Replicative senescence in the control of T cell numbers

Normal somatic cells have a limited replicative capacity after which cells enter a state of replicative senescence mainly characterized by the inability to divide further. This was first demonstrated in human fibroblast cultures by Hayflick and Moorehead in 1961 [217] and has since been confirmed to be true for all normal proliferating somatic cells. Replicative senescence or the “Hayflick limit” has been found to be reached at around 20 to 30 population doublings in vitro, depending on the cell type and culture conditions [37,218-220]. Although the exact mechanisms leading to senescence remain to be elucidated, other factors can lead and/or contribute to the senescent phenotype (reviewed in [221]). Oxidative stress can lead to DNA damage [222], which in turn results in senescence [223-225] either due oxidative damage [222] or breaks in the double strand [226]. Fibroblasts defective in the DNA repair protein ku86 enter senescence prematurely [227], illustrating the importance of DNA mechanisms in cellular life span. Another mechanism thought to be involved in

1. Introduction 31

limiting the replicative capacity of normal cells is telomere shortening, shown to be a key event leading to cell growth arrest [220].

Telomere structure and function

The terminal portions of eukaryotic chromosomes are capped and stabilized by complexes of DNA and protein called telomeres [228-230]. The DNA component of telomeres is synthesized by a reverse transcriptase called telomerase [231,232]. As shown in Figure 3, telomeric DNA terminates in a 3’ overhang [233] composed of G and complementary C-rich repeats which in vertebrates is TTAGGG ranging from 5 to 15kb in length [234].

T elom eric DNA

Sub ond non- telom eric DNA

TTAGGG TTAGGGTTAGGG TTAGGGTTAGGG

A ATCCC A ATCCCA ATCCG

(5-Strand Overhang

T e lo m ere

Figure 3 Schematic representation o f the telomere and telomeric DNA.

This figure shows on the left, the schematic representation of a chromosome, where the telomere, at the terminal portion of the chromosome, is represented in black. Telomeres are complexes of DNA and protein. The DNA component of telomeres is represented on the right of the figure, showing the typical TTAGGG repeats on the 3’ overhang.

The critical importance of telomeric DNA to cellular proliferation comes from the inability of DNA polymerase to fully replicate the 5’ end of the newly synthesized lagging strand, creating what is called the “end-replication problem” leading to telomere shortening [235] (Figure 4). During DNA replication, DNA polymerase synthesizes the lagging strand by adding nucleotides onto a free 3’ end of a short RNA primer. At the end of replication, the RNA primer is removed leaving an unreplicated

1. Introduction 32

segment at the 5’ end of the new DNA strand [235,236]. With each round of cell division less of the 5’ ends of the daughter cell chromosomes are replicated [237]. The presence of the telomeric DNA at the end of chromosomes means that non-coding repeats of DNA rather then coding portions of the genome are left un-replicated. It has been established that in the absence of compensatory mechanisms, such as telomerase activity, telomeres shorten 50 to lOObp with each round of cell division [37,234,238-241]. In this way, telomere length has been described as a mitotic clock that counts the number of times a cell has divided and gives an indication of its remaining replicative capacity [236]. Once telomeres reach a critically short point the signalling machinery of normal cells triggers cell growth arrest as the cell reaches replicative senescence [229,230]. In vitro studies have shown that in human fibroblasts senescence is reached at a mean telomere length of around 5kb [242].

Telomere shortening has been shown to be cell division dependent [220,242], decreasing in in vitro cultures of fibroblasts [220,242] and lymphocytes [37,218] and with increasing age [37,243]. Premature ageing syndromes such as ataxia telangiectasia show increased numbers of telomeric fusions and accelerated telomere shortening [244], indicating a further link not only between telomere length and replicative history but also with ageing. Recently, patients with another premature age syndrome, dyskeratosis congenita, have been shown to have short telomeres and defective telomerase activity [245]. Accelerated telomere shortening has also been found in bone marrow transplant recipients, reflecting the extensive cell division that takes place to reconstitute the host [246].

