High content image-based cytometry as a tool for nuclear fingerprinting

Cytomics aims at understanding the functional relationships between cellular phenotypes (cytome) and metabolic pathways (proteome) that result from a combination of genetically defined mechanisms (genome) and environmental conditions [1,2]. Although flow-cytometry is able to measure the optical properties of single cells at a rate of >1000 cells per minute it has a limited capability of mapping individual events. To accurately quantify (sub-) cellular characteristics within a natural context there is a fast-growing need for image-based cytometry. Images, obtained with fluorescence microscopy, provide the exact information on signal intensity, location and distribution of specific molecules within intact cell systems (tissue or monolayers) and allow for investigating cellular properties in relation to the cell-ecological context [3]. Previously, we have developed a cytometric approach for scoring DNA lesion endpoints in confocal images of murine fibroblasts [3]. We now present a generalized approach for multivariate phenotypic profiling of individual nuclei using automated fluorescence mosaic microscopy and optimized digital image processing tools. An indefinite number of fields, z-slices and channels can be analyzed; the only prerequisite is the presence of a nuclear counterstain, which is used for the generation of masks. To anticipate for erroneous segmentation of clustered nuclei in dense cell cultures we implemented an iterative conditional segmentation (ics) algorithm that uses both morphological and intensity information from the image (Figure 1). The method makes use of a priori knowledge about the size and shape of nuclei in stringent feedback selection of correctly segmented nuclei. Depending on the degree of clustering, segmentation performance varies between 95% and 100%. Complete analysis of nuclei and subnuclear features for a region of 25 images of 1000x1000 pixels, 3 z-slices and 3 channels only takes ~ 3 minutes or ~ 0.7sec/nucleus. Our method is insensitive to scaling, illumination heterogeneity and variability or non-uniformity of staining. We have successfully applied our system in cell cycle analysis, scoring of transfection efficiency and assessment of (localized) DNA damage in response to genotoxic stress and ionizing radiation

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation

Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/mu m) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by gamma-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-kappa B, may be involved in these cellular responses

Radiation-induced protein redistribution and modification involved in DNA damage response and intercellular communication

Human cells are exposed to a wide range of stressors that influence their responses on different levels. There are strong indications that cells endure these stressors as an entity. Increasing evidence suggests that individual cells are not closed systems isolated from their environment but continuously exchange information with each other. Two non-targeted effects were studied: (1) Bystander effects occur when non-treated cells – that are in contact with exposed cells either through the medium or gap junctions – reveal effects similar to those seen in exposed cells. (2) In adaptive response, cells – either directly or indirectly exposed – are influenced by a prior treatment. Both phenomena were studied using high throughput cytometry and multiplex array screening. Central in this investigation was H2AX, a protein belonging to the histon complex that plays an important role in DNA damage response. In response to double stranded breaks (DSB) H2AX is phosphorylated (γH2AX) and is considered as signal enhancer for the DSB repair pathway. The DNA damage response of human fibroblast was analyzed and H2AX analysis revealed the presence of a bystander effect and an adaptive response. In addition, we analyzed the cytokines that were present in the culture medium after exposure to ionizing radiation. We could also link the upregulation of γH2AX in bystander cells to the presence of these secreted cytokines in the culture medium. In line with the above-mentioned results we analyzed the DNA damage response in fibroblasts exposed to cosmic radiation during the Foton M3 space mission. This fully automated mission lasted for 12 days and completed 190 orbits around the earth. During the space experiment, we tested and validated a compact, biological dosimeter, which can be used in future space flights. Finally, we focused on a specific signaling molecule, cyclophilin B (CypB). CypB is secreted into the culture medium, but has the necessary signal peptides for an efficient retrograde transport to the nucleus. In order to identify the various peptide sequences necessary for these relocalizations, different GFP constructs were made. We identified a nucleolar localization sequence that allowed transport of CypB and could link CypB to a role in rDNA transcription

