Frank Traganos

Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis).

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cells propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology.

Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system.

Simultaneous staining of deoxyribonucleic (DNA) and ribonucleic acid (RNA) in nonfixed, but permeable, cells is described. Cells are made permeable by treatment with non-ionic detergent at low pH. RNA is denatured prior to, or during staining, by exposure of cells to chelating agents to ensure that DNA (native) and RNA (dentured) may be stained differentially with the metachromatic dye, acridine orange. The fluorescence of individual cells is measured in a flow cytofluorometer. A comparison between various staining procedures employing acridine orange or other intercalating dyes in unfixed cells is discussed in terms of staining specificity, cell permeability and preservation. Evidence is provided that acridine orange staining of unfixed cells may be used as a simple, fast means of obtaining information on cell ploidy levels and cell cycle status from DNA measurements (green fluorescence), and cell transcriptional activity from RNA staining (red fluorescence), in human and murine cells lines, peripheral blood and bone marrow specimens from patients with leukemia and mitogenically (phytohemagglutinin) or antigenically (mixed lymphocyte culture) stimulated human peripheral blood cultures. Exposure of cells to detergent at low pH as an alternative to cell fixation or hypotonic treatment is proposed as a fast, convenient method of making cells permeable to dyes.

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Abstract Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).

Cytometry of cyclin proteins.

Cyclins are key components of the cell cycle progression machinery. They activate their partner cyclin-dependent kinases (CDKs) and possibly target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. During unperturbed growth of normal cells, the timing of expression of several cyclins is discontinuous, occurring at discrete and well-defined periods of the cell cycle. Immunocytochemical detection of cyclins in relation to cell cycle position (DNA content) by multiparameter flow cytometry has provided a new approach to cell cycle studies. This approach, like no other method, can be used to detect the unscheduled expression of cyclins, namely, the presentation of G1 cyclins by cells in G2/M and of G2/M cyclins by G1 cells, without the need for cell synchronization. Such unscheduled expression of cyclins B1 and A was seen when cell cycle progression was halted, e.g., after synchronization at the G1/S boundary by inhibitors of DNA replication. The unscheduled expression of cyclins B1 or E, but not of A, was also observed in some tumor cell lines even when their growth was unperturbed. Likewise, whereas the expression of cyclins D1 or D3 in nontumor cells was restricted to an early section of G1, the presentation of these proteins in many tumor cell lines also was seen during S and G2/M. This suggests that the partner kinase CDK4 (which upon activation by D-type cyclins phosphorylates pRB committing the cell to enter S) is perpetually active throughout the cell cycle in these tumor lines. Expression of cyclin D also may serve to discriminate G0 vs. G1 cells and, as an activation marker, to identify the mitogenically stimulated cells entering the cell cycle. Differences in cyclin expression make it possible to discriminate between cells having the same DNA content but residing at different phases such as in G2 vs. M or G2/M of a lower DNA ploidy vs. G1 cells of a higher ploidy. The expression of cyclins D, E, A and B1 provides new cell cycle landmarks that can be used to subdivide the cell cycle into several distinct subcompartments. The point of cell cycle arrest by many antitumor agents can be estimated with better accuracy in relation to these compartments compared to the traditional subdivision into four cell cycle phases. The latter applications, however, pertain only to normal cells or to tumor cells whose phenotype is characterized by scheduled expression of cyclins. As sensitive and specific indicators of the cells proliferative potential, the cyclins, in particular D-type cyclins, are expected to be key prognostic markers in neoplasia.

Constitutive Histone H2AX Phosphorylation and ATM Activation, the Reporters of DNA Damage by Endogenous Oxidants

DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

DNA double‐strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis‐associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser‐139; the phosphorylated H2AX is denoted γH2AX. The intensity of immunofluorescence (IF) of γH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug‐induced (DI) IF of γH2AX, and in particular to distinguish it from AA γH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation.

Cytometry of ATM Activation and Histone H2AX Phosphorylation to Estimate Extent of DNA Damage Induced by Exogenous Agents

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double‐strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis‐a‐vis cell cycle phase position and induction of apoptosis that employ flow‐ and laser scanning‐cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide‐releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow‐ and image‐analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis.

Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry

Abstract Secondary structure of RNA was studied in human leukemic SK-L7 cells by their staining with acridine orange (AO) and fluorescence measurements in a flow-through cytofluorimeter. Parallel measurements of RNAse-treated cells made it possible to distinguish and to measure separately those components of total cell fluorescence that are due to AO interaction with RNA. Like DNA, double helical and single-stranded RNA in situ stain differentially with AO. Hence, the extent of RNA in double helical conformation, interacting with AO by intercalation, can be measured. In 10 −3 M phosphate buffer, RNA in situ denatures at 40–60 °C. Mg ions stabilize RNA against thermal denaturation; EDTA markedly decreases stability of RNA in double helical conformation. A slow, progressive denaturation of RNA in situ is seen as a result of cell exposure to AO at low ionic strength at room temperature. It is presumed that the changes described reflect mostly alterations of secondary structure of rRNA. Results of our in situ studies conform with data of others on rRNA denaturation in intact isolated ribosomes. This suggests that the ribosome anchoring in cytoplasm is not associated with double helical rRNA regions. Cell staining with AO under conditions of full RNA denaturation and in the absence of DNA denaturation offers differential, simultaneous staining of RNA and DNA. This approach in cell staining provides a new parameter for automated cell classification based on both quantity and conformation of RNA in situ.

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

Abstract.  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 γH2AX. Also, ATM‐kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of γH2AX 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.

Chapter 12 Lysosomal Proton Pump Activity: Supravital Cell Staining with Acridine Orange Differentiates Leukocyte Subpopulations

Publisher Summary Lysosomes are known to be internally acidic and to require metabolic energy to maintain an intralysosomal pH of about 4.8. It appears that an adenosine tri-phosphate (ATP)-dependent proton (H+) pump is responsible for the internal acidic environment of lysosomes. Many uncharged, lipophilic substances are able to cross biological membranes by an unselective permeation mechanism. Some of these substances, such as the fluorescent dye acridine orange (AO), are weak bases. At acid pH, weak bases will accept a proton and be converted to a positively charged substance that is no longer capable of passing freely through cellular membranes. Thus, AO, like other lysosomotropic agents, will accumulate and become trapped within the lysosomes of living cells. Aggregates of AO are known to luminesce red as compared to the normal green fluorescence associated with the monomer form of the dye that would be expected to predominate at low AO concentrations. Under appropriate staining conditions, the intensity of AO red luminescence is a function of both total lysosomal volume and the capacity of the lysosomal membrane to maintain a proton gradient.