Granulocyte-colony-stimulating factor (G-CSF)-primed allogeneic bone marrow: significantly less graft-versus-host disease and comparable engraftment to G-CSF-mobilized peripheral blood stem cells

Prospective studies have shown rapid engraftment using granulocyte-colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) for allogeneic transplantation, though the risks for graft-versus-host disease (GVHD) may be increased. It was hypothesized that the use of G-CSF to prime bone marrow (GBM) would allow rapid engraftment without increased risk for GVHD compared with G-PBSC. Patients were randomized to receive G-BM or G-PBSCs for allogeneic stem cell transplantation. The study was designed (beta < .8) to detect a difference in the incidence of chronic GVHD of 33% ( < .05). The plan was to recruit 100 patients and to conduct an interim analysis when the 6-month follow-up point was reached for the first 50 patients. Fifty-seven consecutive patients were recruited (G-BM, n = 28; G-PBSC, n = 29). Patients in the G-PBSC group received 3-fold more CD34(+) and 9-fold more CD3(+) cells. Median times to neutrophil (G-BM, 16 days; G-PBSC, 14 days; P < .1) and platelet engraftment (G-BM, 14 days; G-PBSC, 12 days; P < .1) were similar. The use of G-PBSC was associated with steroid refractory acute GVHD (G-BM, 0%; G-PBSC, 32%; P < .001), chronic GVHD (G-BM, 22%; G-PBSC, 80%; P < .02), and prolonged requirement for immunosuppressive therapy (G-BM, 173 days; G-PBSC, 680 days; P < .009). Survival was similar for the 2 groups. Compared with G-PBSC the use of G-BM resulted in comparable engraftment, reduced severity of acute GVHD, and less subsequent chronic GVHD. (Blood. 2001;98:3186-3191) (C) 2001 by The American Society of Hematology.

