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J. Biol. Chem., Vol. 277, Issue 32, 28609-28617, August 9, 2002
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From the Life Sciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720
Most human cells do not express telomerase and
irreversibly arrest proliferation after a finite number of divisions
(replicative senescence). Several lines of evidence suggest that
replicative senescence is caused by short dysfunctional telomeres,
which arise when DNA is replicated in the absence of adequate
telomerase activity. We describe a method to reversibly bypass
replicative senescence and generate mass cultures that have different
average telomere lengths. A retrovirus carrying hTERT
flanked by excision sites for Cre recombinase rendered normal human
fibroblasts telomerase-positive and replicatively immortal.
Superinfection with retroviruses carrying wild-type or mutant forms of
TIN2, a negative regulator of telomere length, created
telomerase-positive, immortal populations with varying average telomere
lengths. Subsequent infection with a Cre-expressing retrovirus
abolished telomerase activity, creating mortal cells with varying
telomere lengths. Using these cell populations, we show that, after
hTERT excision, cells senesce with shorter telomeres than
parental cells. Moreover, long telomeres, but not telomerase, protected
cells from the loss of division potential caused by ionizing radiation.
Finally, although telomerase-negative cells with short telomeres
senesced after fewer doublings than those with long telomeres, telomere
length per se did not correlate with senescence. Our
results support a role for telomere structure, rather than length, in
replicative senescence.
Reversible Manipulation of Telomerase Expression and Telomere
Length
IMPLICATIONS FOR THE IONIZING RADIATION RESPONSE AND REPLICATIVE
SENESCENCE OF HUMAN CELLS*
*
This work was supported by the Ellison Medical Foundation,
by Grant AG17242 from the NIA, National Institutes of Health (to J. C.), and by Grant 7KB-0151 from the University of California Breast
Cancer Research Program (to S. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Lawrence Berkeley
National Laboratory, 1 Cyclotron Rd., Mailstop 84-171, Berkeley, CA
94720. Tel.: 510-486-4416; Fax: 510-486-4545; E-mail:
jcampisi@lbl.gov.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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