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1.  Col-F, a Fluorescent Probe for Ex Vivo Confocal Imaging of Collagen and Elastin in Animal Tissues 
A new low molecular weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials.
doi:10.1002/cyto.a.22264
PMCID: PMC3671577  PMID: 23404939
extracellular matrix; collagen; elastin; fluorescence microscopy; live cell imaging; confocal microscopy
2.  Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells 
Analytical chemistry  2009;81(16):6952-6959.
Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines.
doi:10.1021/ac9010217
PMCID: PMC3977701  PMID: 19572560
3.  Disruption of mutated BRAF signaling modulates thyroid cancer phenotype 
BMC Research Notes  2014;7:187.
Background
Thyroid cancer is the most common endocrine-related cancer in the United States and its incidence is rising rapidly. Since among various genetic lesions identified in thyroid cancer, the BRAFV600E mutation is found in 50% of papillary thyroid cancers and 25% of anaplastic thyroid cancers, this mutation provides an opportunity for targeted drug therapy. Our laboratory evaluated cellular phenotypic effects in response to treatment with PLX4032, a BRAFV600E-specific inhibitor, in normal BRAF-wild-type thyroid cells and in BRAFV600E-positive papillary thyroid cancer cells.
Methods
Normal BRAF-wild-type thyroid cells and BRAFV600E-mutated papillary thyroid cancer cells were subjected to proliferation assays and analyzed for cell death by immunofluorescence. Cell cycle status was determined using an EdU uptake assay followed by laser scanning cytometry. In addition, expression of proteins within the MAPK signal transduction pathway was analyzed by Western blot.
Results
PLX4032 has potent anti-proliferative effects selectively in BRAF-mutated thyroid cancer cells. These effects appear to be mediated by the drug’s activity of inhibiting phosphorylation of signaling molecules downstream of BRAF within the pro-survival MAPK pathway. Interestingly, PLX4032 promotes the phosphorylation of these signaling molecules in BRAF-wild-type thyroid cells.
Conclusions
These findings support further evaluation of combinational therapy that includes BRAFV600E inhibitors in thyroid cancer patients harboring the BRAFV600E mutation.
doi:10.1186/1756-0500-7-187
PMCID: PMC3976539  PMID: 24673746
Thyroid cancer; BRAFV600E mutation; PLX4032; MAPK signal transduction pathway; Targeted therapy; Kinase inhibitors
4.  Real-time cell viability assays using a new anthracycline derivative DRAQ7® 
The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a non-invasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be non-toxic to a panel of cancer cell lines grown continuously for up to 72 hours and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation and drug-induced cytotoxicity. The overall responses to anti-cancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multi-parameter and kinetic fingerprint of anti-cancer drug action.
doi:10.1002/cyto.a.22228
PMCID: PMC3558543  PMID: 23165976
DRAQ7; real-time assays; cell viability; drug; cytotoxicity; DNA damage response; cell cycle; microfluidic; cytometry
5.  Laser Scanning Cytometry 
Summary
The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF- κB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry.
doi:10.1007/978-1-59259-993-6_8
PMCID: PMC3892962  PMID: 16719355
Cytometry; fluorescence; cell cycle; apoptosis; nucleus; nucleolus; micronucleus cytoplasm; enzyme kinetics
6.  Laser Scanning Cytometry: Principles and Applications—An Update 
Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.
doi:10.1007/978-1-62703-056-4_11
PMCID: PMC3488462  PMID: 23027005
Cytometry; Fluorescence; Cell cycle; Apoptosis; Nucleus; Nucleolus; Micronucleus; Cytoplasm; Enzyme kinetics
7.  Biomarkers of Cell Senescence Assessed by Imaging Cytometry 
The characteristic features of senescent cells such as their “flattened” appearance, enlarged nuclei and low saturation density at the plateau phase of cell growth, can be conveniently measured by image-assisted d cytometry such as provided by the laser scanning cytometry (LSC). The “flattening” of senescent cells is reflected by the decline in local density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2- phenylindole (DAPI)] paralleled by an increase in nuclear size (area). Thus, the ratio of the maximal pixel of DAPI fluorescence per nucleus to the nuclear area provides a very sensitive morphometric biomarker of “depth” of senescence, which progressively declines during induction of senescence. Also recorded is cellular DNA content revealing cell cycle phase, as well as the saturation cell density at plateau phase of growth, which is dramatically decreased in cultures of senescent cells. Concurrent immunocytochemical analysis of expression of p21WAF1, p16INK4a or p27KIP1 cyclin kinase inhibitor provides additional markers of senescence. These biomarker indices can be expressed in quantitative terms (“senescence indices”) as a fraction of the same markers of the exponentially growing cells in control cultures.
doi:10.1007/978-1-62703-239-1_5
PMCID: PMC3541526  PMID: 23296652
Cell aging; chromatin structure; cell cycle; apoptosis; nuclear size; laser scanning cytometry; p21WAF1; p16INK4a; p27KIP1; premature cell senescence
8.  Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry 
Proceedings of SPIE  2013;8615:10.1117/12.2001474.
Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells.
doi:10.1117/12.2001474
PMCID: PMC3877312  PMID: 24386542
microfluidics; Lab-on-a-Chip; flow cytometry; cell cycle; apoptosis; programmed cell death; cancer
9.  Activation of Nuclear Factor Kappa B (NF-κB) Assayed by Laser Scanning Cytometry (LSC) 
Cytometry  1998;33(3):376-382.
Nuclear factor kappa B (NF-κB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-κB remains in the cytoplasm sequestered through interaction with IκB protein. Rapid translocation of NF-κB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-κB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor α (TNF-α) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-κB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counter-stained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-κB led to a rapid increase in NF-κB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (FC), which was reflected by an increase in FN/FC ratio. This rapid assay of NF-κB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-κB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.
PMCID: PMC3874872  PMID: 9822350
nucleus; cytoplasm; tumor necrosis factor
10.  Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry 
Experimental cell research  2009;315(10):10.1016/j.yexcr.2009.03.006.
Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry.
Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors.
doi:10.1016/j.yexcr.2009.03.006
PMCID: PMC3874874  PMID: 19298813
SYTO; Apoptosis; Kinetic assays; Antitumor drugs; Microfluidics; Lab-on-a-Chip; Flow cytometry
12.  Forever young, slim and fit 
Cell cycle (Georgetown, Tex.)  2009;8(12):1820-1821.
PMCID: PMC3875229  PMID: 19471126
13.  Kinetic viability assays using DRAQ7 probe 
Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal There is a need therefore for rapid and high-throughput bioassays capable to continuously track viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative DRAQ7 is gaining increasing interest as an easy to use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect cells susceptibility to the death inducing agents. However when the membrane integrity is compromised DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.
doi:10.1002/0471142956.cy0941s65
PMCID: PMC3873765  PMID: 23835805
14.  Cytometric Assessment of DNA Damage Induced by DNA Topoisomerase Inhibitors 
Methods in molecular biology (Clifton, N.J.)  2009;582:10.1007/978-1-60761-340-4_12.
Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is mediated by one of the protein kinases of the phosphoinositide kinase family, namely ATM, ATR, and/ or DNA-PK. The presence of Ser-139 phosphorylated H2AX (γH2AX) is thus a reporter of DNA damage. This protocol describes quantitative assessment of γH2AX detected immunocytochemically in individual cells combined with quantification of cellular DNA content by cytometry. The bivariate analysis of γH2AX expression versus DNA content allows one to correlate DNA damage with the cell cycle phase or DNA ploidy. The protocol can also be used to assess activation (Ser-1981 phosphorylation) of ATM; this event also revealing DNA damage induced by topo I or topo II inhibitors. Examples where DNA damage was induced by topotecan (topo I) and etoposide (topo II) inhibitors are provided.
doi:10.1007/978-1-60761-340-4_12
PMCID: PMC3873872  PMID: 19763948
Histone H2AX phosphorylation; ataxia telangiectasia mutated; ATM; DNA double-strand breaks; flow cytometry; apoptosis; cell cycle; topotecan; etoposide
15.  Cytometric Analysis of DNA Damage: Phosphorylation of Histone H2AX as a Marker of DNA Double-Strand Breaks (DSBs) 
Methods in molecular biology (Clifton, N.J.)  2009;523:10.1007/978-1-59745-190-1_11.
Phosphorylation of histone H2AX on Ser 139 is a sensitive reporter of DNA damage, particularly if the damage involves induction of DNA double-strand breaks (DSBs). Phosphorylated H2AX has been named γH2AX and its presence in the nucleus can be detected immunocytochemically. Multiparameter analysis of γH2AX immunofluorescence by flow or laser-scanning cytometry allows one to measure extent of DNA damage in individual cells and to correlate it with their position in the cell cycle and induction of apoptosis. This chapter presents the protocols and outlines applications of multiparameter cytometry in analysis of H2AX phosphorylation as a reporter of the presence of DSBs.
doi:10.1007/978-1-59745-190-1_11
PMCID: PMC3872964  PMID: 19381940
γH2AX; H2AX phosphorylation; DNA double-strand breaks; Multiparameter flow cytometry; Laser-scanning cytometry; Immunocytochemistry; Apoptosis
16.  DNA damage response as a biomarker in treatment of leukemias 
Cell cycle (Georgetown, Tex.)  2009;8(11):1720-1724.