The role of complex chromosome translocations in leukemia

Resumo A tumorigénese é um processo de transformação celular que se desenrola tipicamente em várias etapas. Os diferentes níveis de evolução tumoral resultam da acumulação sucessiva de mutações genéticas numa célula normal que lhe conferem uma vantagem selectiva no respectivo meio tecidular. As mutações podem manifestar-se sob a forma de alterações nucleotídicas pontuais ao nível da sequência de DNA, levando a uma desregulação da função proteíca ou à formação de proteínas não-funcionais, ou através de alterações cromossómicas numéricas ou estruturais. Na leucemia, por exemplo, os genes híbridos que resultam de translocações cromossómicas desempenham um importante papel no processo tumorigénico. Estes genes são transcritos sob a forma de um RNA mensageiro de fusão, o qual é traduzido numa proteína híbrida com função oncogénica. Frequentemente, os subtipos de doença leucémica estão associados com translocações cromossómicas que envolvem 2 pontos de quebra recorrentes e específicos. É disto exemplo a leucemia mielóide crónica, em que uma translocação recíproca entre os cromossomas 9 e 22 conduz à formação de um gene de fusão BCR-ABL1. Em diferentes subtipos de doença, existe também uma pequena proporção de casos que apresenta translocações cromossómicas complexas, que envolvem um ou mais pontos de quebra adicionais em outras localizações genómicas além das que estão implicadas na formação dos genes de fusão. Por vezes, os pontos de quebra estão também associados a delecções extensas de material genético que se pensa terem uma função importante na tumorigénese. No entanto, o papel destas regiões genómicas no desenvolvimento tumoral não tem sido um motivo recorrente de estudo. Neste contexto, o objectivo desta dissertação foi o de determinar o potencial papel tumorigénico de alterações génicas adicionais ocorridas nos pontos de quebra de translocações cromossómicas complexas. Para a prossecução do objectivo proposto, foram estudados 5 rearranjos cromossómicos distintos associados com diferentes tipos de doença hematológica maligna, nomeadamente a leucemia linfoblástica aguda de células B (2 casos), leucemia mielóide aguda, neoplasma mieloproliferativo e síndrome mielodisplásico/neoplasma ieloproliferativo, não classificável. O mapeamento dos pontos de quebra foi efectuado utilizando a hibridação fluorescente in situ e diferentes metodologias de biologia molecular, tendo como base a informação inicial da análise citogenética. Em casos seleccionados, o papel dos novos genes candidatos foi avaliado in vitro utilizando modelos de linhas celulares, nomeadamente no que respeita às funções de controlo da proliferação celular e de regulação transcricional. De entre os 5 casos estudados, quatro deles evidenciaram translocações complexas envolvendo 3 cromossomas, nomeadamente t(12;21;5)(p13;q22;q13), t(12;6;15)(p13;p24~25;q22), t(9;11;19)(p22;q23;p13) e t(X;20;16)(p11;q13;q23). No caso remanescente, foi observada uma translocação dicêntrica dic(9;12)(p11;p11) acompanhada de delecções extensas em ambos os pontos de quebra. Nos casos com t(12;21;5) e t(9;11;19) as translocações estavam associadas com a presença de genes de fusão recorrentes, nomeadamente TV6(12p13)-RUNX1(21q22) e TLL(11q23)-MLLT3(9p22), indicando que se tratavam de rearranjos complexos das translocações t(12;21) e t(9;11) associadas com a leucemia linfoblástica aguda de células B e a leucemia mielóide aguda, respectivamente. O papel dos pontos de quebra adicionais foi estudado em detalhe no caso com t(9;11;19). Através da metodologia de long distance inverse-polymerase chain reaction, foram identificados os pontos de quebra na sequência de DNA dos 3 cromossomas envolvidos na translocação. Além dos pontos de quebra nos genes MLL e MLLT3, foi observado que o local de quebra no cromossoma 19 interrompeu a sequência de um novo gene, designado CCDC94,conduzindo à sua haplo-insuficiência nas células com t(9;11;19). Através de ensaios de reverse transcription-polymerase chain reaction verificámos que o gene CCDC94 é expresso ubiquitariamente em tecidos humanos normais. A análise informática da sequência prevista da proteína CCDC94 indicou uma elevada identidade de aminoácidos com a proteína cwf16, envolvida na regulação do ciclo celular da levedura Schizosaccharomyces pombe. Através da clonagem do DNA complementar de CCDC94 em vectores de expressão, e após a transfecção destes em culturas de linhas celulares in vitro, observámos que este gene codifica uma proteína de localização exclusivamente nuclear. A expressão ectópica da proteína CCDC94 diminuiu a progressão do ciclo celular e a proliferação das células em