Analysis Of Cellular DNA Content Research Articles

Simple, fast, cost-efficient, reliable, and highly automated DNA content analysis of cells in adherent cultures.

The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double-stranded DNA. This is a laborious and time-consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample-processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well-proven commercial tool-The BD Cycletest™ Plus DNA Reagent Kit-primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.

Testicular Toxicity of Chloroxylenol in Rats: Biochemical, Pathological and Flow Cytometric Study.

BackgroundChloroxylenol (para-chloro-meta-xylenol, PCMX) is claimed to be highly harmful both to humans and the environment. Toxic effects of PCMX on testicular functions are scarcely discussed in the literature.Aim of StudyTo study testicular toxic effects of PCMX on male Sprague-Dawley rats.Materials and MethodsForty animals were randomly distributed into three groups: negative control (G I), vehicle group (G II) and PCMX group (G III). PCMX group was subdivided into three subgroups: GIIIa: received PCMX 100 mg/kg, GIIIb: received PCMX 200 mg/kg and G IIIc: received PCMX 500 mg/kg. Hormonal assay included assessment of serum testosterone and estradiol levels. Histopathological examination of testicular tissue, analysis of cellular viability, necrosis and apoptosis in testicular tissue by flow cytometry, analysis of cellular DNA content and phases of cell cycle analysis by flow cytometry were also performed.ResultsRats in the groups exposed to PCMX (G IIIa, G IIIb and G IIIc) had significantly lower estradiol and testosterone levels in comparison to control groups (G I and GII). Histopathological examination of testicular tissue of PCMX-exposed rats showed irregular crossly sectioned seminiferous tubules with their lumina containing scanty spermatids and spermatozoa. G IIIc animals showed eosinophilic proteinaceous material and vacuolated and necrotic interstitial cells of Leydig. Rats in PCMX-exposed groups (G IIIa, G IIIb and G IIIc) showed significantly lower testicular tissue viability in comparison to control groups (G I and G II). Rats in PCMX-exposed groups (G IIIa, G IIIb and G IIIc) showed significantly lower percentage of cells in the G0/G1 phase in comparison to control groups (G I and G II).ConclusionRats exposed to PCMX had significant reduction in testosterone and estradiol levels with marked histopathological alterations affecting testicular tissues. These effects are dose-dependent.

Assaying Cell Cycle Status Using Flow Cytometry.

In this chapter, four methods are described that can be used to assess cell cycle status in flow cytometry. The first method is based on the simultaneous analysis of cellular DNA content using a fluorescent DNA dye (propidium iodide) and of a nuclear proliferation marker (Ki-67). The second is based on the differential staining of DNA and RNA using Hoechst 33342 and Pyronin Y: this method is particularly useful to distinguish quiescent cells in G0 phase from G1 cells. Finally, two methods are described based on DNA incorporation of the synthetic nucleosides BrdU and EdU.

Analysis of Cellular DNA Content by Flow Cytometry.

Cellular DNA content can be measured by flow cytometry with the aim of: (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

Analysis of Cellular DNA Content by Flow Cytometry.

Cellular DNA content can be measured by flow cytometry with the aim of: (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

The long non-coding RNA BC200 (BCYRN1) is critical for cancer cell survival and proliferation

BackgroundBC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized.MethodsDetailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometryResultsBC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest.ConclusionsBC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.

Inhibitory and apoptosis-inducing effects of Newcastle disease virus strain AF2240 on mammary carcinoma cell line.

