Non-proliferating Cells Research Articles

Aurora A inhibition limits centrosome clustering and promotes mitotic catastrophe in cells with supernumerary centrosomes.

The presence of supernumerary centrosomes is prevalent in cancer, where they promote the formation of transient multipolar mitotic spindles. Active clustering of supernumerary centrosomes enables the formation of a functional bipolar spindle that is competent to complete a bipolar division. Disruption of spindle pole clustering in cancer cells promotes multipolar division and generation of non-proliferative daughter cells with compromised viability. Hence molecular pathways required for spindle pole clustering in cells with supernumerary centrosomes, but dispensable in normal cells, are promising therapeutic targets. Here we demonstrate that Aurora A kinase activity is required for spindle pole clustering in cells with extra centrosomes. While cells with two centrosomes are ultimately able to build a bipolar spindle and proceed through a normal cell division in the presence of Aurora A inhibition, cells with supernumerary centrosomes form multipolar and disorganized spindles that are not competent for chromosome segregation. Instead, following a prolonged mitosis, these cells experience catastrophic divisions that result in grossly aneuploid, and non-proliferative daughter cells. Aurora A inhibition in a panel of Acute Myeloid Leukemia cancer cells has a similarly disparate impact on cells with supernumerary centrosomes, suggesting that centrosome number and spindle polarity may serve as predictive biomarkers for response to therapeutic approaches that target Aurora A kinase function.

Serine Metabolism Supports Macrophage IL-1β Production

Serine is a substrate for nucleotide, NADPH, and glutathione (GSH) synthesis. Previous studies in cancer cells and lymphocytes have shown that serine-dependent one-carbon units are necessary for nucleotide production to support proliferation. Presently, it is unknown whether serine metabolism impacts the function of non-proliferative cells, such as inflammatory macrophages. We find that in macrophages, serine is required for optimal lipopolysaccharide (LPS) induction of IL-1β mRNA expression, but not inflammasome activation. The mechanism involves a requirement for glycine, which is made from serine, to support macrophage GSH synthesis. Cell-permeable GSH, but not the one-carbon donor formate, rescues IL-1β mRNA expression. Pharmacological inhibition of de novo serine synthesis invivo decreased LPS induction of IL-1β levels and improved survival in an LPS-driven model of sepsis in mice. Our study reveals that serine metabolism is necessary for GSH synthesis to support IL-1β cytokine production.

Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos.

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircles for the generation of transgenic animals.

Non-Proliferative Cancer Cells & the Deadly Charge of Metastases

Non-Proliferative Cancer Cells & the Deadly Charge of Metastases

The Comet Assay: Assessment of In Vitro and In Vivo DNA Damage.

Anthropogenic activities, indiscriminate and rapid industrialization as well as pursuance of a better life has led to an increase in the concentration of chemicals, like pesticides, automobile exhausts, and new chemical entities, in the environment, which have an adverse effect on all living organisms including humans. Sensitive and robust test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cell from both in vitro and in vivo sources as well as in humans. The advantages of the in vivo Comet assay are its ability to detect DNA damage in any tissues, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the Comet assay have resulted in establishment of guidelines, and the OECD has adopted a guideline for the in vivo Comet assay as a test for assessing DNA damage in animals. The in vitro Comet assay conducted in cultured cells can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high throughput screening method for new chemical entities, as well as in environmental samples. This chapter details the in vitro Comet assay using the 96-well plate and in vivo Comet assay in multiple organs of the mouse.

Czym jest i czym nie jest starzenie komórki?

Cell senescence is a process that occurs due to telomere erosion or can be induced by various stresses. Senescent cells cease to divide but remain alive, metabolically active and able to secrete many molecules. They also show many hallmarks of senescence, such as enlarged size, increased granularity, increased activity of SA-β-galactosidase, increased level of cyclin-dependent kinase inhibitors, p16 and p21, and DNA damage foci. Originally, cell senescence was attributed to proliferating normal cells, in contrast to cancer cells, which were considered as those endowed with indefinite growth ability. Recently, it has become evident that anticancer treatment induces senescence in cancer cells. Moreover, certain hallmarks of senescence were detected in non-proliferating post-mitotic cells. There are many signalling pathways involved in cell senescence, but the most prevalent is the DNA damage response pathway. In this review we have summarized our long lasting input in the global study of the mechanisms of senescence of normal and cancer cells and discussed the diversity of the concept of cell senescence.

Keeping the Heart Fitm2 during Chemotherapy.

Keeping the Heart Fitm2 during Chemotherapy.

Persistence of fluorescent nanoparticle‐labeled bone marrow mesenchymal stem cells in vitro and after intra‐articular injection

Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra-articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD-labelled MSCs [QD-MSCs]) in vivo after intra-articular injection into normal and osteoarthritic joints. One week after injection of QD-MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD-MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD-MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD-MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle-injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD-MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD-MSCs can be visualized in histological sections 1week after intra-articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long-term tracking of MSCs, especially under proliferative conditions.

