A statistical pattern recognition approach for determining cellular viability and lineage phenotype in cultured cells and murine bone marrow.

BACKGROUND: Cellular binding of annexin V and membrane permeability to 7-aminoactinomycin D (7AAD) are important tools for studying apoptosis and cell death by flow cytometry. Combining viability markers with cell surface marker expression is routinely used to study various cell lineages. Current classification methods using strict thresholds, or "gates," on the fluorescent intensity of these markers are subjective in nature and may not fully describe the phenotypes of interest. We have developed objective criteria for phenotypic boundary recognition through the application of statistical pattern recognition. This task was achieved using artificial neural networks (ANNs) that were trained to recognize subsets of cells with known phenotypes, and then used to determine decision boundaries based on statistical measures of similarity. This approach was then used to test the hypothesis that erythropoietin (EPO) inhibits apoptosis and cell death in erythroid precursor cells in murine bone marrow. METHODS: Our method was developed for classification of viability using an in vitro cell system and then applied to an ex vivo analysis of murine late-stage erythroid progenitors. To induce apoptosis and cell death in vitro, an EPO-dependent human leukemic cell line, UT-7(EPO) cells were incubated without recombinant human erythropoietin (rhEPO) for 72 h. Five different ANNs were trained to recognize live, apoptotic, and dead cells using a "known" subset of the data for training, and a K-fold cross validation procedure for error estimation. The ANNs developed with the in vitro system were then applied to classify cells from an ex vivo study of rhEPO treated mice. Tg197 (human tumor necrosis-alpha transgenic mice, a model of anemia of chronic disease) received a single s.c. dose of 10,000 U/kg rhEPO and femoral bone marrow was collected 1, 2, 4, and 8 days after dosing. Femoral bone marrow cells were stained with TER-119 PE, CD71 APC enable identification of erythroid precursors, and annexin V FITC and 7AAD to identify the apoptotic and dead cells. During classification forward and side angle light scatter were also input to all pattern recognition systems. RESULTS: Similar decision boundaries between live, apoptotic, and dead cells were consistently identified by the neural networks. The best performing network was a radial basis function multi-perceptron that produced an estimated average error rate of 4.5% +/- 0.9%. Using these boundaries, the following results were reached: depriving UT-7(EPO) cells of rhEPO induced apoptosis and cell death while the addition of rhEPO rescued the cells in a dose-dependent manner. In vivo, treatment with rhEPO resulted in an increase of live erythroid cells in the bone marrow to 119.8% +/- 9.8% of control at the 8 day time point. However, a statistically significant transient increase in TER-119(+) CD71(+) 7AAD(+) dead erythroid precursors was observed at the 1 and 2 day time points with a corresponding decrease in TER-119(+) CD71(+) 7AAD(-) Annexin V(-) live erythroid precursors, and no change in the number of TER-119(+) CD71(+) annexin V(+) 7AAD(-) apoptotic erythroid precursors in the bone marrow. CONCLUSIONS: A statistical pattern recognition approach to viability classification provides an objective rationale for setting decision boundaries between "positive" and "negative" intensity measures in cytometric data. Using this approach we have confirmed that rhEPO inhibits apoptosis and cell death in an EPO dependent cell line in vitro, but failed to do so in vivo, suggesting EPO may not act as a simple antiapoptotic agent in the bone marrow. Rather, homeostatic mechanisms may regulate the pharmacodynamic response to rhEPO.     

Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells

OBJECTIVE: To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains. RESULTS: Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis. CONCLUSION: On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.     

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.     

