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Expression of Telomerase after Retroviral Transduction Increases Lifespan of Primary Human Fibroblasts

Abstract 851

Telomeres are the distal portions of the chromosomes. They shorten with cell division. Human cells grown in culture senesce after a finite number of cell divisions (the Hayflick limit). At this timepoint, telomeres have shortened to a minimum length, which may have a role in signaling cell cycle arrest and entry to senescence. Telomerase is a reverse transcriptase that synthesizes telomeric DNA. Primary human fibroblasts do not express telomerase and senesce after 50-70 population doublings (PD) in culture. We introduced a retroviral vector containing the gene for the catalytic subunit of telomerase into primary fetal lung fibroblasts, at PD 25. Single-cell clones were established at PD 50. Control untransduced clones and transduced clones that did not express telomerase senesced, while transduced clones that expressed telomerase continued to proliferate beyond the expected time of senescence. At the present time, several clones have gone through over 100 PD and maintain contact inhibition and growth kinetics similar to those of young fibroblasts. We used the TRAP (Telomeric Repeat Amplification Protocol)assay to measure telomerase activity in each clone. TRAP is a semiquantitative PCR-based assay that is based on elongation of synthetic primers by the telomerase contained in the protein extract. We found that all nonsenescent clones express high levels of telomerase, comparable to or higher than the levels observed in tumor cell lines. We then measured the telomere length of each clone with the TRF assay (Terminal Restriction Fragments). Msp 1 and Rsa 1-digested DNA was electrophoresed, transferred to a nylon membrane and hybridized with the telomeric probe (TTAGGG)4. The resulting signal is a smear of telomeres corresponding to the telomeres of the chromosomes of the assayed cells, and a mean, a range and a peak length is calculated. Control nontransduced fibroblasts shortened their telomeres to a final length of 4 Kb at the time of senescence. Transduced clones stabilized or elongated telomeres. Most transduced clones have telomeres of 20-30 Kb. Telomere length increases at a rate of 30-300 bp/PD, characteristic of each clone. Moreover, the telomere length and the telomerase activity show a direct correlation (r=0.7). We are currently evaluating each clone for cytogenetics, cell cycle dynamics, lack of tumorigenic potential and p53 status. Extension of lifespan of human cells with this strategy could have multiple clinical and laboratory applications.

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(Spon by: Nai-Kong Cheung)

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Franco, S., MacKenzie, K., Tan, C. et al. Expression of Telomerase after Retroviral Transduction Increases Lifespan of Primary Human Fibroblasts. Pediatr Res 45, 146 (1999).

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