Nucleoporin 107 facilitates the nuclear export of Scn5a mRNA to regulate cardiac bioelectricity.

Nucleoporins (Nups) are known to be functional in nucleo‐cytoplasmic transport, but the roles of nucleoporins in nonproliferating cells, such as cardiac myocytes, are still poorly understood. In this study, we report that Nup107 regulates cardiac bioelectricity by controlling the nucleo‐cytoplasmic trafficking of Scn5a mRNA. Overexpression of Nup107 induced the protein expression of Scn5a rather than that of other ion channels, with no effects of their mRNA levels. The analysis for the protein production demonstrated Nup107‐facilitated transport of Scn5a mRNA. Using RIP‐PCR and luciferase assay, we found that the 5′‐UTR of Scn5a mRNA was not involved in the interaction, whereas the spatial interaction between Nup107 protein and Scn5a mRNA was formed when Scn5a mRNA passing through the nuclear pore. Functionally, Nup107 overexpression in neonatal rat ventricle myocytes significantly increased the currents of Scn5a‐encoded INa channel. Moreover, the close correlation between Nup107 and Nav1.5 protein expression was observed in cardiomycytes and heart tissues subjected to hypoxia and ischaemic insults, suggesting a fast regulation of Nup107 on Nav1.5 channel in cardiac myocytes in a posttranscriptional manner. These findings may provide insights into the emergent control of cardiac electrophysiology through Nup‐mediated modulation of ion channels.

Raman and infrared spectroscopy reveal that proliferating and quiescent human fibroblast cells age by biochemically similar but not identical processes.

Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.

Werner syndrome (WRN) DNA helicase and base excision repair (BER) factors maintain endothelial homeostasis

The accelerated ageing disease Werner Syndrome (WRN) is characterized by pronounced atherosclerosis. Here, we investigated the influence of WRN downregulation on the functionality of non-replicating human endothelial cells. RNAi-mediated downregulation of WRN reduces cell motility and enhances the expression of factors regulating adhesion, inflammation, hemostasis and vasomotor tone. Moreover, WRN influences endothelial barrier function and Ca2+-release, while cell adhesion, Dil-acLDL-uptake and the mRNA expression of NO-synthases (eNOS, iNOS) remained unaffected. Regarding motility, we propose that WRN affects Rac1/FAK/ß1-integrin-related mechanisms regulating cell polarity and directed motility. Since oxidative DNA base damage contributes to aging and atherosclerosis and WRN affects DNA repair, we investigated whether downregulation of base excision repair (BER) factors mimics the effects of WRN knock-down. Indeed, downregulation of particular WRN-interacting base excision repair (BER) proteins (APE1, NEIL1, PARP1) imitates the inhibitory effect of WRN on motility. Knock-down of OGG1, which does not interact with WRN, does not influence motility but increases the mRNA expression of E-selectin, ICAM, VCAM, CCL2 and VEGFR and stimulates adhesion. Thus, individual BER factors themselves differently impact endothelial cell functionality and homeostasis. Impairment of endothelial activities caused by genotoxic stressor (tBHQ) remained largely unaffected by WRN. Summarizing, both WRN, WRN-associated BER proteins and OGG1 promote the maintenance of endothelial cell homeostasis, thereby counteracting the development of ageing-related endothelial malfunction in non-proliferating endothelial cells.

Mcl-1 and Bcl-xL are essential for survival of the developing nervous system.

During neurogenesis, proliferating neural precursor cells (NPC) exit the cell cycle and differentiate into postmitotic neurons. The proteins that regulate cell survival through the stages of differentiation, however, are still poorly understood. Here, we examined the roles of the anti-apoptotic Bcl-2 proteins, Mcl-1 and Bcl-xL, in promoting survival as cells progress through the stages of neurogenesis in the mouse embryonic central nervous system. We used Nestin-mediated, nervous system-specific conditional deletion of mcl-1, bcl-x or both to identify their distinct and overlapping roles. Individual conditional deletion of mcl-1 (MKO) and bcl-x (BKO) suggested sequential roles in promoting cell survival during developmental neurogenesis. In the MKO embryo, apoptosis begins at embryonic day 10 (E10) in the proliferating NPC population throughout the entire developing nervous system. In the BKO embryo, apoptosis begins later at E11 within the postmitotic neuron populations. In the double (mcl-1 and bcl-x) conditional knockout (DKO), cell death extended throughout both proliferating and non-proliferating cell populations resulting in embryonic lethality at E12, earlier than in either the MKO or BKO. Apoptotic cell death of the entire central nervous system in the DKO demonstrates that both genes are necessary for cell survival during developmental neurogenesis. To determine whether Mcl-1 and Bcl-xL have overlapping anti-apoptotic roles during neurogenesis, we examined the impact of gene dosage. Loss of a single bcl-x allele in the MKO embryo exasperated apoptotic cell death within the NPC population revealing a novel anti-apoptotic role for Bcl-xL in proliferating NPCs. Cells were rescued from apoptosis in both the MKO and BKO embryos by breeding with the Bax null mouse line indicating that Mcl-1 and Bcl-xL have a common pro-apoptotic target during developmental neurogenesis. Taken together, these findings demonstrate that Mcl-1 and Bcl-xL are the two essential anti-apoptotic Bcl-2 proteins required for the survival of the developing mammalian nervous system.

