RESUMO
Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health.
Assuntos
Dano ao DNA , Anemia de Fanconi/enzimologia , Reparo de DNA por Recombinação , Origem de Replicação , Proteína de Replicação A/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Anemia de Fanconi/genética , Células HeLa , Humanos , Mutação , Ligação Proteica , Interferência de RNA , Proteína de Replicação A/genética , Transfecção , Ubiquitina-Proteína Ligases/genéticaRESUMO
Homeostasis of renewing tissues requires balanced proliferation, differentiation and movement. This is particularly important in the intestinal epithelium where lineage tracing suggests that stochastic differentiation choices are intricately coupled to the position of a cell relative to a niche. To determine how position is achieved, we followed proliferating cells in intestinal organoids and discovered that the behaviour of mitotic sisters predicted long-term positioning. We found that, normally, 70% of sisters remain neighbours, while 30% lose contact and separate after cytokinesis. These post-mitotic placements predict longer term differences in positions assumed by sisters: adjacent sisters reach similar positions over time; in a pair of separating sisters, one remains close to its birthplace while the other is displaced upward. Computationally modelling crypt dynamics confirmed that post-mitotic separation leads to sisters reaching different compartments. We show that interkinetic nuclear migration, cell size and asymmetric tethering by a process extending from the basal side of cells contribute to separations. These processes are altered in adenomatous polyposis coli (Apc) mutant epithelia where separation is lost. We conclude that post-mitotic placement contributes to stochastic niche exit and, when defective, supports the clonal expansion of Apc mutant cells.
Assuntos
Núcleo Celular/fisiologia , Mucosa Intestinal/citologia , Animais , Transporte Biológico , Adesão Celular , Humanos , Cinética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitose , Organoides/citologia , Técnicas de Cultura de TecidosRESUMO
Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants, or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. A detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualize and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured instrument response function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Célula Única , Sobrevivência Celular , Células HEK293 , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Fator 2 Relacionado a NF-E2/química , Imagem ÓpticaRESUMO
The crypts of the intestinal epithelium house the stem cells that ensure the continual renewal of the epithelial cells that line the intestinal tract. Crypt number increases by a process called crypt fission, the division of a single crypt into two daughter crypts. Fission drives normal tissue growth and maintenance. Correspondingly, it becomes less frequent in adulthood. Importantly, fission is reactivated to drive adenoma growth. The mechanisms governing fission are poorly understood. However, only by knowing how normal fission operates can cancer-associated changes be elucidated. We studied normal fission in tissue in three dimensions using high-resolution imaging and used intestinal organoids to identify underlying mechanisms. We discovered that both the number and relative position of Paneth cells and Lgr5+ cells are important for fission. Furthermore, the higher stiffness and increased adhesion of Paneth cells are involved in determining the site of fission. Formation of a cluster of Lgr5+ cells between at least two Paneth-cell-rich domains establishes the site for the upward invagination that initiates fission.
Assuntos
Mucosa Intestinal/citologia , Celulas de Paneth/citologia , Receptores Acoplados a Proteínas G/metabolismo , Nicho de Células-Tronco , Células-Tronco/citologia , Fatores Etários , Animais , Adesão Celular , Contagem de Células , Divisão Celular , Proliferação de Células , Integrina beta4/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Modelos Biológicos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/genética , Células-Tronco/metabolismoRESUMO
Photonics, especially optical coherence elastography (OCE) and second harmonic generation (SHG) imaging are novel high-resolution imaging modalities for characterization of biological tissues. Following our preliminary experience, we hypothesized that OCE and SHG imaging would delineate the microstructure of prostate tissue and aid in distinguishing cancer from the normal benign prostatic tissue. Furthermore, these approaches may assist in characterization of the grade of cancer, as well. In this study, we confirmed a high diagnostic accuracy of OCE and SHG imaging in the detection and characterization of prostate cancer for a large set of biopsy tissues obtained from men suspected to have prostate cancer using transrectal ultrasound (TRUS). The two techniques and methods described here are complementary, one depicts the stiffness of tissues and the other illustrates the orientation of collagen structure around the cancerous lesions. The results showed that stiffness of cancer tissue was ~57.63% higher than that of benign tissue (Young's modulus of 698.43±125.29 kPa for cancerous tissue vs 443.07±88.95 kPa for benign tissue with OCE. Using histology as a reference standard and 600 kPa as a cut-off threshold, the data analysis showed sensitivity and specificity of 89.6 and 99.8%, respectively. Corresponding positive and negative predictive values were 99.5 and 94.6%, respectively. There was a significant difference noticed in terms of Young's modulus for different Gleason scores estimated by OCE (P-value<0.05). For SHG, distinct patterns of collagen distribution were seen for different Gleason grade disease with computed quantification employing a ratio of anisotropic to isotropic (A:I ratio) and this correlated with disease aggressiveness.
