RESUMO
The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.
Assuntos
Proteínas de Ciclo Celular , Centrossomo , Pontos de Checagem da Fase M do Ciclo Celular , Mitose , Centrossomo/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Divisão Celular , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Células HeLaRESUMO
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.
Assuntos
Proteínas Cdc20/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Mitose/fisiologia , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Fuso Acromático/metabolismoRESUMO
Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus â¼9-12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Anáfase , Aurora Quinase B/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Segregação de Cromossomos/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Metáfase , Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , Organoides/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais , Análise Espaço-Temporal , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Spindle checkpoint strength is dictated by the number of unattached kinetochores, cell volume, and cell fate. We show that the conserved AAA-ATPase PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in Caenorhabditis elegans. Having previously established that this function is required for spindle checkpoint activation, we demonstrate that in cells genetically manipulated to decrease in cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role is not limited to large cells: the stronger checkpoint in germline precursor cells also depends on PCH-2. PCH-2 is enriched in germline precursor cells, and this enrichment relies on conserved factors that induce asymmetry in the early embryo. Finally, the stronger checkpoint in germline precursor cells is regulated by CMT-1, the ortholog of p31comet, which is required for both PCH-2's localization to unattached kinetochores and its enrichment in germline precursor cells. Thus, PCH-2, likely by regulating the availability of inactive Mad2 at and near unattached kinetochores, governs checkpoint strength. This requirement may be particularly relevant in oocytes and early embryos enlarged for developmental competence, cells that divide in syncytial tissues, and immortal germline cells.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Mad2/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Fuso Acromático/metabolismoRESUMO
Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.
Assuntos
Antineoplásicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fuso Acromático/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Cães , Feminino , Células HT29 , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Wistar , Fuso Acromático/metabolismo , Resultado do TratamentoRESUMO
Studies have shown that palmatine (PAL) has anti-cancer effects. However, the activity and potential mechanisms of PAL against colorectal cancer remain elusive. The results showed that PAL significantly inhibited the proliferation of colon cancer cells in vitro and in vivo without significant effect on non-tumorigenic colon cells. Target prediction and clinical sample database analysis suggested that PAL may contribute to colon cancer cells phase arrest and apoptosis by targeting aurora kinase A (AURKA). Inhibition and overexpression of AURKA proved that PAL induces G2/M phase arrest and apoptosis in colon cancer cells by targeting AURKA. Moreover, PAL promoted intracellular Reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). PAL reduced the levels of AURKA, Bcl-xl and Bcl2 proteins, and promoted the expression of pro-apoptotic proteins P53, P73, Caspase3 and Caspase9, as well as the increase of cytochrome c (cyt. c) in cell lysates in vitro and in vivo. Together, our study confirmed that PAL induced G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA. PAL may provide a novel solution for the treatment of colon cancer by serving as a new AURKA inhibitor.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Alcaloides de Berberina/administração & dosagem , Neoplasias do Colo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Aurora Quinase A/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Células HCT116 , Células HT29 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: Osteosarcoma is one of the rare but fatal malignancies. The high metastatic rate, late diagnosis, emergence of drug resistance against drugs such as doxorubicin, and the lack of therapeutic targets obstructs the treatment of osteosarcoma. This study was undertaken to investigate the role and therapeutic potential of miR-187 in human osteosarcoma cells. METHODS: The WST-1 proliferation assay was used for investigation of cell viability. Transfections were carried out by Lipofectamine 2000 reagent. The qRT-PCR was used for expression analysis. DAPI, acridine orange (AO)/ethidium bromide (EB) and Annexin V/propidium iodide (PI) assay were used for apoptosis. Western blot analysis was used for the determination of protein expression. RESULTS: The expression of miR-187 was significantly downregulated in human osteosarcoma cells. Out of all osteosarcoma cell lines the SAOS-2 showed the lowest expression of miR-187 and therefore this cell line was selected for further studies. Overexpression of miR-187 caused significant inhibition in the proliferation of SAOS-2 osteosarcoma cells. The miR-187-triggered growth inhibition was found to be mainly due to induction of G2/M phase cell cycle arrest of the SAOS-2 cells. The G2/M cell cycle arrest was also accompanied by depletion of Cyclin-B1 expression. Additionally, miR-187 enhanced the chemosensitivity of the osteosarcoma cells to doxorubicin. The wound healing and transwell assay showed that miR-187 overexpression resulted in the suppression of migration and invasion of the SAOS-2 osteosarcoma cells. In silico analysis showed that miR-187 exerts its effects by inhibiting mitogen activated protein kinase 7 (MAPK7). The expression of MAPK7 was found to be significantly upregulated in osteosarcoma cells and overexpression of MAPK7 could nullify the effects of miR-187 on the proliferation of the osteosarcoma cells.