Lrig1 marks a population of gastric epithelial cells capable of long-term tissue maintenance and growth in vitro

The processes involved in renewal of the epithelium that lines the mouse stomach remain unclear. Apart from the cells in the isthmus, several other populations located deeper in the gastric glands have been suggested to contribute to the maintenance of the gastric epithelium. Here, we reveal that Lrig1 is expressed in the basal layer of the forestomach and the lower part of glands in the corpus and pylorus. In the glandular epithelium of the stomach, Lrig1 marks a heterogeneous population comprising mainly non-proliferative cells. Yet, fate-mapping experiments using a knock-in mouse line expressing Cre specifically in Lrig1+ cells demonstrate that these cells are able to contribute to the long-term maintenance of the gastric epithelium. Moreover, when cultured in vitro, cells expressing high level of Lrig1 have much higher organoid forming potential than the corresponding cellular populations expressing lower levels of Lrig1. Taken together, these observations show that Lrig1 is expressed primarily by differentiated cells, but that these cells can be recruited to contribute to the maintenance of the gastric epithelium. This confirms previous observations that cells located in the lower segments of gastric glands can participate in tissue replenishment.

Nucleolar Division in the Promastigote Stage of Leishmania major Parasite: A Nop56 Point of View.

Nucleogenesis is the cellular event responsible for the formation of the new nucleoli at the end of mitosis. This process depends on the synthesis and processing of ribosomal RNA (rRNA) and, in some eukaryotes, the transfer of nucleolar material contained in prenucleolar bodies (PNBs) to active transcription sites. The lack of a comprehensive description of the nucleolus throughout the cell cycle of the human pathogen Leishmania major prompted us to analyze the distribution of nucleolar protein 56 (Nop56) during interphase and mitosis in the promastigote stage of the parasite. By in silico analysis we show that the orthologue of Nop56 in L. major (LmNop56) contains the three characteristic Nop56 domains and that its predicted three-dimensional structure is also conserved. Fluorescence microscopy observations indicate that the nucleolar localization of LmNop56 is similar, but not identical, to that of the nucleolar protein Elp3b. Notably, unlike other nucleolar proteins, LmNop56 remains associated with the nucleolus in nonproliferative cells. Moreover, epifluorescent images indicate the preservation of the nucleolar structure throughout the closed nuclear division. Experiments performed with the related parasite Trypanosoma brucei show that nucleolar division is carried out by an analogous mechanism.

Mitochondria-driven elimination of cancer and senescent cells.

Mitochondria and oxidative phosphorylation (OXPHOS) are emerging as intriguing targets for the efficient elimination of cancer cells. The specificity of this approach is aided by the capacity of non-proliferating non-cancerous cells to withstand oxidative insult induced by OXPHOS inhibition. Recently we discovered that mitochondrial targeting can also be employed to eliminate senescent cells, where it breaks the interplay between OXPHOS and ATP transporters that appear important for the maintenance of mitochondrial morphology and viability in the senescent setting. Hence, mitochondria/OXPHOS directed pharmacological interventions show promise in several clinically-relevant scenarios that call for selective removal of cancer and senescent cells.

Automatic method for assessment of proliferation index in digital images of DLBCL tissue section

Diffuse large B-cell lymphoma (DLBCL) is a fast-growing and aggressive neoplasm originating from B lymphocytes. Evaluation of proliferation index (PI) based on Ki67 immunohistochemical nuclear staining is used to distinguish proliferating (immunopositive) from nonproliferating (immunonegative) lymphoma cells. Human interpretation of PI varies and is time-consuming, therefore automatic computer-assisted approach may facilitate the performance.Herein we propose a new fully automatic proliferation index estimation (FLAPIE) algorithm, dedicated to detection of immunopositive and immunonegative nuclei, and evaluation of PI in digital microscopy images of DAB&H-stained samples from patients with high-grade DLBCL.FLAPIE performs nuclei detection in original RGB colour space and is independent of image brightness due to its textural-statistical approach. Validation of FLAPIE was performed in 61 non-overlapping whole-slide image fragments and compared to the results of PI estimation by QuPath open-source software, MetPiKi algorithm and manual evaluation by two independent observers. Interobserver agreement was calculated between the nuclei count and PIs by two observers.High concordance was found between both DAB and H-stained nuclei count, and PIs by two observers. Compared to MetPiKi, FLAPIE presented improved results of DAB and H-stained nuclei detection. In contrary to MetPiKi and QuPath, FLAPIE performed nuclei detection in all images and its results closely matched the number of DAB-stained nuclei evaluated by two observers. No significant difference was found between PIs by all computational methods and observers.FLAPIE achieved good results in PI estimation and prospectively aims to serve as a tool for clinical application in support of patients selection and decision to treatment.

