A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1

Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.     

Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.     

Synthesis and evaluation of cytotoxicity of 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles

Several derivatives of 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles were synthesized via Biginelli type reaction and tested for their anti-proliferative activity on human breast cancer (MCF-7) and human colon carcinoma (HT29) cell lines. Malignant and non-malignant cells were cultivated in RPMI medium and incubated with different concentrations of these pyrimidines. Cell viability was evaluated by MTT assay. Apoptotic cells were determined using DAPI (4'-6-diamidino-2-phenylindole) and propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). 6-Amino-4-(4-chlorophenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile and 6-amino-4-[4-dimethylamino)phenyl]-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile decreased the viability of MCF-7 and HT29 cells, in contrast to L929 cells. These compounds induced a sub-G1 peak inflow cytometry histograms of treated cells indicating that apoptosis is involved in their toxicity.     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

A Novel Apoptosis-like Pathway, Independent of Mitochondria and Caspases, Induced by Curcumin in Human Lymphoblastoid T (Jurkat) Cells

We have shown previously [E. Sikora, A. Bielak-Zmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899-907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 microM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Delta psi). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Delta psi, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from "classical" by the lack of mitochondrial depolarization and of the involvement of caspases.     

Ca2+/Mg(2+)-dependent nuclease: tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+.

Ca2+/Mg(2+)-dependent endonuclease has been implicated in the extensive internucleosomal DNA fragmentation that accompanies apoptosis (gene-directed cell death). We present further evidence that this enzyme is involved in apoptosis. Ca2+/Mg2+ nuclease activity was increased about 6-fold during colchicine-induced apoptosis in human chronic lymphocytic leukaemia cells. The increase in activity coincided with onset of DNA fragmentation. Spleen, liver, kidney and thymus expressed high levels of this enzyme while lung, brain, heart and testis contained little activity. Cells from tissues with high Ca2+/Mg2+ nuclease activity underwent rapid DNA fragmentation in response to a Ca2+ flux. Physiological concentrations of Zn2+ known to inhibit both apoptosis and DNA fragmentation also inhibited Ca2+/Mg2+ nuclease activity.     

Simultaneous Measurement of DAPI-Sulforhodamine 101 Stained Nuclear DNA and Protein in Higher Plants by Flow Cytometry

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4', 6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.     

Human fibroblasts undergo oxidative stress-induced apoptosis without internucleosomal DNA fragmentation

In order to evaluate the reliability of fibroblasts as a cell model for studying apoptosis, we tested the response of normal human fibroblasts to the oxidative stress inducers H(2)O(2) and 2-deoxy-D-ribose (dRib). Our results showed that fibroblasts treated with dRib and H(2)O(2) are induced to undergo apoptosis as demonstrated by reduction in total cell number, chromatin condensation, phosphatidylserine (PS) exposure, activation of caspase-3 and 7, changes in mitochondrial membrane potential and increase in the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive nuclei. However we only found a slight increase in the percentage of cells in the sub-G1 region evaluated by flow cytometry, and we did not observe DNA fragmentation by agarose gel electrophoresis. Early in apoptosis, DNA cleavage generates high molecular weight (HMW) fragments which can be detected by TUNEL assay; successively followed by a pronounced DNA brake down into low molecular weight (LMW) fragments, detected as a "DNA ladder" by conventional agarose gel electrophoresis and as an hypodiploid peak by propidium iodide (PI) flow cytometry assay. Our results thus suggest that only HMW fragmentation occurs in fibroblasts exposed to dRib or H(2)O(2) and the lack of internucleosomal DNA fragmentation may depend on the peculiar characteristics of human fibroblasts themselves, irrespective of the apoptotic stimulus used. The existence of distinct events leading to cell death in different cell types makes it necessary to use a combination of strategies and techniques to evaluate the occurrence of apoptosis.     

Ceramide increases steroid hormone production in MA-10 Leydig cells.

