RESUMO
Growing multicellular spheroids recapitulate many features of expanding microtumours, and therefore they are an attractive system for biomechanical studies. Here, we report an original approach to measure and characterize the forces exerted by proliferating multicellular spheroids. As force sensors, we used high aspect ratio PDMS pillars arranged as a ring that supports a growing breast tumour cell spheroid. After optical imaging and determination of the force application zones, we combined 3D reconstruction of the shape of each deformed PDMS pillar with the finite element method to extract the forces responsible for the experimental observation. We found that the force exerted by growing spheroids ranges between 100nN and 300nN. Moreover, the exerted force was dependent on the pillar stiffness and increased over time with spheroid growth.
Assuntos
Neoplasias da Mama/patologia , Técnicas de Cultura de Células/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Esferoides Celulares/patologia , Feminino , Humanos , Estresse Mecânico , Análise Serial de TecidosRESUMO
This paper investigates cell proliferation dynamics in small tumor cell aggregates using an individual-based model (IBM). The simulation model is designed to study the morphology of the cell population and of the cell lineages as well as the impact of the orientation of the division plane on this morphology. Our IBM model is based on the hypothesis that cells are incompressible objects that grow in size and divide once a threshold size is reached, and that newly born cell adhere to the existing cell cluster. We performed comparisons between the simulation model and experimental data by using several statistical indicators. The results suggest that the emergence of particular morphologies can be explained by simple mechanical interactions.
Assuntos
Linhagem da Célula , Modelos Biológicos , Neoplasias/patologia , Algoritmos , Fenômenos Biomecânicos , Divisão Celular , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Proliferação de Células , Tamanho Celular , Simulação por Computador , Células HCT116 , Humanos , Conceitos Matemáticos , Microscopia de Vídeo , Neoplasias/fisiopatologiaRESUMO
Tissue mimics (TMs) on the scale of several hundred microns provide a beneficial cell culture configuration for in vitro engineered tissue and are currently under the spotlight in tissue engineering and regenerative medicine. Due to the cell density and size, TMs are fairly inaccessible to optical observation and imaging within these samples remains challenging. Light Sheet Fluorescence Microscopy (LSFM)- an emerging and attractive technique for 3D optical sectioning of large samples- appears to be a particularly well-suited approach to deal with them. In this work, we compared the effectiveness of different light sheet illumination modalities reported in the literature to improve resolution and/or light exposure for complex 3D samples. In order to provide an acute and fair comparative assessment, we also developed a systematic, computerized benchmarking method. The outcomes of our experiment provide meaningful information for valid comparisons and arises the main differences between the modalities when imaging different types of TMs.
Assuntos
Biomimética/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Animais , Humanos , Miócitos Cardíacos/metabolismo , Ratos , Imagem com Lapso de TempoRESUMO
Cell aggregation is frequently impaired during the growth of primary tumors and the formation of metastatic lesions. Cell aggregation depends on cell-cell adhesion; however, no rigorous approach exists to monitor and quantify it accurately in the absence of the confounding factors of cell-substrate adhesion and the resulting cell motility on the substrate. We report here a highly reproducible, automated, microscopy-based quantification of tumor-cell spheroid formation in the absence of cell-substrate adhesion and use it to characterize cell aggregation dynamics in the early steps of this process. This method is based on fluorescence and bright-field microscopy and on a custom MATLAB program to quantify automatically the cells' aggregation kinetics. We demonstrate that the cell-cell adhesion protein E-cadherin and the desmosome proteins DSG2 and DSC2 are important for aggregation. Furthermore, we show that inhibition or silencing of myosin IIa enhances aggregation, suggesting that cytoskeleton tension inhibits tumor cell aggregation. This work opens new avenues to study the principles that govern multicellular aggregation, to characterize the aggregation properties of various tumor cell types, as well as to screen for drugs that inhibit or promote aggregation.