Clustered DNA Double-Strand Breaks and Neuroradiobiological Effects of Accelerated Charged Particles

The irradiation of brain structures with accelerated heavy charged particles during interplanetary flights or brain tumor therapy raises a number of questions regarding possible neurophysiological disorders in the central nervous system (CNS). Hardly repairable clustered DNA double-strand breaks apparently can have significant influence on the specifics of the development of radiation syndromes in the CNS after heavy charged particle exposure. The mechanisms of these disorders still remain unclear. Taking this into account, we have used immune cyto- and histochemistry techniques to study regularities of the formation of radiationinduced foci in human cell DNA in vitro and in rodent brain neurons in vivo after exposure to charged particles of different energies. It has been found that heavy charged particles induce clustered DNA damage in the genome of proliferating (human fibroblasts) and non-proliferating (Purkinje neurons) cells. We have suggested that changes in genetic structures can affect the conformation of the key proteins participating in neurophysiological processes and violate the normal functioning of the synaptic receptors. As an example, we have considered the action of double point mutations in the gene sequence encoding the proteins of the glutamate receptor NMDA. Using computer molecular dynamics techniques, we have revealed a twofold change in the conductance of the receptor’s ion channel, which incorporates mutant forms of the protein subunits NR2.

Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells

Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognized to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as well as many intracellular proteins, the release of which is of uncertain biological significance. Here, we describe a mass spectrometry-based method for stable isotope dynamic labeling of secretomes (SIDLS) that, by dynamic SILAC, discriminates the secretion kinetics of classical secretory proteins and intracellular proteins released from cancer and stromal cells in culture. SIDLS is a robust classifier of the different cellular origins of proteins within the secretome and should be broadly applicable to nonproliferating cells and cells grown in short term culture.

Mathematical modeling of the proliferation gradient in multicellular tumor spheroids

MultiCellular Tumor Spheroids are 3D cell cultures that can accurately reproduce the behavior of solid tumors. It has been experimentally observed that large spheroids exhibit a decreasing gradient of proliferation from the periphery to the center of these multicellular 3D models: the proportion of proliferating cells is higher in the periphery while the non-proliferating quiescent cells increase in depth. In this paper, we propose to investigate the key mechanisms involved in the establishment of this gradient with a Partial Differential Equations model that mimics the experimental set-up of growing spheroids under different nutrients supply conditions. The model consists of mass balance equations on the two cell populations observed in the data: the proliferating cells and the quiescent cells. The spherical symmetry is used to rewrite the model in radial and relative coordinates. Thanks to a rigorous data postprocessing the model is then fit and compared quantitatively with the experimental quantification of the percentage of proliferating cells from EdU immunodetection on 2D spheroid cryosection images. The results of this calibration show that the proliferation gradient observed in spheroids can be quantitatively reproduced by our model.

Abstract 112: Doxorubicin Alters Cardiac Fibroblast Phenotype

The oldest cohort of childhood cancer survivors are entering into their sixth decade. The milestone comes with the knowledge that this population is at a 15 times greater risk of cardiovascular events than the general population. One factor in this is doxorubicin, a chemotherapeutic drug commonly used to treat pediatric cancers. Our study investigates the effect of doxorubicin on proliferating cells in the heart, specifically, cardiac fibroblasts. Doxorubicin inhibits cardiac fibroblast growth and migration. Cardiac fibroblasts also undergo a shift in gene expression when exposed to doxorubicin. Inflammatory cytokines and their receptors are upregulated, along with extracellular matrix regulatory proteins. ECM structural proteins and adhesion molecules are downregulated compared to control cells. Of note, monocyte chemoattractant protein-1, and its receptor C-C motif chemokine receptor-4, are upregulated over 40-fold compared to control cells. Doxorubicin has multiple mechanisms of actions that target proliferating and non-proliferating cells differently. Cardiac fibroblasts are proliferative cells with a major role in multiple processes of cardiac homeostasis. This study lays the foundation of the phenotypic changes cardiac fibroblasts undergo when exposed to doxorubicin. These changes may create a microenvironment that primes the heart for adverse cardiac remodeling.