Functional cyclophilin B reveals a cell cycle dependent nucleolar pattern with a possible role in rDNA transcription

Cyclophilins are ubiquitously distributed intracellular proteins first identified as cellular binding proteins for the potent immunosuppressive drug, cyclosporin A (CsA). Also known as peptidyl- prolyl cis–trans isomerases, they catalyze the slow cis-trans isomerization of proline peptide bonds in oligopeptides and accelerate folding of several proteins. Cyclophilin B (CypB) belongs to this family with confirmed functions in nucleus, cytoplasm and as intercellular communicator. These different localizations enable CypB to perform distinct functions that vary from chaperone(1), stimulating RNA binding activity during hepatitis C infection(2) to specifically inducing chemotaxis(3). During our research, we investigated the various localization patterns that we found for CypB in both primary fibroblasts as in Hela cells. We found that CypB is able to localize to the different cellular compartments through the presence of a N-terminal nuclear localization signal (NLS) that becomes active after selective removal of the first 33 amino acids that functions as an ER-localization signal. Immunofluorescent staining in human dermal fibroblasts revealed that CypB expression follows a cell cycle dependent pattern with a particular preference for localization at the nucleoli. The fusion of different deletion mutants with GFP allowed reconstruction of the various described phenotypes found in literature. We found that CypB contains in fact a nucleolar localization signal that allows efficient relocation to the nucleoli in a cell cycle (from mid S- to early G2) and energy dependent manner. CypB has a high preference for the fibrillar centers (FC’s). Confocal microscopy revealed that -in these FC’s- CypB colocalized with factors involved in rDNA transcription, such as RNA-polymerase and Upstream binding factor-1 (UBF), but also with coilin - a protein present in both nucleoli and cajal bodies. This protein association was maintained after disruption of the nuclear architecture with adenosine analog DRB. The function of CypB within the nucleoli is part of an ongoing investigation. Within microarray results from a CypB knockout analysis, we screened for proteins localized within the nucleoli and obtained a list of 22 candidate proteins. These proteins are involved in chromatine structure modulation, ribosome biogenisis, etc.. We propose that CypB regulates and interacts with these nucleolar proteins and is involved in the rDNA transcription during the transcriptionally more active phases of the cell cycle. This work is supported by a BOF grant provided by University Gent, Belgium Bibliografie 1. M. A. Rycyzyn et al., Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation. Mol.Endocrinol. 14, 1175-1186 (2000). 2. K. Watashi et al., Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol.Cell 19, 111-122 (2005). 3. F. Allain et al., Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix. Proc.Natl.Acad.Sci.U.S.A 99, 2714-2719 (2002)

Interplanetary space travel and long-term habitation on mars

Robotic planetary exploration has enabled scientists to gather valuable data and understanding about the composition and origin of extraterrestrial structures. However, despite its tremendous possibilities, human exploration is a natural next step. Manned missions to Mars are becoming more feasible and as such the irresistible attraction for mankind to explore this neighbouring planet is growing. Especially since 2004, with the president of the United States outlining specific objectives for future exploration, including missions to the Moon, Mars and beyond these aspirations have become tangible. A prerequisite for a manned mission to Mars, requires an extensive, durable life supporting system which should recycle waste. Preferable also produce additional eatable substances and/or additives in a safe and efficient manner with a high reliability to perform in harsh space conditions

