Heterozygous mutations in the TERT gene impair telomerase activity by haploinsufficiency and may be risk factors for marrow failure.

The t(8;21) translocation between two genes known as AML1 and ETO is seen in approximately 12-15% of all acute myeloid leukemia (AML) and is the second-mostfrequently observed nonrandom genetic alteration associated with AML. AML1 up-regulates a number of target genes critical to normal hematopoiesis, whereas the AML1͞ETO fusion interferes with this trans-activation. We discovered that the fusion partner ETO binds to the human homolog of the murine nuclear receptor corepressor (N-CoR). The interaction is mediated by two unusual zinc finger motifs present at the carboxyl terminus of ETO. Human N-CoR (HuN-CoR), which we cloned and sequenced in its entirety, encodes a 2,440-amino acid polypeptide and has a central domain that binds ETO. N-CoR, mammalian Sin3 (mSin3A and B), and histone deacetylase 1 (HDAC1) form a complex that alters chromatin structure and mediates transcriptional repression by nuclear receptors and by a number of oncoregulatory proteins. We found that ETO, through its interaction with the N-CoR͞mSin3͞HDAC1 complex, is also a potent repressor of transcription. This observation provides a mechanism for how the AML1͞ETO fusion may inhibit expression of AML1-responsive target genes and disturb normal hematopoiesis.

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...

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The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85-c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.

IntroductionA subpopulation of T cells, termed regulatory T cells (Tregs), have been described in humans and animal models; these cells are important because they suppress autoreactive T cells by direct cell contact. [1][2][3][4] Phenotypically, Treg are characterized by cell surface expression of the proteins CD4 and CD25 and by intracellular expression of the transcription factor FOXP3. Only CD4 ϩ CD25hi ϩ FOXP3 ϩ T cells express suppressor functions. FOXP3 is a member of the forkhead/winged-helix family of transcription regulators (FOXP1-4). In humans, mutations in FOXP3 result in an autoimmune syndrome termed immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. 5 The transcription factor NFAT1 induces FOXP3 expression by binding to its promoter. 6 In addition, FOXP3 expresses its repressive effects on cytokine expression and its activating effects on CD25 by cooperation with NFAT1. 7,8 Acquired aplastic anemia is characterized by destruction of hematopoietic stem cells by cytotoxic T lymphocytes. 9 Hematopoietic response and hematologic recovery after successful immunosuppressive treatment represent the most powerful evidence that this rare and complex disease is immune-mediated. 10 Increased T-bet protein levels in T cells probably are responsible for the increased interferon-␥ levels and the Th1 polarization in patients with aplastic anemia. 11 We show that Tregs were decreased in acquired aplastic anemia, as in other autoimmune diseases. [12][13][14][15][16] All patients examined had low levels of FOXP3. Mechanistically, FOXP3 down-regulation appeared to be mediated by the transcription factor NFAT1. Patients, materials, and methods Patients and control subjectsInformed consent was acquired according to protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (NHLBI) and was obtained in accordance with the Declaration of Helsinki for all patients (n ϭ 20; age range, 13-52 years) with acquired aplastic anemia examined (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) and healthy volunteers (n ϭ 14; age range, 18-55 years). Lymphocyte isolation, flow cytometry, and immunoblotsCD4, CD25, and FOXP3 expression was examined in peripheral blood mononuclear cells (PBMC) by 3-color flow cytometry as previously described 11 using an APC-antihuman FOXP3 staining kit (eBioscience, San Diego, CA). 15 Immunoblot experiments were performed as previously described 11 (Document S1). Quantitative real-time polymerase chain reactionFOXP3 gene expression was measured in CD4 ϩ CD25 ϩ T cells as previously described. 15 All polymerase chain reaction assays were performed in duplicate and reported as the mean. Confocal microscopy and T-cell transfectionsNFAT1 and FOXP3 expression was examined by confocal microscopy as previously described 17 (Document S1). Transfections were performed 18,19 using a GFP-wild-type NFAT1 plasmid 20 (a gift from Dr. A. Rao, Harvard University, Cambridge, MA) and examined ...