Early assessment of cancer response to the treatment is of great importance in clinical oncology. Most antitumor drugs, among them DNA topoisomerase (topo) inhibitors, target nuclear DNA. The aim of the present study was to explore feasibility of the assessment of DNA damage response (DDR) as potential biomarker, eventually related to the clinical response, during treatment of human leukemias. We have measured DDR as reported by activation of ATM through its phosphorylation on Ser 1981 (ATM-S1981P) concurrent with histone H2AX phosphorylation on Ser139 (γH2AX) in leukemic blast cells from the blood of twenty patients, 16 children/adolescents and 4 adults, diagnosed with acute leukemias and treated with topo2 inhibitors doxorubicin, daunomycin, mitoxantrone or idarubicin. Phosphorylation of H2AX and ATM was detected using phospho-specific Abs and measured in individual cells by flow cytometry. The increase in the level of ATM-S1981P and γH2AX, varying in extent between the patients, was observed in blasts from the blood collected one hour after completion of the drug infusion with respect to the pre-treatment level. A modest degree of correlation was observed between the induction of ATM activation and H2AX phosphorylation in blasts of individual patients. The number of the studied patients (20) and the number of the clinically non-responding ones (2) was too low to draw a conclusion whether the assessment of DDR can be clinically prognostic. The present findings, however, demonstrate the feasibility of assessment of DDR during the treatment of leukemias with drugs targeting DNA.
PMCID: PMC3863585  PMID: 19411853
histone H2AX phosphorylation; ATM activation; acute leukemias; apoptosis; DNA topoisomerase II inhibitors; mitoxantrone; doxorubicin; idarubicin; daunorubicin; cytometry
17.  Flow cytometry-based apoptosis detection 
Methods in molecular biology (Clifton, N.J.)  2009;559:10.1007/978-1-60327-017-5_2.
An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. A large variety of cytometric methods designed to identify apoptotic cells and probe mechanisms associated with this mode of cell demise have been developed during the past two decades.
In the present chapter we outline a handful of commonly used methods that are based on the assessment of: mitochondrial transmembrane potential, activation of caspases, plasma membrane alterations and DNA fragmentation.
doi:10.1007/978-1-60327-017-5_2
PMCID: PMC3863590  PMID: 19609746
flow cytometry; apoptosis; single cell analysis; mitochondria; caspases; Annexin V; DNA fragmentation
18.  γH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products 
Mutation research  2009;678(1):10.1016/j.mrgentox.2009.06.009.
Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, itwould be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenicDNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previouslywe showed that phosphorylation of H2AX (defined as γH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that γH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring γH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that γH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that γH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.
doi:10.1016/j.mrgentox.2009.06.009
PMCID: PMC3863591  PMID: 19591958
Tobacco smoke; H2AX; Double strand breaks; DNA damage
19.  Cytometric Assessment of DNA Damage by Exogenous and Endogenous Oxidants Reports Aging-related Processes 
The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty- to 60-min exposure of cells to 100 or 200 μM H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24–48 h under hypoxic conditions (3% O2) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20–30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 μM) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.
doi:10.1002/cyto.a.20469
PMCID: PMC3860741  PMID: 17879239
H2AX phosphorylation; ATM activation; reactive oxygen species; hypoxia; hydrogen peroxide; 3-bromopyruvate; dichloroacetate; celecoxib; aging; caloric restriction; mitochondria
20.  Guarding Genome Integrity in Stem Cells 
Cell cycle (Georgetown, Tex.)  2010;9(12):2271-2272.
PMCID: PMC3856174  PMID: 20581438
21.  Cytometric detection of chromatin relaxation, an early reporter of DNA damage response 
Cell cycle (Georgetown, Tex.)  2009;8(14):2233-2237.
One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H2O2. Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon as 10 min after subjecting cells to UV or H2O2. While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H2O2 exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.
PMCID: PMC3856216  PMID: 19502789
UV light; oxidative DNA damage; H2AX phosphorylation; cell cycle; DNA denaturation; acridine orange; metachromasia; ssDNA; lymphocytes
22.  Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide 
Cancer biology & therapy  2009;8(5):452-457.
