Seminario 10

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Review

Telomere maintenance and disease

Judy M Y Wong, Kathleen Collins The proliferative capacity of human cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumour progression and tumour maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. New evidence suggests that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. We review the evidence for premature telomere attrition in proliferative deficiencies of the human haemopoietic system, and discuss the potential use of telomerase activation in telomere-restorative gene therapy.

Telomere biology Telomeres are found at the ends of linear chromosomes. In most eukaryotes, functional telomeres are constituted by short, tandem DNA repeats and a multitude of associated proteins.1,2 The presence of telomeres distinguishes the natural ends of chromosomes from random DNA breaks, thereby preventing unwanted endto-end fusion or nucleolytic degradation. In addition to this physical protection of chromosome ends, eukaryotic telomeres have important roles in cellular processes including chromatin organisation and control of cell proliferation.3,4 DNA-dependent DNA polymerases fail to replicate linear chromosome ends completely. Consequently, up to a few hundred basepairs of telomeric DNA can be lost with each mammalian cell division. Telomere erosion is cumulative. It eventually produces an altered chromatin structure, which either activates a damage-sensing checkpoint to halt cell growth, or escapes checkpoint control and undergoes repair-mediated rearrangement.2,5-7 Cultured normal human fibroblasts with critically short telomere length are restrained from cell division in a quiescent state termed replicative senescence. In other cell types, short telomeres can instead provoke apoptosis. In all cells with an intact telomere checkpoint, telomere attrition can serve as a mitotic clock that safeguards normal somatic cells from deregulated proliferation by counting down the lineage allotment of cell divisions (figure 1). If the cellular pathways monitoring and responding to short telomeres are inactivated, continued proliferation will erode telomeres enough to prevent their endprotective function. Telomeres that become uncovered as double-stranded breaks are subject to DNA repair, creating chromosome fusions or translocations. Aberrant chromosomes are further damaged by rounds of anaphase bridge, breakage, and fusion. Cell cultures with genomic instability caused by unstable telomeres are said to be in crisis phase, which is characterised by a high probability of Lancet 2003; 362: 983–88. Published online May 13, 2003 http://image.thelancet.com/extras/02art7027web.pdf Department of Molecular and Cell Biology, University of California at Berkeley, 401 Barker Hall, Berkeley, CA 94720-3204, USA (J M Y Wong PhD, K Collins PhD) Correspondence to: Dr Kathleen Collins (e-mail: [email protected])

Short telomeres Checkpoint intact Proliferative arrest

Checkpoint bypassed: additional telomere shortening

Apoptosis

Chromosome fusion

Non-reciprocal recombination

Rampant genomic instability (crisis) Reactivation of telomerase: genome stabilisation

Cell death

Cancer

Figure 1: Cellular responses to short telomeres Telomeres shorten with cell proliferation when not balanced by telomere synthesis. In healthy somatic cells, critically short telomeres activate a checkpoint that induces either apoptosis or the proliferative arrest of replicative senescence. In the absence of checkpoint function, telomeres erode until they become substrates for aberrant DNA repair. Infrequently, spontaneous activation of telomerase during the crisis phase of genomic instability stabilises and allows maintenance of the rearranged genome, conferring indefinite renewal capacity.

cell death. Rarely, tumorigenic cells that have activated a mechanism for stable maintenance of telomere length can survive (figure 1).

Telomere maintenance by telomerase Several distinct strategies for telomere length maintenance have evolved in eukaryotes. The physiological pathway for telomere maintenance in human cells involves the ribonucleoprotein enzyme telomerase. This enzyme is a specialised reverse transcriptase that copies a region

Search strategy We searched PubMed with keywords including telomerase, telomeres, proliferative senescence, diseases of the haemopoietic system, gene therapy, and others. We aimed to cite recent studies directly; helpful review articles were cited for greater coverage of articles published before 2002.

