Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.     

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K⁺ and Na⁺ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.     

Phorbol ester inhibits DNA damage-induced apoptosis in U937 cells through activation of protein kinase C

The effects of phorbol 12-myristate 13-acetate (PMA) on DNA damage-induced apoptosis were examined in promyelocytic leukemia cells, U937, in comparison with other differentiation-inducing agents to clarify the role of protein kinase C (PKC) vis-a-vis cellular differentiation in apoptosis. The apoptosis of U937 cells was observed at as early as 1-1.5 h following UV irradiation, with most cells being in apoptotic state at 3 h. Pretreatment with PMA for as short as 5 min was sufficient to inhibit apoptosis induced by UV irradiation, whereas apparent changes in cell cycle distributions and expression of differentiation markers by PMA were not observed until 12 h and 48 h, respectively. The inhibition of apoptosis by PMA was completely abolished by the pretreatment with calphostin C, a PKC inhibitor, and 4 alpha-phorbor 12,13-didecanoate, which is unable to activate PKC, did not protect U937 cells against apoptosis induced by UV irradiation. Other differentiation inducers, such as cyclic AMP and active vitamin D3, did not affect the UV-induced apoptosis of U937 cells. Taken together, it was suggested that PMA inhibits DNA damage-induced apoptosis through the activation of PKC rather than as a result of differentiation of U937 cells.     

Electron probe X-ray microanalysis of cisplatin-induced cell death in rat pheochromocytoma PC12 cells

Several lines of evidence suggest that cisplatin-induced cell death is not always the result of apoptosis. A distinctive feature between apoptosis and necrosis is the alteration in cell volume regulation and ion homeostasis. Here we analyzed the changes in intracellular element content during cell death induced by exposure to therapeutic concentrations of cisplatin in the PC12 cell line. To quantitate Na, Cl and K content, electron probe X-ray microanalysis (EPXMA) was performed in whole freeze-dried cells. We also traced the alterations in morphological features with fluorescence and transmission electron microscopy. EPXMA demonstrated progressive derangement of the absolute intracellular Na, Cl and K contents. Cisplatin-treated cells showed two microanalytical patterns: 1) cells with alterations in elemental content typical of apoptosis, i.e., an increase in intracellular Na and a decrease in intracellular Cl and K, and 2) cells characterized by an increase in Na content and a decrease in K content, with no changes in Cl content. This intracellular profile for Na, Cl, and K was not typical of necrosis or apoptosis. Morphological analysis revealed two cellular phenotypes: 1) cells characterized by a phenotype typical of apoptosis, and 2) cells characterized by a hybrid phenotype combining variable features of apoptosis and necrosis. Taken together, our findings suggest that therapeutic concentrations of cisplatin may cause a hybrid type of cell death characterized by concurrent apoptosis and necrosis in the same individual PC12 cell.     

Apoptosis in leukemia cells is accompanied by alterations in the levels and localization of nucleolin

Molecular defects in apoptotic pathways are thought to often contribute to the abnormal expansion of malignant cells and their resistance to chemotherapy. Therefore, a comprehensive knowledge of the mechanisms controlling induction of apoptosis and subsequent cellular disintegration could result in improved methods for prognosis and treatment of cancer. In this study, we have examined apoptosis-induced alterations in two proteins, nucleolin and poly(ADP-ribose) polymerase-1 (PARP-1), in U937 leukemia cells. Nucleolin is expressed at high levels in malignant cells, and it is a multifunctional and mobile protein that can shuttle among the nucleolus, nucleoplasm, cytoplasm, and plasma membrane. Here, we report our findings that UV irradiation or camptothecin treatment of U937 cells induced apoptosis and caused a significant change in the levels and localization of nucleolin within the nucleus. Additionally, nucleolin levels were dramatically decreased in extracts containing the cytoplasm and plasma membrane. These alterations could be abrogated by pre-incubation with an inhibitor of PARP-1 (3-aminobenzamide), and our data support a potential role for nucleolin in removing cleaved PARP-1 from dying cells. Furthermore, both nucleolin and cleaved PARP-1 were detected in the culture medium of cells undergoing apoptosis, associated with particles of a size consistent with apoptotic bodies. These results indicate that nucleolin plays an important role in apoptosis, and could be a useful marker for assessing apoptosis or detecting apoptotic bodies. In addition, the data provide a possible explanation for the appearance of nucleolin and PARP-1 autoantibodies in some autoimmune diseases.     

