RESUMO
Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.
Assuntos
Proteína Supressora de Tumor p53 , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Imagem Individual de Molécula/métodos , Microscopia de Fluorescência/métodos , Ligação Proteica , Linhagem Celular Tumoral , Mitocôndrias/metabolismoRESUMO
Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow-up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR-associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T-lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non-senescent, non-apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and type I IFN response. Clinicopathological parameters did not differentiate DDR+ cases from the other cases. TILs were equally present in DDR and non-DDR cases. DDR+ MMRd cases were preferentially retaining wild-type MLH1. The outcome after 5FU-based chemotherapy was not different in the two groups. DDR+ COAD represents a subgroup not aligned with known diagnostic, prognostic, or therapeutic categories, with potential new targeted treatment opportunities, exploiting the DNA damage repair pathways.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Humanos , Dano ao DNA/genética , Variações do Número de Cópias de DNA , Neoplasias do Colo/genética , Reparo do DNA/genética , FenótipoRESUMO
In situ multiplexing analysis and in situ transcriptomics are now providing revolutionary tools to achieve the comprehension of the molecular basis of cancer and to progress towards personalized medicine to fight the disease. The complexity of these tasks requires a continuous interplay among different technologies during all the phases of the experimental procedures. New tools are thus needed and their characterization in terms of performances and limits is mandatory to reach the best resolution and sensitivity. We propose here a new experimental pipeline to obtain an optimized costs-to-benefits ratio thanks to the alternate employment of automated and manual procedures during all the phases of a multiplexing experiment from sample preparation to image collection and analysis. A comparison between ultra-fast and automated immunofluorescence staining and standard staining protocols has been carried out to compare the performances in terms of antigen saturation, background, signal-to-noise ratio and total duration. We then developed specific computational tools to collect data by automated analysis-driven fluorescence microscopy. Computer assisted selection of targeted areas with variable magnification and resolution allows employing confocal microscopy for a 3D high resolution analysis. Spatial resolution and sensitivity were thus maximized in a framework where the amount of stored data and the total requested time for the procedure were optimized and reduced with respect to a standard experimental approach.
RESUMO
The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To understand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replication and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARα oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. More specifically, we perform Tau-stimulated emission depletion imaging, a superresolution technique based on the concept of separation of photons by lifetime tuning. Tau-stimulated emission depletion imaging is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability. Upon activation of the oncogene, we detect a significant increase in the fraction of transcription sites colocalized with PML/PML-RARα. This increase of colocalization can be ascribed to oncogene-induced disruption of physiological PML bodies and the abnormal occurrence of a relatively large number of PML-RARα microspeckles. We also detect a significant cell-to-cell variability of this increase of colocalization, which can be ascribed, at least in part, to a heterogeneous response of the cells to the activation of the oncogene. These results prove that our method efficiently reveals oncogene-induced alterations in the spatial organization of nuclear processes and suggest that the abnormal localization of PML-RARα could interfere with the transcription machinery, potentially leading to DNA damage and genomic instability.
Assuntos
Leucemia Promielocítica Aguda , Proteínas de Fusão Oncogênica , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Oncogenes , Análise EspectralRESUMO
53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53-53BP1 interaction on the tens of nanometers scale during the distinct phases of the response.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteína Supressora de Tumor p53 , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Citometria por Imagem , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, poor blood-brain barrier penetration, and resistance to therapy highlight the need for new targets and clinical treatments. A step toward clinical translation includes the eradication of GBM tumor-initiating cells (TICs), responsible for GBM heterogeneity and relapse. By using patient-derived TICs and xenograft orthotopic models, we demonstrated that the selective lysine-specific histone demethylase 1 inhibitor DDP_38003 (LSD1i) is able to penetrate the brain parenchyma in vivo in preclinical models, is well tolerated, and exerts antitumor activity in molecularly different GBMs. LSD1 genetic targeting further strengthens the role of LSD1 in GBM TIC maintenance. GBM TIC plasticity supports their adaptation and survival under a plethora