Assuntos
Adesão Celular/fisiologia , Agregação Celular/fisiologia , Comunicação Celular/fisiologia , Neoplasias/patologia , Caderinas/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/patologia , Desmocolinas/metabolismo , Desmogleína 2/metabolismo , Células HCT116 , Humanos , Neoplasias/metabolismo , TransfecçãoRESUMO
BACKGROUND: MultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D. METHODS: Cell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters. RESULTS: We describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage. CONCLUSION: Our data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Esferoides Celulares/patologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Citotoxinas/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Modelos Biológicos , Neoplasias Pancreáticas/tratamento farmacológico , Esferoides Celulares/efeitos dos fármacos , Células Tumorais Cultivadas , GencitabinaRESUMO
CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage. ΔN-CDC25B isoforms are expressed from a distinct promoter not involved in expression of canonical full-length isoforms. Remarkably, in contrast to the high lability and spatial dynamism of the full-length isoforms, ΔN-CDC25B isoforms are highly stable and exclusively nuclear, strongly suggesting the existence of two pools of CDC25B phosphatases in the cell that have functionally distinct properties. Using isoform-specific siRNA, we found that depleting full-length isoforms, but not ΔN-CDC25B isoforms, delays entry into mitosis. Thus, in an unperturbed cell cycle, the full-length isoforms are exclusively responsible for activating cyclin B-CDK1. Strikingly, in the late response to DNA damage, we found a CHK1-dependent shift in accumulation of CDC25B isoforms toward the ΔN-CDC25B species. Under this physiological stress condition, the ΔN-CDC25B isoform was found to play a crucial, nonredundant function in restarting the cell cycle after DNA damage-induced G2 phase arrest. Our findings reveal the existence of a previously unrecognized CDC25B isoform that operates specifically in the nucleus to reinitiate G2/M transition after DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Fase G2/genética , Fosfatases cdc25/metabolismo , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Separação Celular , Citometria de Fluxo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Fosfatases cdc25/genéticaRESUMO
Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity in response to DNA injury. The aim of this study was to identify new deubiquitinating enzymes (DUBs) involved in the regulation of the G2/M checkpoint. By using an siRNA-based screen to identify DUBs with an inherent ability to enhance a CDC25B-dependent G2/M checkpoint bypass, we have identified 11 candidates whose invalidation compromises checkpoint stringency. We subsequently focused our attention on one of these, the previously uncharacterized USP50. Using a TAP-tag approach associated to mass spectrometry, in addition to a yeast-two-hybrid screen, we identified HSP90 as a major interacting partner for USP50. We also demonstrate USP50 depletion causes a loss in accumulation of the HSP90 client Wee1, which is an essential component of the G2/M cell cycle arrest. Finally, we show that in response to DNA damaging agents, USP50 accumulates in the nucleus. We propose that USP50 may act through a HSP90-dependent mechanism to counteract CDC25B mitotic inducing activity and prevent Wee1 degradation, thereby repressing entry into mitosis following activation of the DNA damage checkpoint.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Endopeptidases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Dano ao DNA , Endopeptidases/genética , Fase G2 , Humanos , Espectrometria de Massas , Estabilidade Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteases Específicas de Ubiquitina , Fosfatases cdc25/metabolismoRESUMO
The CDC25 cell cycle regulators are promising targets for new pharmacologic approaches in cancer therapy. Inhibitory compounds such as BN82685 have proven to be effective in specifically targeting CDC25 in cultured cells and in inhibiting tumor cell growth. Here, we report that BN82685 impairs microtubule dynamic instability and alters microtubule organization and assembly at the centrosome in interphase cells. Treatment of mitotic cells with BN82685 delays mitotic spindle assembly, chromosome capture, and metaphase plate formation. Furthermore, we show that combining low concentrations of both BN82685 and paclitaxel inhibits the proliferation of HT29 human colon cancer cells. Our results show a role for CDC25 phosphatases in regulating microtubule dynamics throughout the cell cycle and suggest that combinations of CDC25 inhibitors with microtubule-targeting agents may be of therapeutic value.
Assuntos
Benzoquinonas/farmacologia , Inibidores Enzimáticos/farmacologia , Interfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Tiazóis/farmacologia , Fosfatases cdc25/antagonistas & inibidores , Cromossomos Humanos/efeitos dos fármacos , Sinergismo Farmacológico , Células HT29 , Células HeLa , Humanos , Metáfase/efeitos dos fármacos , Prometáfase/efeitos dos fármacosRESUMO
CDC25B is one of the three human dual-specificity phosphatases involved in the activation of cyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsible for the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-A kinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/M transition. Here we have investigated this phosphorylation event during the cell cycle, and in response to activation of the G2 DNA damage checkpoint. We show that accumulation of the S353-phosphorylated form of CDC25B at the centrosome correlates with the relocalization of cyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation of the G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated and consequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-A results in a bypass of the checkpoint that was partially overcome by a S353A mutant of CDC25B. Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01 results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These results strongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is an important step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclin B1 activation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Proteínas Serina-Treonina Quinases/química , Fosfatases cdc25/fisiologia , Aurora Quinase A , Aurora Quinases , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Ciclina B/química , Ciclina B1 , Fase G2 , Células HeLa , Histonas/química , Humanos , Microscopia de Fluorescência , Mitose , Mutação , Fosforilação , Conformação Proteica , Serina/química , Fatores de Tempo , Transfecção , Tirosina/química , Fosfatases cdc25/metabolismoRESUMO
Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Centrossomo/metabolismo , Fase G2/fisiologia , Proteínas Quinases/metabolismo , Fosfatases cdc25/metabolismo , Anticorpos/metabolismo , Anticorpos Monoclonais/metabolismo , Aurora Quinases , Proteínas de Ciclo Celular/química , Células HeLa , Humanos , Microinjeções , Fosforilação , Proteínas Serina-Treonina Quinases , Interferência de RNA , Serina/metabolismo , Fatores de Tempo , Proteínas de Xenopus , Fosfatases cdc25/químicaRESUMO
CDC25 dual-specificity phosphatases are essential regulators that dephosphorylate and activate cyclin-dependent kinase/cyclin complexes at key transitions of the cell cycle. CDC25 activity is currently considered to be an interesting target for the development of new antiproliferative agents. Here we report the identification of a new CDC25 inhibitor and the characterization of its effects at the molecular and cellular levels, and in animal models. BN82002 inhibits the phosphatase activity of recombinant human CDC25A, B, and C in vitro. It impairs the proliferation of tumoral cell lines and increases cyclin-dependent kinase 1 inhibitory tyrosine phosphorylation. In synchronized HeLa cells, BN82002 delays cell cycle progression at G1-S, in S phase and at the G2-M transition. In contrast, BN82002 arrests U2OS cell cycle mostly in the G1 phase. Selectivity of this inhibitor is demonstrated: (a) by the reversion of the mitotic-inducing effect observed in HeLa cells upon CDC25B overexpression; and (b) by the partial reversion of cell cycle arrest in U2OS expressing CDC25. We also show that BN82002 reduces growth rate of human tumor xenografts in athymic nude mice. BN82002 is a original CDC25 inhibitor that is active both in cell and animal models. This greatly reinforces the interest in CDC25 as an anticancer target.
