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1.
Nat Immunol ; 18(9): 1016-1024, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28692065

RESUMO

Aberrant population expansion of follicular helper T cells (TFH cells) occurs in patients with lupus. An unanswered question is whether an altered repertoire of T cell antigen receptors (TCRs) is associated with such expansion. Here we found that the transcription factor Blimp-1 (encoded by Prdm1) repressed expression of the gene encoding cathepsin S (Ctss), a cysteine protease that cleaves invariant chains and produces antigenic peptides for loading onto major histocompatibility complex (MHC) class II molecules. The increased CTSS expression in dendritic cells (DCs) from female mice with dendritic cell-specific conditional knockout of Prdm1 (CKO mice) altered the presentation of antigen to CD4+ T cells. Analysis of complementarity-determining region 3 (CDR3) regions containing the ß-chain variable region (Vß) demonstrated a more diverse repertoire of TFH cells from female CKO mice than of those from wild-type mice. In vivo treatment of CKO mice with a CTSS inhibitor abolished the lupus-related phenotype and reduced the diversity of the TFH cell TCR repertoire. Thus, Blimp-1 deficiency in DCs led to loss of appropriate regulation of Ctss expression in female mice and thereby modulated antigen presentation and the TFH cell repertoire to contribute to autoimmunity.


Assuntos
Catepsinas/metabolismo , Células Dendríticas/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Transcrição/genética , Animais , Anticorpos Antinucleares/imunologia , Apresentação do Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Proliferação de Células , DNA/imunologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Rim/patologia , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptores de Antígenos de Linfócitos T alfa-beta/genética
2.
JCI Insight ; 2(1): e89569, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28097234

RESUMO

A SNP identified as rs548234, which is found in PRDM1, the gene that encodes BLIMP1, is a risk allele associated with systemic lupus erythematosus (SLE). BLIMP1 expression was reported to be decreased in women with the PRDM1 rs548234 risk allele compared with women with the nonrisk allele in monocyte-derived DCs (MO-DCs). In this study, we demonstrate that BLIMP1 expression is regulated by the binding of Kruppel-like factor 4 (KLF4) to the risk SNP. KLF4 is highly expressed in MO-DCs but undetectable in B cells, consistent with the lack of altered expression of BLIMP1 in B cells from risk SNP carriers. Female rs548234 risk allele carriers, but not nonrisk allele carriers, exhibited decreased levels of BLIMP1 in MO-DCs, showing that the regulatory function of KLF4 is influenced by the risk allele. In addition, KLF4 directly recruits histone deacetylases (HDAC4, HDAC6, and HDAC7), established negative regulators of gene expression. Finally, the knock down of KLF4 expression reversed the inhibitory effects of the risk SNP on promoter activity and BLIMP1 expression. Therefore, the binding of KLF4 and the subsequent recruitment of HDACs represent a mechanism for reduced BLIMP1 expression in MO-DCs bearing the SLE risk allele rs548234.


Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Lúpus Eritematoso Sistêmico/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Adulto , Alelos , Animais , Linfócitos B , Feminino , Expressão Gênica , Heterozigoto , Histona Desacetilases/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Proteínas Repressoras/metabolismo
3.
PLoS One ; 10(11): e0141523, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26544187

RESUMO

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Senescência Celular , Células Clonais/citologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Deleção de Sequência
4.
PLoS One ; 10(11): e0143240, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26606454

RESUMO

The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.


Assuntos
Genes Homeobox , Leucemia/genética , Proto-Oncogenes , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Linhagem Celular , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Epistasia Genética , Feminino , Deleção de Genes , Expressão Gênica , Biblioteca Gênica , Vetores Genéticos/genética , Imunofenotipagem , Leucemia/mortalidade , Leucemia/patologia , Camundongos , Família Multigênica , Proteínas Proto-Oncogênicas c-myc/genética , Retroviridae/genética , Transdução Genética
5.
Mol Cells ; 34(2): 201-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22843119

