RESUMO
Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Sobrevivência Celular , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
The genomic region encoding the miR-17-92 microRNA (miRNA) cluster is often amplified in lymphoma and other cancers, and cancer cells carrying this amplification have higher expression of miRNA in this cluster. Retroviral expression of miR-17-92 accelerates c-Myc-induced lymphoma development, but precisely how higher expression of miR-17-92 promotes lymphomagenesis remains unclear. Here we generated mice with higher expression of miR-17-92 in lymphocytes. These mice developed lymphoproliferative disease and autoimmunity and died prematurely. Lymphocytes from these mice showed more proliferation and less activation-induced cell death. The miR-17-92 miRNA suppressed expression of the tumor suppressor PTEN and the proapoptotic protein Bim. This mechanism probably contributed to the lymphoproliferative disease and autoimmunity of miR-17-92-transgenic mice and contributes to lymphoma development in patients with amplifications of the miR-17-92 coding region.
Assuntos
Doenças Autoimunes/genética , Linfócitos/imunologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , MicroRNAs/biossíntese , MicroRNAs/genética , Animais , Doenças Autoimunes/patologia , Morte Celular/genética , Morte Celular/imunologia , Proliferação de Células , Células Cultivadas , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Células Jurkat , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfócitos/metabolismo , Linfoma/genética , Linfoma/imunologia , Transtornos Linfoproliferativos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/fisiologiaRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis particularly in the metastatic setting. Treatments with anti-programmed cell death protein-1/programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors (ICI) in combination with chemotherapies have demonstrated promising clinical benefit in metastatic TNBC (mTNBC) but there is still an unmet need, particularly for patients with PD-L1 negative tumors. Mechanisms of resistance to ICIs in mTNBC include the presence of immunosuppressive tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Eganelisib is a potent and selective, small molecule PI3K-γ inhibitor that was shown in preclinical studies to reshape the TME by reducing myeloid cell recruitment to tumors and reprogramming TAMs from an immune-suppressive to an immune-activating phenotype and enhancing activity of ICIs. These studies provided rationale for the clinical evaluation of eganelisib in combination with the anti-PD-L1 atezolizumab and nab-paclitaxel in firstline mTNBC in the phase 2 clinical trial MAcrophage Reprogramming in Immuno-Oncology-3 (MARIO-3, NCT03961698). We present here for the first time, in-depth translational analyses from the MARIO-3 study and supplemental data from eganelisib monotherapy Ph1/b study in solid tumors (MARIO-1, NCT02637531). METHODS: Paired pre-treatment and post-treatment tumor biopsies were analyzed for immunophenotyping by multiplex immunofluorescence (n=11), spatial transcriptomics using GeoMx digital spatial profiling (n=12), and PD-L1 immunohistochemistry, (n=18). Peripheral blood samples were analyzed using flow cytometry and multiplex cytokine analysis. RESULTS: Results from paired tumor biopsies from MARIO-3 revealed gene signatures of TAM reprogramming, immune activation and extracellular matrix (ECM) reorganization. Analysis of PD-L1 negative tumors revealed elevated ECM gene signatures at baseline that decreased after treatment. Gene signatures of immune activation were observed regardless of baseline PD-L1 status and occurred in patients having longer progression-free survival. Peripheral blood analyses revealed systemic immune activation. CONCLUSIONS: This is the first report of translational analyses including paired tumor biopsies from a phase 2 clinical study of the first-in-class PI3K-γ inhibitor eganelisib in combination with atezolizumab and nab-paclitaxel in frontline mTNBC. These results support the mechanism of action of eganelisib as a TAM-reprogramming immunotherapy and support the rationale for combining eganelisib with ICI and chemotherapy in indications with TAM-driven resistance to ICI.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologia , Feminino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/efeitos dos fármacos , Pessoa de Meia-Idade , Metástase Neoplásica , Piridinas/farmacologia , Piridinas/uso terapêutico , Ácidos Picolínicos , BenzotiazóisRESUMO
Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q- syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.