Assuntos
Técnicas de Imagem por Elasticidade , Imagem Óptica , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Microscopia de Geração do Segundo Harmônico , Idoso , Idoso de 80 Anos ou mais , Colágeno/análise , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Próstata/patologia , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgiaRESUMO
During somitogenesis, epithelial somites form from the pre-somitic mesoderm (PSM) in a periodic manner. This periodicity is regulated by a molecular oscillator, known as the 'segmentation clock', that is characterised by an oscillatory pattern of gene expression that sweeps the PSM in a caudal-rostral direction. Key components of the segmentation clock are intracellular components of the Notch, Wnt and FGF pathways, and it is widely accepted that intracellular negative-feedback loops regulate oscillatory gene expression. However, an open question in the field is how intracellular oscillations are coordinated, in the form of spatiotemporal waves of expression, across the PSM. In this study, we provide a potential mechanism for this process. We show at the mRNA level that the Notch1 receptor and Delta-like 1 (Dll1) ligand vary dynamically across the PSM of both chick and mouse. Remarkably, we also demonstrate similar dynamics at the protein level; hence, the pathway components that mediate intercellular coupling themselves exhibit oscillatory dynamics. Moreover, we quantify the dynamic expression patterns of Dll1 and Notch1, and show they are highly correlated with the expression patterns of two known clock components [Lfng mRNA and the activated form of the Notch receptor (cleaved Notch intracellular domain, NICD)]. Lastly, we show that Notch1 is a target of Notch signalling, whereas Dll1 is Wnt regulated. Regulation of Dll1 and Notch1 expression thus links the activity of Wnt and Notch, the two main signalling pathways driving the clock.
Assuntos
Relógios Biológicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/embriologia , Receptor Notch1/metabolismo , Transdução de Sinais/fisiologia , Somitos/embriologia , Algoritmos , Animais , Western Blotting , Proteínas de Ligação ao Cálcio , Embrião de Galinha , Glicosiltransferases/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Hibridização in Situ Fluorescente , Mesoderma/metabolismo , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/metabolismoRESUMO
Colorectal cancers commonly carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein contributes to the stabilization of microtubules. Consistently, microtubules in cells lacking APC depolymerize more readily in response to microtubule-destabilizing drugs. This raises the possibility that such agents are suitable for treatment of APC-deficient cancers. However, APC-deficient cells have a compromised spindle assembly checkpoint, which renders them less sensitive to killing by microtubule poisons whose toxicity relies on the induction of prolonged mitotic arrest. Here, we describe the novel discovery that the clinically used microtubule-depolymerizing drug vinorelbine (Navelbine) kills APC-deficient cells in culture and in intestinal tissue more effectively than it kills wild-type cells. This is due to the ability of vinorelbine to kill cells in interphase independently of mitotic arrest. Consistent with a role for p53 in cell death in interphase, depletion of p53 renders cells less sensitive to vinorelbine, but only in the presence of wild-type APC. The pro-apoptotic protein BIM (also known as BCL2L11) is recruited to mitochondria in response to vinorelbine, where it can inhibit the anti-apoptotic protein BCL2, suggesting that BIM mediates vinorelbine-induced cell death. This recruitment of BIM is enhanced in cells lacking APC. Consistently, BIM depletion dampens the selective effect of vinorelbine on these cells. Our findings reveal that vinorelbine is a potential therapeutic agent for colorectal cancer, but they also illustrate the importance of the APC tumour suppressor status when predicting therapeutic efficacy.
