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Telomeres and Senescence - The History, The Experiment, The Future

Telomeres and Senescence - The History, The Experiment, The Future

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Published by: James Clement on Aug 08, 2010
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Telomeres and senescence:
The history, the experiment, the future
Carol W. Greider
Telomere length has been proposed to signal entry intocellular senescence. Expression of the humantelomerase subunit, hTERT, in primary cells now showsthat these cells bypass senescence.
Address: Department of Molecular Biology and Genetics, JohnsHopkins University School of Medicine, 617 Hunterian Building, 725N. Wolfe Street, Baltimore, Maryland 21205, USA.E-mail: cgreider@bs.jhmi.edu
Current Biology
1998, 8:R178–R181http://biomednet.com/elecref/09609822008R0178 © Current Biology Ltd ISSN 0960-9822
What do chickens, human skin, subway trains andbacteriophage have in common? This unlikely cast of players sets the stage upon which the model that telomerelength signals cellular senescence was built. Two recentpapers provide evidence that telomere shortening inhuman cells does indeed signal cells to enter ‘senescence’[1,2]. I shall examine the development of the hypothesis,the recent evidence supporting it, and its implications.
The cells
In 1908, Alexis Carrel, a Nobel prize-winning surgeon,became interested in the growth of cells in culture. In1912, he established a culture of chick heart fibroblastcells, which he then grew in the laboratory for 34 years.This work led to the general acceptance of the notion thatvertebrate cells can divide indefinitely in culture. As indi-vidual cells were immortal, Carrel reasoned that aging is“an attribute of the multicellular body as a whole”(reviewed in [3]).In 1961, this concept of cell immortality was challengedby experiments published by Hayflick and Moorehead[4]. They found that fibroblast cultures derived fromhuman skin would divide 40 to 50 times, and then stopand “undergo senescence”. Further work showed thatcells from older people underwent fewer divisions thancells from younger people, suggesting that it was the totalnumber of divisions since birth, not total divisions inculture, that was important [5].When subsequent work showed that Carrel’s immortalchicken cell cultures were not reproducible, Hayflick’ssenescence model eventually became accepted (reviewedin [3]). Two important questions emerged from thenascent field of cellular senescence. First, what is the roleof cellular senescence in humans? Does the limitedcapacity of cells to divide relate to human aging, or is it amechanism to prevent tumor formation (reviewed in [6])?And second, what tells cells to stop dividing?
The phage and the train
In 1972, James D. Watson was preparing his lecture for thebiochemistry course at Harvard. In thinking about theobservation that bacteriophage T7 occurs in
 Escherichia coli 
as long concatemers, Watson recognized that the end-to-end joining of individual genomes could allow the com-plete replication of the linear bacteriophage genome (J. D.Watson, personal communication). Watson knew thatDNA polymerases act in the 5
to 3
direction and requirean RNA primer. He recognized that, when the polymerasereached the end of a linear DNA molecule, there wouldbe a problem in completing replication. Linear phagegenomes can avoid this ‘end replication problem’ by joining multiple genomes before replication, reducing thenumber of actual ends and thus minimizing the damageby incomplete replication [7].Far away from Harvard at the Gamelaya Institute in thethen Soviet Union, Alexei Olovnikov was also thinkingabout DNA molecules – while waiting for a subway train[8]. When he heard the train approaching, he suddenlysaw a parallel with DNA replication. If the train enginewere replicating the DNA track, the first bit of DNAwould not be replicated as it would be underneath theengine [8]. Interestingly, although both models recog-nized a problem with DNA ends, Olovnikov’s modelpredicted a problem at the beginning of DNA replication,whereas Watson’s predicted a problem at the end.Olovnikov knew of Hayflick’s work on cellular senescenceand recognized that the end replication problem mighttrigger the Hayflick limit — too much chromosomeshortening might cause cellular senescence [9].
The telomere
In 1978, Blackburn, working in Gall’s laboratory at Yale,was interested in determining the DNA sequence thatallowed the
rDNA molecule to be main-tained as a linear chromosome. Her work led to thefinding that chromosome ends, or telomeres, are made of simple repeated DNA sequences [10]. It soon becameapparent that this motif was conserved throughout evolu-tion and that a common mechanism might exist in eukary-otes for the maintenance of telomeres. In 1984, workingin Blackburn’s laboratory, I identified an enzyme, telom-erase, that added telomere repeats onto chromosomeends. We suggested that telomerase would compensatefor the incomplete replication of chromosome ends. Thiswould explain the telomere length maintenance seen inorganisms such as
and yeast [11]. Telomeraseis an unusual polymerase; it contains an integral RNAcomponent that provides the template for synthesis of telomere repeats [12].
