Extension of Life-Span by Introduction of Telomerase into Normal

Human Cells
Andrea G. Bodnar et al.
Science 279, 349 (1998);
DOI: 10.1126/science.279.5349.349

This copy is for your personal, non-commercial use only.

If you wish to distribute this article to others, you can order high-quality copies for your
colleagues, clients, or customers by clicking here.

Downloaded from www.sciencemag.org on June 1, 2012

Permission to republish or repurpose articles or portions of articles can be obtained by
following the guidelines here.

The following resources related to this article are available online at

www.sciencemag.org (this information is current as of June 1, 2012 ):

Updated information and services, including high-resolution figures, can be found in the online
version of this article at:
http://www.sciencemag.org/content/279/5349/349.full.html
This article cites 29 articles, 12 of which can be accessed free:
http://www.sciencemag.org/content/279/5349/349.full.html#ref-list-1
This article has been cited by 2331 article(s) on the ISI Web of Science
This article has been cited by 100 articles hosted by HighWire Press; see:
http://www.sciencemag.org/content/279/5349/349.full.html#related-urls
This article appears in the following subject collections:
Cell Biology
http://www.sciencemag.org/cgi/collection/cell_biol

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright
1998 by the American Association for the Advancement of Science; all rights reserved. The title Science is a
registered trademark of AAAS.
 RESEARCH ARTICLES
causes cellular senescence.
Extension of Life-Span by Introduction of telomerase into nor-
mal human cells. To determine if telo-
Introduction of Telomerase into merase expression increases cell life-span,
we transfected hTRT2 normal cells with
Normal Human Cells two different hTRT expression constructs.
One construct was engineered for in-
creased translational efficiency by removal
Andrea G. Bodnar,* Michel Ouellette,* Maria Frolkis, of the 59 and 39 untranslated regions of
Shawn E. Holt, Choy-Pik Chiu, Gregg B. Morin, hTRT and creation of a Kozak consensus
Calvin B. Harley, Jerry W. Shay, Serge Lichtsteiner,† sequence. This engineered hTRT cDNA
was cloned downstream of the MPSV pro-
Woodring E. Wright† moter (19). The second construct consist-
ed of the complete (native) hTRT cDNA
Normal human cells undergo a finite number of cell divisions and ultimately enter a cloned downstream of the SV40 promoter
nondividing state called replicative senescence. It has been proposed that telomere in pZeoSV (19). In the first experiments,
shortening is the molecular clock that triggers senescence. To test this hypothesis, two we compared the life-span of stable clones
telomerase-negative normal human cell types, retinal pigment epithelial cells and fore- transfected with MPSV-hTRT versus
skin fibroblasts, were transfected with vectors encoding the human telomerase catalytic “vector only” clones, and in the second,
subunit. In contrast to telomerase-negative control clones, which exhibited telomere we compared the life-span of activity-
shortening and senescence, telomerase-expressing clones had elongated telomeres, positive and activity-negative stable