cultura. Inversamente, a supressão do transcrito do gene CCDC94 através de interferência de RNA conduziu a um aumento significativo da proliferação celular, confirmando que CCDC94 regula negativamente a proliferação e a progressão do ciclo celular. Estes resultados mostram que os pontos de quebra adicionais, presentes em translocações cromossómicas complexas em leucemia, podem resultar na haplo-insuficiência de genes controladores dos mecanismos proliferativos, cooperando desta forma com a acção das proteínas de fusão para proporcionar ao clone leucémico uma proliferação celular descontrolada. Nos restantes 3 casos estudados não foram identificados genes de fusão. Ao invés, todos aqueles apresentaram delecções de extensão variável associadas com os pontos de quebra cromossómicos. No caso com t(12;6;15), identificámos uma delecção de 1.2 megabases de DNA na banda 12p13 que resultou na eliminação de 9 genes incluindo ETV6 e CDKN1B. O gene ETV6 codifica um factor de transcrição que é essencial para a formação das diferentes linhagens hematopoiéticas na medula óssea, enquanto CDKN1B é traduzido numa proteína responsável por bloquear a entrada das células na fase G1 do ciclo celular e,consequentemente, por travar a proliferação celular. Neste contexto, os resultados obtidos indicam que a perda simultânea de ETV6 e de CDKN1B, através de uma translocação cromossómica complexa, constituiu uma acção cooperativa na leucemogénese. A mesma noção pode aplicar-se ao caso com dic(9;12), no qual pelo menos 2 genes que codificam para factores de transcrição importantes na linhagem hematopoiética, PAX5 no cromossoma 9 e ETV6 no cromossoma 12, estavam deleccionados como resultado do rearranjo cromossómico. Dado que o factor de transcrição PAX5 regula negativamente a expressão do gene FLT3, que desempenha uma função pró-proliferativa, é expectável que a haplo-insuficiência de PAX5 no caso com dic(9;12) terá tido como consequência uma elevação dos níveis de expressão de FLT3, contribuindo deste modo para uma proliferação celular aumentada. A t(X;20;16) foi identificada num doente com trombocitémia essencial (TE), uma doença que está intimamente relacionada com alterações de vias intracelulares reguladas por citocinas. Neste caso, através da utilização de um array genómico, identificámos a presença de pequenas delecções associadas com os pontos de quebra nos cromossomas 16 e 20. No cromossoma 16 apenas um gene, MAF, estava deleccionado, enquanto no cromossoma 20 a delecção tinha abrangido 3 genes. Dos genes deleccionados, dois deles, NFATC2 (20q13) e MAF (16q23), codificam proteínas que operam como reguladores transcricionais de citocinas hematopoiéticas. Dado que NFATC2 se localiza numa região que constitui um alvo frequente de delecções em neoplasmas ieloproliferativos, incluindo a trombocitémia essencial,efectuámos um estudo detalhado do papel deste gene na proliferação megacariocítica e na regulação da expressão de uma citocina hematopoiética (GM-CSF), implicada na maturação das diferentes linhagens mielóides. Utilizando um modelo de linha celular de trombocitémia essencial, verificámos que a supressão do transcrito do gene NFATC2 in vitro, por interferência de RNA, estava associada com um aumento da proliferação celular. Em concordância, o bloqueio da activação da proteína NFATC2 através de um inibidor específico da sua interacção com a calcineurina, conduziu a um aumento da proliferação celular in vitro. Utilizando a PCR quantitativa em tempo real, detectou-se um aumento da produção do RNA de GM-CSF em ambos os ensaios celulares, indicando que o factor de transcrição NFATC2 pode regular negativamente a expressão de GM-CSF em células de trombocitémia essencial. No geral, estes resultados mostram que a redução dos níveis fisiológicos do transcrito NFATC2, ou a redução da respectiva actividade proteica, estão relacionados com a proliferação de megacariocitos através do aumento da produção de GM-CSF. De acordo com estes resultados, verificámos que as células dos doentes com TE apresentam níveis mais baixos do transcrito NFATC2 do que a população normal. Dado que o factor de transcrição MAF desempenha igualmente um papel como regular transcricional de citocinas, é plausível que a haplo-insuficiência dos genes NFATC2 e MAF, resultante do rearranjo cromossómico complexo t(X;20;16), teve um efeito cooperativo importante na patogénese da trombocitémia essencial através da alteração do padrão normal de expressão das citocinas hematopoiéticas. Em síntese, efectuámos nesta dissertação um estudo citogenético de 4 translocações cromossómicas complexas incluindo t(12;21;5), t(12;6;15), t(9;11;19) e t(X;20;16), e de uma translocação dicêntrica dic(9;12), associadas com diferentes neoplasmas hematológicos. Em casos seleccionados efectuámos também um estudo molecular detalhado das regiões dos pontos de quebra. Esta análise permitiu-nos identificar 2 genes, CCDC94 no cromossoma 19 e NFATC2 no cromossoma 20, cuja haplo-insuficiência pode promover o aumento da proliferação celular das células leucémicas. A partir destes estudos podem ser retiradas 2 noções principais: (i) Os pontos de quebra adicionais, que ocorrem em translocações complexas associadas com a formação de genes de fusão, podem ter como consequência a desregulação de genes controladores da proliferação celular (e.g., CCDC94); (ii) As translocações complexas caracterizadas pela ausência de genes de fusão recorrentes poderão estar preferencialmente associadas com a presença de delecções, envolvendo um ou mais genes, nos pontos de quebra; nestas situações, serão necessários pelo menos 2 genes com funções celulares semelhantes (e.g., NFATC2 e MAF) ou complementares (e.g., ETV6 e CDKN1B) para, quando deleccionados, promoverem de forma cooperativa a leucemogénese. Nestes termos, o modelo de alterações genéticas sequenciais que caracteriza o desenvolvimento do cancro pode ser substituído por um modelo em que vários genes-alvo são simultaneamente desregulados pela formação de uma translocação cromossómica complexa, evitando deste modo a necessidade de ocorrência de alterações genéticas subsequentes.----------------------ABSTRACT: Tumourigenesis is a multistep process which results from the accumulation of successive genetic mutations in a normal cell. In leukemia for instance, recurrent translocations play a part in this process by generating fusion genes which lead to the production of hybrid proteins with an oncogenic role. However, a minor subset of chromosomal translocations referred to as complex or variant involves extra breakpoints at variable genome locations in addition to those implicated in the formation of fusion genes. We aimed to describe in this work the role, if any, of genes located at extra breakpoint locations or which are affected by breakpoint-adjacent deletions through the study of 5 leukemia patients.Two of the patients presented with TV6(12p13)-RUNX1(21q22) and MLL(11q23)- MLLT3(9p22) fusion genes as a result of a t(12;21;5) and a t(9;11;19), respectively. Detailed molecular characterization of the extra breakpoint at chromosome 19 in the latter case revealed that a novel ubiquitously expressed gene, CCDC94, with a potential role in cell cycle regulation, was disrupted by the breakpoint. We demonstrated using in vitro cellular assays that this gene codifies for a nuclear protein which negatively regulates cell cycle progression. These data shows that extra breakpoint locations of complex translocations may result in haplo-insufficiency of critical proliferation genes, thereby cooperating with the generation of hybrid proteins to provide unrestrained cell proliferation. In the other 3 patients there were reakpoint-associated deletions which precluded the formation of putative fusion genes. In a case with a t(12;6;15) we characterized a deletion at 12p13 which eliminated ETV6 and 8 other genes including CDKN1B. These findings indicate that concomitant loss of ETV6 and CDKN1B, which encodes a cyclin-dependent kinase inhibitor responsible for blocking entry of cells into the G1 phase of the cell cycle, acted cooperatively to promote leukemogenic proliferation. The same notion applied to a case with a dic(9;12) in which 2 genes encoding hematopoietic transcription factors - ETV6 and PAX5 (9p13)- were deleted as a result of breakpoint-adjacent deletions. Similarly, we found that 2 transcription factor genes involved in the regulation of cytokine expression, NFATC2 (20q13) and MAF (16q23), were involved in deletions contiguous to the breakpoints in a patient with a t(X;20;16). In vitro suppression of NFATC2 mRNA or inhibiton of NFATC2 protein activity enhanced cell proliferation as a result of an increase in the production of a myeloid-lineage stimulating hematopoietic cytokine, GM-CSF. These results suggest that haplo-insufficiency of NFATC2 and MAF genes had a cooperative effect in inducing cell proliferation as a result of a disregulation of cytokine production. Two main conclusions may be drawn from our studies: (i) In complex translocations associated with the production of fusion genes, additional breakpoints may cooperate in tumourigenesis by targeting genes that control cell proliferation; (ii) In complex translocations associated with small breakpoint-adjacent deletions, at least 2 genes with similar or complementary functions need to be deregulated to promote tumourigenesis.