Cell shrinkage during apoptosis is not obligatory. Apoptosis of U937 cells induced by staurosporine and etoposide

A study was made of apoptotic cell shrinkage, which is generally believed to be a hallmark of apoptosis. The two conventional models of apoptosis were used for examination of changes in cell water balance--one is apoptosis caused in human lymphoma cell line U937 by staurosporine, and the other by etoposide. Intracellular water was determined by measuring buoyant density of cells in continuous Percoll gradient. Apoptosis was recognized by microscopy and flow cytometry. Apoptosis caused by staurosporine (1 microM, 4 h) was found to be associated with a decrease in cell water content by almost 24%. In contrast, no decrease in cell water content was observed in U937 cells incubated with etoposide (50 microM, 4 h), in spite of the number of features suggesting the presence of apoptosis, such as the appearance of apoptotic bodies, chromatin condensation and fragmentation and disappearance of S-phase cells in DNA histogram. It is concluded that definition of apoptosis as "shrinkage-necrosis" (Kerr, 1971) needs correcting: the distinction of apoptotic cells involves the absence of swelling, rather than cell shrinkage.     

U937 variant cells as a model of apoptosis without cell disintegration

The variant cell line U937_V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937_V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937_V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937_V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937_V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937_V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.     

Novel piperazine induces apoptosis in U937 cells

The effect of 1,4-bis-(4-(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine (BIPP), a newly synthesized piperazine derivative, on U937 leukemia cell viability was investigated. We show that BIPP induces dose-responsive apoptotic cell death in U937 cells by intrinsic mechanisms of apoptosis. Maximum apoptotic effect of BIPP on U937 cells was observed at 12.8μM. BIPP-induced apoptosis was evident by characteristics such as altered annexin-V binding, caspase activation, loss of mitochondrial membrane potential (MMP) and cytochrome c release. BIPP also differentially activates initiator and effector caspases combined with the loss of MMP strongly suggesting that BIPP causes an intrinsic apoptosis in U937 leukemia cells. Due to our observations that BIPP induces leukemia cell death without significantly affecting normal cells, our data suggests that it may be a potential therapeutic agent for human myeloid leukemia.     

Opposite role of changes in mitochondrial membrane potential in different apoptotic processes

We have studied the role of changes in mitochondrial membrane potential (DeltaPsi) in two widely-used models of apoptosis, such as dexamethasone-treated rat thymocytes and U937 human cells treated with tumor necrosis factor-alpha and cycloheximide. To dissipate DeltaPsi, we used low concentrations of valinomycin, unable per se to induce apoptosis, and demonstrated that the decline in DeltaPsi exerts opposite effects in the two models. Indeed, in U937 cells, depolarization of mitochondria increased apoptosis, which decreased in rat thymocytes. This leads to the suggestion that disruption of DeltaPsi plays opposite roles depending on the experimental model. In U937 cells, the drop of DeltaPsi is a possible contributory cause for the apoptotic process; in rat thymocytes, it could be a limiting factor. We propose that these opposite effects could be due to the different ATP requirement of each apoptotic pathway.     

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis

Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P < 0.001) and Cl (P < 0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P < 0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na⁺/K⁺ ATPase. This later phase was also characterized by a significant increase (P < 0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.     

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.     

Multiple Pathways for Apoptotic Nuclear Fragmentation

We analyzed the ultrastructure of apoptotic nuclear fragmentation in U937 cells treated with many different apoptogenic agents. We found that this characteristic apoptotic feature can be achieved through multiple alternative pathways, depending on the apoptogenic inducer, leading to slightly different final nuclear morphologies. In most instances, the irregularly shaped nucleus of U937 rounds up; then, chromatin condenses at the nuclear periphery. Condensed chromatin can form protruding patches, which eventually bud from the nucleus in sealed vesicles through a process which is actin-dependent, since it could be blocked by cytochalasins. Alternatively, chromatin condenses in tiny, nonprotruding crescents, and a cleavage in the nuclear sap forms, beginning from the inner nuclear membrane and growing inward, thus splitting the nucleus. In U937 induced to apoptosis by hydrogen peroxide in the presence of ADP-ribosylation inhibitors, the nuclei fragment in many vesicles before chromatin even begins to condense: chromatin condensation probably occurs as a consequence. While all the apoptotic morphologies described above evolve from interphase cells, a peculiar apoptotic morphology, possibly deriving from mitotic cells, is detected upon oxidative stress, recalling the formation of micronuclei by clastogenic treatments; it shows partially membrane-bound chromatin patches, which look midway between condensed chromosomes and apoptotic condensed chromatin. The existence of these multiple pathways for nuclear fragmentation may indicate an evolutionary convergence, suggesting that this event may play an important physiological role in apoptosis.     

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.     

Apoptotic pathways involved in U937 cells exposed to LDL oxidized by hypochlorous acid

Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. One oxidative pathway of LDL involves myeloperoxidase, which catalyzes the production of hypochlorous acid (HOCl) in monocytes. We investigated the apoptotic mechanism induced by oxLDL, generated by HOCl treatment of native LDL, in human monocytic U937 cell line. The involvement of the mitochondrial apoptotic pathway was analyzed in Bcl-2-overexpressing clones, generated from U937 cells. HOCl-oxLDL induced in U937 cells (i) a marked caspase-dependent increase of apoptosis, (ii) a loss of mitochondrial membrane potential, (iii) a specific activation of caspase-2, -3, -8, and -9, and (iv) a similar degree of apoptosis in presence or absence of anti-Fas and anti-TNF-R1 antibodies. Moreover, the degree of HOCl-oxLDL-induced caspase-3 and -8 activation, and apoptosis was significantly reduced in U937/Bcl-2 cells, with no activation of caspase-9. By contrast, Cu-oxLDL-mediated apoptosis in U937 cells involved exclusively the mitochondrial pathway. In conclusion, the mechanism of HOCl-oxLDL-induced apoptosis in monocytic U937 cells involves the two pathways of apical caspase activation: (i) death receptor-mediated caspase-8 and (ii) mitochondria-mediated caspase-9. This converges in the activation of executing caspases, including caspase-3, and apoptosis. The interference of Bcl-2 overexpression with HOCl-oxLDL-induced apoptosis suggests the importance of mitochondrial involvement in this apoptotic mechanism.     

The sorcin-annexin VII calcium-dependent interaction requires the sorcin N-terminal domain

Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5–20 min), caspase-3-like protease activation (2–4 h), chromatin condensation and DNA ladder formation (3–4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1–3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.     

Bid is not required for Bax translocation during UV-induced apoptosis

UV irradiation triggers apoptosis through both the membrane death receptor and the intrinsic apoptotic signaling pathways. Bax, a member of the Bcl-2 family of proteins, translocates from the cytosol to the mitochondrial membrane during UV-induced apoptosis, but the regulation of Bax translocation by UV irradiation remains elusive. In this study, we show that Bax translocation, caspase-3 activation and cell death by UV irradiation are not affected by Z-IETD-fmk (caspase-8 inhibitor), but delayed by Pifithrin- (p53 inhibitor), although Bid cleavage could be completely abolished by Z-IETD-fmk. Co-transfecting YFP-Bax and Bid-CFP into human lung adenocarcinoma cells, we demonstrate that translocation of YFP-Bax precedes that of Bid-CFP, there is no significant FRET (fluorescence resonance energy transfer) between them. Similar results are obtained in COS-7 cells expressing YFP-Bax and Bid-CFP. Furthermore, using acceptor photobleaching technique, we observe that there is no interaction between YFP-Bax and Bid-CFP in both healthy and apoptotic cells. Additionally, during UV-induced apoptosis there is downregulation of Bcl-x_L, an anti-apoptotic protein. Overexpression of Bcl-x_L in cells susceptible to UV-induced apoptosis prevents Bax translocation and cell death, repression of Bid protein with siRNA (small interfering RNA) do not inhibit cell death by UV irradiation. Taken together, these data strongly suggest that Bax translocation by UV irradiation is a Bid-independent event and inhibited by overexpression of Bcl-x_L.     

Induction of apoptosis in host cells: a survival mechanism for Leishmania parasites?

Leishmania parasites invade host macrophages, causing infections that are either limited to skin or spread to internal organs. In this study, 3 species causing cutaneous leishmaniasis, L. major, L. aethiopica and L. tropica, were tested for their ability to interfere with apoptosis in host macrophages in 2 different lines of human monocyte-derived macrophages (cell lines THP-1 and U937) and the results confirmed in peripheral blood mononuclear cells (PBMC). All 3 species induced early apoptosis 48 h after infection (expression of phosphatidyl serine on the outer membrane). There were significant increases in the percentage of apoptotic cells both for U937 and PBMC following infection with each of the 3 species. Early apoptotic events were confirmed by mitochondrial membrane permeabilization detection and caspase activation 48 and 72 h after infection. Moreover, the percentage of infected THP-1 and U937 macrophages increased significantly (up to 100%) following treatment with an apoptosis inducer. Since phosphatidyl serine externalization on apoptosing cells acts as a signal for engulfment by macrophages, induction of apoptosis in the parasitized cells could actively participate in spreading the infection. In summary, parasite-containing apoptotic bodies with intact membranes could be released and phagocytosed by uninfected macrophages.     

Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells

Ultraviolet (UV) light is a strong apoptotic trigger that induces caspase-dependent biochemical changes in cells. Previously we showed that UV irradiation can activate caspase-3, and the subsequent cleavage and activation of p21(Cdc42/Rac)-activated kinase 2 (PAK2) in human epidermoid carcinoma A431 cells. In this study we demonstrate that curcumin (Cur), the yellow pigment of Curcuma longa with known anti-oxidant and anti-inflammatory properties, can prevent UV irradiation-induced apoptotic changes, including c-Jun N-terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP), mitochondrial release of cytochrome C, caspase-3 activation, and cleavage/activation of PAK2 in A431 cells. Flow cytometric analysis using the cell permeable dye 2',7'-dichlorofluorescin diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation revealed that the increase in intracellular oxidative stress caused by UV irradiation could be abolished by Cur. In addition, we found that SP600125, a JNK-specific inhibitor, reduced UV irradiation-induced JNK activation as well as caspase-3 activation, indicating that JNK activity is required for UV irradiation-induced caspase activation. Collectively, our results demonstrate that Cur significantly attenuates UV irradiation-induced ROS formation, and suggest that ROS triggers JNK activation, which in turn causes MMP change, cytochrome C release, caspase activation, and subsequent apoptotic biochemical changes.     

Preferential externalization of newly synthesized phosphatidylserine in apoptotic U937 cells is dependent on caspase-mediated pathways

Externalization of phosphatidylserine (PtdSer) is a common feature of programmed cell death and plays an important role in the recognition and removal of apoptotic cells. In this study with U937 cells, PtdSer synthesis from [(3)H]serine was stimulated and newly synthesized PtdSer was transferred preferentially to cell-free medium vesicles (CFMV) from cells when apoptosis was induced with a topoisomerase I inhibitor, camptothecin (CAM). When CAM-induced apoptosis was blocked by a caspase inhibitor, z-VAD-fmk, stimulation of PtdSer synthesis and movement to CFMV were abolished. In contrast, changes in synthesis and transport of sphingomyelin (SM) or phosphatidylethanolamine (PtdEtn) were minor; total phosphatidylcholine (PtdCho) synthesis was below control levels. All phospholipids appeared in CFMV but PtdSer displayed a 6-fold increase relative to controls compared to 3-fold for SM, 2-fold for PtdCho and 1.8-fold for PtdEtn. Even greater effects on specificity of PtdSer synthesis, movement to CFMV and inhibition by z-VAD-fmk were observed in apoptotic cells induced by UV irradiation or tumor necrosis factor-alpha/cycloheximide treatment. Thus, PtdSer biosynthesis stimulated during apoptosis in U937 cells was specific for this phospholipid and was correlated with caspase-mediated exposure of PtdSer at the cell surface and preferential movement to vesicles during apoptosis.     

Early changes in intramitochondrial cardiolipin distribution during apoptosis.

Cardiolipin (CL) is essential for the functionality of several mitochondrial proteins. Its distribution between the inner and outer leaflet of the mitochondrial internal membrane is crucial for ATP synthesis. We have investigated alterations in CL distribution during the early phases of apoptosis. Using two classical models (staurosporine-treated HL-60 cells and tumor necrosis factor alpha-treated U937 cells), we found that in apoptotic cells CL moves to the outer leaflet of mitochondrial inner membrane in a time-dependent manner. This occurs before the appearance of apoptosis markers such as plasma-membrane exposure of phosphatidylserine, changes in mitochondrial membrane potential, DNA fragmentation, but after the production of reactive oxygen species. The exposure of a phospholipid on the outer surface during apoptosis thus occurs not only at the plasma membrane level but also in mitochondria, reinforcing the hypothesis of mitoptosis as a crucial regulating system for programmed cell death, also occurring in cancer cells after treatment with antineoplastic agents.