Assuntos
Neoplasias Ósseas/genética , MicroRNAs/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Osteossarcoma/genética , Antibióticos Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Doxorrubicina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , MicroRNAs/biossíntese , MicroRNAs/genética , Invasividade Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/enzimologia , Osteossarcoma/patologiaRESUMO
PURPOSE: Gliomas are aggressive brain tumors accounting for significant mortality across the globe. Biomarkers for early detection and therapeutic targets for efficient treatment are lacking for glioma. This study was undertaken to investigate the role and therapeutic implications of miR-22 in glioma. METHODS: U-87 glioma cell line was used in this study. qRT-PCR was employed for expression analysis. MTT assay was used for determination of cell viability. Lipofectamine 2000 was used for transfection. Flow cytometry was used for cell analysis. Wound healing assay and transwell assay were used for monitoring cell migration and invasion. Western blot analysis was used for estimation of protein expression. RESULTS: The miR-22 expression was found decreased in glioma cells. Overexpression of miR-22 resulted in arrest of the U-87 glioma cells at G2/M checkpoint of the cell cycle. The percentage of apoptotic U-87 cells in G2/M phase were 13.05% in negative control (NC) and 29.06% in miR-22 mimics transfected cells. The cell cycle arrest promoted by miR-22 overexpression was also associated with depletion of cyclin B1 expression in U-87 cells. Furthermore, miR-22 could also significantly increase the sensitivity of glioma U-87 cells to cisplatin. The TargetScan analysis and dual luciferase assay showed SNAIL1 to be the target of miR-22. The expression of SNAIL1 was also enhanced in all the glioma cells and miR-22 overexpression could cause suppression of the SNAIL1 expression in U-87 cells. Furthermore, SNAIL1 silencing could also cause decline in the viability of the U-87 cells. The wound healing assay showed that miR-5 overexpression caused decrease in the migration of U-87 cells, while the transwell assay showed decline in the invasion of miR-22 mimics transfected U-87 cells. CONCLUSION: Taken together, miR-22 may exhibit therapeutic implications in glioma and may prove useful in glioma treatment.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Glioma/genética , Glioma/patologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , MicroRNAs/genética , Metástase Neoplásica , Fatores de Transcrição da Família Snail/biossíntese , Fatores de Transcrição da Família Snail/genética , TransfecçãoRESUMO
During meiosis, diploid progenitor cells undergo one round of DNA replication followed by two rounds of chromosome segregation to form haploid gametes. Once cells initiate the meiotic divisions, it is imperative that they finish meiosis. A failure to maintain meiosis can result in highly aberrant polyploid cells, which could lead to oncogenesis in the germline. How cells stay committed to finishing meiosis, even in the presence of a mitosis-inducing signal, is poorly understood. We addressed this question in budding yeast, in which cells enter meiosis when starved. If nutrient-rich medium is added before a defined commitment point in mid-prometaphase I, they can return to mitosis. Cells in stages beyond the commitment point will finish meiosis, even with nutrient addition. Because checkpoints are signaling pathways known to couple cell-cycle processes with one another, we asked if checkpoints could ensure meiotic commitment. We find that two checkpoints with well-defined functions in mitosis, the DNA damage checkpoint and the spindle position checkpoint, have crucial roles in meiotic commitment. With nutrient-rich medium addition at stages beyond the commitment point, cells that are deficient in both checkpoints because they lack Rad53 and either Bub2, Bfa1, or Kin4 can return to mitotic growth and go on to form polyploid cells. The results demonstrate that the two checkpoints prevent cells from exiting meiosis in the presence of a mitosis-inducing signal. This study reveals a previously unknown function for the DNA damage checkpoint and the spindle position checkpoint in maintaining meiotic commitment.