Expression of pSTAT5 predicts FLT3 internal tandem duplications in acute myeloid leukemia.

Mutations of the Fms-like tyrosine kinase 3 (FLT3) can be detected in a significant number of acute myeloid leukemias (AML). Seventy-five cases of acute myeloid leukemia were evaluated for FLT3-internal tandem duplications (ITD) by polymerase chain reaction. Paraffin-embedded formalin-fixed trephine biopsies of these cases were evaluated for expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT1), pSTAT3, and pSTAT5. Specific expression of pSTAT5 was proven in leukemic blasts in situ by double staining with a blast-specific marker. Expression of pSTAT5 in > or =1% of blasts was highly predictive of FLT3-ITD. Neither expression of pSTAT1 nor pSTAT3 were associated with FLT3 mutations. Altogether we conclude that pSTAT5 expression can precisely be assessed by immunohistochemistry in routinely processed bone marrow trephines, STAT5 is highly likely the preferred second messenger of FLT3-mediated signaling in AML, and expression of pSTAT5 is predictive of FLT3-ITD.

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Natural killer cell mediated missing-self recognition can protect mice from primary chronic myeloid leukemia in vivo.

Background: Natural Killer (NK) cells are thought to protect from residual leukemic cells in patients receiving stem cell transplantation. However, multiple retrospective analyses of patient data have yielded conflicting conclusions regarding a putative role of NK cells and the essential NK cell recognition events mediating a protective effect against leukemia. Further, a NK cell mediated protective effect against primary leukemia in vivo has not been shown directly.Methodology/Principal Findings: Here we addressed whether NK cells have the potential to control chronic myeloid leukemia (CML) arising based on the transplantation of BCR-ABL1 oncogene expressing primary bone marrow precursor cells into lethally irradiated recipient mice. These analyses identified missing-self recognition as the only NK cell-mediated recognition strategy, which is able to significantly protect from the development of CML disease in vivo.Conclusion: Our data provide a proof of principle that NK cells can control primary leukemic cells in vivo. Since the presence of NK cells reduced the abundance of leukemia propagating cancer stem cells, the data raise the possibility that NK cell recognition has the potential to cure CML, which may be difficult using small molecule BCR-ABL1 inhibitors. Finally, our findings validate approaches to treat leukemia using antibody-based blockade of self-specific inhibitory MHC class I receptors.

LEDGF/p75 TATA-less promoter is driven by the transcription factor Sp1.

PSIP1 (PC4 and SFRS1 interacting protein 1) encodes two splice variants: lens epithelium-derived growth factor or p75 (LEDGF/p75) and p52. PSIP1 gene products were shown to be involved in transcriptional regulation, affecting a plethora of cellular processes, including cell proliferation, cell survival, and stress response. Furthermore, LEDGF/p75 has implications for various diseases and infections, including autoimmunity, leukemia, embryo development, psoriasis, and human immunodeficiency virus integration. Here, we reported the first characterization of the PSIP1 promoter. Using 5' RNA ligase-mediated rapid amplification of cDNA ends, we identified novel transcription start sites in different cell types. Using a luciferase reporter system, we identified regulatory elements controlling the expression of LEDGF/p75 and p52. These include (i) minimal promoters (-112/+59 and +609/+781) that drive the basal expression of LEDGF/p75 and of the shorter splice variant p52, respectively; (ii) a sequence (+319/+397) that may control the ratio of LEDGF/p75 expression to p52 expression; and (iii) a strong enhancer (-320/-207) implicated in the modulation of LEDGF/p75 transcriptional activity. Computational, biochemical, and genetic approaches enabled us to identify the transcription factor Sp1 as a key modulator of the PSIP1 promoter, controlling LEDGF/p75 transcription through two binding sites at -72/-64 and -46/-36. Overall, our results provide initial data concerning LEDGF/p75 promoter regulation, giving new insights to further understand its biological function and opening the door for new therapeutic strategies in which LEDGF/p75 is involved.

Cytogenetic characterisation of childhood acute lymphoblastic leukemia in Nicaragua

