Dyskeratosis Congenita: An Inherited Disorder of Telomere Maintenance
Reduced telomere length has been documented in patients withthe progressive BM failure syndrome dyskeratosis congenita (DKC). Abnormalities in these patients include skin pigmentation,nail dystrophy, and leukoplakia, and similarities have beennoted with the telomerase KO mouse. At first sight, it is agenetically heterogeneous condition, with X-linked, autosomalrecessive, and autosomal dominant inheritance all having beendescribed in different pedigrees. The clinical spectrum of thedisease ranges from severely affected individuals in the firstdecade of life (as in Hoyeraal-Hreidarsson syndrome) to asymptomaticpatients with only minor changes in blood counts (reviewed in). It is becoming apparent, however, that disruption oftelomere maintenance may be a final common pathway in the pathogenesisof this disorder. The X-linked form of the disease is due tomissense mutations in the gene DKC1 on the long arm of the Xchromosome . The affected protein, dyskerin, is a nucleolarprotein that is associated with hTR . Furthermore, in patientswith autosomal dominant DKC, both deletions, as well as mutationsinvolving hTR, have recently been described . Mutationsin the DKC1 or hTR gene can be identified in less than 50% ofthe patients ; however, these findings lend considerableweight to a link between BM failure syndromes and failure oftelomere maintenance. More recently, a mutation in a functionalSP1 binding site of telomerase has been identified as an additionalmechanism  in an acquired bone marrow failure syndrome,in this case paroxysmal nocturnal hemoglobinuria (PNH) [66,67]. The possibility of "telomerase therapy" has been proposedfor these individuals and is currently being studied preclinically.
Acquired Bone Marrow Failure Syndromes
Acquired aplastic anemia (AA) is thought to result from damageto the stem and progenitor compartment caused by autoreactiveT cells. Although many patients recover with immunosuppressivetherapy, the majority exhibit a deficit in stem cell numbersdespite apparent hematological recovery . Severe AA alsoconfers an increased risk for development of late clonal marrowdisorders, such as PNH (25% at 15 years), myelodysplastic syndromes(MDS) (probability ranging from 10% to 47%), and acute leukemia.Although this might be due to impaired immune surveillance,a corresponding rise in solid cancers has not been describedas might be expected if this were the primary mechanism. A moreattractive hypothesis might involve disruption of intrinsiccell processes resulting in premature aging or genetic instabilityin the marrow stem cell compartment, ultimately leading to clonalhemopoiesis.
In granulocytes from patients with AA, not only was telomerelength found to be significantly shorter compared with age-adjustedcontrols , but the degree of shortening correlated significantlywith the severity of cytopenia . Intriguingly, telomerelength in granulocytes from AA patients successfully treatedwith immunosuppressive therapy did not differ significantlyfrom controls. Untreated and nonresponding patients with persistentsevere pancytopenia continued to exhibit significant telomereshortening . Although these results imply extensive proliferationof HSC in subgroups of AA patients (reviewed in ), the questionremains as to whether this is a secondary phenomenon (reflectingincreased HSC turnover as a consequence of damage to the stemcell compartment) or indicates telomere-mediated replicativeexhaustion of the HSC pool. Support for the latter has comefrom recently described mutations of the hTR gene in hematopoieticcells from patients with acquired BM failure . However,these mutations occur at such a low frequency (2 of 150 patientswith AA; 0 of 13 patients with PNH) that they would appear notto be the major cause of otherwise clinically typical BM failuresyndromes . More recently, heterozygous mutations in theTERT gene have been detected in patients with AA and shown toimpair telomerase activity via haploinsufficiency .
Telomere Biology and Hematological Malignancy
Progressive telomere shortening is well described in hematologicalmalignancies [37, 74–77] and is thought to result frommarked clonal expansion, although oxidative damage  or telomerasedysregulation may be contributory at least in the early stagesof some leukemias . Furthermore, there is evidence thatcritical telomere shortening with resulting genetic instabilitymay promote tumor evolution and telomerase activation or upregulation,during which critically short telomeres are stabilized and ongoingtumor growth is facilitated. A biphasic pattern of telomereand telomerase kinetics has been proposed, at least as regardsprogression of hematological malignancy such as chronic myeloidleukemia (CML) [37, 75]. Although these are difficult conceptsto prove experimentally, much circumstantial evidence wouldappear to fit this model, as detailed below. Furthermore, givenits widely reported association with advanced disease, criticallyshort telomeres, and genetic instability (reviewed in ),telomerase may be relevant both as a prognostic or "staging"marker and as a therapeutic target, and many investigators continueto address these problems.
Telomerase Expression and Cell Cycle Status
Expression of significant levels of telomerase can dramaticallyincrease proliferative life span and promote cellular immortality,thereby contributing to the malignant phenotype . It istherefore important that its contribution to the burden of humancancer be understood. Initial reports of telomerase expressionseemed to support a specific association with a malignant, immortal,or germline phenotype . However, by using a more sensitiveassay, telomerase has been detected in proliferating normalsomatic tissues. For example, high levels of telomerase activityare detected in activated lymphoid cells  and proliferatingendometrium , with low levels present in other somatic tissues.It would appear that such low-level expression represents raretelomerase-expressing stem or progenitor cells rather than uniformexpression in all cells in the specimen [83–85], althoughthere may be more widespread expression in proliferating endometrium. Telomerase activity appears to be tightly associated withproliferation status, although how this is regulated remainsunclear. One practical consequence is that many observationsof telomerase modulators in vitro or of telomerase activitybetween different primary tissues (including tumors) may beconfounded by altered or inherently different cell cycle activationstatus rather than by direct modulation of telomerase activityper se. In this regard, studies of telomerase regulation duringcell cycle progression have produced conflicting results. Zhuet al. demonstrated upregulation of telomerase activity in humantumor cells during S-phase, using phase-selective cell cycleinhibitors . However, a subsequent study, using selectedcycling cell populations based on DNA content, demonstratedno change in telomerase activity during cycle progression .When cells entered G0, however, telomerase was repressed andtelomerase activity generally correlated with growth rate. Byanalogy, cells that are postmitotic should not express telomerase;rapid downregulation of activity has been described in severalcell lines during terminal differentiation [89, 90], apparentlymediated by a rapid reduction in hTERT mRNA levels occurringindependently of simple cell cycle arrest and requiring de novoprotein synthesis .
The implications of these observations are frequently overlooked.They would imply that a (if not the) primary determinant ofTRAP activity in a tumor or tissue sample is the proportionof cycling cells (i.e., stem or progenitor cells, whether theyare normal or malignant). Studies on hematological malignancysupport this notion; for example, in early chronic lymphocyticleukemia (a disorder characterized by slow accumulation of matureB lymphocytes displaying resistance to apoptosis rather thanincreased turnover), TRAP activity is not raised until the diseasetransforms or accelerates (when cell turnover is increased). In keeping with this, our recent study (using purifiedprimary CD34+BCR-ABL+ cells from chronic-phase CML at diagnosis)demonstrated increased cell cycle activation compared with controls,with a significant inverse correlation between the proportionof G0 cells and TRAP activity . These data also explaina well-documented paradox, namely reduced telomere length inthe face of increased telomerase activity; overall, an increasedproportion of cycling cells would appear to elevate "whole tissue"TRAP levels; however, telomere maintenance in any individualcycling cell must remain suboptimal as shortening continues.