Analysis of radiation-induced DNA-damage generated by X-rays

Ionizing radiation poses a severe threat to chromosomal integrity and genome stability by inducing both single stranded and double stranded breaks. When exposed to radiation, cells rapidly phosphorylate the nucleosomal histone protein H2AX at sites surrounding double-stranded breaks (DSB). The phosphorylation of H2AX (γH2AX) functions as a signal enhancer for DSB-repair. XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSB) by coordinating the enzymatic processing in base excision repair. Using XRCC1 as a representative of global SSB- and γH2AX as marker for DSB-repair, we assessed the global DNA repair response in human primary fibroblasts and cell lines after exposure to ionizing radiation. Using immunocytochemistry and automated high-content cytometry we were able to extract statistical information on global population response as well as individual variations at the level of the single cell. Cell lines were found to have higher basal levels of both DNA damage markers than fibroblasts. In fibroblasts, a cell cycle specific pattern for γH2AX was observed: γH2AX is significantly upregulated during S-phase. Although XRCC1 showed no cell cycle dependency, a distinct dose-dependent relocation to the nucleoli was observed 4-6 hours after X-ray exposure. We hypothesize that these nucleoli function as factories where XRCC1 is processed

Functional analysis of Cyclophilin B reveals a cell cycle dependent nucleolar pattern with a possible role in rDNA transcription

Cyclophilin B (CypB) belongs to the family of Cyclophilins, ubiquitously distributed proteins with confirmed functions in nucleus, cytoplasm and as intercellular communicator. CypB is able to combine these functions through the presence of a N-terminal nuclear localization signal (NLS) that becomes active after selective removal of the first 33 amino acids. Immunofluorescent staining in human dermal fibroblasts revealed that CypB expression follows a cell cycle dependent pattern with a particular preference for localization at the nucleoli. The fusion of different deletion mutants with GFP allowed reconstruction of the various described phenotypes found in literature. We found that CypB contains in fact a nucleolar localization signal that allows efficient relocation to the nucleoli in a cell cycle dependent manner. CypB revealed a high preference for the fibrillar centers (FC’s). In these FC’s CypB colocalized with factors involved in rDNA transcription, such as RNA-polymerase, Upstream binding factor-1 (UBF) and fibrillarin. This protein association was maintained after disruption of the nuclear architecture with adenosine analog DRB and became associated with cajal bodies. We propose that CypB interacts with these proteins and is involved in the rDNA transcription during the transcriptionally more active phases of the cell cycle. This work is supported by a BOF grant provided by University Gent, Belgiu

Medium-mediated bystander response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts

For years research concerning radiation-induced damage initiated from the reasoning that in order for cells to experience malignant effects of ionizing radiation, direct contact of specific targets (DNA, RNA,..) with the radiation itself or with short-lived free radicals is required. It is becoming increasingly clear however, that cells experience a plethora of influences that are not solely restricted to close proximity effects. Apart from direct damage in target cells, ionizing radiation can invoke secondary effects in non-targeted cells. These secondary effects, collectively referred to as bystander effects, rely on communication between cells. Regarding this communication, two non-exclusive and probably complementary mechanisms have been proposed: direct cell-cell contact via gap junctions and secondly through secretion of small signaling components into the medium [1, 2]. We investigated the medium-mediated bystander response in human dermal fibroblasts (HDF) after exposure to ionizing irradiation. We looked at histone modifications, more specifically phosphorylation of H2AX. When a double stranded break (DSB) occurs H2AX is phosphorylated within minutes acting as a signal enhancer molecule in the DSB repair cascade. We showed that HDF experienced an elevated level of double stranded DNA damage repair foci, when incubated with conditioned growth medium of irradiated cells. The magnitude of this response is much lower than that of directly irradiated cells and is proportional to the irradiation dose. Using multiplex analysis, four cytokines IL-6, IL-8, MCP-1 and RANTES were identified in the growth medium of irradiated cells that were significantly upregulated and each with differential kinetics. These soluble proteins could function as potential bystander signaling mediators providing new insights to the complex domain of (in-)direct radiation biology. [1] M.V.Sokolov, J.S.Dickey, W.M.Bonner, O.A.Sedelnikova. gamma-H2AX in bystander cells: not just a radiation-triggered event, a cellular response to stress mediated by intercellular communication, Cell Cycle 6 (2007) 2210-2212. [2] H.Yang, N.Asaad, K.D.Held. Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts, Oncogene 24 (2005) 2096-2103