Assuntos
Anemia Macrocítica/genética , MicroRNAs/genética , Fases de Leitura Aberta/genética , Anemia Macrocítica/etiologia , Animais , Estudos de Casos e Controles , Diferenciação Celular/genética , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Células Eritroides/metabolismo , Eritropoese/genética , Eritropoese/fisiologia , Humanos , Perda de Heterozigosidade , Megacariócitos/metabolismo , Megacariócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/fisiologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/fisiologia , Células Tumorais CultivadasRESUMO
The prognosis for adults with precursor B-cell acute lymphoblastic leukemia (B-ALL) remains poor, in part from a lack of therapeutic targets. We identified the type I cytokine receptor subunit CRLF2 in a functional screen for B-ALL-derived mRNA transcripts that can substitute for IL3 signaling. We demonstrate that CRLF2 is overexpressed in approximately 15% of adult and high-risk pediatric B-ALL that lack MLL, TCF3, TEL, and BCR/ABL rearrangements, but not in B-ALL with these rearrangements or other lymphoid malignancies. CRLF2 overexpression can result from translocation with the IGH locus or intrachromosomal deletion and is associated with poor outcome. CRLF2 overexpressing B-ALLs share a transcriptional signature that significantly overlaps with a BCR/ABL signature, and is enriched for genes involved in cytokine receptor and JAK-STAT signaling. In a subset of cases, CRLF2 harbors a Phe232Cys gain-of-function mutation that promotes constitutive dimerization and cytokine independent growth. A mutually exclusive subset harbors activating mutations in JAK2. In fact, all 22 B-ALLs with mutant JAK2 that we analyzed overexpress CRLF2, distinguishing CRLF2 as the key scaffold for mutant JAK2 signaling in B-ALL. Expression of WT CRLF2 with mutant JAK2 also promotes cytokine independent growth that, unlike CRLF2 Phe232Cys or ligand-induced signaling by WT CRLF2, is accompanied by JAK2 phosphorylation. Finally, cells dependent on CRLF2 signaling are sensitive to small molecule inhibitors of either JAKs or protein kinase C family kinases. Together, these findings implicate CRLF2 as an important factor in B-ALL with diagnostic, prognostic, and therapeutic implications.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Receptores de Citocinas/genética , Transdução de Sinais/fisiologia , Adulto , Criança , Citocinas/metabolismo , Análise Mutacional de DNA , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Prognóstico , Receptores de Citocinas/metabolismo , Taxa de Sobrevida , Transcrição GênicaRESUMO
SETD2, a lysine N-methyltransferase, is a histone methyltransferase that plays an important role in various cellular processes and was identified as a target of interest in multiple myeloma that features a t(4,14) translocation. We recently reported the discovery of a novel small-molecule SETD2 inhibitor tool compound that is suitable for preclinical studies. Herein we describe the conformational-design-driven evolution of the advanced chemistry lead, which resulted in compounds appropriate for clinical evaluation. Further optimization of this chemical series led to the discovery of EZM0414, which is a potent, selective, and orally bioavailable inhibitor of SETD2 with good pharmacokinetic properties and robust pharmacodynamic activity in a mouse xenograft model.
RESUMO
The human fetus is not rejected by the maternal immune system despite expressing paternal antigens. Natural killer cells, the major lymphocyte population of the human decidua (dNKs), express genes with immunomodulatory potential. These include galectin-1 (gal1), a lectin with apoptotic activity on activated CD8(+) T cells, Th1 and Th17 CD4(+) cells. Although many cell types at the maternal-fetal interface also produce gal1, its production by dNKs has been used here to study its function in pregnancy. Media conditioned by dNKs containing gal1 induced apoptosis of activated T cells. This effect was blocked by anti-gal1 antibodies. Decidual T (dT) cells but not peripheral T (pT) cells bound gal1 and presented a distinct glycophenotype compatible with sensitivity to gal1. Annexin V staining, TUNEL, and hypodiploidy showed a substantial proportion of apoptotic dT cells. Immunohistochemistry revealed widespread expression of gal1 as well as periglandular apoptotic dT foci that colocalized with dNKs. Thus, secretion of gal1 by dNKs and other decidual cells contributes to the generation of an immune-privileged environment at the maternal-fetal interface.
