Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34 ؉ cells to androgens increased telomerase activity, coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity, which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function, and letrozole, an aromatase inhibitor, blocked androgen effects on telomerase activity. Conversely, flutamide, an androgen receptor antagonist, did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-␣ (ER␣), but not ER␤, inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER␣. IntroductionTelomere attrition has been associated with the process of normal aging and as etiologic of aneuploid malignancies (in mouse "knockout" models) and of a variety of human diseases (due to mutations in relevant genes). 1 Telomeres consist of T 2 AG 3 repeats and proximate proteins located at the end of chromosomes that serve to prevent recombination, end-to-end fusion, and activation of DNA damage responses. 2 As DNA polymerase is unable to fully duplicate telomeres during cell divisionthe "end replication problem" 3 -telomeres are eroded until reaching critically short lengths, signaling the cell to cease proliferation (cellular senescence) and apoptosis. 2 To maintain telomeres, some highly proliferative cells, including hematopoietic progenitor and stem cells, express telomerase (TERT), a specialized reverse transcriptase capable of adding DNA repeats to the 3Ј end of telomeric leading strand using an RNA molecule (TERC) as a template. Telomerase also is expressed in the majority of malignant cells of many tissues. 4 Abnormal telomere maintenance is a feature of a variety of human diseases. Dyskeratosis congenita, a constitutional type of aplastic anemia, is caused by mutations in genes involved in telomere maintenance (DKC1 is mutated in X-linked dyskeratosis congenita 5,6 ; TERC, TERT, and TINF2 are mutated in autosomal dominant dyskeratosis congenita [7][8][9] ; and TERT, NOP10, and NHP2 are mutated in autosomal recessive dyskeratosis congenita 10,11 ). Mutations in TERT and TERC also are genetic risk factors for acquired aplastic anemia. 12,13 Although most acquired aplastic anemia is the result of an immune process destroying hematopoietic stem and progenitor cells, 14 predisposit...

BackgroundTelomerase is an enzyme specialized in maintaining telomere lengths in highly proliferative cells. Loss-of-function mutations cause critical telomere shortening and are associated with the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia and with idiopathic pulmonary fibrosis. Here, we sought to determine the spectrum of clinical manifestations associated with telomerase loss-of-function mutations.Methodology/Principal FindingsSixty-nine individuals from five unrelated families with a variety of hematologic, hepatic, and autoimmune disorders were screened for telomerase complex gene mutations; leukocyte telomere length was measured by flow fluorescence in situ hybridization in mutation carriers and some non-carriers; the effects of the identified mutations on telomerase activity were determined; and genetic and clinical data were correlated. In six generations of a large family, a loss-of-function mutation in the telomerase enzyme gene TERT associated with severe telomere shortening and a range of hematologic manifestations, from macrocytosis to acute myeloid leukemia, with severe liver diseases marked by fibrosis and inflammation, and one case of idiopathic pulmonary fibrosis but not with autoimmune disorders. Additionally, we identified four unrelated families in which loss-of-function TERC or TERT gene mutations tracked with marrow failure, pulmonary fibrosis, and a spectrum of liver disorders.Conclusions/SignificanceThese results indicate that heterozygous telomerase loss-of-function mutations associate with but are not determinant of a large spectrum of hematologic and liver abnormalities, with the latter sometimes occurring in the absence of marrow failure. Our findings, along with the link between pulmonary fibrosis and telomerase mutations, also suggest a common pathogenic mechanism for fibrotic diseases in which defective telomere repair plays important role.

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P < 0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n ‫؍‬ 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n ‫؍‬ 1,110; P ‫؍‬ 0.0009). Introduction of TERT mutants into telomerasedeficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.risk factor ͉ telomere ͉ dyskeratosis congenita ͉ cancer