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Figure 2: The human telomerase ribonucleoprotein complex Telomerase RNA (red) must interact with H/ACA proteins (including dyskerin, green) and with other unknown proteins, to accumulate in cells as stable, fully processed functional RNA. A stable but inactive telomerase ribonucleoprotein is present in most human somatic cells. To become catalytically active, TERT (blue) and probably other proteins must join the ribonucleoprotein complex. Additional subunits may be required to mediate interaction of telomerase with the telomeric repeats at a chromosome 3 end (black).

within its integral RNA component to extend chromosome 3 ends by synthesis of telomeric simple sequence repeats.8 An alternative mechanism for lengthening of telomeres, which is dependent on homologous recombination, can arise in human cells under selective pressure. This second pathway is detectable in some transformed fibroblast cultures and a small minority of cancers.9 Telomerase ribonucleoproteins are only partly characterised in subunit composition. Our knowledge of human telomerase-associated proteins has been gained from candidate approaches (figure 2). The telomerase RNA component is ubiquitously transcribed and assembled into a stable but catalytically inactive ribonucleo protein.10 Precursor processing and stability of human telomerase RNA (hTR) require binding of small nucleolar ribonucleoproteins such as dyskerin, NHP2, NOP10, and GAR1 to the H/ACA motif of hTR.11-15 Catalytic activity is acquired by binding of the telomerase reverse transcriptase subunit (TERT)16 and is modulated by additional factors including chaperones and nucleic acid binding proteins.17 Finally, unknown additional conformational changes or interaction partners are necessary to recruit telomerase to telomeres during the DNA synthesis phase of the cell cycle. Telomerase activity is absent from most human somatic cells beyond the early stages of fetal development,18 although telomerase-positive cells are more generally present in mouse tissues.19 Telomerase inactivation in human somatic cells is mainly due to transcriptional repression of TERT, and can be overcome by constitutive expression of a human TERT (hTERT) transgene.20 Constitutive expression of hTERT in presenescent fibroblasts, lymphocytes, epithelial cells, and other cell types can extend their replicative lifespan.4 Other mechanisms of telomerase inactivation, including alternative mRNA splicing to express a catalytically inactive form of hTERT, have been noted in early development.21,22 The general somatic inhibition of telomerase activity may function as a tumour suppression mechanism, which is perhaps most relevant in organisms with long lives. Because telomere erosion in telomerasenegative cells limits proliferative capacity, the likelihood of accumulation of the multiple spontaneous DNA mutations essential for carcinogenesis should be diminished. This hypothesis is consistent with data from some mouse models, although the short telomeres resulting from telomerase disruption can also enhance tumorigenesis by promoting genomic instability.23

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Telomerase inactivation in the human somatic tissues has exceptions, in a highly restricted subset of adult tissues that are undergoing rapid proliferation.22 Telomerase is known to be active in some germline, epithelial, and haemopoietic cells. In telomerase-positive stages of these cell lineages, telomerase activation correlates with induction of hTERT gene transcription. In some cases, additional stimulation is provided by an increase in the amount of ribonucleoprotein, posttranslational modification of hTERT, or subnuclear shuttling of catalytically active ribonuclearprotein.22,24,25 Telomerase in somatic stem cells does not compensate for proliferation-dependent telomere loss; it does so only in a restricted phase of progenitor cell proliferation, and is subsequently downregulated by terminal differentiation. Intuitively, telomerase activation could be thought to replenish every telomeric repeat lost during multiple preceding rounds of DNA replication. Such telomere length homoeostasis has been extensively characterised in single-celled organisms such as yeasts1 and is being intensively studied in long-term cultures of mammalian cells.26 In cancer cells, the balance between telomere loss and telomere addition can maintain stable telomere lengths over an extended period of proliferation. In normal human somatic tissues, however, telomerase activation does not equate with the acquisition or maintenance of stable telomere lengths. The transient nature of telomerase activation in normal somatic cell lineages differs strikingly from the constitutive activation of telomerase in tumour cells. The amount of telomere shortening offset by somatic cell telomerase activation depends on telomere length before activation, rate of telomere shortening with each round of cell division, amount of telomerase activity, and cellular regulation of telomere-telomerase interaction. Much remains unknown about mechanisms that establish telomere length to account for findings such as proliferation-linked activation of telomerase in cells that nonetheless undergo rapid erosion of telomeres.27