BACKGROUND: Within the frame of a twinning programme with Nicaragua, The La Mascota project, we evaluated in our study the contribution of cytogenetic characterization of acute lymphoblastic leukemia (ALL) as prognostic factor compared to clinical, morphological, and immunohistochemical parameters. METHODS: All patients with ALL treated at the only cancer pediatric hospital in Nicaragua during 2006 were studied prospectively. Diagnostic immunophenotyping was performed locally and bone marrow or blood samples were sent to the cytogenetic laboratory of Zurich for fluorescence in situ hybridization (FISH) analysis and G-banding. RESULTS: Sixty-six patients with ALL were evaluated. Their mean age at diagnosis was 7.3 years, 31.8% were >or=10 years. Thirty-four patients (51.5%) presented with hyperleucocytosis >or=50 x 10(9)/L, 45 (68.2%) had hepatosplenomegaly. Immunophenotypically 63/66 patients (95%) had a B-precursor, 2 (3%) a T- and 1 (1.5%) a B-mature ALL. FISH analysis demonstrated a TEL/AML1 fusion in 9/66 (14%), BCR/ABL fusion in 1 (1.5%), MLL rearrangement in 2 (3.1%), iAMP21 in 2 (3.1%), MYC rearrangement in 1 (1.5%), and high-hyperdiploidy in 16 (24%). All patients but two with TEL/AML1 fusion and high-hyperdiploidy were clinically and hematologically in the standard risk group whereas those with poor cytogenetic factors had clinical high-risk features and were treated intensively. CONCLUSIONS: Compared to Europe, the ALL population in Nicaragua is older, has a higher proportion of poor prognostic clinical and hematological features and receives more intensive treatment, while patients with TEL/AML1 translocations and high-hyperdiploidy are clinically in the standard risk group. Cytogenetics did not contribute as an additional prognostic factor in this setting.

Implication of DNA methylation and PAX5 factor in the transcriptional regulation of hTERT, the human telomerase reverse transcriptase gene

RESUME La télomérase est une enzyme dite "d'immortalité" qui permet aux cellules de maintenir la longueur de leurs télomères, ce qui confère une capacité de réplication illimitée aux cellules reproductrices et cancéreuses. A l'inverse, les cellules somatiques normales, qui n'expriment pas la télomérase, ont une capacité de réplication limitée. La sous-unité catalytique de la télomérase, hTERT, est définie comme le facteur limitant l'activité télomérasique. Entre activateurs et répresseurs, le rôle de la méthylation de l'ADN et de l'acétylation des histones, de nombreux modèles ont été suggérés. La découverte de l'implication de CTCF dans la régulation transcriptionnelle de hTERT explique en partie le mécanisme de répression de la télomérase dans la plupart des cellules somatiques et sa réactivation dans les cellules tumorales. Dans les cellules télomérase-positives, l'activité inhibitrice de CTCF est bloquée par un mécanisme dépendent ou non de la méthylation. Dans la plupart des carcinomes, une hyperméthylation de la région 5' de hTERT bloque l'effet inhibiteur de CTCF, alors qu'une petite région hypométhylée permet un faible niveau de transcription du gène. Nous avons démontré que la protéine MBD2 se lie spécifiquement sur la région 5' méthylée de hTERT dans différentes lignées cellulaires et qu'elle est impliquée dans la répression partielle de la transcription de hTERT dans les cellules tumorales méthylées. Par contre, nous avons montré que dans les lymphocytes B normaux et néoplasiques, la régulation de hTERT est indépendante de la méthylation. Dans ces cellules, le facteur PAX5 se lie sur la région 5' de hTERT en aval du site d'initiation de la traduction (ATG). L'expression exogène de PAX5 dans les cellules télomérase-négatives active la transcription de hTERT, alors que la répression de PAX5 dans les cellules lymphomateuses inhibe la transcription du gène. PAX5 est donc directement impliqué dans l'activation de l'expression de hTERT dans les lymphocytes B exprimant la télomérase. Ces résultats révèlent des différences entre les niveaux de méthylation de hTERT dans les cellules de carcinomes et les lymphocytes B exprimant la télomérase. La méthylation de hTERT en tant que biomarqueur de cancer a été évaluée, puis appliquée à la détection de métastases. Nous avons ainsi montré que la méthylation de hTERT est positivement corrélée au diagnostic cytologique dans les liquides céphalorachidiens. Nos résultats conduisent à un modèle de régulation de hTERT, qui aide à comprendre comment la transcription de ce gène est régulée par CTCF, avec un mécanisme lié ou non à la méthylation du gène hTERT. La méthylation de hTERT s'est aussi révélée être un nouveau et prometteur biomarqueur de cancer. SUMMARY Human telomerase is an "immortalizing" enzyme that enables cells to maintain telomere length, allowing unlimited replicative capacity to reproductive and cancer cells. Conversely, normal somatic cells that do not express telomerase have a finite replicative capacity. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors, and the role of DNA methylation and histone acetylation, an abundance of hTERT regulatory models have been suggested. The discovery of the implication of CTCF in the transcriptional regulation of hTERT in part explained the mechanism of silencing of telomerase in most somatic cells and its reactivation in neoplastic cells. In telomerase-positive cells, the inhibitory activity of CTCF is blocked by methylation-dependent and -independent mechanisms. In most carcinoma cells, hypermethylation of the hTERT 5' region has been shown to block the inhibitory effect of CTCF, while a short hypomethylated region allows a low transcription level of the gene. We have demonstrated that MBD2 protein specifically binds the methylated 5' region of hTERT in different cell lines and is therefore involved in the partial repression of hTERT transcription in methylated tumor cells. In contrast, we have shown that in normal and neoplastic B cells, hTERT regulation is methylation-independent. The PAX5 factor has been shown to bind to the hTERT 5'region downstream of the ATG translational start site. Ectopic expression of PAX5 in telomerase-negative cells or repression of PAX5 expression in B lymphoma cells respectively activated and repressed hTERT transcription. Thus, PAX5 is strongly implicated in hTERT expression activation in telomerase-positive B cells. These results reveal differences between the hTERT methylation patterns in telomerase-positive carcinoma cells and telomerase-positive normal B cells. The potential of hTERT methylation as a cancer biomarker was evaluated and applied to the detection of metastasis. We have shown that hTERT methylation correlates with the cytological diagnosis in cerebrospinal fluids. Our results suggest a model of hTERT gene regulation, which helps us to better understand how hTERT transcription is regulated by CTCF in methylation-dependant and independent mechanisms. Our data also indicate that hTERT methylation is a promising new cancer biomarker.

