RESUMO
Proper chromosome segregation is essential for maintenance of genomic integrity and instability resulting from failure of this process may contribute to cancer. Here, we demonstrate that a mutation in the mitotic regulator separase is responsible for the cell cycle defects seen in the zebrafish mutant, cease&desist (cds). Analysis of cds homozygous mutant embryos reveals high levels of polyploidy and aneuploidy, spindle defects, and a mitotic exit delay. Carcinogenesis studies demonstrated that cds heterozygous adults have a shift in tumor spectrum with an eightfold increase in the percentage of fish bearing epithelial tumors, indicating that separase is a tumor suppressor gene in vertebrates. These data strongly support a conserved cross-species role for mitotic checkpoint genes in genetic stability and epithelial carcinogenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Suscetibilidade a Doenças , Endopeptidases/genética , Instabilidade Genômica , Mitose , Mutação , Neoplasias Epiteliais e Glandulares/etiologia , Animais , Bromodesoxiuridina , Carcinoma Ductal Pancreático/etiologia , Carcinoma Ductal Pancreático/patologia , Ciclo Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Heterozigoto , Homozigoto , Neoplasias Intestinais/etiologia , Neoplasias Intestinais/patologia , Neoplasias Epiteliais e Glandulares/patologia , Ploidias , Separase , Fuso Acromático/genética , Fuso Acromático/patologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4-DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4-DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiologia , Mitose/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas Culina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/anormalidades , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos da radiação , Fase G2/efeitos da radiação , Testes Genéticos , Células HCT116 , Células HeLa , Humanos , Mitose/efeitos da radiação , Modelos Biológicos , Mutagênese Insercional , Mutação/genética , Proteínas Nucleares , Ligação Proteica/efeitos da radiação , Radiação Ionizante , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra/embriologiaRESUMO
Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse approximately 16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy-treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia--and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes.
Assuntos
Adamantano/análogos & derivados , Mutação , Proteínas Proto-Oncogênicas c-myb/genética , Piridinas/farmacologia , Fase S/genética , Peixe-Zebra/genética , Adamantano/química , Adamantano/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclina B/efeitos dos fármacos , Ciclina B/genética , Ciclina B1 , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mitose/efeitos dos fármacos , Fenótipo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Piridinas/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Fase S/efeitos dos fármacos , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
A major goal of cancer research has been to identify genes that contribute to cancer formation. The similar pathology between zebrafish and human tumors, as well as the past success of large-scale genetic screens in uncovering human disease genes, makes zebrafish an ideal system in which to find such new genes. Here, we show that a zebrafish forward genetic screen uncovered multiple cell proliferation mutants including one mutant, crash&burn (crb), that represents a loss-of-function mutation in bmyb, a transcriptional regulator and member of a putative proto-oncogene family. crb mutant embryos have defects in mitotic progression and spindle formation, and exhibit genome instability. Regulation of cyclin B levels by bmyb appears to be the mechanism of mitotic accumulation in crb. Carcinogenesis studies reveal increased cancer susceptibility in adult crb heterozygotes. Gene-expression signatures associated with loss of bmyb in zebrafish are also correlated with conserved signatures in human tumor samples, and down-regulation of the B-myb signature genes is associated with retention of p53 function. Our findings show that zebrafish screens can uncover cancer pathways, and demonstrate that loss of function of bmyb is associated with cancer.
Assuntos
Instabilidade Genômica , Mutação , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myb/genética , Animais , Ciclina B/metabolismo , Embrião não Mamífero , Predisposição Genética para Doença , Mitose , Neoplasias/etiologia , Proto-Oncogene Mas , Fuso Acromático , Proteína Supressora de Tumor p53 , Peixe-ZebraRESUMO
Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo.
Assuntos
Linhagem da Célula/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Aorta/citologia , Aorta/fisiologia , Células Sanguíneas/citologia , Células Sanguíneas/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/efeitos da radiação , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Desenvolvimento Embrionário/fisiologia , Desenvolvimento Embrionário/efeitos da radiação , Raios gama , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Gônadas/citologia , Gônadas/fisiologia , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Homeostase/fisiologia , Homeostase/efeitos da radiação , Mesonefro/citologia , Mesonefro/fisiologia , Mutação , Irradiação Corporal Total/métodos , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaAssuntos
Ciclo Celular/fisiologia , Embrião não Mamífero/fisiologia , Peixe-Zebra/fisiologia , Laranja de Acridina/química , Animais , Apoptose , Bromodesoxiuridina/análise , Bromodesoxiuridina/metabolismo , Ciclo Celular/genética , Centrossomo/química , DNA/análise , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Citometria de Fluxo , Histonas/metabolismo , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Mutação , Fosforilação , Propídio/química , Fuso Acromático/química , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The zebrafish, with its combination of forward genetics and vertebrate biology, has great potential as a cancer model system.