RESUMO

The hematopoietic cell malignancy is one of the most prevalent type of cancer and the disease has multiple pathologic molecular signatures. Research on the origin of hematopoietic cancer stem cells and the mode of subsequent maintenance and differentiation needs robust animal models that can reproduce the transformation and differentiation event in vivo. Here, we show that co-transduction of MYC and PIM2 proto-oncogenes into mouse bone marrow cells readily establishes permanent cell lines that can induce lethal myeloid sarcoma in vivo. Unlike the previous doubly transgenic mouse model in which coexpression of MYC and PIM2 transgenes exclusively induced B cell lymphoma, we were able to show that the same combination of genes can also transform primary bone marrow myeloid cells in vitro resulting in permanent cell lines which induce myeloid sarcoma upon in vivo transplantation. By inducing cancerous transformation of fresh bone marrow cells in a controlled environment, the model we established will be useful for detailed study of the molecular events involved in initial transformation process of primary myeloid bone marrow cells and provides a model that can give insight to the molecular pathologic characteristics of human myeloid sarcoma, a rare presentation of solid tumors of undifferentiated myeloid blast cells associated with various types of myeloid leukemia.


Assuntos
Células da Medula Óssea/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Sarcoma Mieloide/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/patologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Sarcoma Mieloide/patologia
6.
Mol Endocrinol ; 24(7): 1441-52, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20444885

RESUMO

In obesity, dysregulation of adipocytokines is involved in several pathological conditions including diabetes and certain cancers. As a member of the adipocytokines, adiponectin plays crucial roles in whole-body energy homeostasis. Recently, it has been reported that the level of plasma adiponectin is reduced in several types of cancer patients. However, it is largely unknown whether and how adiponectin affects colon cancer cell growth. Here, we show that adiponectin suppresses the proliferation of colon cancer cells including HCT116, HT29, and LoVo. In colon cancer cells, adiponectin attenuated cell cycle progression at the G(1)/S boundary and concurrently increased expression of cyclin-dependent kinase inhibitors such as p21 and p27. Adiponectin stimulated AMP-activated protein kinase (AMPK) phosphorylation whereas inhibition of AMPK activity blunted the effect of adiponectin on the proliferation of colon cancer cells. Furthermore, knockdown of adiponectin receptors such as AdipoR1 and AdipoR2 relieved the suppressive effect of adiponectin on the growth of colon cancer cells. In addition, adiponectin repressed the expression of sterol regulatory element binding protein-1c, which is a key lipogenic transcription factor associated with colon cancers. These results suggest that adiponectin could inhibit the growth of colon cancer cells through stimulating AMPK activity.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adiponectina/farmacologia , Proliferação de Células/efeitos dos fármacos , Receptores de Adiponectina/metabolismo , Adiponectina/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Células CHO , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Cricetinae , Cricetulus , Células HCT116 , Células HT29 , Humanos , Reação em Cadeia da Polimerase , Receptores de Adiponectina/genética
7.
Mol Cancer Res ; 7(3): 371-82, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19276188

RESUMO

Functional suppression of spindle checkpoint protein activity results in apoptotic cell death arising from mitotic failure, including defective spindle formation, chromosome missegregation, and premature mitotic exit. The recently identified p31(comet) protein acts as a spindle checkpoint silencer via communication with the transient Mad2 complex. In the present study, we found that p31(comet) overexpression led to two distinct phenotypic changes, cellular apoptosis and senescence. Because of a paucity of direct molecular link of spindle checkpoint to cellular senescence, however, the present report focuses on the relationship between abnormal spindle checkpoint formation and p31(comet)-induced senescence by using susceptible tumor cell lines. p31(comet)-induced senescence was accompanied by mitotic catastrophe with massive nuclear and chromosomal abnormalities. The progression of the senescence was completely inhibited by the depletion of p21(Waf1/Cip1) and partly inhibited by the depletion of the tumor suppressor protein p53. Notably, p21(Waf1/Cip1) depletion caused a dramatic phenotypic conversion of p31(comet)-induced senescence into cell death through mitotic catastrophe, indicating that p21(Waf1/Cip1) is a major mediator of p31(comet)-induced cellular senescence. In contrast to wild-type p31(comet), overexpression of a p31 mutant lacking the Mad2 binding region did not cause senescence. Moreover, depletion of Mad2 by small interfering RNA induced senescence. Here, we show that p31(comet) induces tumor cell senescence by mediating p21(Waf1/Cip1) accumulation and Mad2 disruption and that these effects are dependent on a direct interaction of p31(comet) with Mad2. Our results could be used to control tumor growth.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/metabolismo , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , DNA/metabolismo , Humanos , Proteínas Mad2 , Mitose/fisiologia , Proteínas Nucleares/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais , Fuso Acromático/genética , Fuso Acromático/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/biossíntese
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