1. Introduction 33 5'_ 3'" 3' P a r m td C>NA 5' Leading s tr a n d sy n th e sis 5 RNA Prim er 3' Logging s tr a n d sy n th e sis -f 3' U n-replicated 5' end on lagging s tra n d 5' + 3' 5' Lagging s tra n d : Removal o f RNA prim er and ligation o f O kakosi fra g m e n ts

3'

S '

Figure 4 Schematic representation o f the end-replication problem.

The lagging strand synthesis by DNA polymerase requires a RNA primer, that once removed, leads to the generation of an unreplicated 5’ end. Continuous divisions, if left unchecked, lead to telomere shortening.

Telomeric DNA and its associated proteins organize into a higher order structure that caps and confers stability to the chromosomes [247]. This higher order complex encompasses a structure called a t-loop in which the 3’ overhang invades the duplex [248] (see Figure 5). T-loops are thought to be generated and stabilized by telomere associated proteins that can bind both single and double stranded telomeric DNA [248].

1. Introduction 34

T-loop Formation

Figure 5 The presence o f a 3 ' end overhang leads to the formation o f a T-loop at the chromosomes.

The 3’ end overhang at the end of chromosomes has been shown to invade the duplex to form a loop allowing the stabilization of chromosomes.

Telomeric DNA-binding proteins

Two mammalian double strand-binding telomeric proteins have so far been described. Telomere Repeat Binding Factors 1 and 2 (TRFl and TRF2, respectively) [249]. These are shown in Figure 6. TRFl is a negative regulator of telomere length as its over-expression leads to telomere shortening [250,251]. It is thought to promote the three-dimensional t-loop structure by pairing double stranded repeats [252]. TRF2 has been shown to remodel the telomeric DNA into the t-loop, preventing the activation of the p53 DNA repair and damage response pathway and protecting the telomere from non-homologous end-joining [248,253,254]. Like TRFl, TRF2 over-expression leads to telomere shortening [255] possibly due to the inability of telomerase to access the telomere.

Mammalian single strand-binding proteins include hpot Ip, hnRNPAl and telomerase. Pot 1 has been described as a single strand-binding protein in fission yeast that protects telomeres from degradation [256]. Its depletion results in complete telomere loss and reduced cell growth [256]. Its human homolog, hpot Ip, also binds specifically to the G-rich strand of the telomere (Figure 6) and has been described as a housekeeping gene that is required to maintain chromosomal integrity, possibly by

being involved in recruiting telomerase to the telomere [256].

The Heterogeneous Nuclear Wbonuclear Protein A l, hnRNPAl or A1 is the candidate homolog for Cdcl3p in yeast. Like Cdcl3p [257,258], Al has been described to protect the telomere ends from degradation and to mediate telomerase access to its substrate [259]. Mouse cell lines deficient in hnRNPAl show accelerated telomere

1. Introduction_______________________________________________________ ^

shortening while its restoration results in telomere elongation [259]. Single stranded DNA and RNA binding sites have been found at the amino-terminal fragment of A l, named U Pl, making A l/U Pl able to mediate both binding to the telomere and telomerase access [259]. In fact, A l/U Pl has been found to interact directly with the RNA component of telomerase and this complex can bind telomeric DNA, suggesting that hnRNPAl may be involved in the recruitment of telomerase to the telomere [260- 262] (Figure 6).

The third, and best described single stranded DNA binding protein at the telomere is telomerase (Figure 6). Telomerase is a reverse transcriptase that synthesizes and therefore elongates telomeric DNA, counteracting telomere shortening [231,232]. Human telomerase is composed of two moieties, the catalytic unit hTERT [263,264] and the RNA component, hTER [265]. Both components are required to obtain enzymatic activity where hTER serves as a template for the synthesis of new telomeric sequences by hTERT [232,266]. The Telomerase-Associated Protein 1 TEP-1 has been found to bind mammalian telomerase RNA and to co-precipitate with telomerase activity [267,268]. TEP-1 does not seem to be required to obtain enzymatic activity and, like hTER, is constitutively expressed [267-270].

