RESUMO
Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart.
Assuntos
Reparo do DNA , Replicação do DNA , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Quinases/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histonas , Humanos , Metilação , Mutação , Fosforilação , Proteínas Quinases/genéticaRESUMO
A total of 50 consecutive patients (median age, 57.5 years) with AML (n=30) or myelodysplasia (MDS, n=20) underwent HLA matched related donor (MRD, n=27) or unrelated donor (MUD, n=23) peripheral blood hematopoietic cell transplantation after nonmyeloablative CY/fludarabine (Flu) conditioning. GVHD prophylaxis included CsA (n=19)+/-mycophenolate mofetil (n=31). At a median follow-up of 59 months, 21 patients (42%) were alive without evidence of disease. By Kaplan-Meier analysis, year 1-4 disease-free survival (DFS) and OS estimates were 0.50/0.58, 0.40/0.46, 0.37/0.43 and 0.37/0.41. MUD recipients were engrafted quickly (13.5 days) compared to MRD recipients (16 days) and relapsed/progressed less frequently (P=0.005). Overall grade 3/4 acute GVHD (aGVHD) occurred in 26% in the absence of antecedent mucositis and was associated with chronic GVHD (cGVHD) and poor OS. Extensive cGVHD developed in 51.2% of 100 day survivors. Rates of aGVHD, cGVHD and survival were similar between MRD and MUD recipients. Of 14 survivors with cGVHD, 5 (35.7%) experienced resolution off immunosuppression, suggesting that tolerance with HLA matched grafts is possible at an advanced age by this method. This study provides further evidence for prolonged DFS after CY/Flu MRD allotransplantation for AML/MDS, and extends the findings to older patients and those with unrelated donors.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicas/terapia , Condicionamento Pré-Transplante/métodos , Adolescente , Adulto , Fatores Etários , Idoso , Ciclofosfamida/uso terapêutico , Intervalo Livre de Doença , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Transplante Homólogo , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico , Adulto JovemAssuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Idoso , Sequência de Bases , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Subunidade alfa 2 de Fator de Ligação ao Core , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patologia , Masculino , Dados de Sequência Molecular , Translocação GenéticaRESUMO
Chemokines are a large family of cytokines that direct normal leukocyte migration. They also have been implicated in leukocyte development and in the pathogenesis of many diseases. The CC chemokine CCL21, also known as Exodus-2, SLC, 6Ckine, and TCA4 induces both the adhesion and migration of human T cells. CCL21 is hypothesized to regulate the trafficking of T cells through secondary lymphoid tissues. To test this hypothesis, a transgenic mouse model was generated that placed the expression of mouse CCL21 (mCCL21) under the control of the T cell-specific lck promoter to abrogate the concentration gradient to which T cells normally respond. Overexpression of mCCL21 in T cells resulted in defects in CCL21- and CCL19-induced T-cell chemotaxis, node T-cell subpopulations, and lymph node architecture. The regulation of T-cell trafficking in secondary lymphoid tissues by CCL21 is therefore a tightly regulated system that can be altered by changes in the level of environmental CCL21 protein.
