Arnaoutova I.,Trevigen, Inc. |
Kleinman H.K.,U.S. National Institutes of Health
Nature Protocols | Year: 2010
A protocol is presented here for a rapid, quantitative and reliable in vitro angiogenesis assay that can be adapted for high throughput use. Endothelial cells are plated on a gelled basement matrix, their natural substrate, and form capillary-like structures with a lumen. The assay can be used to identify inhibitors or stimulators of angiogenesis, as well as genes and signaling pathways involved in angiogenesis. It has also been used to identify endothelial progenitor cells. This assay involves endothelial cell adhesion, migration, protease activity and tubule formation. This tube formation assay is preferred, as other in vitro assays for angiogenesis, such as cell adhesion, migration and invasion, measure limited steps in the angiogenesis process. The tube formation assay on basement membrane can be completed in a day because transformed endothelial cells form tubes within 3 h, whereas non-transformed endothelial cells form tubes within 6 h. © 2010 Nature Publlshing Group. Source
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 153.00K | Year: 2016
DESCRIPTION provided by applicant The objective of this Phase STTR research is to quantify genotoxicity in basal cell keratinocytes from organotypic cultures Epiderm in response to commonly used chemical agents The proposed product is a Comet Chip assay that measures DNA damage in basal cells derived from a reconstructed human epidermis Technical questions that will be addressed are Can we modify our currently used Comet Chip assay to incorporate extracellular matrix proteins or antibodies Can we isolate individual basal keratinocytes from a D organotypic skin culture on the basis of their preferential adhesion to these matrix proteins including collagen I and IV or by using immobilized antibodies to integrin Can we confirm their identity by quantum dot coupled antibodies specific for or collagen ligand integrins Can we use the isolated antibody labeled epidermal basal cells to detect and quantify levels of DNA damage in response to known environmental genotoxic agents Can we use our Immuno CometChip assay to screen large numbers of agents currently or proposed to be marketed The impact of the proposed research will be to reduce animal model use for toxic agent screening since human organotypic culture has been shown to be almost identical to human skin with respect to its cytokine profile in response to corrosive or irritating agents The market for screening skin genotoxic agents is immense since the current screening procedures cannot keep pace with the number of new agents currently being introduced Aim will be to develop a new method for the isolation of basal keratinocytes and an immunostaining method for simultaneous visualization of specific antigens including integrin and DNA damage Aim will be to validate the Immuno CometChip assay using known DNA damaging agents including H O This adds three new parameters to the Comet assay The first is obtaining treatment groups of single basal epidermal keratinocytes from an organotypic culture on a single chip the second is verifying their identity with surface markers and the third is the simultaneous reproducible assay for DNA damage PUBLIC HEALTH RELEVANCE The Comet assay has appeal for many reasons The assay is rapid simple sensitive reliable and fairly inexpensive Its use for testing for skin toxicity hs been hampered by a number of factors including background DNA damage incurred during the normal process of differentiation in organotypic skin models which mimic human skin We therefore propose to develop a Comet Chip assay a high throughput reproducible and quantitative method to isolate basal keratinocytes and measure genotoxicity in response to commonly used chemical agents
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 318.78K | Year: 2011
DESCRIPTION (provided by applicant): Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. DNA damaging agents and other genotoxins that arise from cellular metabolism, environmental sources or disease-related cellular defects contribute to cell death (e.g., neurodegeneration), gene mutations, gene rearrangements and in many cases, the onset of cancer, disease and aging phenotypes. In addition, many exogenous exposures suchas chemotherapy and radiation treatment rely on the induction of tumor cell genotoxicity to mediate therapeutic response. Further, the ability to effectively and accurately repair spontaneous or induced DNA damage depends on the cellular DNA repair capacity. Therefore, the ability to quantify DNA damage and the rate of repair of the damage to the nuclear genome directly in human cells is critical in applications ranging from epidemiology to drug development. To address this technological need in the research community, to be better positioned to characterize the genotoxicity of newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair gene defects, we propose the development of the next generationin DNA damage detection and quantification technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose-based Microwell arrays, spatially- encoded cellular recognition, human tumor cell lines with genetically-defined DNA repair status and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays that will function as a tool to quantify DNA damage and measure cellular DNA Repair capacity at baseline and following genotoxin exposure on a single