Dominant-Negative Form of SIGIRR: SIGIRRΔE8 Promotes Tumor Growth Through Regulation of Metabolic Pathways.

Colorectal carcinoma is the leading cause of cancer-related death. Previously we have shown that tumor suppressor single immunoglobulin interleukin-1-related receptor (SIGIRR) is frequently inactivated in human colorectal cancer by the increased expression of a novel SIGIRR isoform (SIGIRRΔE8). SIGIRRΔE8 showed increased retention in the cytoplasm and loss of complex glycan modification compared to the full-length SIGIRR. Now we found that the arginine residues located in the C-terminus of SIGIRRΔE8 serve as an endoplasmic reticulum retention signal and are required for resident protein ribophorin 1 (RPN1) interaction. In addition, we found that SIGIRRΔE8 exerts a direct impact on cell metabolism through interaction with the adenosine triphosphate synthase in the colorectal cancer cells. SIGIRRΔE8 expression promoted the metabolic shift through upregulation of mammalian target of rapamycin signaling pathway and dysregulation of mitochondrial function to promote survival and proliferation of colon cancer cells in xenograft model.

MCPIP1 inhibits Wnt/ß-catenin signaling pathway activity and modulates epithelial-mesenchymal transition during clear cell renal cell carcinoma progression by targeting miRNAs.

Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active ß-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/ß-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors (SFRP4, KREMEN1, CXXC4, CSNK1A1 and ZNFR3). Thus, the level of active nuclear ß-catenin was increased, leading to increased levels of EMT inducers (SNAI1, SNAI2, ZEB1 and TWIST) and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active ß-catenin and EMT inducers.

Fatty Acids and a High-Fat Diet Induce Epithelial-Mesenchymal Transition by Activating TGFß and ß-Catenin in Liver Cells.

Nonalcoholic fatty liver disease is defined as the accumulation of excessive fat in the liver in the absence of excessive alcohol consumption or any secondary cause. Although the disease generally remains asymptomatic, chronic liver inflammation leads to fibrosis, liver cirrhosis, and even to the development of hepatocellular carcinoma (HCC). Fibrosis results from epithelial-mesenchymal transition (EMT), which leads to dedifferentiation of epithelial cells into cells with a mesenchymal-like phenotype. During EMT, epithelial cells with high expression of E-cadherin, influenced by growth factors, cytokines, and inflammatory processes, undergo morphological changes via enhanced expression of, e.g., vimentin, fibronectin, and N-cadherin. An inducer of EMT and, consequently, of fibrosis development is transforming growth factor beta (TGFß), a pleiotropic cytokine associated with the progression of hepatocarcinogenesis. However, the understanding of the molecular events that direct the development of steatosis into steatohepatitis and liver fibrosis remains incomplete. Our study revealed that both prolonged exposure of hepatocarcinoma cells to fatty acids in vitro and high-fat diet in mice (20 weeks) result in inflammation. Prolonged treatment with fatty acids increased the levels of TGFß, MMP9, and ß-catenin, important EMT inducers. Moreover, the livers of mice fed a high-fat diet exhibited features of liver fibrosis with increased TGFß and IL-1 levels. Increased expression of IL-1 correlated with a decrease in monocyte chemoattractant protein-induced protein 1 (MCPIP1), a negative regulator of the inflammatory response that regulates the stability of proinflammatory transcripts encoding IL-1. Our study showed that a high-fat diet induced EMT by increasing the levels of EMT-activating transcription factors, including Zeb1, Zeb2, and Snail and changed the protein profile to a profile characteristic of the mesenchymal phenotype.

PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP-ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and -2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1-contaning foci constitute newly recognized PML-like nuclear bodies that accrete and locally deliver essential factors for repair of single-strand DNA breaks in replication regions.-Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann-Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

Aqueous mounting media increasing tissue translucence improve image quality in Structured Illumination Microscopy of thick biological specimen.

Structured Illumination Microscopy (SIM) is a super-resolution microscopy method that has significantly advanced studies of cellular structures. It relies on projection of illumination patterns onto a fluorescently labelled biological sample. The information derived from the sample is then shifted to a detectable band, and in the process of image calculation in Fourier space the resolution is doubled. Refractive index homogeneity along the optical path is crucial to maintain a highly modulated illumination pattern necessary for high-quality SIM. This applies in particular to thick samples consisting of large cells and tissues. Surprisingly, sample mounting media for SIM have not undergone a significant evolution for almost a decade. Through identification and systematic evaluation of a number of non-hazardous, water-soluble chemical components of mounting media, we demonstrate an unprecedented improvement in SIM-image quality. Mounting solutions presented in this research are capable of reducing abundant light scattering which constitutes the limiting factor in 3D-SIM imaging of large Hodgkin's lymphoma and embryonic stem cells as well as 10 µm tissue sections. Moreover, we demonstrate usefulness of some of the media in single molecule localisation microscopy. The results presented here are of importance for standardisation of 3D-SIM data acquisition pipelines for an expanding community of users.

Changes in lipid membrane mechanics induced by di- and tri-phenyltins.

Organotin compounds, being biologically active, affect a variety of cellular functions due to their ability to accumulate in and penetrate biological membranes. These compounds influence the distribution of electrostatic charges, alter organization, disrupt molecular dynamics and change mechanical properties of biological membranes. It was found that the membrane/water partition coefficient equals 4, a value significantly higher than octanol/water partition coefficient. In addition, the effect of di- and tri-phenyltin chlorides on the mechanics of model lipid membranes was measured for the first time. It has been determined that phenyltins affect the global model lipid bilayer properties by reducing the membrane expansion modulus, when measured using micromanipulation technique, and elevating the bending rigidity coefficient of the lipid bilayer, as determined with the flickering noise spectroscopy. In addition, the elevated water permeability shows that phenyltins also cause the local defects formation in the lipid bilayer, i.e. lipid pores. These data shows that phenyltins may interfere indirectly with variety cellular processes by altering non-specifically the entire cellular membrane system. Accordingly, when phenyltins are added to macrophages in culture, they inflict massive alterations of cell morphology and interfere with membrane-associated processes, as visualized using fluorescence labelling of selected subcellular compartments.

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations.

Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions.

Two stages of XRCC1 recruitment and two classes of XRCC1 foci formed in response to low level DNA damage induced by visible light, or stress triggered by heat shock.

Induction of local photosensitised DNA damage has been used to study recruitment of repair factors, spatial organisation and subsequent stages of the repair processes. However, the damage induced by a focused laser beam interacting with a photosensitiser may not fully reflect the types of damage and repair encountered in cells of an animal under typical conditions in vivo. We report on two characteristic stages of recruitment of XRCC1 (a protein engaged in BER and SSB repair pathways), in response to low level DNA damage induced by visible light. We demonstrate that, when just a few DNA breaks are induced in a small region of the nucleus, the recruited XRCC1 is initially distributed uniformly throughout this region, and rearranges into several small stationary foci within minutes. In contrast, when heavy damage of various types (including oxidative damage) is induced in cells pre-sensitized with a DNA-binding drug ethidium bromide, XRCC1 is also recruited but fails to rearrange from the stage of the uniform distribution to the stage of several small foci, indicating that this heavy damage interferes with the progress and completion of the repair processes. We hypothesize that that first stage may reflect recruitment of XRCC1 to poly(ADP-ribose) moieties in the region surrounding the single-strand break, while the second-binding directly to the DNA lesions. We also show that moderate damage or stress induces formation of two types of XRCC1-containing foci differing in their mobility. A large subset of DNA damage-induced XRCC1 foci is associated with a major component of PML nuclear bodies--the Sp100 protein.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells.

BACKGROUND: Tumour cells release membrane micro(nano)fragments called tumour-derived microvesicles (TMV) that are believed to play an important role in cancer progression. TMV suppress/modify antitumour response of the host, but there is also some evidence for their direct interaction with cancer cells. In cancer patients TMV are present in body fluid and tumour microenvironment. The present study aimed at characterization of whole types/subpopulations, but not only exosomes, of TMV from newly established gastric cancer cell line (called GC1415) and to define their interactions with autologous cells. METHODS: TMV were isolated from cell cultures supernatants by centrifugation at 50,000×g and their phenotype was determined by flow cytometry. The size of TMV was analysed by dynamic light scattering and nanoparticle tracking analysis, while morphology by transmission electron microscopy and atomic force microscopy. Interactions of TMV with cancer cells were visualized using fluorescence-activated cell sorter, confocal and atomic force microscopy, biological effects by xenografts in NOD SCID mice. RESULTS: Isolated TMV showed expression of CD44H, CD44v6 (hyaluronian receptors), CCR6 (chemokine receptor) and HER-2/neu molecules, exhibited different shapes and sizes (range 60-900 nm, highest frequency of particles with size range of 80-120 nm). TMV attached to autologous cancer cells within 2 h and then were internalized by them at 24 h. CD44H, CD44v6 and CCR6 molecules may play a role in attachment of TMV to cancer cells, while HER-2 associated with CD24 be involved in promoting cancer cells growth. Pre-exposure of cancer cells to TMV resulted in enhancement of tumour growth and cancer cell-induced angiogenesis in NOD SCID mice model. CONCLUSIONS: TMV interact directly with cancer cells serving as macro-messengers and molecular cargo transfer between gastric cancer cells resulting in enhancement of tumour growth. TMV should be considered in future as target of anticancer therapy.

Different rates of DNA replication at early versus late S-phase sections: multiscale modeling of stochastic events related to DNA content/EdU (5-ethynyl-2'deoxyuridine) incorporation distributions.

Mathematical modeling allows relating molecular events to single-cell characteristics assessed by multiparameter cytometry. In the present study we labeled newly synthesized DNA in A549 human lung carcinoma cells with 15-120 min pulses of EdU. All DNA was stained with DAPI and cellular fluorescence was measured by laser scanning cytometry. The frequency of cells in the ascending (left) side of the "horseshoe"-shaped EdU/DAPI bivariate distributions reports the rate of DNA replication at the time of entrance to S phase while their frequency in the descending (right) side is a marker of DNA replication rate at the time of transition from S to G2 phase. To understand the connection between molecular-scale events and scatterplot asymmetry, we developed a multiscale stochastic model, which simulates DNA replication and cell cycle progression of individual cells and produces in silico EdU/DAPI scatterplots. For each S-phase cell the time points at which replication origins are fired are modeled by a non-homogeneous Poisson Process (NHPP). Shifted gamma distributions are assumed for durations of cell cycle phases (G1, S and G2 M), Depending on the rate of DNA synthesis being an increasing or decreasing function, simulated EdU/DAPI bivariate graphs show predominance of cells in left (early-S) or right (late-S) side of the horseshoe distribution. Assuming NHPP rate estimated from independent experiments, simulated EdU/DAPI graphs are nearly indistinguishable from those experimentally observed. This finding proves consistency between the S-phase DNA-replication rate based on molecular-scale analyses, and cell population kinetics ascertained from EdU/DAPI scatterplots and demonstrates that DNA replication rate at entrance to S is relatively slow compared with its rather abrupt termination during S to G2 transition. Our approach opens a possibility of similar modeling to study the effect of anticancer drugs on DNA replication/cell cycle progression and also to quantify other kinetic events that can be measured during S-phase.

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.

Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions.

Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25-250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

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 noninvasive 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 nontoxic to a panel of cancer cell lines grown continuously for up to 72 h and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. The 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 anticancer 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 (ΔΨ(m) ) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multiparameter and kinetic fingerprint of anticancer drug action.

Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide.

DNA topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors are widely used to treat a variety of cancers. Their mechanism of action involves stabilization of otherwise transient ("cleavable") complexes between Top1 or Top2 and DNA; collisions of DNA replication forks with such stabilized complexes lead to formation of DNA double-strand breaks (DSBs). In this study, using 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor, we directly assessed the relationship between DNA replication and induction of DSBs revealed as γH2AX foci in A549 cells treated with Top1 inhibitors topotecan (Tpt) or camptothecin (Cpt) and Top2 inhibitors mitoxantrone (Mxt) and etoposide (Etp). Analysis of cells by multiparameter laser scanning cytometry following treatment with Tpt or Cpt revealed that only DNA replicating cells showed induction of γH2AX and a strong correlation between DNA replication and formation of DSBs (r = 0.86). In cells treated with Mxt or Etp, the correlation was weaker (r = 0.52 and 0.64). In addition, both Mtx and Etp caused induction of γH2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of γH2AX foci predominantly in DNA replicating cells and close association and co-localization of γH2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the γH2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of γH2AX foci and DNA replication sites was also apparent. The data are consistent with the view that collision of DNA replication forks with cleavable Top1-DNA complexes stabilized by Tpt/Cpt is the sole cause of induction of DSBs. Additional mechanisms such as involvement of transcription and/or generation of oxidative stress may contribute to DSBs induction by Mxt and Etp. The confocal analysis of the association between DNA replication sites and the sites of DSBs (γH2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage.

Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry".

Induction of DNA damage by oxidants such as H(2) O(2) activates the complex network of DNA damage response (DDR) pathways present in cells to initiate DNA repair, halt cell cycle progression, and prepare an apoptotic reaction. We have previously reported that activation of Ataxia Telangiectasia Mutated protein kinase (ATM) and induction of γH2AX are among the early events of the DDR induced by exposure of cells to H(2) O(2) , and in human pulmonary carcinoma A549 cells, both events were expressed predominantly during S-phase. This study was designed to further explore a correlation between these events and DNA replication. Toward this end, we utilized 5-ethynyl-2'deoxyuridine (EdU) and the "click chemistry" approach to label DNA during replication, followed by exposure of A549 cells to H(2) O(2) . Multiparameter laser scanning cytometric analysis of these cells made it possible to identify DNA replicating cells and directly correlate H(2) O(2) -induced ATM activation and induction of γH2AX with DNA replication on a cell by cell basis. After pulse-labeling with EdU and exposure to H(2) O(2) , confocal microscopy was also used to examine the localization of DNA replication sites ("replication factories") versus the H2AX phosphorylation sites (γH2AX foci) in nuclear chromatin in an attempt to observe the absence or presence of colocalization. The data indicate a close association between DNA replication and H2AX phosphorylation in A549 cells, suggesting that these DNA damage response events may be triggered by stalled replication forks and perhaps also by induction of DNA double-strand breaks at the primary DNA lesions induced by H(2) O(2) .

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

Rationale for the real-time and dynamic cell death assays using propidium iodide.

We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. This study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long-term proliferation capacity, DNA damage response, and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and laser scanning cytometry. Exposure of human carcinomic alveolar basal epithelial A549 cells in cultures to 1.5 or 7.5 microM of PI for 24 h had minimal effect on cell cycle progression including DNA replication as measured by incorporation of 5'-ethynyl-2-deoxyuridine (EdU) detected by the "click chemistry" approach and measured by laser scanning cytometry. A modest reduction, from 44 to 40% or 33%, in frequency of DNA replicating cells was seen after 48 h at 1.5 or 7.5 microM concentration of PI. There was no evidence of increased phosphorylation of histone gammaH2AX in cells growing in the presence of 1.5 or 7.5 microM of PI for up to 48 h. Confocal image analysis of HeLa and NIH 3T3 mouse embryonic fibroblasts growing in the presence of PI showed granular distribution in cell cytoplasm suggesting PI accumulation in endosomes and progressive increase in fluorescence of nucleoli reflecting PI binding to nucleolar RNA. The overall responses of cells to cytotoxic agents were also not affected by the growth in the presence PI. Our data lend further support to the notion that PI can be effectively used in real-time, kinetic viability assays.

Recruitment of heterochromatin protein 1 to DNA repair sites.

Heterochromatin protein 1 (HP1) was originally identified as a constitutive component of heterochromatin. However it is recognized now that it plays an important role in a number of dynamic processes in the cell nucleus, including transcriptional repression and regulation of euchromatic genes. Recent reports demonstrate that HP1 may be involved in the DNA damage response. Two seemingly contradictory phenomena have been observed-HP1 detachment from chromatin and HP1 recruitment to damaged DNA foci. Based on quantitative FRAP and FLIP studies carefully designed to minimize phototoxicity, we demonstrate that HP1 is recruited to the damaged regions in hetero- as well as euchromatin within a few minutes after damage.

Induction of DNA damage response by the supravital probes of nucleic acids.

The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G(2) progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2, and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G(2)M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis.

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 microM 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 microM) 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.