Assuntos
Proteína da Polipose Adenomatosa do Colo/deficiência , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Vimblastina/análogos & derivados , Adenoma/tratamento farmacológico , Adenoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Humanos , Interfase/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/deficiência , Vimblastina/farmacologia , VinorelbinaRESUMO
The role of the basement membrane is vital in maintaining the integrity and structure of an epithelial layer, acting as both a mechanical support and forming the physical interface between epithelial cells and the surrounding connective tissue. The function of this membrane is explored here in the context of the epithelial monolayer that lines the colonic crypt, test-tube shaped invaginations that punctuate the lining of the intestine and coordinate a regular turnover of cells to replenish the epithelial layer every few days. To investigate the consequence of genetic mutations that perturb the system dynamics and can lead to colorectal cancer, it must be possible to track the emerging tissue level changes that arise in the crypt. To that end, a theoretical crypt model with a realistic, deformable geometry is required. A new discrete crypt model is presented, which focuses on the interaction between cell- and tissue-level behaviour, while incorporating key subcellular components. The model contains a novel description of the role of the surrounding tissue and musculature, based upon experimental observations of the tissue structure of the crypt, which are also reported. A two-dimensional (2D) cross-sectional geometry is considered, and the shape of the crypt is allowed to evolve and deform. Simulation results reveal how the shape of the crypt may contribute mechanically to the asymmetric division events typically associated with the stem cells at the base. The model predicts that epithelial cell migration may arise due to feedback between cell loss at the crypt collar and density-dependent cell division, an hypothesis which can be investigated in a wet lab. This work forms the basis for investigation of the deformation of the crypt structure that can occur due to proliferation of cells exhibiting mutant phenotypes, experiments that would not be possible in vivo or in vitro.
Assuntos
Membrana Basal/citologia , Membrana Basal/fisiologia , Comunicação Celular/fisiologia , Colo/citologia , Colo/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Modelos Biológicos , Animais , Simulação por Computador , HumanosRESUMO
OBJECTIVES: To investigate the effect of dab-on or brushing of stannous-fluoride SnF2 or sodium-fluoride NaF dentifrice on eroded dentine tubule patency, surface and inter-tubular dentine roughness, using Confocal-Laser-Scanning-Microscopy (CLSM), Atomic-Force-Microscopy (AFM), Energy-Dispersive-X-ray-Spectroscopy (EDX), Scanning-Electron-Microscopy (SEM) and Contact-Profilometry (CP). METHODS: 75-polished human dentine samples were prepared and eroded in agitated 6% citric acid to expose patent tubules and 'initiate' DH. Samples were randomly allocated into 5 intervention groups; artificial saliva control (1); electric tooth-brushing with NaF (2) or SnF2 (3), and dab-on application of NaF (4) or SnF2 (5). Samples underwent three cycles of intervention followed by further acid challenge. Patent tubules, likely to cause DH clinically, were measured using validated biocomputational methods with CLSM images of dentine surfaces taken baseline and post-intervention. Randomised samples (nâ¯=â¯15, 20 %) were investigated using AFM, EDX and SEM to study surface and sub-surface tubular occlusion. Dentine surface and inter-tubular roughness were measured using CP and AFM respectively. RESULTS: At baseline, mean tubule patency in all samples was 216 (SD 58) with no significant inter-group differences. Post-intervention, the mean patency was 220 (40) and 208 (35) in groups 1 and 2 respectively (pâ¯≥â¯0.06), but decreased to 62 (41), 62 (21) and 63 (19) in groups 3, 4 and 5 respectively (pâ¯<â¯0.0001). Patency was confirmed using AFM, SEM and EDX. SnF2 interventions created greater sub-surface occlusion (pâ¯<â¯0.01), and increased CP surface roughness (pâ¯=â¯0.015). Significant negative correlation (-0.6) existed between CP surface roughness and tubule patency (pâ¯=â¯0.009). CONCLUSIONS: Dab-on with NaF and SnF2 or brushing with SnF2 reduces DH in eroded dentine with ongoing acid challenges. Contacting surface roughness measures indicate risk of DH. CLINICAL SIGNIFICANCE: Dab-on is a convenient supplementary method of dentifrice application to reduce DH; it beneficially avoids brushing post-erosion or overzealous brushing, enables re-establishment of an appropriate brushing regime post-DH and supports oral health. Significant modes of action of SnF2 in reducing DH are revealed. Finally, CP roughness measures provide indication of dentine lesions that may cause DH clinically.