/Al/UPl hTERT

hTERT rv c ru itin c n t?

Figure 6 The t-loop and some o f its associated proteins.

The t-loop (see also Figure 5) is thought to be stabilized by several proteins. TRFl and TRF2 are double strand binding proteins, while hpot-1, like A l/U Pl binds the 3’ overhang. Telomerase also binds and furthermore extends the 3’.

Stable transfection of hTERT into telomerase negative fibroblasts leads to telomere maintenance and extension of the proliferative capacity of the transfected cells [271,272]. In contrast to tumours which generally express telomerase activity, most somatic cells do not[273-277]. The exceptions are cell populations found in highly proliferating tissues and organs such as the gut epithelium, the proliferating cells in

1. Introduction________________________________________________________%

hair follicles [278-283] and lymphocytes [238,284,285]. The importance of telomerase as a compensatory mechanism for telomere shortening in proliferative organs is best revealed in studies using mice where the RNA component of telomerase was deleted (mTER/ mice) [286-289]. Although in the first generations the animals did not present any abnormalities, generation 6 showed signs of premature ageing, decreased longevity and defects in highly proliferative organs. These included defective spermatogenesis, compromised proliferative capacity of hematopoietic cells in the bone marrow and spleen, increased hair graying and hair loss in young animals and a higher incidence of severe ulcerative skin lesions. In addition there was a greater than 50% decrease in telomere length in PBMC and a dramatic reduction in the number of germinal centres formed following immunization. This was paralleled by a further decrease in telomere length upon immunization, which was in clear contrast to the telomere elongation observed in control mice.

Capping of chromosome ends by telomeric structures has recently been described as a dynamic process. Capping prevents the chromosome ends from being seen as broken DNA and allows cell cycle to proceed, while a controlled degree of uncapping allows telomerase to elongate the telomere [247]. The dynamic equilibrium between capping and uncapping is summarised in Figure 7. The molecular mechanisms controlling capping and uncapping have not yet been elucidated, but one of the factors leading to telomere uncapping is thought to be telomere shortening. A short telomeric sequence would provide less binding sites for telomeric proteins leading to more frequent uncapping. Uncapping in turn, is a danger signal, which, if left for too long leads to cell cycle arrest and senescence (see Figure 7). Prolonged capping can also have deleterious effects in that the telomere becomes inaccessible to telomerase, leading to telomere shortening.

1. Introduction 37

A ccessib le to te lo m era se

Telom ere Elongation

i

I n c r e o s ^ Protein Binding S ite s

Uncapped Telomere

i

O.K. For Cell Division

A

Telom ere P53 A ctivation In sta b ility

Prolonged Telomere Stability tapping

In a cc essib le t o telom erase

Cell Cycle A rre st

D ecrea sed Protein

Binding S it e s Telom ere Sh ortening

Figure 7 Telomeres are at a constant equilibrium between capping and uncapping. Telomeres are thought to alternate between capped and uncapped states. The capped state, shown on the right, confers stability to the telomere that is not seen as broken DNA. Telomere stability is thought to positively signal cell division. Uncapping of the telomere is necessary if telomere elongation by telomerase is to take place. The balance between capping and uncapping has to be fmely controlled as both prolonged capping an uncapping have deleterious consequences. Prolonged uncapping causes the telomere to be seen as broken DNA and telomere instability. This leads to the activation of the p53 pathway causing cell cycle arrest On the other hand if the telomere is capped for too long it becomes inaccessible to telomerase, leading to telomere shortening, which in turn,

increases the likelihood of uncapping.