Dynamic properties of signaling pathways control their behavior and function. We undertook an iterative computational and experimental investigation of the dynamic properties of tumor necrosis factor (TNF)␣-mediated activation of the transcription factor NF-B. Surprisingly, we found that the temporal profile of the NF-B activity is invariant to the TNF␣ dose. We reverse engineered a computational model of the signaling pathway to identify mechanisms that impart this important response characteristic, thus predicting that the IKK activity profile must transiently peak at all TNF␣ doses to generate the observed NF-B dynamics. Experimental confirmation of this prediction emphasizes the importance of mechanisms that rapidly down-regulate IKK following TNF␣ activation. A refined computational model further revealed signaling characteristics that ensure robust TNF␣-mediated cell-cell communication over considerable distances, allowing for fidelity of cellular inflammatory responses in infected tissue.The transcription factor NF-B 3 is a key mediator of physiologic processes such as inflammation and adaptive immunity and has been implicated in numerous pathologic states such as cancer, rheumatoid arthritis, and sepsis (1). Consequently, understanding the mechanisms of NF-B activation and regulation is of prime importance. One major activator of NF-B is the potent inflammatory cytokine TNF␣. TNF␣ binds to and trimerizes its receptor, TNFR1, which leads to a receptorassociated signalosome that activates the kinase IKK (2). IKK phosphorylates IB proteins, which normally sequester NF-B in the cytoplasm; phosphorylated IBs are rapidly polyubiquitinated and proteasomally degraded, releasing free NF-B, which translocates to the nucleus and modulates gene expression (2).Detailed biochemical and genetic analyses over the past 25 years have helped elucidate the components that connect TNF␣ to NF-B. However, relatively little is known about how these molecular players act together as a signaling system, whose complex dynamics control the time-variable activity of NF-B and subsequent gene expression (3-6).Recently, it has become apparent that analysis of the systems properties of complex biochemical pathways can benefit from an integrated approach combining systematic experimental perturbations with an associated computational analysis of molecular interactions (5, 7, 8, 10 -13). This type of analysis applied to TNF␣-induced NF-B activity demonstrated that the ␣, ␤, and ⑀ isoforms of IB cooperate to produce a biphasic NF-B response (5). Varying the duration of the TNF␣ stimulus had no effect on the duration of the initial response, thus ensuring expression of some NF-B-regulated genes even in response to very short stimuli (5). This analysis, however, did not address the question of how other types of signaling inputs are processed.In this study, we analyze in detail a different type of inputs, constant stimulations at different TNF␣ doses, and experimentally and computationally analyze the resulting pathway characteristics. Surp...

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 dis- IntroductionTelomerase is a specialized reverse transcriptase (RT) that adds long, repetitive stretches of simple telomeric DNA sequence (ie, TTAGGG in the vertebrates) onto chromosomal termini. 1 This cellular RT protein (TERT) copies a short stretch of nucleotides located within the template region of an integral RNA component (TERC) into telomeric DNA repeats. 1 Vertebrate TERCs are believed to adopt a complex, folded secondary structure 2 as depicted for human TERC (hTERC) in Figure 1A. We have recently conducted extensive site-directed mutagenesis analysis of hTERC to show that much of the structure folded as predicted. 3 As is true of many biologically active RNA molecules, most of the internally base-paired regions of hTERC can be extensively mutated without loss of function, provided that the normal base-pairing pattern is preserved. 3 However, at certain locations, especially of the single-stranded template region that is copied into telomeric DNA, specific RNA base sequences have been shown to be required for biologic activity. 4 Telomerase catalytic proteins (TERTs) from evolutionary distant organisms share a conserved structural organization that can be divided into 3 functional domains ( Figure 1C). 5 The telomerasespecific domains exist at both the N and C termini of TERTs that are not present in any of the viral RTs. 6 The N-terminal region is required to participate in enzymatic function, 7,8 in assembly of the protein with its integral hTERC RNA component,7,9 and in the homodimerization of the protein (ie, hTERT protein-protein interaction), 7,9,10 whereas the C-terminal domain is required for telomerase-specific activity other than its catalytic function 11,12 as well as in the telomeric nucleotide addition processivity process. [13][14][15] The functional RT domain with the universally conserved RT motifs is almost centrally located in the protein primary sequence ( Figure 1C). The fact that mutations of key residues that are known to affect its conventional RT catalytic activity also negatively influence telomerase activity strongly argues that telomerase RT domain is the catalytic domain of the enzyme complex. 13,[15][16][17][18] Inherited mutations in both hTERC RNA and hTERT protein underlie rare bone marrow failure syndromes, autosomal dominant dyskeratosis congenita (DC) and acquired aplastic anemia (AA). [19][20][21] DC is characterized by abnormal skin pigmentation, nail dystrophy, and oral leukoplakia and is often complicated by life-threatening bone marrow failure and immunodeficiency. 22 Lymphocytes from patients show decreased hTERC expression, decreased t...