Ceramide is known to have major roles in the control of cell proliferation, differentiation, and apoptosis. Recent studies also have shown that ceramide affects steroid production by JEG-3 choriocarcinoma cells, acutely dispersed rat Leydig cells, and ovarian granulosa cells, but the mechanism by which this occurs is unknown. Because ceramide induces apoptosis in many different cell types, we hypothesized that ceramide might affect steroidogenesis and/or induce apoptosis in MA-10 murine Leydig cells. To test this, MA-10 cells were incubated with either the water soluble C2-ceramide, (N-acetyl-sphingosine, 0.01-10 cm); bacterial sphingomyelinase (1-100 mU/ml); or C2-dihydroceramide (N-acetyl-sphinganine, 0.1-10 microM). The data show that N-acetyl-sphingosine significantly increased basal (0.87 +/- 0.2 vs. 0.42 +/- 0.09 ng/mg cell protein, P < 0.01) and human chorionic gonadotropin (hCG) stimulated progesterone (P) synthesis (204 +/- 12 vs. 120 +/- 5 ng/mg cell protein, P < 0.001); as did sphingomyelinase (basal P = 0.83 +/- 0.1 ng/mg cell protein, P < 0.01; hCG stimulated P = 173 +/- 7 ng/mg cell protein, P < 0.001). C2-dihydroceramide also increased basal P synthesis but was less effective than ceramide on a molar basis. Neither sphingomyelinase (100 mU/ml) nor ceramide (10 microM) had any effect on cAMP production or human chorionic gonadotropin binding; and neither induced any signs of apoptosis (FragEL DNA fragmentation assay and electron microscopy). Cells incubated with anti-Fas (300 ng/ml) demonstrated DNA fragmentation, nuclear condensation, and frequent apoptotic bodies, but had no change in P synthesis. These data show that ceramide significantly increases MA-10 Leydig cell P synthesis but does not induce apoptosis. The mechanism by which ceramide increases steroid hormone synthesis remains unknown but does not appear to be linked to the induction of apoptosis in MA-10 cells.     

Cell cycle G2/M arrest and activation of cyclin-dependent kinases associated with low-dose paclitaxel-induced sub-G1 apoptosis

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 μM) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase. This revised version was published online in November 2006 with corrections to the Cover Date.     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.     

Relationships between DNA Fragmentation, Chromatin Condensation, and Changes in Flow Cytometry Profiles Detected during Apoptosis

The determination of whether a cell dies by apoptosis as opposed to necrosis is usually best made on the basis of distinct structural changes in the chromatin. These changes include extensive condensation of the chromatin and DNA fragmentation. We have shown that the cytotoxic drug bleomycin (BLM) is able to cleave the DNA between the nucleosomes when it enters into the cell. If sufficient amounts of BLM are internalized, the nuclear morphological changes characteristic of apoptosis are detected. In this work, we describe the nuclear changes that occurred after DNA fragmentation as a function of the number of DNA double-strand breaks generated per cell and of the time after their generation. Our results show that DNA fragmentation and degradation of higher-order DNA structure were directly responsible for the nuclear morphological changes associated with apoptosis. During apoptosis reduced fluorescence with respect to the G₀/G₁ cell cycle region (the sub-G₁ region) is often detected if fixed cells from cultures undergoing apoptosis are analyzed by flow cytometry. We demonstrate here that, depending on the extent of the DNA fragmentation and on ulterior changes in chromatin structure, the content of the fluorescent sub-G₁ region can be either soluble pieces of DNA or apoptotic bodies or cells depleted in the DNA content by partial loss of fragmented DNA dissolved in the washing media and/or by the release of apoptotic bodies.     

DNA fragmentation in microorganisms assessed in situ

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.     

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3 end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN₃, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3end labelling of DNA breaks.     

Studies of apoptosis of malignant lymphoma cells induced by arsenic trioxide

The present study investigated the effect of As(2)O(3)on malignant lymphoma cells. Cell apoptosis was detected by cell staining and TdT-mediated dUTP Nick-end Labelling (TUNEL). Cellular DNA and protein expression content were determined by immunohistochemistry and flow cytometry. It was found that 0.5-2.0 microm /l As(2)O(3)could inhibit cell growth, including Raji cells and lymphoma cells from patients, and induce apoptosis, such as condensed chromatin and nuclear fragmentation with intact cell membrane, i.e. apoptotic body. It was also found that the cells of the sub-G(1)phase increased significantly and bcl-2 gene expression was greatly downregulated. However, this effect was not observed for Jurkat cells under the same conditions. We concluded that As(2)O(3)at a range of 0.5-2.0 microm /l can inhibit the growth and induce apoptosis in malignant lymphoma cells, which may have therapeutic potential.