The haemopoietic system: telomerase and telomere dynamics The haemopoietic system provides an excellent model for the study of human telomerase and telomere dynamics in a physiological context.28,29 Haemopoietic stem cells have longer telomeres than descendant lineages. The low telomerase activity in stem cells does not seem to compensate for proliferation, resulting in progressive telomere loss with age. In the differentiation of myeloid cells, including granulocytes and monocytes, telomere length in progenitor cells sets an upper limit on the proliferative capacity of lineage descendants. Telomere lengths in differentiated myeloid populations correlate with their anticipated replicative history.30 By contrast, lymphoid cell lineages have a complicated telomerase and telomere length dynamic. Naive and memory T cells and B cells from peripheral blood undergo an age-progressive erosion of telomere length.31 However, antigen challenge can induce telomerase activation and telomere elongation in these cells. Germinal centre B cells provide the most striking example of telomerase-dependent telomere elongation in the normal human soma.32 Strong telomerase activation in centroblasts and centrocytes leads to a net gain in telomere length of 3–4 Kb for germinal centre B cells relative to naive B cells. Memory B cells downregulate telomerase, returning the lineage to proliferationdependent telomere loss. Although CD4+ memory T cells possess shorter telomeres than their naive counterparts,33

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memory and naive B cells can have similar telomere lengths.32 In culture, mitogenic stimulation of B and T cells induces telomerase activation in a wide range of conditions.29 However, telomerase activation in cultured lymphocytes does not increase telomere length; it can offset the rate of telomere loss for only a restricted number of cell cycles after the initial stimulus to proliferation.34

Haemopoietic proliferative failure induced by telomerase deficiency Mice without genes for telomerase survive for several generations despite progressive telomere attrition, since some inbred strains have especially long telomeres.35 After several generations of knockout breeding, with successive telomere shortening transmitted through the germline, knockout mice develop haemopoietic deficiencies. These problems include small spleen size, decreased follicle numbers, reduced lymphocyte counts, and impaired lymphocyte proliferation.36 These findings could not predict whether telomerase function would be required for somatic cell renewal within a person’s lifespan. Dyskeratosis congenita was the first primary telomere maintenance disorder to be identified in man. The major cause of death is progressive bone marrow failure. Data from studies of this disease suggest that the restricted amount of telomerase-dependent maintenance of telomeres in human somatic cells is essential for health and viability, serving to boost cellular renewal in highly proliferative epithelial and haemopoietic tissues.22 Dyskeratosis congenita is mainly inherited as an X-linked disorder, and is characterised by reticulate skin pigmentation, nail dystrophy, mucosal leucoplakia, and pancytopenia.37 Genetic linkage analysis in many families with X-linked disease showed that a mutant gene termed dyskerin might cause the disorder.38 Dyskerin has homologues in all eukaryotic organisms. The highly related protein Cbf5p in budding yeast catalyses the posttranscriptional conversion of uridine to pseudouridine at target RNA sites, which are specified by hybridisation with tightly bound H/ACA small nucleolar RNAs.39 In vertebrate cells, human dyskerin is associated with H/ACA small nucleolar RNAs and also with hTR12 via the hTR H/ACA motif (figure 2).11,13 Most mutations in the dyskerin gene result in substitutions of a single aminoacid.40 Since the null allele is lethal for all organisms investigated (including mice41) alleles of dyskerin in dyskeratosis congenita must alter but not eliminate the protein’s function. Dyskerin gene mutations noted in patients with the disease do not seem to affect amounts of H/ACA small nucleolar RNA, pseudouridine synthesis in ribosomal RNA, or any other aspect of ribosome biogenesis tested to date.12,42 Consistent with a partial loss of function in disease-associated isoforms of dyskerin, telomerase RNA is decreased but not absent in cells from X-linked dyskeratosis congenita patients with dyskerin gene mutations,12 with a 3–5 fold reduction in steady-state RNA (unpublished). A reduced concentration of telomerase ribonucleoprotein restricts maximum catalytic activity of telomerase, inducing premature telomere shortening that varies in extent according to the type of cell and its proliferative history. Mutations in the telomerase RNA gene are a genetic basis for autosomal dominant dyskeratosis congenita. In a large family with autosomal dominant inheritance of this disorder, a heterozygous deletion of the 3 region of the telomerase RNA gene cosegregated with disease.43 This deletion removes important regions of the H/ACA motif that are essential for RNA accumulation, and such a mutation should therefore create a null allele. If