Downloaded from www.sciencemag.org on June 1, 2012

divided vigorously, and showed reduced staining for b-galactosidase, a biomarker for clones containing integrated SV40-hTRT
senescence. Notably, the telomerase-expressing clones have a normal karyotype and constructs.
have already exceeded their normal life-span by at least 20 doublings, thus establishing hTRT2 normal retinal pigment epithe-
a causal relationship between telomere shortening and in vitro cellular senescence. The lial cells (RPE-340) were transfected with
ability to maintain normal human cells in a phenotypically youthful state could have the MPSV-hTRT vector at population
important applications in research and medicine. doubling (PD) 37, and 27 of the 39 result-
ant stable clones (69%) expressed telo-
merase activity. BJ foreskin fibroblasts were
transfected with the MPSV-hTRT vector
Normal human diploid cells placed in cul- expressed in most human somatic tissues at PD 58, and 3 of the 22 stable clones
ture have a finite proliferative life-span and (13, 14), and telomere length is signifi- (14%) expressed telomerase activity. Re-
enter a nondividing state termed senes- cantly shorter (15). The telomere hypoth- verse transcriptase–polymerase chain reac-
cence, which is characterized by altered esis of cellular aging (16) proposes that tion experiments demonstrated that the
gene expression (1, 2). Replicative senes- cells become senescent when progressive hTRT mRNA originated from the trans-
cence is dependent upon cumulative cell telomere shortening during each division fected cDNA and not the endogenous
divisions and not chronologic or metabolic produces a threshold telomere length. gene (20). Telomerase activity, measured
time, indicating that proliferation is limited The human telomerase reverse tran- relative to that in the lung cancer–derived
by a “mitotic clock” (3). The reduction in scriptase subunit (hTRT) has been cloned human cell line H1299, ranged from 65 to
proliferative capacity of cells from old do- (17). We recently demonstrated that telo- 360% in the RPE clones (Fig. 1) and 86 to
nors and patients with premature aging syn- merase activity can be reconstituted by 95% in the BJ clones. This range of
dromes (4), and the accumulation in vivo of transient expression of hTRT in normal telomerase activity is similar to that ob-
senescent cells with altered patterns of gene human diploid cells, which express low served for tumor cell lines (13). Thirty-
expression (5, 6), implicate cellular senes- levels of the template RNA component of three RPE clones and 24 BJ clones trans-
cence in aging and age-related pathologies telomerase (hTR) but do not express fected with the control plasmid were also
(1, 2). hTRT (18). This provided the opportuni- isolated; RPE clones that generated suffi-
Telomere loss is thought to control ty to manipulate telomere length and test cient cells for the TRAP assay (n 5 15)
entry into senescence (7–10). Human the hypothesis that telomere shortening (Fig. 1) and control BJ clones (n 5 15)
telomeres consist of repeats of the se-
quence TTAGGG/CCCTAA at chromo-
some ends; these repeats are synthesized by Fig. 1. Telomerase activity in stable
99
C 7
3
18
20

12
0

the ribonucleoprotein enzyme telomerase RPE clones. Stable human RPE

PD

T3

T7

T8

T1

T4

T5
C

(11, 12). Telomerase is active in germline Cell # 500 500 500 500 100 500 100 500 100 500 100 500 100 500 0 100 500 clones obtained by transfection
cells and, in humans, telomeres in these with a control vector (clone num-
cells are maintained at about 15 kilobase bers prefixed with “C”) or with a
vector expressing the hTRT cDNA
pairs (kbp). In contrast, telomerase is not (“T” clones) were analyzed for te-
lomerase activity by the TRAP as-
A. G. Bodnar, M. Frolkis, C.-P. Chiu, G. B. Morin, C. B.
Harley, and S. Lichtsteiner are at Geron Corporation, 230 say (19). “PD37” represents the cell
Constitution Drive, Menlo Park, CA 94025, USA. M. population at the time of transfec-
Ouellette, S. E. Holt, J. W. Shay, and W. E. Wright are in tion. The number of cells assayed
the Department of Cell Biology and Neuroscience, Uni- for each clone is indicated above
versity of Texas Southwestern Medical Center, 5323 Har- each lane. “IC” is the internal control
ry Hines Boulevard, Dallas, T X 75235 –9039, USA.
in the TRAP assay. The positive
* These authors contributed equally to this work. IC — control was the telomerase activity
†To whom correspondence should be addressed. E-mail:
extracted from H1299 cells (20).
slichtste@geron.com; wright@utsw.swmed.edu

www.sciencemag.org z SCIENCE z VOL. 279 z 16 JANUARY 1998 349

were negative for telomerase activity. BJ by a ribonuclease protection assay, hTRT lengths to determine if the hTRT-reconsti-
fibroblasts were also transfected with the mRNA was undetectable in activity-neg- tuted telomerase acts on its normal chro-
pZeoSV-hTRT construct at PD 44. Six of ative BJ cells but readily observed in mosomal substrate (21). Telomeres in the
76 clones (8%) expressed telomerase ac- hTRT1 clones (20). hTRT2 cells decreased by 0.4 to 1.3 kbp
tivity ranging from 10 to 30% of that in Telomere lengthening in hTRT1 nor- (Fig. 2), comparable to the shortening seen
the reference H1299 cell line. As assessed mal cells. We then measured telomere in mass cultures at equivalent PDs, whereas
telomeres in the hTRT1 RPE and BJ clones
transfected with the MPSV-hTRT vector
A RPE BJ increased by 3.7 kbp (6 1.4 kbp, n 5 26)
and 7.1 kbp (6 0.3 kbp, n 5 3), respective-