Assuntos
Dentina , Erosão Dentária , Humanos , Saliva Artificial , Fluoreto de Sódio , Fluoretos de Estanho , Erosão Dentária/prevenção & controle , Escovação DentáriaRESUMO
OBJECTIVES: To validate novel non-contacting Confocal-Laser-Scanning-Microscopy (CLSM) methodology with conventional Contacting Profilometry (CP) measures investigating brushing or dab-on of stannous-fluoride dentifrice on early aggressive dentine erosion. METHODS: 75 polished human dentine samples were prepared and eroded in agitated 6% citric acid then randomly allocated into 5 intervention groups; artificial saliva control (1); controlled use of a pressure sensitive counter-rotating oscillatory powered toothbrush with sodium-fluoride NaF (2) or stannous-fluoride SnF2 (3), and dab-on application of NaF (4) or SnF2 (5). Samples underwent three cycles of intervention and 2-min agitated 6 % citric acid challenges. CLSM images were taken and 3D reconstructions produced of step height using a developed software algorithm. In addition, 20 % samples were randomised and profiled using CP to measure step height and surface roughness. Vickers's diamond micro-hardness testing was carried out on all samples. RESULTS: Comparing CLSM and CP; Pearson correlation was 0.77 and Intra-class correlation 0.81 (p = 0.01). There were no significant statistical differences in step height between groups using both CLSM and CP. From baseline, SnF2 brushing (3) increased micro-hardness more than control (1) (p = 0.03), NaF (4) and SnF2 dab-on (5) (p ≤ 0.001), and increased surface roughness more than control (p = 0.02), NaF brushing (2) and NaF dab-on (4) (p ≤ 0.017). Dab-on of SnF2 (5) produced rougher surfaces than control (1) (p = 0.014) and reduced hardness compared with NaF brushing (p = 0.04). CONCLUSIONS: Good agreement and correlation exists between CLSM and CP measures in dentine. There were no significant differences in surface loss after interventions between groups. Compared with control, SnF2 application increased dentine surface roughness and SnF2 controlled powered brushing application increased dentine hardness, likely caused by exposure of uneroded dentine. CLINICAL SIGNIFICANCE: Isosurfaces produced using CLSM can be used to represent dentine step height loss. They show good correlation and agreement with conventional CP measures, following early aggressive erosion-abrasion cycles of dentine. The CLSM and computer algorithm therefore provides an accurate, standalone and non-contacting three-dimensional measurement of early dentine wear. Stannous-fluoride brushing, and dab-on application offer no benefits following early aggressive erosion in dentine. To reduce dentine wear, limiting erosive challenges and avoiding brushing post-erosion is advised.
RESUMO
Prostate cancer is a multifocal disease with characteristic heterogeneity and foci that can range from low grade indolent to aggressive disease. The latter is characterised by the well-established histopathological Gleason grading system used in the current clinical care. Nevertheless, a large discrepancy exists on initial biopsy and after the final radical prostatectomy. Moreover, there is no reliable imaging modality to study these foci, in particular at the level of the cells and surrounding matrix. Extracellular matrix (ECM) remodelling is significant in cancer progression with collagen as the dominant structural component providing mechanical strength and flexibility of tissue. In this study, the collagen assembly in prostate tissue was investigated with second harmonic generation (SHG) microscopy: malignant foci demonstrated a reticular pattern, with a typical collagen pattern for each Gleason score. The orientation of collagen for each biopsy was computed by applying a ratio of the anisotropic and isotropic collagen fibres. This value was found to be distinct for each Gleason score. The findings suggest that this approach can not only be used to detect prostate cancer, but also can act as a potential biomarker for cancer aggressiveness.
