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 plas mid telomeres with T(2)AG(3) tracts of 1600 and 3200 bp. Seeding occurred o nly in immortal cells, indicating that transformed preimmortal cells behave like normal cells vis a vis formation of new telomeres and that T-antigen inhibition of cellular checkpoints is insufficient to allow seeding. Telome rase 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 p rotein that is catalytically active but unable to maintain endogenous telom eres. No seeding occurred with catalytically inactive hTERT. Given that tel omerase expression was transient and that longer seeds did not form telomer es in the absence of the enzyme, seeding may not be elicited merely by elon gation of telomeric sequences. We propose that modification of the telomeri c terminus by telomerase may contribute to telomere seeding by leading to f ormation of a structure that impedes rejoining of this terminus with chromo somal sequences. (C) 2001 Academic Press.