C 8
PD37
C 55

5
18

T70

1
1
4
8
4
3
2
T61
2
3
6

PD
ly. Telomeres in six hTRT1 clones trans-

1
2
3
PD

T5
T6
T7
M M M M M M
T3

T8
T1
T4
T2
T2
T3
T4
T5
T6

T6
T8

C
C
—19
fected with the pZeoSV-hTRT vector, in-
creased by 0.4 kbp (6 0.3 kbp, n 5 6).
—12
—10 Because two hTRT1 clones expressing only
—8 5 to 7% relative telomerase activity (RPE
—6
clone T30 and BJ clone B13) did not main-
tain telomere length, they were considered
—4
to be functionally hTRT2 (Fig. 2B). These
results demonstrate that hTRT-reconstitut-
ed telomerase extends the endogenous telo-
meres in a normal cell.

Downloaded from www.sciencemag.org on June 1, 2012

—2 Life-span, karyotype, and phenotype.
To investigate the effect of telomerase ex-
pression on the life-span of normal cells, we
—1
compared the growth of hTRT1 and
hTRT2 clones. hTRT2 RPE clones showed
the expected slowing of growth that is as-
Fig. 2. Telomere length in stable RPE and BJ clones. (A)
Terminal restriction fragment (TRF ) length of DNA from rep-
sociated with aging in vitro, and 30 out of
resentative RPE and BJ clones (21). “C” clones are telo- 33 senesced (22) by an age typical for mass
merase-negative and “T” clones are telomerase-positive. RPE cultures (Fig. 3). In contrast, hTRT1
“PD37” and “PD58” represent cells at the time of transfection RPE clones transfected with MPSV-hTRT
for RPE and BJ cells, respectively, and “PD55” represents exceeded the mean life-span of the hTRT2
the RPE mass culture at the time of senescence. “M” indi- clones by ;20 doublings (P , 10224; Stu-
cates molecular size markers in kbp. (B) Mean TRF length at dent’s T test). These clones have exceeded
the indicated population doublings of the hTRT1 (triangles) the maximal RPE life-span (PD 55 to 57),
and hTRT2 (circles) RPE clones. “T30” refers to clone T30. and continue to divide at the rate of young
The gray horizontal bar represents the mean TRF of the cell
RPEs (Fig. 3). Similarly, most of the
population at the time of transfection. The dashed horizontal
lines indicate the average TRF values for the hTRT1 and hTRT2 BJ fibroblast clones senesced or are
hTRT2 clones. (C) Mean TRF length at the indicated popu- near senescent (64 of 70 clones), whereas
lation doublings of the BJ clones transfected with pZeoSV-hTRT; designations are as in (B). “B13” refers all six of the hTRT1 clones transfected
to clone B13. Closed symbols represent cells that senesced; half-filled symbols correspond to cells near with the pZeoSV-hTRT vector exceeded
senescence (dividing less than once per week). the maximal BJ life-span (85 to 90 PD)
(Fig. 3). The average PD of these six rapidly
dividing hTRT1 clones is already 36 dou-
blings beyond the average life-span of the
70 hTRT2 clones (P , 1026). Similar re-
sults were obtained with human vascular
endothelial cells (23). Thus, expression of
functional hTRT in normal cells extends
their life-span.
Senescence-associated b-galactosidase
(SA–b-Gal) is an established biomarker as-
sociated with cellular aging (6). We stained
hTRT2 RPE clones at or near senescence
and compared the level of SA–b-Gal stain-
ing to that in hTRT1 clones that had un-
dergone a similar or greater number of cell
divisions (Fig. 4, A and B). A majority of
the cells in the hTRT2 clones showed
Fig. 3. Effect of telomerase expression on cell life-span. The proliferative status of each RPE (upper
strong staining; by contrast, few of the cells
panel) and BJ (lower panel; pZeoSV-hTRT experiment) clone is shown. The hTRT1 clones (triangles) and
the hTRT2 clones (circles) are plotted (35). Closed symbols represent senescent clones (dividing less in hTRT1 clones at equivalent or greater
than once per 2 weeks); half-filled symbols correspond to cells near senescence (dividing less than once PD showed staining. The cells of the
per week); open symbols represent clones dividing more than once per week. The shaded vertical area hTRT2 clones that had stopped dividing
indicates the typical PD range where the mass population of cells senesce. Dashed vertical lines exhibited SA–b-Gal staining levels equiv-
represent the mean PD of: (a) the hTRT2 and (b) the hTRT1 clones. alent to that observed in senescent mass