Clinical and immunological consequences of human T cell leukemia virus type-I and Schistosoma mansoni co-infection

Human T cell leukemia virus type-I (HTLV-I) infection is associated with spontaneous T cell activation and uncontrolled lymphocyte proliferation. An exacerbated type-1 immune response with production of pro-inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) is significantly higher in patients with myelopathy associated to HTLV-I than in HTLV-I asymptomatic carriers. In contrast with HTLV-I, a chronic Schistosoma mansoni infection is associated with a type-2 immune response with high levels of interleukin (IL-4, IL-5, and IL-10) and low levels of IFN-gamma. In this study, clinical and immunological consequences of the HTLV-I and S. mansoni infection were evaluated. The immune response in patients with schistosomiasis co-infected with HTLV-I showed low levels of IL-5 (p < 0.05) in peripheral blood mononuclear cells cultures stimulated with S. mansoni antigen (SWAP) and decreased SWAP-specific IgE levels when compared with patients with only schistosomiasis (p < 0.05). Liver fibrosis was mild in all HTLV-I co-infected patients. Immunological response was also compared in individuals who had only HTLV-I infection with those who were co-infected with HTLV-I and helminths (S. mansoni and Strongyloides stercoralis). In patients HTLV-I positive co-infected with helminths the IFN-gamma levels were lower than in individuals who had only HTLV-I. Moreover, there were fewer cells expressing IFN-gamma and more cells expressing IL-10 in individuals co-infected with HTLV-I and helminths. These dates indicate that HTLV-I infection decrease type 2-response and IgE synthesis and are inversely associated with the development of liver fibrosis. Moreover, helminths may protect HTLV-I infected patients to produce large quantities of pro-inflammatory cytokines such as IFN-gamma.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Arenavirus nucleoproteins prevent activation of nuclear factor kappa B.

Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

The lincRNA HOTAIRM1, located in the HOXA genomic region, is expressed in acute myeloid leukemia, impacts prognosis in patients in the intermediate-risk cytogenetic category, and is associated with a distinctive microRNA signature.

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown.We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients.The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6;9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival (OR:2.04;P = 0.001), shorter leukemia-free survival (OR:2.56; P < 0.001) and a higher cumulative incidence of relapse (OR:1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33-microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004).Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature.