Preferential Fas-mediated apoptotic execution at G1 phase: the resistance of mitotic cells to the cell death

Apoptosis is induced by various stresses generated from the extracellular and intracellular environments. The fidelity of the cell cycle is monitored by surveillance mechanisms that arrest its further progression if any crucial process has not been completed or damages are sustained, and then the cells with problems undergo apoptosis. Although the molecular mechanisms involved in the regulation of the cell cycle and that of apoptosis have been elucidated, the links between them are not clear, especially that between cell cycle and death receptor-mediated apoptosis. By using the HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we investigated the relationship between the cell cycle progression and apoptotic execution. To monitor apoptotic execution during cell cycle progression, we observed the cells after induction of apoptosis with time-lapse fluorescent microscopy. About 70% of Fas-mediated apoptotic cells were present at G₁ phase and about 20% of cells died immediately after cytokinesis, whereas more than 60% of etoposide-induced apoptotic cells were at S/G₂ phases in random culture of the cells. These results were confirmed by using synchronized culture of the cells. Furthermore, mitotic cells showed the resistance to Fas-mediated apoptosis. In conclusion, these findings suggest that apoptotic execution is dependent on cell cycle phase and Fas-mediated apoptosis preferentially occurs at G₁ phase.     

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.     

Lectin-based three-color flow cytometric approach for studying cell surface glycosylation changes that occur during apoptosis.

BACKGROUND: Changes in cell surface glycosylation that accompany apoptosis are thought to be involved in the recognition and removal of apoptotic cells by phagocytes, but in most instances these changes are ill defined. To improve our understanding of this phenomenon, we designed a trivariate flow cytometry procedure that allows direct comparison of cell surface glycosylation in apoptotic and viable cells. METHODS: The annexin V/propidium iodide assay has been adapted for cell surface glycosylation analysis by combining the use of these two reagents with biotinylated lectins, and this has been used to investigate camptothecin-induced apoptosis in U-937 cells. RESULTS: Although numerous lectins are potent inducers of apoptosis, we found that it is possible to determine lectin concentrations that produce interpretable data without inducing significant cytotoxicity even when using apoptogenic lectins. That apoptosis is associated with a marked decrease in cell surface sialylation was confirmed by using the sialic acid-specific lectins Maackia amurensis agglutinin and Sambucus nigra agglutinin. These observations were corroborated by lectin blotting analysis with the same lectins. CONCLUSIONS: Species- and cell-dependent altered glycosylation patterns are likely to be associated with different modes of apoptosis. The easy and versatile method described in this report should be useful for exploring this field.     

BCL-2 does not control programmed cell death in the IPLB-LdFB cell line from the insect Lymantria dispar

In the insect Lymantria dispar cell line IPLB-LdFB the presence of a Bcl-2-like molecule has been demonstrated. The Western blot analysis performed on the cells incubated with 2-deoxy-D-ribose (dRib), an apoptotic inducer, revealed that, in comparison with the control, the Bcl-2 expression was unaffected. Furthermore, incubation of the insect cells with an anti-Bcl-2 polyclonal antibody inhibited the apoptotic effect induced by dRib, and provoked mitochondrial membrane depolarization without any apoptotic phenomena. Similar behaviour was observed using the K+ ionophore valinomycin. From these findings, we hypothesize that the L. dispar Bcl-2-like protein is essential for maintenance of the mitochondrial membrane potential, but not, as usually thought, for the regulation of programmed cell death.     

Isolation of acetylcholinesterase from apoptotic human lung fibroblast cells by antibody affinity chromatography

Acetylcholinesterase (AChE; EC3.1.1.7) is well known for its role in the hydrolysis of acetylcholine at cholinergic synapses to terminate neurotransmission. In addition to its synaptic presence, AChE has been found to be in non-cholinergic cells such as hematopoietic and osteogenic cells. We have recently reported that AChE is expressed in various cells undergoing apoptosis. To characterize AChE in apoptotic cells and to investigate the role of AChE expression in apoptosis, we devised a method to purify AChE expressed in apoptotic human lung fibroblast cell line HLF. The isolation of this enzyme is mainly based on inhibitor ligand affinity chromatography using immobilized tacrine. However, this method is only effective in isolating active AChE. Here we employed antibody-based chromatography and found that both active and inactive AChE were present in apoptotic HLF cells. Active AChE was predominantly observed in the nuclei of apoptotic cells, while inactive AChE was mainly present in the cytoplasm. Therefore, our method provides an opportunity to investigate further the role of AChE, especially inactive AChE, in apoptosis.     

ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.     

Effects of phenformin on the proliferation of human tumor cell lines

Phenformin is a biguanide that has been largely used in the past for its anti-diabetic activity. A large body of evidence suggests additional effects of phenformin including antitumoral activity in different animal models in vivo. Thus, the present study has been conducted in order to elucidate possible mechanisms involved in the antitumoral effects of phenformin. In various tumoral cell lines (SH-SY5Y neuroblastoma and LNCaP prostate adenocarcinoma cells), increasing concentrations of phenformin (50-500 microM) induced a concentration-dependent inhibition of cell proliferation. This effect was not dependent on the ability of the drug to reduce glucose levels and was accompanied by induction of apoptotic cell death as measured by cytofluorometric analysis. In addition, a short-time incubation of SH-SY5Y cells with phenformin induced enhanced and transient expression of the cell cycle inhibitor p21 suggesting that phenformin causes inhibition of cell cycle progression prior to induction of apoptosis. These results demonstrate an activity at the cellular level of phenformin that supports its antitumoral effect observed in vivo.     

Hydra, a versatile model to study the homeostatic and developmental functions of cell death

In the freshwater cnidarian polyp Hydra, cell death takes place in multiple contexts. Indeed apoptosis occurs during oogenesis and spermatogenesis, during starvation, and in early head regenerating tips, promoting local compensatory proliferation at the boundary between heterografts. Apoptosis can also be induced upon exposure to pro-apoptotic agents (colchicine, wortmannin), upon heat-shock in the thermosensitive sf-1 mutant, and upon wounding. In all these contexts, the cells that undergo cell death belong predominantly to the interstitial cell lineage, whereas the epithelial cells, which are rather resistant to pro-apoptotic signals, engulf the apoptotic bodies. Beside this clear difference between the interstitial and the epithelial cell lineages, the different interstitial cell derivatives also show noticeable variations in their respective apoptotic sensitivity, with the precursor cells appearing as the most sensitive to pro-apoptotic signals. The apoptotic machinery has been well conserved across evolution. However, its specific role and regulation in each context are not known yet. Tools that help characterize apoptotic activity in Hydra have recently been developed. Among them, the aposensor Apoliner initially designed in Drosophila reliably measures wortmannin-induced apoptotic activity in a biochemical assay. Also, flow cytometry and TUNEL analyses help identify distinctive features between wortmannin-induced and heat-shock induced apoptosis in the sf-1 strain. Thanks to the live imaging tools already available, Hydra now offers a model system with which the functions of the apoptotic machinery to maintain long-term homeostasis, stem cell renewal, germ cell production, active developmental processes and non-self response can be deciphered.     

Clusterin protects granulosa cells from apoptotic cell death during follicular atresia

Clusterin expression is associated with programmed cell death (apoptosis) in many cell types but its exact role has not yet been defined. This study was carried out to determine the cellular localization of clusterin in the ovary and its functional role in the apoptotic cell death of ovarian follicles. A homogenous population of healthy and atretic follicles was obtained by treating immature rats with pregnant mare serum gonadotropin (PMSG). Apoptotic cell death was evaluated by TUNEL. Clusterin expression in the healthy and atretic follicles was examined by immunohistochemical and Western blot analyses, and gene expression was examined by Northern blot analysis. Clusterin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healthy follicles from PMSG-treated rats on day 2 of the treatment do not express clusterin. Theca and stroma cells of both healthy and atretic follicles showed no signs of apoptosis and did not express clusterin. Withdrawal of trophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish the functional role of clusterin in the apoptotic cell death of ovarian follicles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cDNA. Treatment of granulosa cells with the antisense oligonucleotide resulted in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed cell death in ovary, suggesting a functional role for this protein in follicular atresia.     

Fenretinide induces autophagic cell death in caspase-defective breast cancer cells

The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displaying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumor cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.     

Fenretinide induces autophagic cell death in caspase-defective breast cancer cells

The elimination of tumour cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumour cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.     

A histochemical comparison of methyl green-pyronin,and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma,oral leukoplakia,oral submucous fibrosis and normal oral mucosa

Abstract

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.     

Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines

ABSTRACT: BACKGROUND: Lycopene, a major carotenoid component of tomato, has a potential anticancer activity in many types of cancer. Epidemiological and clinical trials rarely provide evidence for mechanisms of the compound's action, and studies on its effect on cancer of different cell origins are now being done. The aim of the present study was to determine the effect of lycopene on cell cycle and cell viability in eight human cancer cell lines. METHODS: Human cell lines were treated with lycopene (1-5 uM) for 48 and 96 h. Cell viability was monitored using the method of MTT. The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) and by DAPI. RESULTS: Our data showed a significant decrease in the number of viable cells in three cancer cells lines (HT-29, T84 and MCF-7) after 48 h treatment with lycopene, and changes in the fraction of cells retained in different cell cycle phases. Lycopene promoted also cell cycle arrest followed by decreased cell viability in majority of cell lines after 96 h, as compared to controls. Furthermore, an increase in apoptosis was observed in four cell lines (T-84, HT-29, MCF-7 and DU145) when cells were treated with lycopene. CONCLUSIONS: Our findings show the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate lines after 96 h. These observations suggest that lycopene may alter cell cycle regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the antiproliferative effect of lycopene was cellular type, time and dose-dependent. KEY WORDS: lycopene, cancer, bioactive compounds, cell cycle.     

Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content

BACKGROUND: Induction of apoptosis in adherent cell lines is associated with cell loss from the substratum. In this study the adenocarcinoma cell line, HT29, treated with indomethacin (400microM) has been employed as a model system to demonstrate how flow cytometric analysis can be used to quantify the changes that occur during this process. METHODS: Adherent and floating cell populations have been analyzed independently for effects on cell number, cell cycle characteristics and apoptosis using TUNEL assay and Annexin V binding. In addition apoptosis has been assessed using DNA laddering and morphology. RESULTS: Apoptosis was detected in adherent cells treated with indomethacin using Annexin V binding but not by other techniques employed in this study. In contrast, analysis of "floating" cells revealed the presence of apoptotic cells both in control and indomethacin treated cells using all the techniques employed. However quantification by flow cytometry showed that a significantly higher proportion of control "floaters" were late apoptotic/necrotic rather than apoptotic. DISCUSSION: The data here illustrate the need to interpret measures of apoptosis in adherent cell lines with care and the value of using flow cytometric techniques in the quantitative evaluation of the process.     

Lack of galectin-3 disturbs mesenteric lymph node homeostasis and B cell niches in the course of Schistosoma mansoni infection

Galectin-3 is a β-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAcβ1-4(Fucα1-3)GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine β1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in non-lymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S.mansoni.     

Effects of three-dimensional culturing on osteosarcoma cells grown in a fibrous matrix: analyses of cell morphology, cell cycle, and apoptosis

Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three-dimensional (3-D) fibrous culture had better cell growth and higher metabolic rates than the two-dimensional (2-D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long-term (1 month) T-flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at approximately 15%. These results could explain why the fibrous bed bioreactor had good long-term stability and was advantageous for production of non-growth-associated proteins by animal cell cultures.     

Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF)

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines.     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

PR-000350, a novel hypoxic radiosensitizer, enhances tumor cell killing by promoting apoptosis preferentially in the S-phase fraction

PR-000350, a novel hypoxic radiosensitizer, is a 2-nitroimidazole nucleoside analog and has begun to be used for clinical cancer therapy. In this study, using U937 monoblastoid cells we investigated the mechanisms of enhanced cell killing by PR-000350. When cells were irradiated under an extremely hypoxic condition, the apoptotic rate was strongly suppressed. However, a remarkable increase in the DNA fragmentation rate as well as in the ladder formation was observed when hypoxic cells were irradiated in the presence of 5 mM PR-000350. DNA histograms of the PR-000350 treated group showed enhancement of the sub-G₁ fraction and simultaneous suppression of the progression of the cell cycle from the S to G₂/M phase at 4–8 h after X-irradiation, suggesting the importance of the S phase in the induction of apoptotic cell death. Flow cytometric and immunohistochemical analyses after BrdU labelling revealed that apoptotic cell death is induced mainly in the BrdU-positive cells. In addition, by using cell synchronization technique it was proved that the S phase is the most sensitive fraction to the radiosensitizing effect of PR-000350. These results suggest that PR-000350 strongly enhances tumor cell killing by promoting X-ray induced-apoptosis preferentially in the S-phase fraction. PR-000350 is a new type radiosensitizer and promise to provide an effective anti-cancer activity against hypoxic tumor cells that are resistant to the usual radiotherapy.