350 SCIENCE z VOL. 279 z 16 JANUARY 1998 z www.sciencemag.org

 RESEARCH ARTICLES
cultures. Their large size and increased ratio Telomere homeostasis is likely to result factors that may affect the functional level
of cytoplasm:nucleus also indicates that the from a balance of lengthening and shorten- of telomerase. This hypothesis is supported
clones had senesced (Fig. 4A). The remain- ing activities. Although certain proteins in by our finding that hTRT1 clones derived
der of the slowly dividing hTRT2 clones yeast are thought to facilitate the interac- from different cell types and transfected
exhibited SA–b-Gal staining typical of tion of telomerase with the telomere (25), with different vectors showed marked dif-
cells close to senescence. The same result our results indicate that if analogous mam- ferences in telomere lengths.
was found for fibroblasts: Six of six hTRT1 malian factors are required, they are already Certain stem cells or germline popula-
clones showed low levels of staining typical present in hTRT2 human cells. The telom- tions are telomerase positive (13, 27, 28)
of young fibroblast cultures, whereas all of erase catalytic subunit produces the length- and have long or indefinite life-spans, illus-
the hTRT2 clones showed elevated SA–b- ening activity, but other factors including trating that telomerase expression per se is
Gal staining (Fig. 4C). Detailed G-banding telomere binding proteins such as hTRF-1 not oncogenic. Cellular transformation with
of two hTRT1 RPE clones and two hTRT1 and -2 (26) might be involved in establish- viral oncoproteins can also extend cell life-
BJ clones revealed that the cells had the ing a telomere length equilibrium. Very low span, but through mechanisms that reduce
normal complement of 46 chromosomes levels of telomerase activity, such as that checkpoint control, increase genomic insta-
and no abnormalities (24). hTRT1 cells exhibited by RPE clone T30 and BJ clone bility, and fail to prevent telomere loss (29,
with an extended life-span therefore appear B13, are apparently insufficient to prevent 30). We have not observed any gross pheno-
to have a normal karyotype and phenotype telomere shortening. This is consistent with typic or morphological characteristics of
similar to young cells. the observation that stem cells have low but transformed cells (such as loss of contact
Implications. Our results indicate that detectable telomerase activity, yet continue inhibition or growth in low serum) that
telomere loss in the absence of telomerase is to exhibit shortening of their telomeres might account for the extended proliferative
the intrinsic timing mechanism that con- throughout life (27). Thus, we believe that capacity of the hTRT1 cells. The normal

Downloaded from www.sciencemag.org on June 1, 2012

trols the number of cell divisions prior to a threshold level of telomerase activity is karyotype and the absolute correlation be-
senescence. The long-term effects of exog- required for life-span extension. Promoter tween extended life-span and telomerase ac-
enous telomerase expression on telomere strength, structure of untranslated regions, tivity suggest that stochastic mutagenesis
maintenance and the life-span of these cells site of integration, levels of hTR and does not account for the life-span extension.
remain to be determined in studies of longer hTRT, and telomere- or telomerase-associ- Cellular senescence is believed to con-
duration. ated proteins in specific cell types are all tribute to multiple conditions in the elderly