Assuntos
Quimiocinas CC/genética , Quimiocinas CC/fisiologia , Quimiotaxia de Leucócito , Expressão Gênica , Linfócitos T/fisiologia , Animais , Linfócitos B , Western Blotting , Adesão Celular , Quimiocina CCL21 , Quimiocinas CC/análise , DNA/análise , Imuno-Histoquímica , Linfonodos/citologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Subpopulações de Linfócitos , Camundongos , Camundongos Transgênicos , Regiões Promotoras GenéticasRESUMO
A novel secreted cytokine, termed IL-17F, was cloned using nested RACE PCR. This cytokine bears homology to IL-17. IL-17F was expressed only in activated CD4(+) T cells and activated monocytes. Recombinant human IL-17F did not stimulate the proliferation of hematopoietic progenitors or the migration of mature leukocytes. However, it markedly inhibited the angiogenesis of human endothelial cells and induced endothelial cells to produce IL-2, TGF-beta, and monocyte chemoattractant protein-1.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Interleucina-17/farmacologia , Monócitos/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Linfócitos T/metabolismo , Sequência de Aminoácidos , Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/genética , Sequência de Bases , Clonagem Molecular , Citocinas/biossíntese , Humanos , Interleucina-17/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Homologia de Sequência de AminoácidosRESUMO
Chemokines are small basic proteins that are the major mediators of all leukocyte migration. There are at least 46 distinct chemokines, and 19 chemokine receptors, making it easily the largest cytokine family. Chemokines can be both beneficial and harmful, by either stimulating an appropriate immune response to microbial invasion, or by mediating pathologic tissue destruction in many types of human disease. Chemokines have been implicated in the tissue destruction seen in autoimmune diseases, atherosclerosis, allograft rejection, and neoplasia. Chemokines also play essential roles in normal lymphocyte trafficking to primary and secondary lymphoid organs for antigen presentation and lymphocyte maturation. Chemokines also regulate hematopoietic stem and progenitor cell homing and proliferation. Therefore, it is likely that chemokines will become important targets for pharmacologic intervention in a wide variety of human diseases in the future.
Assuntos
Quimiocinas/metabolismo , Hematopoese , Sistema Imunitário/metabolismo , Receptores de Quimiocinas/fisiologia , Animais , Northern Blotting , HumanosRESUMO
MZF1 is a transcription factor belonging to the Krüppel family of zinc finger proteins, expressed in totipotent hemopoietic cells as well as in myeloid progenitors. Here we have inactivated Mzfi1 by gene targeting. Mzf1(-/-) mice develop lethal neoplasias characterized by the infiltration and complete disruption of the liver architecture by a monomorphic population of cells of myeloid origin reminiscent of human chloromas. Mzf1 inactivation results in a striking increase of the autonomous cell proliferation and of the ability of Mzf1(-/-) hemopoietic progenitors to sustain long-term hemopoiesis. These findings demonstrate that Mzf1 can act as a tumor/growth suppressor in the hemopoietic compartment.
Assuntos
Divisão Celular/fisiologia , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Genes Supressores de Tumor , Fatores de Transcrição/fisiologia , Dedos de Zinco , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Knockout , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
AML1 is a transcription factor that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of radiation-associated secondary acute myelogenous leukemia that had AML1 translocations. Using polymerase chain reaction, the AML1 fusion transcript was isolated from the patient who had a t(19;21) radiation-associated leukemia. The AML1 gene is fused out of frame to chromosome 19 sequences, resulting in a truncated AML protein bearing the DNA binding domain but not the transcriptional activation domain. This fusion AML1 protein functions as an inhibitor of the normal AML1 protein. (Blood. 2001;97:2168-2170)
Assuntos
Cromossomos Humanos Par 19/ultraestrutura , Cromossomos Humanos Par 21/ultraestrutura , Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Leucemia Induzida por Radiação/genética , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Translocação Genética/genética , Doença Aguda , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 21/genética , Subunidade alfa 2 de Fator de Ligação ao Core , DNA Complementar/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Humanos , Hibridização In Situ , Leucemia Mieloide/etiologia , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Deleção de Sequência , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/químicaRESUMO
The AML1 gene, located at chromosome 21q22, encodes a component (CBFalpha2) of a heterodimeric transcription factor complex termed core binding factor (CBF), which binds to DNA and activates gene expression. Chromosomal rearrangements may lead to disruption of this gene and development of acute leukemia. Twelve AML1 translocations have been identified to date, and include sites on chromosomes 1, 2, 3, 5, 8, 12, 14, 15, 16, 17, 18, and 19. Here we report two new translocations involving AML1 in acute myeloid leukemia, in which the disruption of the AML1 gene was documented by GTG banding cytogenetic studies and metaphase and interphase FISH analysis. These chromosomal breakpoints identified as harboring new fusion partners for AML1 are at 2p11.2 and 20q13.1. The two patients in who these translocation were identified were elderly males with newly diagnosed AML. These patients shared the same poor outcomes reported for other rare AML1 translocations.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Translocação Genética , Doença Aguda , Idoso , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 21/genética , Subunidade alfa 2 de Fator de Ligação ao Core , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , MasculinoRESUMO
Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.