transcription limits expression of hTR, steady state amounts of RNA would be reduced by 50%. Additional telomerase RNA gene mutations were identified in individual patients with autosomal dominant dyskeratosis congenita.43 These mutations would be predicted to have a range of different effects on telomerase function, because some mutations arise within hTR regions that are important for RNA accumulation, whereas others occur within hTR regions important for catalytic activity but dispensable for accumulation.13 Defects in different structural parts of the telomerase enzyme can lead to different phenotypes. The large degree of cell type-specific telomerase enzyme regulation means that a particular mechanism of inhibition could markedly affect enzyme function in one tissue but not in another.22 Disease onset is generally later in patients with autosomal dominant dyskeratosis congenita than in those with X-linked disease; this difference might result from a less severe telomerase deficiency in autosomal dominant disease inheritance. Different penetrance of dermatological phenotypes relative to bone marrow deficiency is also noted among patients with dyskeratosis congenita,37 suggesting that different amounts of telomerase ribonucleoprotein loss can have differential effects on epithelial and haemopoietic tissue renewal.22 The enhanced risk of carcinoma development in some patients probably indicates that short telomeres in human epithelial cells have a role in promotion of tumorigenic genomic instability.44,45 Dyskerin gene mutations have also been noted in association with X-linked Hoyeraal-Hreidarsson syndrome.46-48 Patients with this disorder have prenatalonset growth retardation, cerebellar hypoplasia, microcephaly, and immunodeficiency in the first few years of life. However, they do not present with the set of epithelial features that is typically diagnostic of dyskeratosis congenita. Additionally, telomerase RNA gene mutations have been described in a subset of patients with aplastic anaemia.49 Consistent with a primary defect in telomerase function, peripheral blood leukocytes in these patients can have substantially shorter telomeres than age-matched controls.50-52 Possibly, a large range of proliferative deficiency phenotypes could result from primary deficiencies of telomerase function, with variations dependent on genetic background, environmental challenge, and the mechanism of telomerase inhibition.

Disease with premature telomere loss due to increased proliferative demand Defects in genes other than those encoding telomerase subunits themselves can result in premature telomere loss. For example, an increased burden of proliferative demand can drive chronologically premature telomere shortening, even with an unaltered rate of telomere loss per cell division. Phenotypes distinct from dyskeratosis congenita would arise dependent on which tissues are subject to the highest rates of turnover and which cell types have the least telomerase-dependent compensation for proliferation. Some examples relevant to the human haemopoietic system are described below. Chronic infection The chronic phase of HIV infection is associated with substantial loss of telomere length in the CD8+ subset of T lymphocytes.53 The reason why telomere loss is specific to this group of T cells is unknown. Excessive telomere shortening has also been shown in patients with chronic hepatitis or liver cirrhosis,54,55 consistent with accelerated liver cirrhosis in telomerase knockout mice.56 In liver disease, and probably in other chronic infections, the rate of

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Telomere erosion with proliferation Normal genome structure Normal physiology Extended proliferative lifespan

Telomere length

Early telomerase activation Genome rearrangements Transformed phenotypes Extended proliferative lifespan

Short telomere checkpoint

Late telomerase activation Cell divisions

Senescence, apoptosis

Genomic instability

Crisis Cell death

Figure 3: Cellular responses to telomerase activation Most human cells do not express telomerase and lose terminal telomeric repeats with each round of cell division (blue line). Short telomeres trigger the proliferative arrest of replicative senescence or apoptosis. Telomerase activation before this checkpoint allows indefinite proliferation with normal cellular physiology (green curve). Activation of telomerase after onset of genomic instability promotes acquisition of transformed and tumorigenic phenotypes (red curve, dashed to denote the small proportion of cells that evade death).

cellular renewal rises to meet higher demand from the increase in cell death. Bone marrow transplant Bone marrow transplant is currently the only therapeutic option for some patients with haematological disorders. For long-term engraftment after transplantation, an expansion of the transplanted cells must happen to repopulate the host’s marrow. This unusually high demand for haemopoietic system renewal, although transient, could substantially deplete the telomere reserves of stem and progenitor cells. Shorter telomere lengths have been shown in transplant recipients than in their donors.57-60 Although clinical data is not yet available to implicate telomere shortening as an important factor in the failure of bone marrow transplant, some relevant evidence has been reported.61 DNA damage repair syndromes Damaged DNA and aberrant DNA replication intermediates must be repaired to protect genome stability. When gene mutations compromise the function of DNA repair, an increase in cell death occurs and the proliferative demands for renewal are increased. Cell lines derived from patients with DNA-repair gene mutations undergo premature senescence, which is causally linked to chronologically accelerated telomere shortening. Ataxia telangiectasia is an autosomal recessive disorder. Patients with this disease have several defects, including hypersensitivity to ionising radiation. Death is mostly due to infection, with increased predisposition to lymphoid malignant diseases.62 The ataxia telangiectasia gene locus encodes ATM, a protein kinase involved in