Fig. 4. SA–b-galactosidase staining of stable clones. (A) Bright-field photomicrograph

of representative RPE hTRT1 and hTRT2 clones stained for SA–b-Gal (6). Clones T8
(PD60) and T 71 (PD60) are hTRT1; clones C22 (PD54) and C23 (PD56) are hTRT2.
Scale bar, 100 mm. (B) SA–b-Gal staining at the indicated population doublings of
RPE clones. Each point represents one clone. hTRT1 clones (triangles) and hTRT2
clones (circles) are plotted. Closed symbols represent senescent clones (dividing less
than once per 2 weeks), half-filled symbols correspond to cells near senescence
(dividing less than once per week); open symbols represent clones dividing at least
once per week. (C) SA–b-Gal staining at the indicated population doublings of BJ
clones; designations are as in (B).

www.sciencemag.org z SCIENCE z VOL. 279 z 16 JANUARY 1998 351

that could in principle be remedied by cell Am. J. Hum. Genet. 52, 661 (1993). weeks; near-senescence is defined as less than one
8. N. D. Hastie et al., Nature 346, 866 (1990); W. E. PD per week. Young BJ and RPE cells typically dou-
life-span extension in situ. Examples in- Wright and J. W. Shay, Exp. Gerontol. 27, 383 ble in 1 to 2 days.
clude atrophy of the skin through loss of (1992); W. E. Wright and J. W. Shay, Trends Cell Biol. 23. A. G. Bodnar et al., unpublished data.
extracellular matrix homeostasis in dermal 5, 293 (1995); R. B. Effros et al., AIDS 10, F17 (1996); 24. Chromosomes were analyzed by G-banding with
D. Wynford-Thomas, Oncology Res. 8, 387 (1996). trypsin and Wright’s stain (GT W) by the Clinical Cy-
fibroblasts (31); age-related macular degen- 9. E. Chang and C. B. Harley, Proc. Natl. Acad. Sci. togenetics Laboratory, Stanford Health Services
eration caused by accumulation of lipofus- U.S.A. 92, 11190 (1995). (RPE clones) and the Cytogenetics Laboratory, Uni-
cin and downregulation of a neuronal sur- 10. N.-P. Weng, B. L. Levine, C. H. June, R. J. Hodes, versity of Texas Southwestern Medical Center (BJ
vival factor in RPE cells (32); and athero- Proc. Natl. Acad. Sci. U.S.A. 92, 11091 (1995). clones).
11. J. Lingner et al., Science 276, 561 (1997 ). 25. L. M. C. Konkel et al., Proc. Natl. Acad. Sci. U.S.A.
sclerosis caused by loss of proliferative ca- 12. C. W. Greider and E. H. Blackburn Nature 337, 331 92, 5558 (1995); P. W. Greenwell et al., Cell 82, 823
pacity and overexpression of hypertensive (1989); J. Feng et al., Science 269, 1236 (1995). (1995); T. Lendvay, D. Morris, J. Sah, B. Balasubra-
and thrombotic factors in endothelial cells 13. N. W. Kim et al., Science 266, 2011 (1994). manian, V. Lundblad, Genetics 144, 1399 (1996); D.
14. J. W. Shay and S. Bacchetti, Eur. J. Cancer 33, 777 Wotton and D. Shore, Genes Dev. 11, 748 (1997 ); S.
(9, 33). Extended life-span cells also have (1997 ). Marcand, E. Gilson, D. Shore, Science 275, 986
potential applications ex vivo. Cloned nor- 15. H. J. Cooke and B. A. Smith, Cold Spring Harb. Lab. (1997 ).
mal diploid cells could replace established Symp. Quant. Biol. 51, 213 (1986); T. de Lange et al., 26. D. Broccoli, A. Smogorzewska, L. Chong, T. de
Mol. Cell Biol. 10, 518 (1990). Lange, Nature Genet. 17, 231 (1997 ); B. van
tumor cell lines in studies of biochemical 16. C. B. Harley, Mutat. Res. 256, 271 (1991). Steensel and T. de Lange, Nature 385, 740 (1997 ).
and physiological aspects of growth and dif- 17. T. M. Nakamura et al., Science 277, 955 (1997 ); M. 27. C.-P. Chiu et al., Stem Cells 14, 239 (1996).
ferentiation; long-lived normal human cells Meyerson et al., Cell 90, 78 (1997 ); A. Kilian et al., 28. A. Bodnar, N. W. Kim, R. B. Effros, C.-P. Chiu, Exp.
Hum. Mol. Genet. 6, 2011 (1997 ).
could be used for the production of normal 18. S. L. Weinrich et al., Nature Genet. 17, 498 (1997 ).
Cell Res. 228, 58 (1996); W. E. Wright, M. A. Pia-
tyszek, W. E. Rainey, W. Byrd, J. W. Shay, Dev.
or engineered biotechnology products; and 19. RPE-340 cells and BJ fibroblasts were cultured as Genet. 18, 173 (1996); M. Engelhardt et al., Blood
expanded populations of normal or geneti- previously described (18). In one set of experiments, 90, 182 (1997 ).
RPE and BJ cells were subjected to electroporation
cally engineered rejuvenated cells could be 29. C. M. Counter et al., EMBO J. 11, 1921 (1992); J. W.