Assuntos
Dano ao DNA/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Meiose/fisiologia , Saccharomyces cerevisiae/fisiologia , Segregação de Cromossomos/fisiologia , Mitose/fisiologiaRESUMO
Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of these viruses using an array of cell lines with different levels of impairment of antiviral signaling and a panel of chemical compounds arresting the cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G2/M phase strongly enhanced the replication of VSV-ΔM51 in cells with functional antiviral signaling. G2/M arrest strongly inhibited type I and type III interferon (IFN) production as well as expression of IFN-stimulated genes in response to exogenously added IFN. Moreover, G2/M arrest enhanced the replication of Sendai virus (a paramyxovirus), which is also highly sensitive to the type I IFN response but did not stimulate the replication of a wild-type VSV that is more effective at evading antiviral responses. In contrast, the positive effect of G2/M arrest on virus replication was not observed in cells defective in IFN signaling. Altogether, our data show that replication of IFN-sensitive cytoplasmic viruses can be strongly stimulated during G2/M phase as a result of inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. The G2/M phase thus could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest.IMPORTANCE Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of VSV and VSV-ΔM51. We show that G2/M cell cycle arrest strongly enhances the replication of VSV-ΔM51 (but not of wild-type VSV) and Sendai virus (a paramyxovirus) via inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. Our data suggest that the G2/M phase could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest, and it has important implications for oncolytic virotherapy, suggesting that frequent cell cycle progression in cancer cells could make them more permissive to viruses.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Vesiculovirus/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Citoplasma , Fase G2/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Expressão Gênica/genética , Humanos , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Interferons , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus de RNA/imunologia , Vírus de RNA/metabolismo , Vírus Sendai/genética , Vírus Sendai/metabolismo , Transdução de Sinais , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus/metabolismo , Proteínas da Matriz Viral/genética , Replicação Viral/imunologia , Interferon lambdaRESUMO
PURPOSE: Ovarian cancer is responsible for a significant number of deaths in women and there is urgent need to develop efficient treatment strategies for this disease. Studies have shown microRNAs (miRs) are involved in diverse cellular processes and exhibit therapeutic implications. Herein, the role of miR-375 in ovarian cancer was explored. METHODS: OVACAR-3 cell line was mainly used in this research. Expression analysis was performed by qRT-PCR. Cell viability was determined by MTT assay. Cell cycle analysis was carried out by flow cytometry. Transwell assay was used for cell migration and invasion. Western blot analysis was used to determine the protein expression. RESULTS: Gene expression analysis carried out by qRT-PCR of ovarian cancer cell lines and tissues revealed significant downregulation of miR-375. Ectopic expression of miR-375 halted the growth of the OVACAR-3 cells by triggering G2/M cell cycle arrest. Moreover, miR-375 also caused a significant decrease in the migratory and invasive potential of the OAVACAR-3 cells and enhanced their chemosensitivity to cisplatin. Bioinformatic analysis and the dual luciferase showed that miR-375 targets PAX2 in OVACAR-3 cells. Suppression of PAX2 inhibits the growth of the OVACAR-3 cells while PAX2 overexpression could avoid the growth inhibitory effects of miR-375 in OVACAR-3 cells. CONCLUSION: miR-375 may prove to be an important therapeutic target in ovarian cancer and warrants further research endeavors.
Assuntos
MicroRNAs/metabolismo , Neoplasias Ovarianas/genética , Fator de Transcrição PAX2/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , MicroRNAs/genética , Metástase Neoplásica , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fator de Transcrição PAX2/metabolismo , TransfecçãoRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, including gefitinib, are first-line drugs against advanced non-small cell lung cancer with activating EGFR mutations. However, the development of resistance to such drugs is a major clinical challenge. METHODS: The role of annexin A5 in resistance to EGFR tyrosine kinase inhibitors was investigated by qPCR and western blot of relevant molecules, by CCK8 and EdU assay of cell proliferation and viability, by annexin V/propidium iodide assay of apoptosis and cell cycle distribution, by JC-1 assay of mitochondrial integrity, and by xenograft assay of tumorigenicity. RESULTS: We found that annexin A5 is upregulated in gefitinib-resistant cell lines, as well as in clinical specimens resistant to EGFR tyrosine kinase inhibitors. Accordingly, knockdown of the gene from gefitinib-resistant cells restores gefitinib sensitivity in vitro and in vivo by downregulating polo-like kinase 1 signal pathway, thereby inducing mitochondrial damage, caspase activation, cell cycle arrest at G2/M, and, finally, apoptosis. CONCLUSIONS: The data indicate that annexin A5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and G2/M cell cycle arrest, and is thus a potential therapeutic target in non-small cell lung cancers resistant to EGFR tyrosine kinase inhibitors.
Assuntos
Anexina A5/deficiência , Antineoplásicos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Técnicas de Silenciamento de Genes , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Quinazolinas/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Anexina A5/biossíntese , Anexina A5/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Gefitinibe , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.
Assuntos
Cinetocoros/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Cisteína/análogos & derivados , Cisteína/farmacologia , Feminino , Cinesinas/antagonistas & inibidores , Cinetocoros/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Paclitaxel/farmacologia , Pirimidinas/farmacologia , Tionas/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
Aneuploidy, a condition that results from unequal partitioning of chromosomes during mitosis, is a hallmark of many cancers, including those caused by human papillomaviruses (HPVs). E6 and E7 are the primary transforming proteins in HPV that drive tumor progression. In this study, we stably expressed E6 and E7 in noncancerous RPE1 cells and analyzed the specific mitotic defects that contribute to aneuploidy in each cell line. We find that E6 expression results in multiple chromosomes associated with one or both spindle poles, causing a significant mitotic delay. In most cells, the misaligned chromosomes eventually migrated to the spindle equator, leading to mitotic exit. In some cells, however, mitotic exit occurred in the presence of pole-associated chromosomes. We determined that this premature mitotic exit is due to defects in spindle assembly checkpoint (SAC) signaling, such that cells are unable to maintain a prolonged mitotic arrest in the presence of unaligned chromosomes. This SAC defect is caused in part by a loss of kinetochore-associated Mad2 in E6-expressing cells. Our results demonstrate that E6-expressing cells exhibit previously unappreciated mitotic defects that likely contribute to HPV-mediated cancer progression.
Assuntos
Transformação Celular Viral , Cromátides/metabolismo , Papillomavirus Humano 16/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Polos do Fuso/metabolismo , Aneuploidia , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Humanos , Cinetocoros/metabolismo , Mitose , Proteínas E7 de Papillomavirus/metabolismo , Transdução de Sinais , Fuso Acromático/metabolismo , Polos do Fuso/fisiologiaRESUMO
Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αß'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dineínas/metabolismo , Células HeLa , Humanos , Cinetocoros/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Microtúbulos/metabolismo , Mitose/fisiologia , Transporte Proteico/fisiologiaRESUMO
The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31comet Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.
Assuntos
Chaperonina com TCP-1/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/fisiologia , Células HeLa , Humanos , Proteínas Mad2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Células Sf9 , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Spodoptera , Estaurosporina/farmacologiaRESUMO
In mitosis, for each daughter cell to inherit an accurate copy of the genome from the mother cell, sister chromatids in the mother cell must attach to microtubules emanating from opposite poles of the mitotic spindle, a process known as bi-orientation. A surveillance mechanism, termed the spindle assembly checkpoint (SAC), monitors the microtubule attachment process and can temporarily halt the separation of sister chromatids and the completion of mitosis until bi-orientation is complete. SAC failure results in abnormal chromosome numbers, termed aneuploidy, in the daughter cells, a hallmark of many tumours. The HORMA-domain-containing protein mitotic arrest deficient 2 (MAD2) is a subunit of the SAC effector mitotic checkpoint complex (MCC). Structural conversion from the open to the closed conformation of MAD2 is required for MAD2 to be incorporated into the MCC. In vitro, MAD2 conversion and MCC assembly take several hours, but in cells the SAC response is established in a few minutes. Here, to address this discrepancy, we reconstituted a near-complete SAC signalling system with purified components and monitored assembly of the MCC in real time. A marked acceleration in MAD2 conversion and MCC assembly was observed when monopolar spindle 1 (MPS1) kinase phosphorylated the MAD1-MAD2 complex, triggering it to act as the template for MAD2 conversion and therefore contributing to the establishment of a physical platform for MCC assembly. Thus, catalytic activation of the SAC depends on regulated protein-protein interactions that accelerate the spontaneous but rate-limiting conversion of MAD2 required for MCC assembly.