Assuntos
Inibidores Enzimáticos/farmacologia , Fosfatases cdc25/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Etilaminas , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Nitrocompostos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Fosfatases cdc25/biossíntese , Fosfatases cdc25/genéticaRESUMO
Regulation of the intracellular localisation of its actors is one of the key mechanisms underlying cell cycle control. CDC25 phosphatases are activators of Cyclin-Dependent Kinases (CDK) that undergo nucleo-cytoplasmic shuttling during the cell cycle and in response to checkpoint activation. Here we report that the protein kinase PKB/Akt phosphorylates CDC25B on serine 353, resulting in a nuclear export-dependent cytoplasmic accumulation of the phosphatase. Oxidative stress activates PKB/Akt and reproduces the effect on CDC25B phosphorylation and localisation. However, inhibition of PKB/Akt activity only partially reverted the effect of oxidative stress on CDC25B localisation and mutation of serine 353 abolishes phosphorylation but only delays nuclear exclusion. These results indicate that additional mechanisms are also involved in preventing nuclear import of CDC25B. Our findings identify CDC25B as a target of PKB/Akt and provide new insight into the regulation of its localisation in response to stress-activated signalling pathways.
Assuntos
Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Citoplasmáticos e Nucleares , Fosfatases cdc25/análise , Fosfatases cdc25/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/química , Vetores Genéticos , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Carioferinas/fisiologia , Sinais de Localização Nuclear/fisiologia , Estresse Oxidativo , Plasmídeos , Mutação Puntual , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/genética , Fosfatases cdc25/genética , Proteína Exportina 1RESUMO
Human dual-specificity phosphatases CDC25 (A, B and C) play an important role in the control of cell cycle progression by activating the cyclin-dependent kinases (CDKs). Regulation of these phosphatases during the cell cycle involves post-translational modifications such as phosphorylation and protein-protein interactions. Given the suspected involvement of the protein kinase CK2 at the G2/M transition, we have investigated its effects on the CDC25B phosphatase. We show that in vitro CK2 phosphorylates CDC25B, but not CDC25C. Mass spectrometry analysis demonstrates that at least two serine residues, Ser-186 and Ser-187, are phosphorylated in vivo. We also report that CDC25B interacts with CK2, and this interaction, mediated by the CK2beta regulatory subunit, involves domains that are located within the first 55 amino acids of CK2beta and between amino acids 122 and 200 on CDC25B. This association was confirmed in vivo, in Sf9 insect cells and in U(2)OS human cells expressing an HA epitope-tagged CDC25B. Finally, we demonstrate that phosphorylation of CDC25B by protein kinase CK2 increases the catalytic activity of the phosphatase in vitro as well as in vivo. We discuss the possibility that CDC25B phosphorylation by CK2 could play a role in the regulation of the activity of CDC25B as a starter of mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fosfatases cdc25/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II , Proteínas de Ciclo Celular/genética , Células Cultivadas , Epitopos/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Spodoptera/citologia , Regulação para Cima , Fosfatases cdc25/genéticaRESUMO
CDC25B phosphatases are essential regulators that control cyclin-dependent kinases activities at the entry into mitosis. In this study, we demonstrate that serine 146 is required for two crucial features of CDC25B1. It is essential for CDC25B1 to function as a mitotic inducer and to prevent CDC25B1 export from the nucleus. We also show that serine 146 is phosphorylated in vitro by CDK1-cyclin B. However, phosphorylation of CDC25B does not stimulate its phosphatase activity, and mutation of serine 146 had no effect on its catalytic activity. Serine 146 phosphorylation is proposed to be a key event in the regulation of the CDC25B function in the initiation of mammalian mitosis.