Assuntos
Fibronectinas/genética , Técnicas de Transferência de Genes , Genes MDR , Vetores Genéticos , Germinoma/terapia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Retroviridae , Adolescente , Adulto , Antígenos CD34 , Seguimentos , Terapia Genética/métodos , Humanos , Pessoa de Meia-Idade , Projetos Piloto , Fatores de Tempo , Resultado do TratamentoRESUMO
AML1 is a transcriptional activator that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. We recently identified 3 patients in whom pancytopenia developed almost 50 years after high-level radiation exposure from nuclear explosions during or after World War II. In all 3 patients, acute myeloid leukemia (AML) eventually developed that had similar characteristics and clinical courses. Cytogenetics from the 3 patients revealed a t(1;21)(p36;q22), a t(18;21)(q21;q22), and a t(19;21)(q13.4;q22). By fluorescent in situ hybridization (FISH), all 3 translocations disrupted the AML1 gene. Two of these AML1 translocations, the t(18;21) and the t(19;21), have not been reported previously. It is possible that the AML1 gene is a target for radiation-induced AML. (Blood. 2000;95:4011-4013)
Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Leucemia Induzida por Radiação/genética , Proteínas Proto-Oncogênicas , Cinza Radioativa , Fatores de Transcrição/genética , Translocação Genética , Doença Aguda , Idoso , Medula Óssea/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 21 , Subunidade alfa 2 de Fator de Ligação ao Core , Humanos , Leucemia Mieloide/patologia , Leucemia Induzida por Radiação/patologia , Masculino , Proteínas de Neoplasias/genética , Guerra Nuclear , SíndromeRESUMO
Natural killer (NK) cells play an important role in innate and adaptive immune responses to obligate intracellular pathogens. Nevertheless, the regulation of NK cell trafficking and migration to inflammatory sites is poorly understood. Exodus-1/MIP-3alpha/LARC, Exodus-2/6Ckine/SLC, and Exodus-3/MIP-3beta/ELC/CKbeta-11 are CC chemokines that share a unique aspartate-cysteine-cysteine-leucine motif near their amino terminus and preferentially stimulate the migration of T lymphocytes. The effects of Exodus chemokines on human NK cells were examined. Exodus-1, -2, and -3 did not induce detectable chemotaxis of resting peripheral blood NK cells. In contrast, Exodus-2 and -3 stimulated migration of polyclonal activated peripheral blood NK cells in a dose-dependent fashion. Exodus-2 and -3 also induced dose-dependent chemotaxis of NKL, an IL-2-dependent human NK cell line. Results of modified checkerboard assays indicate that migration of NKL cells in response to Exodus-2 and -3 represents true chemotaxis and not simply chemokinesis. Exodus-1, -2, and -3 did not induce NK cell proliferation in the absence of other stimuli. Nevertheless, Exodus-2 and -3 significantly augmented IL-2-induced proliferation of normal human CD56(dim) NK cells. In contrast, Exodus-1, -2, and -3 did not affect the cytolytic activity of resting or activated peripheral blood NK cells. Expression of message for CCR7, a shared receptor for Exodus-2 and -3, was detected in activated polyclonal NK cells and NKL cells but not resting NK cells. Taken together, these results indicate that Exodus-2 and -3 can participate in the recruitment and proliferation of activated NK cells. Exodus-2 and -3 may regulate interactions between T cells and NK cells that are crucial for the generation of optimal immune responses.
