

[13]  J. Shay,et al.  Telomerase activity does not always imply telomere maintenance. , 1999, Biochemical and biophysical research communications.

[14]  W. Hahn,et al.  Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  David Shore.  Telomeres--Unsticky Ends , 1998, Science.

[16]  V. Lundblad,et al.  The telomerase reverse transcriptase: components and regulation. , 1998, Genes & development.

[17]  R. Weinberg,et al.  Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase , 1998, Oncogene.

[18]  S. Benchimol,et al.  Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span , 1998, Current Biology.

[19]  M. Robinson,et al.  Reconstitution of human telomerase activity in vitro , 1998, Current Biology.

[20]  C. Harley,et al.  Extension of life-span by introduction of telomerase into normal human cells. , 1998, Science.

[21]  M. Bass,et al.  Human telomerase contains evolutionarily conserved catalytic and structural subunits. , 1997, Genes & development.

[22]  S. Bacchetti,et al.  Reprogramming of telomerase by expression of mutant telomerase RNA template in human cells leads to altered telomeres that correlate with reduced cell viability , 1997, Molecular and cellular biology.

[23]  D. Bowtell,et al.  Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. , 1997, Human molecular genetics.

[24]  D. Broccoli,et al.  Human telomeres contain two distinct Myb–related proteins, TRF1 and TRF2 , 1997, Nature Genetics.

[25]  E. Gilson,et al.  Telomeric localization of TRF2, a novel human telobox protein , 1997, Nature Genetics.

[26]  R. Weinberg,et al.  hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization , 1997, Cell.

[27]  C B Harley,et al.  Telomerase catalytic subunit homologs from fission yeast and human. , 1997, Science.

[28]  R. Wellinger,et al.  The DNA structures at the ends of eukaryotic chromosomes. , 1997, European journal of cancer.

[29]  Gregg B. Morin,et al.  Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT , 1997, Nature Genetics.

[30]  C. Farr.  Mammalian telomeres and chromosome fragmentation , 1996 .

[31]  H. Erdjument-Bromage,et al.  A Human Telomeric Protein , 1995, Science.

[32]  C B Harley,et al.  Specific association of human telomerase activity with immortal cells and cancer. , 1994, Science.

[33]  F. Graham,et al.  An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Yanowitz,et al.  Stringent sequence requirements for the formation of human telomeres. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Harley,et al.  Telomerase activity in human ovarian carcinoma. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. Murnane,et al.  Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break , 1993, Molecular and cellular biology.

[37]  Michael A. Barnett,et al.  Telomere directed fragmentation of mammalian chromosomes , 1993, Nucleic Acids Res..

[38]  W. Brown,et al.  Targeted breakage of a human chromosome mediated by cloned human telomeric DNA , 1992, Nature Genetics.

[39]  P. Goodfellow,et al.  Telomere–associated chromosome fragmentation: applications in genome manipulation and analysis , 1992, Nature Genetics.

[40]  J. Steitz,et al.  Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. , 1992, The EMBO journal.

[41]  J. Shay,et al.  A role for both RB and p53 in the regulation of human cellular senescence. , 1991, Experimental cell research.

[42]  E. Hara,et al.  Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1. , 1991, Biochemical and biophysical research communications.

[43]  P. Goodfellow,et al.  Functional reintroduction of human telomeres into mammalian cells. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[44]  S. Bacchetti,et al.  Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells. , 1991, Virology.

[45]  C. Harley,et al.  Telomeres shorten during ageing of human fibroblasts , 1990, Nature.

[46]  Carol W. Greider,et al.  Identification of a specific telomere terminal transferase activity in tetrahymena extracts , 1985, Cell.

[47]  A M Olovnikov,et al.  A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. , 1973, Journal of theoretical biology.

[48]  J. D. Watson.  Origin of Concatemeric T7DNA , 1972 .

[49]  Olovnikov Am.  Principle of marginotomy in template synthesis of polynucleotides , 1971 .