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DNA damage-triggered signal transduction to p53 activation and checkpoint arrest. T cells from patients with ataxia telangiectasia have shorter telomeres than age-matched controls, consistent with a greater rate of cell turnover due to unrepaired genome damage.63 Constitutive expression of hTERT in patients’ cells increases telomere length and prevents proliferative senescence, but does not fully rescue telomere dysfunction; these findings suggest that ATM may have a direct role at the telomere as well.64 Similar findings have been reported for cells from patients with Nijmegen breakage syndrome.65 This disorder derives from mutations of an ATM target gene, and shares with ataxia telangiectasia the features of radiosensitivity, cancer predisposition, and immunodeficiency. Fanconi anaemia is an autosomal recessive disorder characterised by progressive bone marrow failure and an increased risk of cancer, most commonly acute myeloid leukaemia.62 Patients with this disease have chronologically accelerated telomere shortening.66 Although the spectrum of disease phenotypes has some similarities to dyskeratosis congenita, only cells from patients with Fanconi anaemia have increased sensitivity to DNA crosslinking agents. Fanconi anaemia can arise from mutations in different loci, including the breast cancer susceptibility gene BRCA2.67 These and other findings firmly establish a direct role for Fanconi anaemia group proteins in the response to DNA damage.68 In view of the raised oxidative stress sensitivity of Fanconi anaemia cells, an increased rate of cell turnover could chronologically accelerate telomere shortening. Alternatively, a faster rate of telomere shortening with each cell division could result from increased telomere damage.69 Cells of patients with ataxia telangiectasia show

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increased telomere damage,70 but immortal ataxia telangiectasia cell lines do not differ from controls in any measured variable of telomere length maintenance.71 Therefore, rates of telomere shortening will have to be measured directly in different cell types from individuals with Fanconi anaemia and from healthy people, to establish a direct effect of disease on rate of telomere loss.

Clinical applications Expanded proliferative capacity would reverse or prevent many adverse symptoms of haemopoietic deficiencies, regardless of their precise molecular cause. Patients with primary genetic defects in telomere maintenance pathways or with a higher burden of demand for cellular renewal would both be served by boosting proliferative capacity. Patients who might be helped by a telomerebased treatment could be identified by use of genetic tests or telomere length measurements, well before the onset of severe haemopoietic failure. Constitutive activation of telomerase before activation of the short-telomere checkpoint (figure 3, green curve), instead of as a selection in the crisis phase of telomere dysfunction (red curve), has been shown to leave typical cellular physiology unperturbed.4 Several features of hTERT transgene expression as a gene therapy protocol to allow telomerase activation in haemopoietic cells seem favourable. First, constitutive hTERT expression with a standard retroviral vector is sufficient for telomere length maintenance or gain in most cell types, including T cells72,73 and fibroblasts from patients with dyskeratosis congenita (unpublished). Second, hTERT expression can become downregulated after restoration of telomere reserves and still provide an adequate expansion of renewal capacity. Third, because cells that regain telomere length have a proliferative advantage, they should compete well after engraftment. Finally, trangene-mediated expression of hTERT protein should not provoke rejection because this molecule is expressed widely in development and in some adult somatic cells as well. Because many cell types in the human haemopoietic system can activate telomerase endogenously, constitutive activation of the enzyme might not increase cancer risk, and could even decrease risk by reduction of the genomic instability caused by short telomeres.4,22 These favourable features should provide a stimulus for clinical trials. However, putative telomereindependent roles of telomerase in the stimulation of cell growth and the prevention of apoptosis will also need to be considered.74 Experimental analysis will be needed to show whether manipulation of telomere length by telomerase activation is clinically useful.

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Conflict of interest statement None declared.

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Acknowledgments We thank staff of the Collins Laboratory for comments about the manuscript, and many colleagues for general discussion. Both authors contributed to all aspects of manuscript production. JMYW was funded in part by a postdoctoral fellowship from the National Cancer Institute of Canada, with additional funding from a grant of the American Cancer Society to the laboratory of KC. This review does not reflects the interests of any funding agency.

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