Assuntos
Biópsia Guiada por Imagem , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Microscopia de Geração do Segundo Harmônico , Ultrassonografia , Humanos , Masculino , Gradação de TumoresRESUMO
Biomedical research and drug development increasingly involve the extraction of quantitative data from digital microscope images, such as those obtained using fluorescence microscopy. Here, we describe a novel approach for both managing and analyzing such images. The Open Microscopy Environment (OME) is a sophisticated open-source scientific image management database that coordinates the organization, storage, and analysis of the large volumes of image data typically generated by modern imaging methods. We describe FindSpots, a powerful image-analysis package integrated in OME that will be of use to those who wish to identify and measure objects within microscope images or time-lapse movies. The algorithm used in FindSpots is in fact only one of many possible segmentation (object detection) algorithms, and the underlying data model used by OME to capture and store its results can also be used to store results from other segmentation algorithms. In this report, we illustrate how image segmentation can be achieved in OME using one such implementation of a segmentation algorithm, and how this output subsequently can be displayed graphically or processed numerically using a spreadsheet.
Assuntos
Biologia Computacional , Interpretação de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Microscopia de Fluorescência , Algoritmos , Animais , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Fibroblastos/metabolismo , Corantes Fluorescentes , Indóis , Armazenamento e Recuperação da Informação , Cinetocoros/metabolismo , Camundongos , Proteínas Quinases/análise , Proteínas Serina-Treonina Quinases , Reprodutibilidade dos Testes , Interface Usuário-ComputadorRESUMO
A flagellate predator, Aurigamonas solis n. gen., n. sp., with numerous radiating axopodia-like appendages, has been isolated in culture from soils. Despite its heliozoan-like appearance, Aurigamonas is not a sit-and-wait predator but a mobile hunter and its stiff appendages are not microtubule-supported axopodia but elongate haptopodia, each supported by a cylindrical core of microfilaments and bearing at its capitate tip a single extrusome-like body (haptosome). Prey flagellates are trapped on the sticky tips of the haptopodia and a large funnel-shaped pseudopodium then emerges to engulf the prey or suck out part of it for internal digestion. Pseudopodial contact is accompanied by killing, possibly as a result of the injection of spicules by the predator. Cytoplasmic haptosomes appear to induce formation of a haptopodium on making contact with the plasma membrane. Propulsion of the organism along the substratum is effected by beating of a long trailing flagellum, the short and inconspicuous second flagellum lacks motility. Small subunit rDNA sequencing shows that Aurigamonas arose within the Cercozoa. Its closest relatives are Cercobodo agilis and several flagellates currently known only as environmental sequences. This conclusion is supported further by the presence of only a single amino acid insertion in the polyubiquitin sequence of Aurigamonas solis.
Assuntos
Eucariotos/classificação , Eucariotos/citologia , Solo/parasitologia , Animais , Extensões da Superfície Celular/ultraestrutura , Eucariotos/genética , Eucariotos/isolamento & purificação , Eucariotos/ultraestrutura , Comportamento Alimentar , Flagelos/ultraestrutura , Genes de RNAr/genética , Dados de Sequência Molecular , Organelas/ultraestrutura , Filogenia , Poliubiquitina/genética , Escócia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Epidemiological data reveals the gastrointestinal (GI) tract as one of the main sites for low-LET radiation-induced cancers. Importantly, the use of particle therapy is increasing, but cancer risk by high-LET particles is still poorly understood. This gap in our knowledge also remains a major limiting factor in planning long-term space missions. Therefore, assessing risks and identifying predisposing factors for carcinogenesis induced by particle radiation is crucial for both astronauts and cancer survivors. We have previously shown that exposure to relatively high doses of high-energy (56)Fe ions induced higher intestinal tumor frequency and grade in the small intestine of Apc(Min/+) mice than γ rays. However, due to the high number of spontaneous lesions (â¼30) that develop in Apc(Min/+) animals, this Apc mutant model is not suitable to investigate effects of cumulative doses <1 Gy, which are relevant for risk assessment in astronauts and particle radiotherapy patients. However, Apc(1638N/+) mice develop a relatively small number of spontaneous lesions (â¼3 per animal) in both small intestine and colon, and thus we propose a better model for studies on radiation-induced carcinogenesis. Here, we investigated model particle radiation increases tumor frequency and grade in the entire gastrointestinal tract (stomach and more distal intestine) after high- and low-radiation doses whether in the Apc(1638N/+). We have previously reported that an increase in small intestinal tumor multiplicity after exposure to γ rays was dependent on gender in Apc(1638N/+) mice, and here we investigated responses to particle radiation in the same model. Phenotypical and histopathological observations were accompanied by late changes in number and position of mitotic cells in intestinal crypts from animals exposed to different radiation types.
Assuntos
Carcinogênese/efeitos da radiação , Trato Gastrointestinal/patologia , Trato Gastrointestinal/efeitos da radiação , Transferência Linear de Energia , Animais , Relação Dose-Resposta à Radiação , Feminino , Ferro/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitose/efeitos da radiação , Gradação de Tumores , Caracteres Sexuais , Carga Tumoral/efeitos da radiaçãoRESUMO
Cell and tissue polarity are tightly coupled and are vital for normal tissue homeostasis. Changes in cellular and tissue organization are common to even early stages of disease, particularly cancer. The digestive tract is the site of the second most common cause of cancer deaths in the developed world. Tumours in this tissue arise in an epithelium that has a number of axes of cell and tissue polarity. Changes in cell and tissue polarity in response to genetic changes that are known to underpin disease progression provide clues about the link between molecular-, cellular- and tissue-based mechanisms that accompany cancer. Mutations in adenomatous polyposis coli (APC) are common to most colorectal cancers in humans and are sufficient to cause tumours in mouse intestine. Tissue organoids mimic many features of whole tissue and permit identifying changes at different times after inactivation of APC. Using gut organoids, we show that tissue polarity is lost very early during cancer progression, whereas cell polarity, at least apical-basal polarity, is maintained and changes only at later stages. These observations reflect the situation in tumours and validate tissue organoids as a useful system to investigate the relationship between cell polarity and tissue organization.
Assuntos
Polipose Adenomatosa do Colo/fisiopatologia , Carcinogênese/patologia , Polaridade Celular/fisiologia , Trato Gastrointestinal/citologia , Organoides/citologia , Animais , Adesão Celular/fisiologia , Imunofluorescência , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos C57BL , Organoides/fisiopatologiaRESUMO
Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating ß-catenin-binding sites from APC, which leads to upregulation of ß-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in ß-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APC(Min/+); multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APC(Min/+) intestines are overpopulated with cells, suggesting that a lack of migration might cause cell accumulation in a precancerous state.
Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/metabolismo , Movimento Celular , Transformação Celular Neoplásica/patologia , Genes Dominantes , Modelos Animais , Fragmentos de Peptídeos/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Transformação Celular Neoplásica/metabolismo , Embrião de Galinha , Dictyostelium/citologia , Dictyostelium/metabolismo , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linha Primitiva/metabolismo , Linha Primitiva/patologiaRESUMO
The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of ß-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α-dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of ß-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.
Assuntos
Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes APC , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , NF-kappa B/metabolismo , Transcrição Gênica , beta Catenina/metabolismoRESUMO
The importance of asymmetric divisions for stem cell function and maintenance is well established in the developing nervous system and the skin; however, its role in gut epithelium and its importance for tumorigenesis is still debated. We demonstrate alignment of mitotic spindles perpendicular to the apical surface specifically in the stem cell compartments of mouse and human intestine and colon. This orientation correlates with the asymmetric retention of label-retaining DNA. Both the preference for perpendicular spindle alignment and asymmetric label retention are lost in precancerous tissue heterozygous for the adenomatous polyposis coli tumor suppressor (Apc). This loss correlates with cell shape changes specifically in the stem cell compartment. Our data suggest that loss of asymmetric division in stem cells might contribute to the oncogenic effect of Apc mutations in gut epithelium.
Assuntos
Colo/citologia , Células Epiteliais/citologia , Intestinos/citologia , Lesões Pré-Cancerosas/patologia , Fuso Acromático/metabolismo , Células-Tronco/citologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Divisão Celular , Forma Celular , Colo/metabolismo , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Células-Tronco/metabolismoRESUMO
In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is 'better', in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.