The regulation of telomere length in lymphocytes

Telomere length has been shown to decrease with increasing age in T (CD4^ and CD8^) and B lymphocytes, reflecting the proliferative history of lymphocytes [243.290]. CD4 CD45RA^ T lymphocytes have consistently been found to have longer telomeres than CD4 CD45RO^ primed lymphocytes [37,239,290], illustrating that CD45RA^ to CD45RO^ conversion is associated with proliferation (see section 1.2.1). Furthermore, it has been found that although the difference in telomere length between CD4 CD45RA^ and CD4 CD45RO^ T cells remains constant throughout life, both these CD4^ T cell subsets decrease telomere length with increasing age

1. Introduction________________________________________________________38

potential, CD4^CD45RO^ T cells have been confirmed to undergo less population doublings and to reach replicative senescence earlier than CD4^CD45RA^ T cells following in vitro stimulation [37]. Primitive hematopoietic stem cells have also been shown to decrease telomere length with increasing age, as cells fi'om foetal liver and cord blood have longer telomeres than those from adult bone marrow [234,291]. Within CDS^ T lymphocytes, the CD28‘ fraction, present during acute viral infections and thought to contain more differentiated cells has been shown to have shorter telomeres than the CD28^ fraction [292-295]. Whether telomere shortening observed in this subset is a reflection of their longer proliferative history or whether it indicates that these cells have proliferated in the absence of adequate co-stimulation and perhaps inducing sub-optimal telomerase activity remains to be elucidated. It reveals however, that human CD8^ memory T lymphocytes may require additional or alternative co­ stimulatory signals to become re-activated. In this respect, it has been found that these cells can be co-stimulated in vitro by CD 11 a/CD 18 [296]. Whether such co­

stimulation induces telomerase activity remains to be investigated. In addition, CD8 RA^CD27^ naive T cells have been shown to have longer telomeres than the clonally expanded CD8 RA^CD27 effectors and CD8^RACD27^ memory lymphocytes [239,297].

The regulation of telomerase activity in lymphocytes.

Telomerase activity has been found to be tightly regulated during T and B lymphocyte development and activation. In vitro studies have shown that both B [298-300] and T cell activation leads to telomerase up-regulation, peaking at around 48 to 72 hours [218,238,243,284,285], decreasing thereafter. These kinetics have been reported to be mirrored by the expression of the RNA component [238,301]. Ex vivo analysis of telomerase activity during T cell development has further revealed increased activity in proliferating populations. Thymocytes have been shown to express high levels of telomerase [301] while tonsilar CD4^ and CD8^ T lymphocytes express intermediate levels [301]. The enzymatic activity then becomes low to undetectable on resting peripheral blood T lymphocytes [238].

This profile of high telomerase in dividing and low in resting populations is also followed during B cell development [300]. In this way, actively dividing B cells in the germinal centres express high telomerase activity, while plasma cells express

1. Introduction________________________________________________________39

intermediate levels and activated and memory B cells express low activity. Virgin B cells express low to undetectable levels [298-300,302].

Telomerase activity has been shown to depend on T cell activation and mediated by PKC activity [238]. In CD4^ T lymphocytes, telomerase has been shown to be phosphorylated and to translocate from the cytoplasm to the nucleus following TCR stimulation [303]. Telomerase activity has been reported to be independent of levels of hTERT transcripts [302]. The correlation between activity and protein levels remains subject to debate, with some recent reports indicating no correlation between protein levels and activity [303], while others disagree [299,302].

The transient increase in telomerase activity following T cell activation is likely to delay replicative senescence resulting in the maintenance or elongation of telomeres [243,285], but telomere erosion does take place following repeated stimulation in vitro [218,238].

Further confirming the role of telomerase in determining the replicative capacity of lymphocytes, two different reports have recently shown extension of the lifespan of human CD8^ lymphocytes by ectopic expression of telomerase [304,305]. Telomerase expression in these cells not only prolonged the replicative capacity of the cells, but this was associated with the maintenance and in some cases elongation of telomeres. These studies were preceded by investigations in other cell types, showing extension of the life span in telomerase-transfected cells [271,272]. Another report [304] shows maintenance of telomere length but no increase in the replicative capacity of the transfected cells, suggesting that further studies are required to settle this issue.