B19 parvovirus is pathogenic in humans, causing fifth disease, transient aplastic crisis, some cases of hydrops fetalis, and acquired pure red cell aplasia. Efforts to develop serologic assays and vaccine development have been hampered by the virus's extreme tropism for human bone marrow and the absence of a convenient culture system. We constructed recombinants containing either the major (VP2) or minor (VP1) structural proteins of B19 in a baculovirus-based plasmid, from which the polyhedrin gene had been deleted; these recombinant plasmids were used to generate recombinant infectious baculovirus. Subsequent infection of insect cells in vitro resulted in high-level expression ofeither B19 VP1 or VP2. Parvovirus capsids were obtained by self-assembly in cell cultures coinfected with either VP1-and VP2-containing baculoviruses or, surprisingly, VP2-containing baculoviruses alone. Empty B19 capsids composed of VP1 and VP2 could replace serum virus as a source of antigen in a conventional inmmunoassay for detection ofeither IgG or IgM antiparvovirus antibodies in human serum. Immunization of rabbits with capsids composed of VP1 and VP2 resulted in production of antisera that recognized serum parvovirus on immunoblot and neutralized parvovirus infectivity for human erythroid progenitor cells. Baculovirus-derived parvovirus antigen can substitute for scarce viral antigen in immunoassays and should be suitable as a human vaccine.B19 parvovirus is the only member ofthe family Parvoviridae pathogenic in humans (1, 2). Acute infection results in fifth disease, a common childhood exanthem that in adults more usually manifests as an arthralgia/arthritis syndrome. In persons with underlying hemolysis, acute infection produces transient aplastic crisis, precipitous anemia due to hypoproliferative erythropoiesis. B19 infection can be persistent (3). In the setting of immunodeficiency, due to congenital, acquired, or iatrogenic immunodeficiency, persistent parvovirus results in chronic pure red cell aplasia. In utero infection causes hydrops fetalis, with fetal death due to severe anemia and congestive heart failure (4).Clinical studies of B19 parvovirus infection have been hampered by limited supplies of viral antigen. The virus has extraordinary tropism for human erythroid progenitor cells and has only been propagated in explanted human bone marrow (5-8), fetal liver (9), and (to a lesser degree) erythroleukemia cells (10). We describe here expression of the virus's structural proteins in a baculovirus system. Large quantities of empty viral capsids were produced, which can substitute for serum virus in clinical assays and stimulate the production of neutralizing antibodies in animals. Only the major protein was required for capsid assembly; the minor capsid protein serves an important role in virus function independent of virion assembly. MATERIALS AND METHODSCell Culture and Virus Stocks. Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant viruses were grown in monolayers of Sf9 cells. Sf9 c...

Transplanted donor lymphocytes infused during hematopoietic stem cell transplantation (HSCT) have been shown to cure patients with hematological malignancies. However, less is known about the effects of HSCT on metastatic solid tumors. Thus, a better understanding of the immune cells and their target antigens that mediate tumor regression is urgently needed to develop more effective HSCT approaches for solid tumors. Here we report regression of metastatic renal cell carcinoma (RCC) in patients following nonmyeloablative HSCT consistent with a graft-versus-tumor effect. We detected RCC-reactive donor-derived CD8 + T cells in the blood of patients following nonmyeloablative HSCT. Using cDNA expression cloning, we identified a 10-mer peptide (CT-RCC-1) as a target antigen of RCC-specific CD8 + T cells. The genes encoding this antigen were found to be derived from human endogenous retrovirus (HERV) type E and were expressed in RCC cell lines and fresh RCC tissue but not in normal kidney or other tissues. We believe this to be the first solid tumor antigen identified using allogeneic T cells from a patient undergoing HSCT. These data suggest that HERV-E is activated in RCC and that it encodes an overexpressed immunogenic antigen, therefore providing a potential target for cellular immunity.