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...

Shwachman-Diamond syndrome (SDS; OMIM 260400), an inherited bone marrow failure syndrome, is caused by mutations in both alleles of the SBDS gene, which encodes a protein of unknown function. Here we report heterozygosity for the 258 ؉ 2 T>C SBDS gene mutation previously identified in SDS patients in 4 of 91 patients with apparently acquired aplastic anemia (AA) but not in 276 ethnically matched controls (Fisher exact test, P < .004). Affected patients were young and had a poor outcome; they had reduced SBDS expression but no evidence of the pancreatic exocrine failure or skeletal abnormalities typical of SDS. Length of telomeres in granulocytes of SBDS heterozygous patients was short for their age, and in SDS patients with both SBDS alleles affected further analyzed, granulocytes' telomeres were even shorter, correlating in length with SBDS expression. Higher heterogeneity in telomere length also was observed in SDS patients. Telomerase activity of SBDS-deficient patients' lymphocytes was comparable with controls, and no physical interaction between SBDS protein and telomerase complex components (TERT or TERC) was established. We propose that heterozygosity for the 258 ؉ 2 T>C SBDS mutation predisposes to AA by accelerating telomere shortening of leukocytes via a telomerase-independent mechanism.

Mutations in components of the telomerase complex (TERT and TERC) are risk factors for acquired aplastic anemia. Telomerase is a reverse transcriptase responsible for the maintenance of telomeres, repetitive nucleotide sequences capping the chromosome ends. Patients with marrow failure with TERT or TERC mutations have low telomerase activity, short telomere length in leukocytes, reduced hematopoietic function, and they do not respond adequately to immunosuppressive therapy. The constitutional aplastic anemia dyskeratosis congenita is also caused by mutations in TERC (autosomal recessive type) or in an additional component of the telomerase complex, dyskerin, encoded by the DKC1 gene (X-linked type). The majority of patients with dyskeratosis congenita and some patients with acquired aplastic anemia benefit from androgen therapy, but the mechanism by which this hormone stimulates hematopoiesis is not well understood. We investigated whether sex hormones stimulated telomerase activity in hematopoietic tissue. Peripheral blood lymphocytes from either healthy controls or mutation carriers were cultured with phytohemagglutinin and interleukin-2 for 1–7 days in the presence of methyltrienolone, estradiol, hydroxycortisone, and/or tamoxifen. Telomerase enzymatic activity was measured using a fluorescent telomere-repeating amplification protocol (TRAP), and TERT mRNA expression was assayed by real-time PCR. Androgens and estradiol up-regulated telomerase activity in lymphocytes from normal subjects in a dose-dependent fashion (controls, 124±24 total product generated [TGP] units per reaction; methyltrienolone 1 μM, 242±16 TGP units/reaction; methyltrienolone 10 μM, 757±87 TGP units/reaction; P&lt;0.01). Increased telomerase activity correlated to higher TERT mRNA levels (P&lt;0.05). Steroidal effects on telomerase activity were neutralized by tamoxifen, an estrogen antagonist. TERT mutation carriers had lower telomerase activity compared to controls (P&lt;0.05), but their enzymatic function was restored to normal levels when cultured with methyltrienolone. As a control, hydrocortisone at 1 μM inhibited telomerase activity without influencing cell cycle or inducing apoptosis. We also investigated the effects of androgen on hematopoietic progenitor cells. Bone marrow CD34+ cells from normal subjects were immunomagnetically separated and cultured for eight days in StemSpan supplemented with Flt-3 ligand, stem cell factor, and interleukins 3 and 6, in the presence methyltrienolone. Up-regulation of telomerase activity was observed in CD34+ cells cultured with methyltrienolone (P&lt;0.05), the increase correlated with greater cell numbers, a higher proportion of CD34+ cells after eight days of culture (from 10% to 15% at 10 μM methyltrienolone and to 19% at 100 μM), without changes in cell cycle or cell differentiation, based on CD45, CD71, and CD33 expression. These results indicate that sex hormones stimulate TERT expression via the estrogen receptors, leading to increased telomerase activity in hematopoietic cells and increased proliferation of hematopoietic progenitors. Androgens were able to restore telomerase activity to normal levels in TERT mutation carriers. Sex hormone effects on telomerase in normal cells may have implications for the normal aging process and in hematological malignancy (leukemic cells have high telomerase activity), and our results suggest that androgens might be useful in the treatment of patients bearing telomerase complex mutations.

Aplastic anemia is characterized by peripheral blood pancytopenia and a hypocellular bone marrow. In the majority of patients, bone marrow destruction is immune-mediated by a type-1 T cell response, dominated by oligoclonal expansion of CD8+ cells and targeting hematopoietic stem cells. Some patients with acquired aplastic anemia are heterozygous for mutations in genes encoding the major components of the telomerase complex, telomerase reverse transcriptase (encoded by TERT; Yamaguchi et al., N Engl J Med2005;352:1413) and the RNA component (encoded by TERC; Fogarty et al., Lancet2003;362:1628). These mutations lead to shortened telomeres of leukocytes, low telomerase activity and reduced hematopoietic function. Relatives carrying the same mutations also have short telomeres and reduced hematopoietic function, but they do not inevitably develop overt marrow failure. In contrast, dyskeratosis congenita, a constitutional type of marrow failure, is caused by mutations in TERC (autosomal dominant type) or in the DKC1 gene, which encodes an additional component of the telomerase complex, dyskerin. In addition to marrow failure, patients with dyskeratosis congenita also often have physical anomalies, such as leukoplakia, nail dystrophy, and hyperpigmentation as well as hepatic or pulmonary fibrosis. In dyskeratosis congenita, family members bearing the genetic mutation present variable degrees of physical abnormalities or organ damage. However, in patients with acquired aplastic anemia carrying telomerase mutations without physical anomalies, it is unclear whether these mutations are sufficient for the development of marrow failure. We hypothesized that telomerase complex mutations reduce the size of the hematopoietic stem cell compartment and affect its regenerative capacity, making carriers more vulnerable to environmental insults or autoimmune damage. We analyzed the distribution of the T-cell repertoire (T-cell receptor [TCR] Vβ subfamily) and expansion of particular Vβ subsets by flow cytometry in peripheral blood of six aplastic anemia patients carrying telomerase complex mutations (five with TERT mutations, one with a TERC mutation), two patients with Fanconi anemia, and 15 healthy subjects. The expression of 22 Vβ subfamilies were evaluated in CD8+CD28− cells, and expansion was defined when the percentage of a given Vβ subfamily was above two standard deviations based on the control group (Risitano et al. Lancet2004;364:355). We also evaluated interferon-γ levels in serum of nine telomerase mutant patients and 10 controls by ELISA. Expanded Vβ subsets were observed in all six aplastic patients carrying telomerase complex mutations analyzed. Five patients (with TERT mutations) had two clones expanded and one (with TERC mutation) showed three overrepresented clones. The Vβ subsets 9, 13.6, 17, and 20 were expanded in more than one patient. Oligoclonal Vβ expansion was not observed in Fanconi anemia. Telomerase mutant patients also had significantly increased interferon-γ serum levels in comparison to controls (27 pg/mL; range, 0–95 vs. 7.6, 0–16, respectively; P&lt;0.02). These results indicate that an immune process targeting hematopoiesis may operate in patients carrying telomerase complex gene mutations. Limited responsed to immunosuppression may reflect the poor hematopoietic reserve rather than a nonimmunologic mechanism of marrow destruction.