Breast cancer is the malignant tumour that developed from cells of the breast and is the first leading cause of cancer death among women worldwide. Surgery, radiotherapy, and chemotherapy are the available treatments for breast cancer, but these were reported to have side effects. Newcastle disease virus (NDV) known as Avian paramyxovirus type-1 (APMV1) belongs to the genus Avulavirus in a family Paramyxoviridae. NDV is shown to be a promising anticancer agent, killing tumour cells while sparing normal cells unharmed. In this study, the oncolytic and cytotoxic activities of NDV AF2240 strain were evaluated on MDA-MB-231, human mammary carcinoma cell line, using MTT assay, and its inhibitory effects were further studied using proliferation and migration assays. Morphological and apoptotic-inducing effects of NDV on MD-MB-231 cells were observed using phase contrast and fluorescence microscopes. Detection of DNA fragmentation was done following terminal deoxyribonucleotide transferase-mediated Br-dUTP nick end labeling staining (TUNEL) assay, which confirmed that the mode of death was through apoptosis and was quantified by flow cytometry. Furthermore, analysis of cellular DNA content demonstrated that the virus caused an increase in the sub-G1 phase (apoptotic peak) of the cell cycle. It appears that NDV AF2240 strain is a potent anticancer agent that induced apoptosis in time-dependent manner.

Cell cycle synchronization and flow cytometry analysis of mammalian cells.

Analysis of cellular DNA content and measurement of pulse-labeled newly replicated DNA by flow cytometry are useful techniques for cell cycle studies. In this chapter, we describe the protocols for cell cycle synchronization of mammalian cells, including time course designs and consideration of cell types to achieve successful experiments, along with the methods for detection of DNA. Some selected applications dealing with siRNA-mediated knockdown are also presented.

Aromatic Bis-N-hydroxyguanidinium Derivatives: Synthesis, Biophysical, and Biochemical Evaluations

In this paper we report the synthesis of a new family of hydroxyguanidinium aromatic derivatives (4a-g) as potential minor groove binders and cytotoxic agents. Their DNA affinity was evaluated by thermal denaturation experiments using salmon sperm DNA. The antiproliferative effects of derivatives 4a, 4d, and 4f were evaluated in human promyelocytic HL-60, breast carcinoma MCF-7, and neuroblastoma Kelly cell lines using the AlamarBlue viability assay, and IC(50) values were obtained. All three compounds were active in the HL-60 cell line. In particular, 4b exhibits antiproliferative effects in all three cell lines while 4d reduced HL-60 and Kelly viability. Both 4b and 4d produced considerable antiproliferative activity in the Kelly cell line. Derivative 4d was chosen for further cell cycle and apoptosis studies using flow cytometric analysis of cellular DNA content.

Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol

Earlier studies showed that 2-methoxyestradiol (2ME2), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G2/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME2-induced cell cycle arrest. We found that 2ME2 can selectively induce mitotic prometaphase arrest, but not G2 phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12–24h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME2-induced prometaphase arrest. The 2ME2-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME2-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME2 causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.

Effects of Newcastle Disease Virus Strains AF2240 and V4-UPM on Cytolysis and Apoptosis of Leukemia Cell Lines

Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD50 values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line.

Critical aspects in analysis of cellular DNA content.

This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.

Critical Aspects in Analysis of Cellular DNA Content

This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.

Flow cytometry analysis of cellular DNA content from formalin fixed paraffin embedded tissues: a useful technique in barrett’s surveillance?

Demonstration of dysplasia on histology is the best current indicator of risk of developing cancer in Barrett's oesophagus. <h3>Aim</h3> To determine if cellular DNA determined by flow cytometry can be used in stratifying patients at greater risk of developing high grade dysplasia/adenocarcinoma (HGD/AC). <h3>Methods</h3> 165 oesophageal biopsies [40 no dysplasia, 50 indefinite for dysplasia (ID), 55 low grade dysplasia (LGD), 10 HGD and 10 AC) from 108 patients were analysed by flow cytometry using formalin fixed paraffin embedded tissue. <h3>Results</h3> Cellular DNA content showed a strong relationship with grade of histological dysplasia, with higher grades more likely to show abnormal ploidy (χ²=81.2, df=3, <i>p</i><0.0001). DNA analysis differentiated HGD/AC from no dysplasia, with only 5% (2/40) of biopsies showing no dysplasia having abnormal DNA cellular content compared to 100% (20/20) with HGD/AC (<i>p</i>≤0.0001). LGD was more likely to show abnormal ploidy (18%, 10/55) compared to no dysplasia (<i>p</i>=0.06), but ID (10% abnormal ploidy, 5/50) were similar to no dysplasia (<i>p</i>=0.38). Six patients with initially no dysplasia/ ID and diploid DNA developed HGD/AC over 2–7 years with concomitant development of aneuploidy. <h3>Conclusions</h3> Abnormal ploidy on flow cytometry is strongly associated with worsening dysplasia. However, HGD/AC can still develop in patients when initial biopsies show diploid DNA and no dysplasia.