Assuntos
Quimiocinas CC/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Proteínas Inflamatórias de Macrófagos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CCL19 , Quimiocina CCL20 , Quimiocina CCL21 , Quimiotaxia de Leucócito/imunologia , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Relação Dose-Resposta Imunológica , Expressão Gênica , Humanos , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores CCR6 , Receptores CCR7 , Receptores de Quimiocinas/genéticaRESUMO
Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in disease processes. Among the biologic activities of chemokines is inhibition of proliferation of normal hematopoietic progenitors. However, chemokines that inhibit normal progenitors rarely inhibit proliferation of hematopoietic progenitors from patients with chronic myelogenous leukemia (CML). We and others recently cloned a subfamily of CC chemokines that share similar amino-terminal peptide sequences and a remarkable ability to chemoattract T cells. These chemokines, Exodus-1/LARC/MIP-3alpha, Exodus-2/SLC/6Ckine/TCA4, and Exodus-3/CKbeta11/MIP-3beta, were found to inhibit proliferation of normal human marrow progenitors. The study described here found that these chemokines also inhibited the proliferation of progenitors in every sample of marrow from patients with CML that was tested. This demonstration of consistent inhibition of CML progenitor proliferation makes the 3 Exodus chemokines unique among chemokines. (Blood. 2000;95:1506-1508)
Assuntos
Quimiocinas CC/farmacologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/fisiopatologia , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL19 , Quimiocina CCL20 , Quimiocina CCL21 , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Receptores CCR6RESUMO
CK beta-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CK beta-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CK beta 11)SN, the murine breast cancer cell line C3L5 (C3L5-CK beta 11) showed expression of retroviral mRNA by Northern analysis and production of functional CK beta-11 by chemotaxis of human NK cells to C3L5-CK beta 11 supernatant. Only 10% of mice injected with C3L5-CK beta 11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CK beta-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CK beta 11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CK beta 11 vaccination significantly reduced CK beta-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CK beta 11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CK beta-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CK beta-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CK beta-11 as an adjuvant in stimulating antitumor responses.
Assuntos
Quimiocinas CC/fisiologia , Rejeição de Enxerto/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Quimiocina CCL19 , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/imunologia , Humanos , Soros Imunes/administração & dosagem , Injeções Intraperitoneais , Injeções Subcutâneas , Depleção Linfocítica , Subpopulações de Linfócitos/imunologia , Transfusão de Linfócitos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Baço/citologia , Baço/transplante , Células Tumorais CultivadasRESUMO
Acute promyelocytic leukemia (APL) is characterized by a block in myeloid cell differentiation. As a result of a chromosomal translocation in these patients, the promyelocytic leukemia protein PML is disrupted as are the nuclear bodies it forms. Disruption of PML and PML nuclear bodies in APL is linked to a loss of growth control and subsequent leukemogenesis. PML contains a zinc-binding domain known as the RING which is required for formation of these bodies. Using yeast 2-hybrid techniques, we found that PML and a related RING protein, Z, bind the proline rich homeodomain protein (PRH) through their RING domains. Previous reports indicate that PRH functions in hematopoiesis and may act as a transcriptional repressor. Our data indicate that PML and Z both bind the repressor domain of PRH and are the first protein partners reported for PRH. We observe that PRH has a punctate pattern in both the nucleus and cytoplasm of chronic myelogenous leukemia K562 cells and in the APL cell line, NB4. Immunoprecipitation and co-localization studies indicate that PML and PRH interact in both cell lines. The effect on cell growth by PML and the hematopoietic actions of PRH raises the possibility that the interaction between PML and PRH represents a link between growth control and hematopoiesis.
Assuntos
Divisão Celular , Hematopoese , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Prolina/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/química , Humanos , Proteína da Leucemia Promielocítica , Ligação Proteica , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
We and other workers have recently isolated three novel CC chemokines termed Exodus-1/LARC/Mip-3alpha, Exodus-2/6Ckine/SLC/TCA4, and Exodus-3/Mip-3beta/CKbeta11/ELC. These chemokines share an amino terminal Asp-Cys-Cys-Leu sequence, unique among all chemokines. They also selectively regulate migration of adult T cells. Indeed, there is evidence that Exodus-2 and -3 are critical for adult T-cell adhesion to high endothelial venules in lymph nodes, a rate-limiting step for T-cell trafficking through nodal tissue. Less is known of the factors controlling migration of naïve human fetal T cells. We tested whether these chemokines could regulate chemotaxis in cord blood T-cell populations, and compared that efficacy with normal peripheral blood adult T cells. The findings indicated that naive CD45RA+ cord blood T-cell migration is stimulated by Exodus-2 and -3, and CD4+ cord blood T cells are attracted preferentially by Exodus-2 or -3 as compared with CD8+. Exodus-2 and -3 are likely to be critical in regulating the flux of naive CD4 + fetal T-cell population of secondary lymphoid tissue.