Phpsphorylation of histone H2AX is a sensitive marker of DNA damage, particularly of DNA double strand breaks. Using multiparameter cytometry we explored effects of etoposide and temozolomide (TMZ) on three glioblastoma cell lines with different p53 status (A172, T98G, YKG-1) and on normal human astrocytes (NHA) correlating the drug-induced phosphorylated H2AX (γH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced γH2AX in all phases of the cell cycle in all three glioblastoma lines and led to an arrest of T98G and YKG-1 cells in S and G2/M. NHA cells were arrested in G1 with no evidence of γH2AX induction. A172 responded by rise in γH2AX throughout all phases of the cycle, arrest at the late S- to G2/M-phase, and appearance of senescence features: induction of p53, p21WAF1/CIP1, p16INK4A and β-galactosidase, accompanied by morphological changes typical of senescence. T98G cells showed the presence of γH2AX in S phase with no evidence of cell cycle arrest. A modest degree of arrest in G1 was seen in YKG-1 cells with no rise in γH2AX. While frequency of apoptotic cells in all four TMZ-treated cell cultures was relatively low it is conceivable that the cells with extensive DNA damage were reproductively dead. The data show that neither the status of p53 (wild-type vs. mutated, or inhibited by pifithrin-α) nor the expression of O6-methylguanine-DNA methyltransferase significantly affected the cell response to TMZ. Because of diversity in response to TMZ between individual glioblastoma lines our data suggest that with better understanding of the mechanisms, the treatment may have to be customized to individual patients.
PMCID: PMC3855308  PMID: 19305157
glioblastoma; temozolomide; etoposide; DNA double strand break; DNA damage; senescence; cell cycle
23.  Cytometry of ATM Activation and Histone H2AX Phosphorylation to Estimate Extent of DNA Damage Induced by Exogenous Agents 
This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis.
doi:10.1002/cyto.a.20426
PMCID: PMC3855668  PMID: 17622968
ionizing radiation; DNA topoisomerase inhibitors; DNA double-strand breaks; carcinogens; tobacco smoke; replication stress; genotoxins; DNA photolysis
24.  DNA Damage Signaling Assessed in Individual Cells in Relation to the Cell Cycle Phase and Induction of Apoptosis 
Reviewed are the phosphorylation events reporting activation of protein kinases and the key substrates critical for the DNA damage signaling (DDS). These DDS events are detected immunocytochemically using phospho-specific Abs; flow cytometry or image-assisted cytometry provide the means to quantitatively assess them on a cell by cell basis. The multiparameter analysis of the data is used to correlate these events with each other and relate to the cell cycle phase, DNA replication and induction of apoptosis. Expression of γH2AX as a possible marker of induction of DNA double strand breaks is the most widely studied event of DDS. Reviewed are applications of this multiparameter approach to investigate constitutive DDS reporting DNA damage by endogenous oxidants byproducts of oxidative phosphorylation. Also reviewed are its applications to detect and explore mechanisms of DDS induced by variety of exogenous agents targeting DNA such as exogenous oxidants, ionizing radiation, radiomimetic drugs, UV light, DNA topoisomerase I and II inhibitors, DNA crosslinking drugs and variety of environmental genotoxins. Analysis of DDS induced by these agents provides often a wealth of information about mechanism of induction and the type of DNA damage (lesion) and is reviewed in the context of cell cycle phase specificity, DNA replication, and induction of apoptosis or cell senescence. Critically assessed is interpretation of the data as to whether the observed DDS events report induction of a particular type of DNA lesion.
doi:10.3109/10408363.2012.738808
PMCID: PMC3522801  PMID: 23137030
ATM; γH2AX; Chk2; DNA double stand breaks; chromatin relaxation; DNA replication; DNA topoisomerase inhibitors; UV; cytometry; confocal microscopy
25.  Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence 
Cell Cycle  2012;11(16):3132-3140.
Mutations of oncogenes and tumor suppressor genes which activate mTOR through several downstream signaling pathways are common to cancer. Activation of mTOR when combined with inhibition of cell cycle progression or DNA replication stress has previously been shown to promote cell senescence. In the present study, we examined the conditions under which human non-small cell lung carcinoma A549 cells can undergo senescence when treated with the DNA alkylating agent mitomycin C (MMC). While exposure of A549 cells to 0.1 or 0.5 µg/ml of MMC led to their arrest in S phase of the cell cycle and subsequent apoptosis, exposure to 0.01 or 0.02 µg/ml for 6 d resulted in induction of cell senescence and near total (0.01 µg/ml) or total (0.02 µg/ml) elimination of their reproductive potential. During exposure to these low concentrations of MMC, the cells demonstrated evidence of DNA replication stress manifested by expression of γH2AX, p21WAF1 and a very low level of EdU incorporation into DNA. The data are consistent with the notion that enduring DNA replication stress in cells known to have activated oncogenes leads to their senescence. It is reasonable to expect that tumors having constitutive activation of oncogenes triggering mTOR signaling may be particularly predisposed to undergoing senescence following prolonged treatment with low doses of DNA damaging drugs.
doi:10.4161/cc.21506
PMCID: PMC3442923  PMID: 22871735
cell cycle; human non-small cell lung carcinoma; mTOR; metronomic chemotherapy; oncogenes; personalized cancer treatment; senescence; γH2AX

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