PKH26 as a fluorescent label for live human umbilical mesenchymal stem cells

To determine whether PKH26 labeling affects the morphologies, phenotypes, proliferation, and secretion abilities of human umbilical mesenchymal stromal cells (HUMSCs) were investigated. Isolated HUMSCs were labeled with PKH26, and cell morphology was observed under microscope. Cell cycle, apoptotic cell death, expression of PKH26, and the proliferation rate were evaluated. Additionally, fluorescence intensity of PKH26 labeling at different passage times was quantified. There were no detectable differences in cell morphology, cell growth, and proliferation rate after PKH26 labeling. In addition, fluorescence intensity of PKH26 labeling was gradually reduced with increase of the passage times. The PKH26 labeling disappeared after passage six times. In summary, PKH26 labeling is a safe and effective way to label live HUMSCs.     

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in gamma irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5 Gy and 20 Gy gamma irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5 Gy gamma irradiation of PBMCs mainly causes apoptosis, whereas a 20 Gy gamma irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.     

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.     

Decrease in CD93 (C1qRp) expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.     

Phosphatidylserine externalization during bone marrow cell death after treatment of mice with WR-2721 and gamma-rays

The cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment were assessed in the bone marrow of adult male Swiss mice exposed to a single 6 Gy dose of 60 Co gamma-rays, and treated intraperitoneally with the aminothiol WR-2721 (Amifostine, S-2-/3-aminopropylamino/ethyl phosphorothioic acid), at a dose of 400 mg/kg body weight, 30 min prior to gamma-irradiation. The bone marrow cells were stained with a combination of fluoresceinated annexin V (annexin V--FITC) and propidium iodide (PI) at 3 h, 7 h, and 24 h after treatment of mice with WR-2721 and 60Co gamma-irradiation. The number of early apoptotic cells (annexin V--FITC positive/PI negative), and late apoptotic and necrotic cells (annexin V--FITC positive/PI positive), was increased at 3 h after exposure of mice to 60Co gamma-rays and thereafter declined with the frequency of apoptotic and necrotic cells remaining lower in WR-2721 pre-treated mice. Using the annexin V--FITC flow cytometric assay, the radioprotective effect of WR-2721 against induction of apoptosis and necrosis in normal cells of the haematopoietic system was shown.     

EGCG 对人结肠癌HT-29 细胞凋亡的影响以及对MMP-2，RECK 的 调节作用

目的:观察表没食子儿茶素没食子酸酯(Epigallaocatechin-3-gallate,EGCG)对人结肠癌HT-29细胞增殖和凋亡的影响,并探讨其对MMP-2,RECK的调节作用。方法:体外培养人结肠癌HT-29细胞,MTT比色法检测EGCG对HT-29细胞的生长抑制作用;Histone/DNA ELISA检测细胞凋亡;FITC标记Annexin-V/PI双染流式细胞术分析凋亡细胞百分率;Western Blot和RT-PCR方法检测EGCG对MMP-2,RECK蛋白和mRNA表达的影响。结果:EGCG呈浓度和时间依赖性抑制HT-29细胞的增殖,并且增加HT-29细胞Histone/DNA碎片的渗漏;EGCG诱导HT-29细胞凋亡百分率增高;EGCG抑制MMP-2蛋白和mRNA的表达,促进RECK蛋白和mRNA的表达。结论:EGCG抑制人结肠癌HT-29细胞的增殖,促进其凋亡,并且呈浓度和时间依赖性;其作用机制可能与其下调MMP-2蛋白和mRNA的表达、上调RECK蛋白和mRNA的表达有关。     

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.     

There is substantial nuclear and cellular disintegration before detectable phosphatidylserine exposure during the camptothecin-induced apoptosis of HL-60 cells

BACKGROUND: An early sign of apoptosis in many cells is the appearance of phosphatidylserine (PS) on the outside of the plasma membrane, whilst the cells still retain the ability to exclude DNA-binding molecules such as propidium iodide and 7-aminoactinomycin D (7-AAD). The protein annexin V binds preferentially to PS and has often been used to monitor the early phase of apoptosis. There have been some conflicting results concerning whether annexin V binds to camptothecin (CAM)-treated HL-60 cells, a commonly used model for apoptosis. We investigated the effects of culturing HL-60 cells for up to 8 h with a range of CAM concentrations. METHODS: We used flow cytometry to measure cellular light scatter, annexin V-FITC binding, and 7-AAD uptake, and DNA content after fixation and permeabilization. We also used microscopy to examine the morphology of cells (both unsorted and sorted according to their light scatter) after cytocentrifugation. RESULTS: We found that CAM caused the rapid appearance of low light scatter apoptotic bodies. Even among cells with "normal" light scatter, there was widespread DNA cleavage and nuclear fragmentation by 3 h. The percentage of apoptotic bodies peaked at about 4 h and it was only afterward that annexin V binding could be detected to both intact cells and to apoptotic bodies. When they first appeared, the intact annexin V+ cells had S-phase DNA content. CONCLUSIONS: During CAM-induced apoptosis of HL-60 cells, the external exposure of PS can either precede or follow DNA cleavage, which suggests that PS exposure is not always an indicator of early apoptosis.     

Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation.

BACKGROUND: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. METHODS: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). RESULTS: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. CONCLUSIONS: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.     

Magneto/optical annexin V, a multimodal protein

Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC(50) of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO-Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.     

Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction. Accepted: 2 June 1999     

Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells

Malignant glioblastoma is one of the most common malignant tumors in the neurological system. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, appears to exhibit various biological activities, including antitumor effect, but the function and mechanism of this new agent on glioblastoma cells has not previously been determined. In the present study, we investigated the proliferation change of human glioblastoma U87MG cells exposured to different concentrations (0.9-14.8 microM) of Tubeimoside V (1) for a certain time. The results showed that Tubeimoside V (1) significantly suppressed U87MG cell proliferation in a time- and dose-dependent manner (IC(50) = 3.6 microM). Flow cytometric analysis of DNA in U87MG cells showed that Tubeimoside V (1) induces the prominent appearance of a sub-G1 peak in the cell cycle suggestive of apoptosis. Furthermore, U87MG cells' treatment with Tubeimoside V (1) resulted in nuclear condensation with apoptotic bodies observed by both fluorescence and electron microscopy. The result of annexin V/PI assay showed that phosphatidylserine externalization began after treatment, and then increased in the following 24h. Molecular changes explored through Western-blot staining showed Tubeimoside V (1) decreased the expression levels of Bcl-2 protein and increased the expression levels of Bax protein. The novel findings suggest that the cytotoxic actions of Tubeimoside V (1) toward U87MG cells result from the induction of cell apoptosis. Overall, our data demonstrate that Tubeimoside V (1) is an efficient apoptotic killing agent of glioblastoma cells and suggest that this mechanism may play a critical role in anti-tumor chemotherapy.     

Molecular mechanism of apoptosis induction by Gaillardin,a sesquiterpene lactone,in breast cancer cell lines

Medicinal plant extracts have been widely used for cancer treatment. Gaillardin is a natural sesquiterpene lactone that has recently been reported to have anticancer properties. The ability to induce apoptosis is an important property of a candidate anticancer drug, which discriminates between anticancer drugs and toxic compounds. The current study was therefore carried out to address the issue if Gaillardin is able to induce apoptosis in the breast cancer cell lines MCF-7 and MDA-MB-468 and to determine the underlying mechanism of its anticancer effects. Apoptosis induction by Gaillardin treatment was confirmed by annexin V–FITC/PI staining, and caspase-3,-6, and-9 activation. Using Western blot analysis, we found that Gaillardin upregulated the pro-apoptotic protein Bax and p53 and downregulated the anti-apoptotic protein Bcl-2. Moreover, the apoptotic effect of Gaillardin was also related to ROS production and loss of mitochondrial membrane potential (ΔΨm). Taken together, these results demonstrate that Gaillardin can inhibit proliferation of breast cancer cells via inducing mitochondrial apoptotic pathway and therefore, might be a promising molecule in cancer chemoprevention or chemotherapy.     

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.