Assuntos
Quimiocinas CC/farmacologia , Quimiotaxia de Leucócito/fisiologia , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas , Subpopulações de Linfócitos T/efeitos dos fármacos , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Adesão Celular/efeitos dos fármacos , Quimiocina CCL19 , Quimiocina CCL20 , Quimiocina CCL21 , Quimiotaxia de Leucócito/efeitos dos fármacos , Sangue Fetal/citologia , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/análise , Imunofenotipagem , Recém-Nascido , Antígenos Comuns de Leucócito/análise , Receptores CCR6 , Homologia de Sequência de Aminoácidos , Subpopulações de Linfócitos T/imunologiaRESUMO
A novel repressor of the Winged Helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis (also called HFH2), was previously found to be exclusively expressed in primitive embryonic cell lines. In this study in situ cRNA hybridization experiments revealed that Genesis was expressed during embryogenesis only in developing neural crest cells. Its expression diminished upon their terminal differentiation into sympathetic and parasympathetic neurons. Based on that finding, Genesis was retrovirally transduced into pluripotent N-Tera-2 clone D1 (NT2/D1) teratocarcinoma cells, which are a well-described in vitro model of neural development. Retinoic acid (RA) treatment will drive these cells to differentiation toward the neuronal lineage and cause an increase in expression of the cyclin-dependent kinase inhibitor p21 protein, which leads to an inhibition in cellular proliferation. Although RA-induced expression of neuronal differentiation markers was not influenced by forced overexpression of Genesis in NT2-D1 cells, proliferation of Genesis-transduced cells continued following RA treatment. RA was unable to induce the expression of the cyclin-dependent kinase inhibitor p21 in the Genesis-transduced cells, but Go/G1 tumor suppressor p53 expression was induced normally. Therefore, Genesis may play a role in the regulation of primitive neural crest development by preventing terminal quiescence through inhibition of p21 protein expression. These data also lend evidence for the hypothesis that proliferation and differentiation pathways are not irrevocably linked, but can function independently.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/embriologia , Proteínas Repressoras/genética , Animais , Diferenciação Celular/genética , Divisão Celular/genética , Desenvolvimento Embrionário e Fetal/genética , Fatores de Transcrição Forkhead , Sequências Hélice-Volta-Hélice/genética , Camundongos , Especificidade de Órgãos , Transativadores/genéticaRESUMO
Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF-1-mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP-4/CK beta-10, MIP-4/CK beta-7, I-309, TECK, GCP-2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP-2, MCP-3, eotaxin-1, MCIF/HCC-1/CK beta-1, TARC, MDC, MPIF-2/eotaxin-2/CK beta-6, SDF-1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF-1-induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of beta 1-, and possibly alpha 4-, integrins.
Assuntos
Quimiocinas CXC/farmacologia , Quimiocinas/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Quimiocina CXCL12 , Quimiocinas/fisiologia , Quimiocinas C/farmacologia , Quimiocinas CC/farmacologia , Quimiotaxia , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , HumanosRESUMO
Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report here, the isolation of a novel murine CC chemokine termed ALP for its amino terminal peptide sequence. This novel chemokine is distantly related to other CC chemokines (37% identity with murine Exodus-1/LARC/Mip-3alpha), but has a unique carboxy terminal extension. It is expressed preferentially in testis, heart, and liver, which is atypical for CC chemokines.
Assuntos
Quimiocinas CC/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Quimiocina CCL27 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Quimiotaxia , Humanos , Fígado/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Miocárdio/metabolismo , Homologia de Sequência de Aminoácidos , Testículo/metabolismoRESUMO
Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report the PCR cloning of a novel human chemokine termed BRAK for its initial isolation from breast and kidney cells. This novel chemokine is distantly related to other CXC chemokines (30% identity with MIP-2alpha and beta) and shares several biological activities. BRAK is expressed ubiquitously and highly in normal tissue. However, it was expressed in only 2 of 18 cancer cell lines. BRAK is located on human chromosome 5q31.