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1.  The tail that wags the dog 
Cell Cycle  2013;13(1):23-31.
DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication. Human Pol δ is a heterotetramer whose p12 subunit is degraded in response to DNA damage, leading to the in vivo conversion of Pol δ4 to Pol δ3. Two E3 ubiquitin ligases, RNF8 and CRL4Cdt2, participate in the DNA damage-induced degradation of p12. We discuss how these E3 ligases integrate the formation of Pol δ3 and ubiquitinated PCNA for DNA repair processes. CRL4Cdt2 partially degrades p12 during normal cell cycle progression, thereby generating Pol δ3 during S phase. This novel finding extends the current view of the role of Pol δ3 in DNA repair and leads to the hypothesis that it participates in DNA replication. The coordinated regulation of licensing factors and Pol δ3 by CRL4Cdt2 now opens new avenues for control of DNA replication. A parallel study of Pol δ4 and Pol δ3 in Okazaki fragment processing provides evidence for a role of Pol δ3 in DNA replication. We discuss several new perspectives of the role of the 2 forms of Pol δ in DNA replication and repair, as well the significance of the integration of p12 regulation in DNA repair and cell cycle progression.
PMCID: PMC3925730  PMID: 24300032
CRL4Cdt2; DNA damage; DNA polymerase δ; DNA replication; RNF8; cell cycle; cell cycle progression; p12 subunit
2.  DNA Damage Signaling, Impairment of Cell Cycle Progression, and Apoptosis Triggered by 5-Ethynyl-2′-deoxyuridine Incorporated into DNA 
The “click chemistry” approach utilizing 5-ethynyl-2′-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2, likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.
PMCID: PMC3846616  PMID: 24115313
click chemistry; DNA strand breaks; p53 activation; Chk2 activation; ATM activation; γH2AX foci; p53BP1 foci; caspase-3 activation; laser scanning cytometry; flow cytometry; confocal microscopy; SBIP methodology
3.  PDIP38 is translocated to the spliceosomes/nuclear speckles in response to UV-induced DNA damage and is required for UV-induced alternative splicing of MDM2 
Cell Cycle  2013;12(19):3184-3193.
PDIP38 (polymerase delta interacting protein 38) was originally discovered as a protein that interacts with DNA polymerase δ and PCNA. PDIP38 is present in multiple intracellular locations and is a multifunctional protein that has been implicated in several diverse cellular functions. We investigated the nuclear localization of PDIP38 in order to gain insights to its response to UV damage. PDIP38 was found to form distinct nuclear foci in response to UV irradiation in several cell lines, including HeLa S3 and A549 cells. However, these foci were not those associated with UV repair foci. Using various markers for different nuclear subcompartments, the UV-induced PDIP38 foci were identified as spliceosomes/nuclear speckles, the storage and assembly sites for mRNA splicing factors. To assess the role of PDIP38 in the regulation of splicing events, the effects of PDIP38 depletion on the UV-induced alternate splicing of MDM2 transcripts were examined by nested RT-PCR. Alternatively spliced MDM2 products were induced by UV treatment but were greatly reduced in cells expressing shRNA targeting PDIP38. These findings indicate that upon UV-induced DNA damage, PDIP38 is translocated to spliceosomes and contributes to the UV-induced alternative splicing of MDM2 transcripts. Similar results were obtained when cells were subjected to transcriptional stresses with actinomycin D or α-amanitin. Taken together, these studies show that PDIP38 is a protein regulated in a dynamic manner in response to genotoxic stress, as evidenced by its translocation to the spliceosomes. Moreover, PDIP38 is required for the induction of the alternative splicing of MDM2 in response to UV irradiation.
PMCID: PMC3865014  PMID: 23989611
DNA damage; DNA polymerase δ; MDM2; PDIP38; Poldip2; alternative splicing; nuclear speckles; spliceosomes
4.  New insights into cell cycle and DNA damage response machineries through the high resolution AMICO quantitative imaging cytometry 
Cell proliferation  2013;46(5):497-500.
Progress in biology and medicine research is being driven by development of new instrumentation and associated methodologies which open analytical capabilities that expand understanding of complexity of biological systems. Application of cytometry, which is now widely used in so many disciplines of biology, is the best example of such a progress. In the recent publications in the journal Cytometry A Furia et al., from the European Institute of Oncology in Milano, Italy, push the envelope in expanding capabilities of cytometry by introducing a high resolution imaging cytometry defined by them as Automated Microscopy for Image CytOmetry (AMICO). They utilize this approach to further elucidate mechanisms of the cell cycle progression and also the DNA damage response. This approach is going beyond the presently possible analytical technologies regarding throughput and depth of information. The possibility of multiparametric analysis combined with the high resolution mapping of individual constituents of cell cycle and DNA damage response machineries provides new tools to probe molecular mechanism of these processes. The capability of analysis of proximity of these constituents to each other offered by AMICO is a novel and potentially important approach that can be used to elucidate mechanisms of other biological processes,
PMCID: PMC3788027  PMID: 23952744
5.  Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes 
Nucleic Acids Research  2014;42(16):10245-10264.
Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.
PMCID: PMC4176373  PMID: 25150148
6.  Pre-Clinical Evaluation of 1-Nitroacridine Derived Chemotherapeutic Agent that has Preferential Cytotoxic Activity Towards Prostate Cancer 
Cancer biology & therapy  2007;6(10):1632-1637.
Chemotherapy in prostate cancer (CaP) even as an adjunct has not been a success. In this communication, we report the pre-clinical efficacy of a nitroacridine derivative, C-1748 (9[2′-hydroxyethylamino]-4-methyl-1-nitroacridine) in CaP cell culture and human xenograft animal models. C-1748, a DNA intercalating agent has been derived from its precursor C-857 that was a potent anti-cancer drug, but failed clinical development “methyl” group imparted novel properties, the most interesting of which is the difference in the IC50 values between LnCaP (22.5 nM), a CaP cell line and HL-60, a leukemia cell line (>100 nM). Using γH2AX as an intervention marker of DNA double strand breaks, we concluded that C-1748 is more efficacious in CaP cells than in HL-60 cells. In hormone dependent cells, the androgen receptor (AR) was identified as an additional target of C-1748. In xenograft studies, administration of C-1748 intra-peritoneally inhibited tumor growth by 80–90% with minimal toxicity. These studies identify C-1748 as a novel acridine drug that has a high therapeutic index and low cytotoxicity on myelocytic cells with potential for clinical development.
PMCID: PMC4134887  PMID: 17921700
chemotherapy; xenografts; 1-nitroacridine derivative; systemic treatment; DNA double strand breaks; γH2AX
7.  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.
PMCID: PMC3671577  PMID: 23404939
extracellular matrix; collagen; elastin; fluorescence microscopy; live cell imaging; confocal microscopy
8.  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.
PMCID: PMC3977701  PMID: 19572560
9.  Disruption of mutated BRAF signaling modulates thyroid cancer phenotype 
BMC Research Notes  2014;7:187.
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.
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.
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.
These findings support further evaluation of combinational therapy that includes BRAFV600E inhibitors in thyroid cancer patients harboring the BRAFV600E mutation.
PMCID: PMC3976539  PMID: 24673746
Thyroid cancer; BRAFV600E mutation; PLX4032; MAPK signal transduction pathway; Targeted therapy; Kinase inhibitors
10.  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.
PMCID: PMC3558543  PMID: 23165976
DRAQ7; real-time assays; cell viability; drug; cytotoxicity; DNA damage response; cell cycle; microfluidic; cytometry
11.  Laser Scanning Cytometry 
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.
PMCID: PMC3892962  PMID: 16719355
Cytometry; fluorescence; cell cycle; apoptosis; nucleus; nucleolus; micronucleus cytoplasm; enzyme kinetics
12.  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.
PMCID: PMC3488462  PMID: 23027005
Cytometry; Fluorescence; Cell cycle; Apoptosis; Nucleus; Nucleolus; Micronucleus; Cytoplasm; Enzyme kinetics
13.  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.
PMCID: PMC3541526  PMID: 23296652
Cell aging; chromatin structure; cell cycle; apoptosis; nuclear size; laser scanning cytometry; p21WAF1; p16INK4a; p27KIP1; premature cell senescence
14.  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.
PMCID: PMC3877312  PMID: 24386542
microfluidics; Lab-on-a-Chip; flow cytometry; cell cycle; apoptosis; programmed cell death; cancer
15.  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
16.  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.
PMCID: PMC3874874  PMID: 19298813
SYTO; Apoptosis; Kinetic assays; Antitumor drugs; Microfluidics; Lab-on-a-Chip; Flow cytometry
18.  Forever young, slim and fit 
Cell cycle (Georgetown, Tex.)  2009;8(12):1820-1821.
PMCID: PMC3875229  PMID: 19471126
19.  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.
PMCID: PMC3873765  PMID: 23835805
20.  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.
PMCID: PMC3873872  PMID: 19763948
Histone H2AX phosphorylation; ataxia telangiectasia mutated; ATM; DNA double-strand breaks; flow cytometry; apoptosis; cell cycle; topotecan; etoposide
21.  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.
PMCID: PMC3872964  PMID: 19381940
γH2AX; H2AX phosphorylation; DNA double-strand breaks; Multiparameter flow cytometry; Laser-scanning cytometry; Immunocytochemistry; Apoptosis
22.  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
23.  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.
PMCID: PMC3863590  PMID: 19609746
flow cytometry; apoptosis; single cell analysis; mitochondria; caspases; Annexin V; DNA fragmentation
24.  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.
PMCID: PMC3863591  PMID: 19591958
Tobacco smoke; H2AX; Double strand breaks; DNA damage
25.  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.
PMCID: PMC3860741  PMID: 17879239
H2AX phosphorylation; ATM activation; reactive oxygen species; hypoxia; hydrogen peroxide; 3-bromopyruvate; dichloroacetate; celecoxib; aging; caloric restriction; mitochondria

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