The connection
Insight into the molecular structure of the ends of humanchromosomes came from using a probe representing asequence on the Y chromosome that was near enough tothe telomere to follow the terminal chromosome fragment.Using this probe, Cooke found that telomeres in germlinetissue (sperm) were longer than telomeres in somatictissue (blood cells). He speculated that telomere length istissue-specific and that telomere length shortens betweengermline and somatic tissues [13]. Subsequently, the iden-tification of the simple-sequence telomere repeat fromhuman cells [14] allowed a simple analysis of telomerelength and cell senescence. Harley, Futcher, and I thenfound that telomeres indeed shorten as normal humanfibroblasts grow in culture [15]. Telomeres were alsoshorter in skin samples from older people than fromyounger people, suggesting that the shortening was not atissue culture phenomenon [15,16]. At about the sametime, Hastie
et al.
[16] and de Lange
et al.
[17] found thattumor samples had shorter telomeres than adjacent normaltissue, providing the first suggestion of a connectionbetween telomeres and cancer.
The synthesis
In the early 1990s, a model emerged which had two parts:one that related telomeres to cell senescence, and the otherthat linked telomeres, telomerase and cancer (reviewed in[18]; Figure 1). The model stated that telomeres shortenduring growth of primary fibroblasts, because of the endreplication problem and the absence of telomerase.Germline cells, in contrast, have telomerase and thus main-tain telomere length. Telomere shortening signals cells tosenesce. Senescence, however, can be bypassed by expres-sion of viral oncogenes. During this extended life span,telomeres continue to shorten until the cell culture reachesa crisis. At crisis many cells die. The few cells that survive,can divide indefinitely. These cells have activated telom-erase and maintain or even elongate telomeres. With thedemonstration that tumor cells, like immortal cells inculture, express the enzyme, telomerase was proposed as apotential target for anti-cancer therapy [15]. The recentreports by Bodner
et al.
[1] and Vaziri and Benchimol [2]provide strong evidence for the first part of this hypothesis— that telomere shortening is a mechanism that limits celldivision capacity.
The experiment
To test whether telomere length signals cell senescence,several groups have sought to elongate telomeres artifi-cially and determine whether cellular life-span would beextended. The recent isolation of the catalytic componentof telomerase allowed telomere length to be altered inhuman cells. The catalytic component of 
telom-erase was isolated by Lingner in Cech’s laboratory [19],and the sequence of the 123kDa protein was shown to besimilar to Est2p of budding yeast, which is required fortelomere maintenance [20]. The homology of p123 andEst2p to reverse transcriptase motifs allowed the elegantdemonstration that these proteins represent the catalyticsubunits of telomerase [21]. When a human cDNA frag-ment encoding a protein sequence similar to p123 wasdeposited in Genbank, several groups quickly cloned thecorresponding gene,
[22–25] and showed that itencodes an essential component of human telomerase[24,26]. This set the stage for testing the role of telomeresin cell senescence by forcing telomerase expression inprimary human cells.The cloning of 
prompted a flurry of experimentsthat resulted in a number of remarkable findings. Theinitial surprise was that simply expressing
inprimary cells led to telomerase activity [2,26,27]. Althoughtelomerase is composed of both protein and RNA, theRNA component is often present even in cells that do notnormally have activity [28]. Apparently this level of RNAis sufficient for telomerase activity when hTERT ispresent. The next surprise was that the presence of telomerase activity in primary cells was sufficient fortelomere elongation [1,2]. Although this was the resultthat many had hoped for, recent evidence suggested thatthe availability of telomere-binding proteins might limittelomere growth [29]. At last the experiment could bedone: the frequency of senescent primary cell clones wascompared between cell that had telomerase activity and
Figure 1
The telomere hypothesis of cell senescence and immortalizationproposes that:
Telomeres are maintained in the germline becauseof the presence of telomerase.
Telomeres shorten due to the endreplication problem in many somatic cells that do not expresstelomerase.
Agents such as oncogenes which extend cell life spanbypass the senescence signal.
Cultures containing cells with shorttelomeres undergo a growth crisis where many cells die.
Immortalcell clones that emerge from crisis have activated telomerase and somaintain, or even lengthen, telomeres.