Effects of L-Acetylcarnitine (LAC) on the Post-Injury Recovery of Mouse Spermatogenesis Monitored by Flow Cytometry 1. Recovery after X-Irradiation/Über den Einfluß von L-Acetylcarnitin (LAC) auf die Wiederherstellung der Mäusespermatogenese mittels Überw

L-acetylcarnitine plays a key role in sperm metabolism and in the whole spermatogenetic process. In the present work, the influence of L-acetylcarnitine, administered i.p. (100 mg/kg body weight), on the recovery processes of mouse spermatogenesis after local acute irradiation with 10 Gy X-rays has been investigated. The effects were monitored 28, 35, 40, 45, 50, 55, and 60 days after irradiation by flow cytometric analysis of cellular DNA content. In the LAC-treated animals, the fraction of tetraploid cells is higher at 28 (p less than 0.05) and 45 days (p less than 0.02). Corresponding with the timing of the stages of murine spermatogenesis, the round spermatid fraction is higher at 45 days (p less than 0.1) and the elongated spermatid fraction is higher at 50 days (p less than 0.1) after irradiation. In addition, the LAC-treated animals show a faster recovery throughout the maturation process, from tetraploid to round and elongated spermatids. These results indicate that the presence of exogenous LAC could enhance the recovery of spermatogonial cells.

Lycopene differentially induces quiescence and apoptosis in androgen-responsive and -independent prostate cancer cell lines

Lycopene has been credited with a number of health benefits including a decrease in prostate cancer risk. Our study investigates the molecular mechanism underlying anti-cancer activity of lycopene-based products in androgen-responsive (LNCaP) and androgen-independent (PC3) cells. The effect of lycopene-based agents on prostate cancer growth and survival were examined using proliferation assays, bromodeoxyuridine incorporation and flow cytometric analysis of cellular DNA content. Biochemical effects of lycopene treatment were investigated by immunoblotting for changes in the absolute levels and phosphorylation states of cell cycle regulatory and signalling proteins. LNCaP and PC3 cells treated with the lycopene-based agents undergo mitotic arrest, accumulating in G0/G1 phase. Immunoblot screening indicated that lycopene's antiproliferative effects are likely achieved through a block in G1/S transition mediated by decreased levels of cyclins D1 and E and cyclin dependent kinase 4 and suppressed Retinoblastoma phosphorylation. These responses correlated with decreased insulin-like growth factor-I receptor expression and activation, increased insulin-like growth factor binding protein 2 expression and decreased AKT activation. Exposure to lycopene at doses as low as 10 nM for 48 h induced a profound apoptotic response in LNCaP cells. In contrast PC3 cells were resistant to apoptosis at doses up to 1 microM. Lycopene exposure can suppress phosphatidylinositol 3-kinase-dependent proliferative and survival signalling in androgen-responsive LNCaP and androgen-independent PC3 cells suggesting that the molecular mechanisms for the cytostatic and cytotoxic actions of lycopene involve induction of G0/G1 cell cycle arrest. This study supports further examination of lycopene as a potential agent for both the prevention and treatment of prostate cancer.

Sequential phosphorylation of Ser‐10 on histone H3 and ser‐139 on histone H2AX and ATM activation during premature chromosome condensation: Relationship to cell‐cycle phase and apoptosis

Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis. To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry. Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells. Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.

Assessment of ATM phosphorylation on Ser‐1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser‐139‐histone H2AX phosphorylation, cell cycle phase, and apoptosis

The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-à-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor. Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATM-S1981(P)) or for H2AX (gammaH2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry. Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells. The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topo1 (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the "cleavable complexes" stabilized by topo1 inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells.

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.

Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

Among numerous inflammatory mediators a nitric oxide molecule is supposed to be important in the modulation of neutrophil survival in vivo and in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD) but not by catalase (CAT) was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.