of environmental stresses, including nutrient deficiency and proteostasis perturbation. By mimicking these stresses in vitro, we found that LSD1 inhibition hampers the induction of the activating transcription factor 4 (ATF4), the master regulator of the integrated stress response (ISR). The resulting aberrant ISR sensitizes GBM TICs to stress-induced cell death, hampering tumor aggressiveness. Functionally, LSD1i interferes with LSD1 scaffolding function and prevents its interaction with CREBBP, a critical ATF4 activator. By disrupting the interaction between CREBBP and LSD1-ATF4 axis, LSD1 inhibition prevents GBM TICs from overcoming stress and sustaining GBM progression. The effectiveness of the LSD1 inhibition in preclinical models shown here places a strong rationale toward its clinical translation for GBM treatment.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Fator 4 Ativador da Transcrição/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Histona Desmetilases/metabolismo , Humanos , Recidiva Local de Neoplasia/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
Reactivation of the anti-tumor response has shown substantial progress in aggressive tumors such as melanoma and lung cancer. Data on less common histotypes are scanty. Immune checkpoint inhibitor therapy has been applied to few cases of uterine leiomyosarcomas, of which the immune cell composition was not examined in detail. We analyzed the inflammatory infiltrate of 21 such cases in high-dimensional, single cell phenotyping on routinely processed tissue. T-lymphoid cells displayed a composite phenotype common to all tumors, suggestive of antigen-exposure, acute and chronic exhaustion. To the contrary, myelomonocytic cells had case-specific individual combinations of phenotypes and subsets. We identified five distinct monocyte-macrophage cell types, some not described before, bearing immunosuppressive molecules (TIM3, B7H3, VISTA, PD1, PDL1). Detailed in situ analysis of routinely processed tissue yields comprehensive information about the immune status of sarcomas. The method employed provides equivalent information to extractive single-cell technology, with spatial contexture and a modest investment.
Assuntos
Imunidade Adaptativa , Biomarcadores Tumorais/imunologia , Imunidade Inata , Leiomiossarcoma/imunologia , Análise de Célula Única/métodos , Neoplasias Uterinas/imunologia , Adulto , Idoso , Antígenos B7/metabolismo , Antígeno B7-H1/metabolismo , Feminino , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Pessoa de Meia-Idade , Monócitos/metabolismo , Receptor de Morte Celular Programada 1 , Linfócitos T/metabolismoRESUMO
Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.
Assuntos
Núcleo Celular/metabolismo , Microscopia/métodos , Nanotecnologia/métodos , Análise Espectral , Cor , Humanos , Células MCF-7RESUMO
It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.
Assuntos
Quebras de DNA de Cadeia Dupla , Loci Gênicos , Neoplasias/genética , Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Elementos Facilitadores Genéticos , Etoposídeo/farmacologia , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Íntrons , Neoplasias/patologia , Regiões Promotoras Genéticas , Sítios de Splice de RNA , Inibidores da Topoisomerase/farmacologia , Sítio de Iniciação de TranscriçãoRESUMO
Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.
Assuntos
Anticorpos/química , Antígenos/análise , Imuno-Histoquímica/métodos , Animais , Antígenos/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Cabras , Ensaios de Triagem em Larga Escala , Humanos , Rim/química , Camundongos , Placenta/química , Gravidez , Renaturação Proteica , Coelhos , Pele/química , Inclusão do Tecido , Fixação de TecidosRESUMO
Epigenetic regulation plays an essential role in tumor development and epigenetic modifiers are considered optimal potential druggable candidates. In order to identify new breast cancer vulnerabilities and improve therapeutic chances for patients, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model (MCF10DCIS.com cell line) using epigenetic libraries. Among the genes identified in our screening, we deeply investigated the role of Chromodomain Helicase DNA binding Protein 4 (CHD4) in breast cancer tumorigenesis. CHD4 silencing significantly reduced tumor growth in vivo and proliferation in vitro of MCF10DCIS.com cells. Similarly, in vivo breast cancer growth was decreased in a spontaneous mouse model of breast carcinoma (MMTV-NeuT system) and in metastatic patient-derived xenograft models. Conversely, no reduction in proliferative ability of non-transformed mammary epithelial cells (MCF10A) was detected. Moreover, we showed that CHD4 depletion arrests proliferation by inducing a G0/G1 block of cell cycle associated with up-regulation of CDKN1A (p21). These results highlight the relevance of genetic screens in the identification of tumor frailties and the role of CHD4 as a potential pharmacological target to inhibit breast cancer growth.
Assuntos
Neoplasias da Mama/genética , Proliferação de Células , DNA Helicases/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Interferência de RNA , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Biologia Computacional , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA Helicases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fenótipo , Transdução de Sinais , Fatores de Tempo , Carga TumoralRESUMO
Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.