array or chip (DNA Repair on a Chip). The studies described in Aim 1 involve the development of a series of 24-well Spatially Encoded Microwell Arrays, with Microwells ranging from 10-50 5M in diameter and 20-50 5M indepth, suitable for gravity capture of a single cell of various sizes. Efficacy of the Microwell Arrays will be validated using radiation and small molecule inhibitors. Further, the sensitivity of the Microwell Arrays for analysis of cellular DNA Repair capacity will be evaluated using an isogenic panel of human tumor cell lines with defined defects in DNA Repair gene expression and following genotoxic stress. Iterative analysis and Microwell characterization will inform to finalize a set of 24-well Microwell Arrays for production and distribution. The studies described in Aim 2 involve additives to the Microwell Arrays that will enhance cell growth and attachment, providing optimal analysis of baseline DNA damage and most importantly, critical data on cellular capacity for in vivo repair post-damage. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses. Phase II of the project will beexpanded to offer 96-well capability, end-user software for spatial recognition and quantitation plus micro-well additive options for specialized cell growth and attachment. PUBLIC HEALTH RELEVANCE: We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 200.26K | Year: 2009
DESCRIPTION (provided by applicant): Human illness results from the complex interactions of integrated processes and factors, including genetic predispositions and environmental agents. The environmental genome project (EGP) was formally initiated to systematically and comprehensively evaluate how genetic polymorphisms impact our susceptibility to environmentally founded disease. The EGP has identified eight categories of environmentally responsive genes (ERG) that have been shown to react to environmental agents. These categories include cell cycle, DNA repair, cell division, cell signaling, cell structure, gene expression, apoptosis and metabolism. The genome is under continuous assault by a combination of both environmental and endogenous DNA damaging agents requiring a complex set of DNA repair proteins to resolve these genetic insults. However, there are clear inter-individual differences between humans in their susceptibility to DNA damaging agents that result from either pre-existing environmental exposures or genetic factors such as sequence variation or single- nucleotide polymorphisms, SNPs. Evaluating the functional impact of individual polymorphisms will require novel approaches and new reagents. During the first phase of this proposal, we will develop and characterize a series of isogenic DNA glycosylase deficient human cell lines for future studies towards evaluation of the functional significance of DNA repair gene SNPs and genetic variants in human cells. These studies are designed to provide essential reagents to aid in understanding the biological significance of human DNA polymorphisms and the role of these SNPs either alone or in combination with specific environmental stressors in disease outcomes as varied as cancer, aging-related disorders, stroke and diabetes. Upon successful completion of the 1st phase, we intend to have demonstrated the feasibility of producing stable human cell lines with complete deficiency in DNA repair proteins, specifically DNA glycosylases. Further, we propose to characterize each newly developed cell line with respect to mRNA and protein expression and DNA glycosylase activity and finally, each will be evaluated for the impact of the depletion of a single DNA repair gene product on the global transcriptome. The development of such isogenic human cells for an additional 140 DNA repair genes will be the topic of the second phase of this proposal, covering genes involved in Base Excision Repair (BER), Direct Reversal of Damage, Mismatch excision repair (MMR), Nucleotide Excision Repair (NER), Homologous Recombination, Non- homologous end-joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA repair genes defective in diseases and conserved DNA damage response genes. PUBLIC HEALTH RELEVANCE: The over all goal of the phase I project is to develop cell lines each depleted of the known DNA repair associated glycosylases. In the proposal we plan to develop real time in vivo assays to monitor glycosylase activity. Additionally we intend to determine the effect of depletion of a single glycosylases on the global transcriptome.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 202.05K | Year: 2014
Project Summary / Abstract DNA repair pathways maintain the integrity of the genome and thereby help prevent the onset of cancer, disease and aging phenotypes. Further, many cancer treatments function by inducing genomic DNA damage. As such, the critical requirement for DNA repair proteins and pathways in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer regimens. Essential to the development of specific DNA repair inhibitors is the availability of robust, highly sensitive assays to measure DNA repair capacity. In addition, defects in critical DNA repair pathways or proteins can predispose to cancer onset and may also provide an option for therapeutic selectivity. Many of these defects in the 150 or more DNA repair proteins can be detected using current omics technologies. However, there are many defects that can only be detected using functional assays such as those described herein. To effectivel