Assuntos
Apoptose/fisiologia , Galectina 1/fisiologia , Troca Materno-Fetal , Linfócitos T/citologia , Meios de Cultivo Condicionados , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Células Matadoras Naturais/imunologia , GravidezRESUMO
Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia-A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor-ß (TGF-ß), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell- and natural killer cell-dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Oxigênio/uso terapêutico , Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Hiperóxia/complicações , Hiperóxia/patologia , Hipóxia/complicações , Hipóxia/imunologia , Hipóxia/patologia , Terapia de Imunossupressão , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neoplasias/patologia , Oxigênio/farmacologia , Indução de Remissão , Respiração/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
The marginal zone non-Hodgkin's lymphomas are a recently defined group of related low-grade B cell malignancies whose natural history is heterogeneous. The optimal therapy is often unclear, particularly for the subset of patients with disseminated disease that behaves aggressively. We have retrospectively analyzed the outcomes of 11 patients with chemosensitive but disseminated marginal zone lymphomas who underwent uniform conditioning with cyclophosphamide and total body irradiation followed by bone marrow transplantation (BMT) with anti-B cell monoclonal antibody-purged autologous bone marrow between January 1994 and September 1999. All patients had stage IV disease and received multiple chemotherapy regimens prior to autologous BMT. Only 36% were in complete remission at the time of bone marrow harvest, and 36% had overt bone marrow infiltration at that time. Two treatment-related deaths occurred between 100 days and 6 months. Three patients relapsed and died of disease. One patient developed and died of myelodysplasia. Five patients remain in continuous complete remission at a median follow-up of 52 months (45%). The median progression-free survival for these patients was 56 months, with median overall survival 58 months. The only significant predictor of disease-free and overall survival was age at the time of transplant; no patient under 45 at the time of transplant has relapsed or died of any cause (P = 0.003). Outcomes of autologous BMT in patients with disseminated marginal zone NHL are similar to those in follicular NHL, and suggest that certain patients may experience prolonged disease-free survival.
Assuntos
Transplante de Medula Óssea/métodos , Linfoma não Hodgkin/terapia , Transplante Autólogo/métodos , Adulto , Fatores Etários , Células da Medula Óssea/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Linfoma não Hodgkin/mortalidade , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Fatores de Tempo , Resultado do TratamentoRESUMO
UNLABELLED: Intratumoral hypoxia and hypoxia inducible factor-1α (HIF-1-α)-dependent CD39/CD73 ectoenzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60 % O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomic studies in mice. Importantly, oxygenation also downregulated the expression of adenosine-generating ectoenzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the upregulation of antigen-presenting MHC class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60 % oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer. KEY MESSAGES: Oxygenation decreases levels of tumor protecting hypoxia. Oxygenation decreases levels of tumor protecting extracellular adenosine. Oxygenation decreases expression of HIF-1alpha dependent tumor-protecting proteins. Oxygenation increases MHC class I expression and enables tumor regression.
Assuntos
Adenosina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/complicações , Hipóxia/terapia , Neoplasias/complicações , Neoplasias/terapia , Oxigênio/uso terapêutico , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Feminino , Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Microambiente TumoralRESUMO
Fanconi anemia (FA) is a human genetic disease characterized by chromosome instability, cancer predisposition, and cellular hypersensitivity to DNA crosslinking agents. The FA pathway regulates the repair of DNA crosslinks. A critical step in this pathway is the monoubiquitination and deubiquitination of FANCD2. Deubiquitination of FANCD2 is mediated by the ubiquitin protease, USP1. Here, we demonstrate that targeted deletion of mouse Usp1 results in elevated perinatal lethality, male infertility, crosslinker hypersensitivity, and an FA phenotype. Usp1(-/-) mouse embryonic fibroblasts had heightened levels of monoubiquitinated Fancd2 in chromatin. Usp1(-/-) cells exhibited impaired Fancd2 foci assembly and a defect in homologous recombination repair. Double knockout of Usp1 and Fancd2 resulted in a more severe phenotype than either single knockout. Our results indicate that mouse Usp1 functions downstream in the FA pathway. Deubiquitination is a critical event required for Fancd2 nuclear foci assembly, release from chromatin, and function in DNA repair.