Assuntos
Biocatálise , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas Mad2/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Cinética , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Proteínas Tirosina Quinases/metabolismo , Fuso Acromático/metabolismo , Fatores de TempoRESUMO
Cell cycle checkpoints are surveillance mechanisms that sequentially and continuously monitor cell cycle progression thereby contributing to the preservation of genetic stability. Among them, the spindle assembly checkpoint (SAC) prevents the occurrence of abnormal divisions by halting the metaphase to anaphase transition following the detection of erroneous microtubules-kinetochore attachment(s). Most synchronization strategies are based on the activation of cell cycle checkpoints to enrich the population of cells in a specific phase of the cell cycle. Here, we develop a two-step protocol of sequential cell synchronization and desynchronization employing antimitotic SAC-inducing agents (i.e., nocodazole or paclitaxel) in combination with the depletion of the SAC kinase MPS1. We describe cytofluorometric and time-lapse videomicroscopy methods to detect cell cycle progression, including the assessment of cell cycle distribution, quantification of mitotic cell fraction, and analysis of single cell fate profile of living cells. We applied these methods to validate the synchronization-desynchronization protocol and to qualitatively and quantitatively determine the impact of SAC inactivation on the activity of antimitotic agents.
Assuntos
Ciclo Celular/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Anáfase/genética , Ciclo Celular/genética , Citometria de Fluxo , Células HCT116 , Histonas/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Microscopia de Vídeo , Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , Fosforilação , Poliploidia , RNA Interferente Pequeno/genética , Software , TetraploidiaRESUMO
The spindle checkpoint, also called spindle assembly checkpoint (SAC), is a crucial control instance in animals and yeast that surveys the correct attachment of chromosomes to the spindle assuring their equal distribution in mitosis and meiosis. The presence of homologs of all core SAC components in plants indicates that these regulators have an ancient function. However, the fact that mutants of SAC components in plants are usually fully viable together with the observation that plants can be readily made polyploid raises the question whether plants have an efficient SAC. Recently, the role and regulation of a putative SAC in plants has been addressed. Interestingly, these studies also revealed that SAC genes are involved in several other cellular and developmental processes outside of chromosome distribution control.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular/genética , Cromossomos de Plantas/genética , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Meiose/genética , Meiose/fisiologia , Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , PoliploidiaRESUMO
In mammals, a finite population of oocytes is generated during embryogenesis, and proper oocyte meiotic divisions are crucial for fertility. Sperm-associated antigen 1 (SPAG-1) has been implicated in infertility and tumorigenesis; however, its relevance in cell cycle programs remains rudimentary. Here we explore a novel role of SPAG-1 during oocyte meiotic progression. SPAG-1 associated with meiotic spindles and its depletion severely compromised M-phase entry (germinal vesicle breakdown [GVBD]) and polar body extrusion. The GVBD defect observed was due to an increase in intraoocyte cAMP abundance and decrease in ATP production, as confirmed by the activation of AMP-dependent kinase (AMPK). SPAG-1 RNA interference (RNAi)-elicited defective spindle morphogenesis was evidenced by the dysfunction of γ-tubulin, which resulted from substantially reduced phosphorylation of MAPK and irregularly dispersed distribution of phospho-MAPK around spindles instead of concentration at spindle poles. Significantly, actin expression abruptly decreased and formation of cortical granule-free domains, actin caps, and contractile ring disrupted by SPAG-1 RNAi. In addition, the spindle assembly checkpoint remained functional upon SPAG-1 depletion. The findings broaden our knowledge of SPAG-1, showing that it exerts a role in oocyte meiotic execution via its involvement in AMPK and MAPK signaling pathways.