(1) Germline:
telomeraseon; telomeres maintained
(2) Soma:
telomeraseoff; telomeres shorten
(5) Immortal cells:
telomerase on;telomeres maintained
(3) Extendedlife span(4) Crisis
CrisisSenescenceCell divisions
   T  e   l  o  m  e  r  e   f  r  a  g  m  e  n   t   l  e  n  g   t   h   (   k   b   )
Current Biology
Current Biology
, Vol 8 No 5
those that did not. Cells with longer telomeres did notundergo senescence, whereas those with short telomeresdid [1,2]. Telomere length is therefore one criterion thatdetermines entry into cell senescence (Figure 2).
The interpretation and the future
Like all good experiments, this discovery raises more ques-tions than it answers. At the simplest level there aredetailed questions about how telomere length might signalentry into senescence. What is measured? The length of the shortest telomere? The average telomere length, or thetotal number of telomere repeats? How is the signal sentand what are the pathways that respond? The medicalimplications of the experiment are also important toexplore. Extending the life span of primary cells could sig-nificantly enhance the ability to grow cells
ex vivo
for autol-ogous transplantation. This technique could be useful ingrowing extra skin cells for burn victims, for example.Telomerase has also been proposed for gene therapy toextend the life span of cells
in vivo
[1]. Before such app-roaches are implemented, it will be important to under-stand better why cells senesce. If cellular senescenceindeed is a mechanism of tumor suppression (reviewed in[6]), activation of telomerase might pre-dispose cells totumor formation by overcoming one of the many stepsrequired for transformation.Recent experiments have shown that mouse cells lackingtelomerase become transformed and cause tumors [30].Does this suggest that telomerase activation will not play arole in the transformation of human cells? Probably not,because there are important differences between humanand mouse cells. First the length of telomere repeatsequence on mouse chromosomes is two-to-five timeslonger than human telomere tracts [31–33], suggestingmouse cells may be less sensitive than human cells totelomere shortening. Second, mouse cells immortalizemuch more readily than human cells. Humans, as a long-lived species, may have evolved have additional mecha-nisms to protect against cell immortalization and cancer.Human cells may use telomeres to signal senescence andthus limit cells growth, while mouse cells do not. With therecent advances in understanding the basic structure andsynthesis of telomeres, it is now possible to test whetherextending cell life span might predispose human cells totumor formation.
I thank members of the Greider laboratory research group and Titia deLange, Jef Boeke and Nathaniel Comfort for helpful discussions and usefulcomments on the manuscript. Work in my lab is supported by the NationalInstitutes of Health.
1.Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu C, Morin GB,Harley CB, Shay JW, Lichtsteiner S, Wright WE:
Extension of lifespan by introduction of telomerase into normal human cells.
349-352.2.Vaziri H, Benchimol S:
Reconstitution of telomerase activity innormal human cells leads to elongation of telomeres andextended replicative life span.
Curr Biol 
279-282.3.Witkowski J:
The myth of cell immortality.
Trends Biochem Sci 
258-260.4.Hayflick L, Moorhead PS:
The serial cultivation of human diploidstrains.
Exp Cell Res 
585-621.5.Hayflick L:
The limited
in vitro 
lifetime of human diploid strains.
Exp Cell Res 
614-636.6.Campisi J:
Replicative senescence: an old lives’ tale?
497-500.7.Watson JD:
Origin of concatameric T4 DNA.
Nature New Biol 
197-201.8.Olovnikov AM:
Telomeres, telomerase, and aging: origin of thetheory.
Exp Gerontol 
443-448.9.Olovnikov AM:
A theory of marginotomy.
J Theor Biol 
181-190.10.Blackburn EH, Gall JG:
A tandemly repeated sequence at thetermini of the extrachromosomal ribosomal RNA genes in
J Mol Biol 
33-53.11.Greider CW, Blackburn EH:
Identification of a specific telomereterminal transferase activity in
405-413.12.Greider CW, Blackburn EH:
A telomeric sequence in the RNA of
telomerase required for telomere repeat synthesis.
Figure 2
In cell strains that do not express telomerase, telomere repeats arelost during cell division. When telomeres reach a certain length, asignal is sent to initiate a program of cellular senescence.
In cellstrains that are forced to make telomerase by expression of
,telomere length does not shorten (it even lengthens, but this is notshown). These cells do not enter senescence, even after undergoingmore cell doubling than their telomerase-negative cousins.
Cells withouttelomerase
No senescence
Cells withtelomerase
SenescenceTelomere repeat
   C  e   l   l   d   i  v   i  s   i  o  n  s
Current Biology

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