Downloaded from www.sciencemag.org on June 1, 2012

with control vector (pBBS212) or vector encoding
used for autologous or allogeneic cell and Shay, W. E. Wright, H. Werbin, Int. J. Oncol. 3, 559
hTRT with a consensus Kozak sequence down-
(1993); J. W. Shay, W. E. Wright, D. Brasiskyte, B. A.
gene therapy. Thus the ability to extend stream of the myeloproliferative sarcoma virus
Van Der Haegen, Oncogene 8, 1407 (1993); J. W.
cellular life-span, while maintaining the (MPSV ) promoter (pGRN145) (18). After 48 hours,
Shay, B. A. Van der Haegen, Y. Ying, W. E. Wright,
transfected cells were placed into medium contain-
diploid status, growth characteristics, and ing Hygromycin-B (50 mg/ml) for 2 to 3 weeks, at
Exp. Cell Res. 209, 45 (1993).
30. J. W. Shay, O. M. Pereira-Smith, W. E. Wright, Exp.
gene expression pattern typical of young which time the concentration was reduced to 10
Cell Res. 196, 33 (1991).
normal cells, has important implications for mg/ml. Individual stable clones were selected and
analyzed for telomerase activity by the telomeric re- 31. K. Takeda, A. Gosiewska, B. Peterkofsky, J. Cell.
biological research, the pharmaceutical in- peat amplification protocol (TRAP) (13, 18). In a sep- Physiol. 153, 450 (1992); M. D. West, Arch. Derma-
dustry, and medicine. arate experiment, BJ fibroblasts were transfected tol. 130, 87 (1994).
with pZeoSV-hTRT, a derivative of pZeoSV (Invitro- 32. M. Boulton, F. Docchio, P. Dayhaw-Barker, R. Ram-
Note added in proof: As of the time of poni, R. Cubeddu, Vision Res. 30, 1291 (1990); J.
gen, Carlsbad, CA) encoding hTRT downstream of
galley proofs, virtually all of the hTRT2 the simian virus 40 (SV40) promoter. After electropo- Tombran-Tink, S. M. Shivaram, G. J. Chader, L. V.
clones were senescent or near senescent, ration, the BJ cells were cultured in zeocin (200 mg/ Johnson, D. Bok, J. Neurosci. 15, 4992 (1995).
whereas all of the hTRT1 clones continued ml). hTRT1 and hTRT2 clones from each transfec- 33. T. Kumazaki, Hiroshima J. Med. Sci. 42, 97 (1993).
tion were obtained and expanded. 34. S. Lichtsteiner, I. Savre-Train, M. Ouellette, unpub-
to divide rapidly. 20. Reverse transcriptase–polymerase chain reaction lished results.
(RT-PCR) was performed on cells transfected with 35. To accumulate doublings as rapidly as possible, we
REFERENCES AND NOTES pGRN145. Endogenous hTRT mRNA was detected shifted all six hTRT1 BJ clones and the six fastest
___________________________ with the primer set RA58 (59-GGCTGAAGTGTCA- growing hTRT2 BJ clones from 10% to 20% serum
1. L. Hayflick and P. S. Moorhead, Exp. Cell Res. 25, CAG-39) and hTRT39UTR (59-GGCTGCTGGTGTCT- and maintained them in continuous log growth as of
585 (1961); W. E. Wright, O. M. Periera-Smith, J. W. GCTCTCGGCC-39). Exogenous hTRT mRNA was PD 66 to 78 (hTRT2) or PD 74 to 80 (hTRT1). Neither
Shay, Mol. Cell. Biol. 9, 3088 (1989); S. Goldstein, detected with the primer set RA58 and RA55 (59- increased serum nor exponential growth conditions