Assuntos
Endopeptidases/genética , Endopeptidases/fisiologia , Anemia de Fanconi/genética , Instabilidade Genômica , Animais , Proteínas de Arabidopsis , Cruzamentos Genéticos , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Feminino , Fibroblastos/metabolismo , Deleção de Genes , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Proteases Específicas de UbiquitinaRESUMO
FLT3 receptor tyrosine kinase is expressed on lymphoid and myeloid progenitors in the hematopoietic system. Activating mutations in FLT3 have been identified in approximately 30% of patients with acute myelogenous leukemia, making it one of the most common mutations observed in this disease. Frequently, the mutation is an in-frame internal tandem duplication (ITD) in the juxtamembrane region that results in constitutive activation of FLT3, and confers interleukin-3 (IL-3)-independent growth to Ba/F3 and 32D cells. FLT3-ITD mutants were cloned from primary human leukemia samples and assayed for transformation of primary hematopoietic cells using a murine bone marrow transplantation assay. FLT3-ITDs induced an oligoclonal myeloproliferative disorder in mice, characterized by splenomegaly and leukocytosis. The myeloproliferative phenotype, which was associated with extramedullary hematopoiesis in the spleen and liver, was confirmed by histopathologic and flow cytometric analysis. The disease latency of 40 to 60 days with FLT3-ITDs contrasted with wild-type FLT3 and enhanced green fluorescent protein (EGFP) controls, which did not develop hematologic disease (> 200 days). These results demonstrate that FLT3-ITD mutant proteins are sufficient to induce a myeloproliferative disorder, but are insufficient to recapitulate the AML phenotype observed in humans. Additional mutations that impair hematopoietic differentiation may be required for the development of FLT3-ITD-associated acute myeloid leukemias. This model system should be useful to assess the contribution of additional cooperating mutations and to evaluate specific FLT3 inhibitors in vivo.
Assuntos
Transplante de Medula Óssea , Leucemia Mieloide Aguda/genética , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Citometria de Fluxo , Expressão Gênica , Humanos , Imunofenotipagem , Leucocitose , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transtornos Mieloproliferativos/patologia , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Esplenomegalia , Sequências de Repetição em Tandem , Transfecção , Células Tumorais Cultivadas , Receptor 1 de Fatores de Crescimento do Endotélio VascularRESUMO
Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor alpha (RARalpha). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARalpha in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARalpha from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARalpha can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARalpha transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARalpha transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARalpha and FLT3-ITD in development of the murine APL phenotype.
Assuntos
Leucemia Promielocítica Aguda/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Animais , Catepsina G , Catepsinas/genética , Cruzamentos Genéticos , Humanos , Imunofenotipagem , Leucemia Promielocítica Aguda/imunologia , Leucemia Promielocítica Aguda/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Camundongos Transgênicos , Transplante de Neoplasias , Serina Endopeptidases , Tretinoína/farmacologia , Ensaio Tumoral de Célula-TroncoRESUMO
The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell (HSC) biology, immune cell function, and leukemogenesis in zebrafish, we have developed hematopoietic cell transplantation (HCT) into adult recipient animals conditioned by gamma irradiation. Dose-response experiments showed that the minimum lethal dose (MLD) of 40 Gy led to the specific ablation of hematolymphoid cells and death by 14 days after irradiation. Sublethal irradiation doses of 20 Gy predominantly ablated lymphocytes and permitted transplantation of a lethal T-cell leukemia. Finally, transplantation of hematopoietic cells carrying transgenes yielding red fluorescent erythrocytes and green fluorescent leukocytes showed that HCT is sufficient to rescue the MLD, that recipient hematolymphoid tissues were repopulated by donor-derived cells, and that donor blood cell lineages can be independently visualized in living recipients. Together, these results establish transplantation assays to test for HSC function and oncogenic transformation in zebrafish.