Assuntos
Neoplasias Encefálicas/patologia , Canais de Cloreto/metabolismo , Exossomos/patologia , Vesículas Extracelulares/patologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Animais , Apoptose , Western Blotting , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Nanopartículas/química , Células-Tronco Neoplásicas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1 - an aberrant tyrosine kinase - in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours. We postulate that care has to be taken when distinguishing between coexisting, secondary-to-treatment and second in sequence, but independent tumourigenic events, in order to achieve an unbiased picture of the adverse effects of novel treatments. To illustrate this point, we present a case of a patient in which CML and peripheral T-cell lymphoma (PTCL) coexisted, although the clinical presentation of the latter followed the achievement of major molecular response of CML to TKIs.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Linfoma de Células T Periférico/patologia , Antineoplásicos/uso terapêutico , Proteínas de Fusão bcr-abl/sangue , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib/uso terapêutico , Hibridização in Situ Fluorescente , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Linfoma de Células T Periférico/genética , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Indóis/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Leucemia/tratamento farmacológico , Leucemia/mortalidade , Leucemia/patologia , Masculino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Light-sheet microscopy is a useful tool for performing biological investigations of thick samples and it has recently been demonstrated that it can also act as a suitable architecture for super-resolution imaging of thick biological samples by means of individual molecule localization. However, imaging in depth is still limited since it suffers from a reduction in image quality caused by scattering effects. This paper sets out to investigate the advantages of non-linear photoactivation implemented in a selective plane illumination configuration when imaging scattering samples. In particular, two-photon excitation is proven to improve imaging capabilities in terms of imaging depth and is expected to reduce light-sample interactions and sample photo-damage. Here, two-photon photoactivation is coupled to individual molecule localization methods based on light-sheet illumination (IML-SPIM), allowing super-resolution imaging of nuclear pH2AX in NB4 cells.
Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Fótons , Linhagem Celular Tumoral , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes , Histonas/ultraestrutura , Humanos , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentaçãoRESUMO
Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (γH2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of γH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Replicação do DNA/genética , DNA/análise , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Software , Linhagem Celular , Dano ao DNA , Células Epiteliais/citologia , Feminino , Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Citometria por Imagem/instrumentação , Citometria por Imagem/normas , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Glândulas Mamárias Humanas/citologia , Microscopia Confocal/instrumentação , Microscopia Confocal/normas , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/normas , Robótica/instrumentação , Robótica/normas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
Hardware automation and software development have allowed a dramatic increase of throughput in both acquisition and analysis of images by associating an optimized statistical significance with fluorescence microscopy. Despite the numerous common points between fluorescence microscopy and flow cytometry (FCM), the enormous amount of applications developed for the latter have found relatively low space among the modern high-resolution imaging techniques. With the aim to fulfill this gap, we developed a novel computational platform named A.M.I.CO. (Automated Microscopy for Image-Cytometry) for the quantitative analysis of images from widefield and confocal robotized microscopes. Thanks to the setting up of both staining protocols and analysis procedures, we were able to recapitulate many FCM assays. In particular, we focused on the measurement of DNA content and the reconstruction of cell-cycle profiles with optimal parameters. Standard automated microscopes were employed at the highest optical resolution (200 nm), and white-light sources made it possible to perform an efficient multiparameter analysis. DNA- and protein-content measurements were complemented with image-derived information on their intracellular spatial distribution. Notably, the developed tools create a direct link between image-analysis and acquisition. It is therefore possible to isolate target populations according to a definite quantitative profile, and to relocate physically them for diffraction-limited data acquisition. Thanks to its flexibility and analysis-driven acquisition, A.M.I.CO. can integrate flow, image-stream and laser-scanning cytometry analysis, providing high-resolution intracellular analysis with a previously unreached statistical relevance.
Assuntos
Proteínas de Ciclo Celular/análise , Ciclo Celular/genética , DNA/análise , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Software , Linhagem Celular , Células Epiteliais/citologia , Feminino , Humanos , Citometria por Imagem/instrumentação , Citometria por Imagem/normas , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Glândulas Mamárias Humanas/citologia , Microscopia Confocal/instrumentação , Microscopia Confocal/normas , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/normas , Robótica/instrumentação , Robótica/normasRESUMO
DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.
Assuntos
Divisão Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/efeitos da radiação , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Pontos de Checagem do Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Reparo do DNA , Feminino , Células-Tronco Hematopoéticas/efeitos da radiação , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/efeitos da radiação , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima/efeitos da radiaçãoRESUMO
The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity.