Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence.

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).

Attenuation of constitutive DNA damage signaling by 1,25-dihydroxyvitamin D3.

In addition to its traditional role in the regulation of calcium homeostasis and bone metabolism, vitamin D also exhibits immunomodulatory, anti-proliferative and cancer preventive activities. Molecular mechanisms that confer the chemo-preventive properties to vitamin D are poorly understood. We previously reported that constitutive phosphorylation of histone H2AX on Ser139 (γH2AX) and activation of ATM (Ser1981 phosphorylation), seen in untreated normal or tumor cells predominantly in S phase of the cell cycle, is to a large extent indicative of DNA replication stress occurring as a result of persistent DNA damage caused by endogenous oxidants, by-products of oxidative metabolism.

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).

Genome protective effect of metformin as revealed by reduced level of constitutive DNA damage signaling.

We have shown before that constitutive DNA damage signaling represented by H2AX-Ser139 phosphorylation and ATM activation in untreated normal and tumor cells is a reporter of the persistent DNA replication stress induced by endogenous oxidants, the by-products of aerobic respiration. In the present study we observed that exposure of normal mitogenically stimulated lymphocytes or tumor cell lines A549, TK6 and A431 to metformin, the specific activator of 5'AMP-activated protein kinase (AMPK) and an inhibitor of mTOR signaling, resulted in attenuation of constitutive H2AX phosphorylation and ATM activation. The effects were metformin-concentration dependent and seen even at the pharmacologically pertinent 0.

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.

Analysis of individual molecular events of DNA damage response by flow- and image-assisted cytometry.

This chapter describes molecular mechanisms of DNA damage response (DDR) and presents flow- and image-assisted cytometric approaches to assess these mechanisms and measure the extent of DDR in individual cells. DNA damage was induced by cell treatment with oxidizing agents, UV light, DNA topoisomerase I or II inhibitors, cisplatin, tobacco smoke, and by exogenous and endogenous oxidants. Chromatin relaxation (decondensation) is an early event of DDR chromatin that involves modification of high mobility group proteins (HMGs) and histone H1 and was detected by cytometry by analysis of the susceptibility of DNA in situ to denaturation using the metachromatic fluorochrome acridine orange.

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.

Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on "click chemistry".

This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of (3) H- or (14) C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G(1) , S, G(2) , and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of (3) H-uridine incorporation and RNA content provided the means to distinguish quiescent G(0) from cycling G(1) cells.

New biomarkers probing depth of cell senescence assessed by laser scanning cytometry.

The imaging analytical capabilities of laser scanning cytometer (LSC) have been used to assess morphological features considered to be typical of the senescent phenotype. The characteristic "flattening" of senescent cells was reflected by the decline in the 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). The decrease in ratio of maximal pixel to nuclear area was even more sensitive senescence biomarker than the change in maximal pixel or nuclear area, each alone.

DNA damage response induced by exposure of human lung adenocarcinoma cells to smoke from tobacco- and nicotine-free cigarettes.

Cigarette smoke (CS) is the major cause of lung cancer and contributes to the development of other malignancies. Attempts have been made to construct reduced toxicity cigarettes, presumed to have diminished genotoxic potential. One such product on the market is the tobacco and nicotine free (T&N-free) cigarette type made from lettuce and herbal extracts.

DNA damage detected with gammaH2AX in endometrioid adenocarcinoma cell lines.

Phosphorylation of histone H2AX (gammaH2AX) is a sensitive marker of DNA damage, particularly induction of DNA double-strand breaks. Using multiparameter cytometry we explored the effects of doxorubicin (DOX), cisplatin (CDDP) and 5-fluorouracil (5-FU) on four types of endometrioid adenocarcinoma cell lines (HEC-1A, HEC-1B, Ishikawa, KLE) correlating the drug-induced increases in phosphorylated H2AX (gammaH2AX) with cell cycle phase, induction of apoptosis and induction of cell senescence, the latter detected by analysis of beta-galactosidase. The study revealed significant differences among the cell lines in the effects of DNA damage vis-a-vis cell cycle phase specificity, induction of apoptosis or senescence following drug treatment.

DNA damage signaling is activated during cancer progression in human colorectal carcinoma.

Purpose: Recent studies have shown that the DNA damage response (DDR) is activated in precancerous lesions, suggesting that neoplastic cells may avoid apoptosis by impairing the DDR which acts as a barrier against tumor progression. To define the role of the DDR pathway in human colorectal carcinoma, we investigated the level of phosphorylated proteins of the DDR pathway.

Results: Immunostaining for pATM, gammaH2AX and pChk2 revealed that all were significantly expressed during tumor progression in advanced carcinoma (vs.

Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication.

It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of gammaH2AX not only in S- but also in G(1)-phase cells. In the present study, in addition to gammaH2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 J/m(2) of UV-B. Phosphorylation of these proteins detected with phospho-specific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to S-phase cells.

Impaired DNA damage response--an Achilles' heel sensitizing cancer to chemotherapy and radiotherapy.

Despite the progress in targeting particular molecular abnormalities specific to different cancers (targeted therapy), chemo- and radiotherapies are still the most effective of all anticancer modalities. Induction of DNA damage and inhibition of cell proliferation are the objects of most chemotherapeutic agents and radiation. Their effectiveness was initially thought to be due to the high rate of proliferation of cancer cells.

Fluoroquinolones lower constitutive H2AX and ATM phosphorylation in TK6 lymphoblastoid cells via modulation of the intracellular redox status.

Accumulation of reactive oxygen species (ROS)-induced damage and mutations in the genomic DNA is considered the primary etiology of aging and age-related pathologies including cancer. Strategies aimed at slowing these conditions often involve protecting against oxidative DNA damage via modulation of the intracellular redox state. Recently, a biomarker of DNA double-strand breaks (DSBs), serine 139-phosphorylated histone H2AX (gammaH2AX), and its upstream mediator, activated PI-3-related kinase, ATM (ATM(P1981)), were shown to be constitutively expressed in cells and modulated by antioxidant treatment.

DNA damage response induced by tobacco smoke in normal human bronchial epithelial and A549 pulmonary adenocarcinoma cells assessed by laser scanning cytometry.

Cigarette smoke (CS) is a major cause of lung cancer and a contributor to the development of a wide range of other malignancies. There is an acute need to develop a methodology that can rapidly assess the potential carcinogenic properties of the genotoxic agents present in CS. We recently reported that exposure of normal human bronchial epithelial cells (NHBEs) or A549 pulmonary carcinoma cells to CS induces the activation of ATM through its phosphorylation on Ser1981 and phosphorylation of histone H2AX on Ser139 (gammaH2AX) most likely in response to the formation of potentially carcinogenic DNA double-strand breaks (DSBs).

gammaH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products.

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.

Cytometric detection of chromatin relaxation, an early reporter of DNA damage response.

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 damage response as a biomarker in treatment of leukemias.

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.

Cytometric analysis of DNA damage: phosphorylation of histone H2AX as a marker of DNA double-strand breaks (DSBs).

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 gammaH2AX and its presence in the nucleus can be detected immunocytochemically. Multiparameter analysis of gammaH2AX 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.

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.

Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide.

Phosphorylation 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 (gammaH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced gammaH2AX 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 G(2)/M.

Phosphorylation of p53 on Ser15 during cell cycle caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation.

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15(P)) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT-induced p53-Ser15(P) with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase.

Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase.

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitors doxorubicin, etoposide and mitoxantrone (MXT) are widely used antitumor drugs. They stabilize otherwise transient ("cleavable") complexes of topo1 or topo2 with DNA, respectively. Collisions of DNA replication forks (during replication) or progressing RNA polymerase molecules (during transcription) with these complexes convert them into double-strand DNA breaks (DSBs).

Assessment of DNA double-strand breaks and gammaH2AX induced by the topoisomerase II poisons etoposide and mitoxantrone.

Double-strand breaks (DSBs) are highly deleterious DNA lesions as they lead to chromosome aberrations and/or apoptosis. The formation of nuclear DSBs triggers phosphorylation of histone H2AX on Ser-139 (defined as gammaH2AX), which participates in the repair of such DNA damage. Our aim was to compare the induction of gammaH2AX in relation to DSBs induced by topoisomerase II (TOPO II) poisons, etoposide (ETOP) and mitoxantrone (MXT), in V79 cells.