Science 249, 1129 (1990). TCCGCACGTGAGAAT-39). RT-PCR showed that significantly extends life-span [T. Ohno, Mech. Age-
2. J. Campisi, Cell 84, 497 (1996); J. Campisi, Eur. J. the hTRT mRNA derived from the transfected cDNA, ing Devel. 11, 179 (1979); J. R. Smith and K. I.
Cancer 33, 703 (1997 ); R. G. A. Faragher and S. but not the endogenous hTRT mRNA, was present Braunschweiger, J. Cell Physiol. 98, 597 (1979)] par-
Shall, Drug Discovery Today 2, 64 (1997 ). in telomerase-positive RPE (n 5 4) and BJ (n 5 3) ticularly if instituted near the proliferative limit of the
3. R. T. Dell’Orco, J. G. Mertens, P. F. J. Kruse, Exp. clones (34). Ribonuclease protection assays were culture.
Cell Res. 77, 356 (1973); C. B. Harley and S. Gold- performed on BJ cells transfected with pZeoSV- 36. We thank M. Liao, J.-F. Train, V. Tesmer, B. Frank, Y.
stein, J. Cell. Physiol. 97, 509 (1978). hTRT and on H1299 cells (NCI-H1299, American Oei, S. Gitin, S. Wong, V. Votin, B. Lastelic, R. Ad-
4. G. M. Martin, C. A. Sprague, C. J. Epstein, Lab. Type Culture Collection). The probe corresponded to ams, S. Amshey, D. Choi, and N. Spiff-Robinson for
Invest. 23, 86 (1970); E. L. Schneider and Y. Mitsui, hTRT sequences spanning amino acids 541 to 647. excellent technical assistance; W. Andrews and R.
Proc. Natl. Acad. Sci. U.S.A. 73, 3584 (1976); E. L. The abundance of the catalytic subunit was compa- Adams for the construction of the MPSV vector; L.
Schneider and C. J. Epstein, Proc. Soc. Exp. Biol. rable to that in H1299 cells (34). Hjelmeland for the RPE-340 cells; J. Smith for the BJ
Med. 141, 1092 (1972); E. Elmore and M. Swift, J. 21. DNA isolation and TRF analyses for the RPE and BJ fibroblasts; M. Kozlowski for helpful discussions; L.
Cell Physiol. 87, 229 (1976). clones were performed essentially as described (7 ), Hayflick and T. R. Cech for critical reading of the
5. B. M. Stanulis-Praeger, Mech. Ageing Dev. 38, 1 except that in some cases the DNA was resolved on manuscript; I. Savre-Train for the ribonuclease pro-
(1987 ). 0.6% agarose gels and electroblotted to nylon mem- tection results; and W. O’Wheels for support during
6. G. P. Dimri et al., Proc. Natl. Acad. Sci. U.S.A. 92, branes (18), and for mean TRF calculations, the av- manuscript preparation. Supported in part by NIH
9363 (1995). erage of the weighted and unweighted means was grant AGO7992 (W.E.W.) and NIH postdoctoral fel-
7. C. B. Harley, A. B. Futcher, C. W. Greider, Nature used [M. Levy, R. C. Allsopp, A. B. Futcher, C. W. lowship AG05747 (S.E.H.).
345, 458 (1990); R. C. Allsopp et al., Proc. Natl. Greider, C. B. Harley, J. Mol. Biol. 225, 951 (1992)].
Acad. Sci. U.S.A. 89, 10114 (1992); H. Vaziri et al., 22. Senescence is defined as less than one PD in 2 1 December 1997; accepted 22 December 1997

352 SCIENCE z VOL. 279 z 16 JANUARY 1998 z www.sciencemag.org