Annexin V staining during reperfusion detects cardiomyocytes with unique properties

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and further elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in approximately 3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.     

Cellular imaging assay for early evaluation of an apoptosis inducer

The objective of this study was to propose a feasibility of a cellular imaging assay as an alternative to the conventional cytotoxicity assay, such as MTS assay, for apoptosis monitoring. As an apoptosis monitoring parameter, affinity interaction between phosphatidylserine (PS) and annexin V was chosen. First, the specific binding affinity between annexin V and PS in phospholipid bilayers consisting of various molar (0–15%) composition of PS was measured using a surface plasmon resonance biosensor. As PS composition increased, the binding level of annexin V increased proportionally. Second, various concentrations (0.1–10 μM) of staurosporine were used as to induce apoptosis and introduced to MCF-7 breast carcinoma cells. The cellular fluorescence images from annexin V-FITC conjugate were obtained by confocal microscopy, and their fluorescence intensities were quantified by image scanning. Dose–apoptosis (or cell death) relationships were very similar to those from MTS and FACS assays. In summary, our cellular imaging method could serve as a quicker and simpler alternative to MTS (end point assay) and FACS (flow cytometry) to screen potential apoptosis inducers.     

Assessment of cardiovascular apoptosis in the isolated rat heart by magnetic resonance molecular imaging

Apoptosis, an active process of cell self-destruction, is associated with myocardial ischemia. The redistribution of phosphatidylserine (PS) from the inner to the outer leaflet of the cell membrane is an early event in apoptosis. Annexin V, a protein with high specificity and tight binding to PS, was used to identify and localize apoptosis in the ischemic heart.Fluorescein-labeled annexin V has been used routinely for the assessment of apoptosis in vitro. For the detection of apoptosis in vivo, positron emission tomography and single-photon emission computed tomography have been shown to be suitable tools. In view of the relatively low spatial resolution of nuclear imaging techniques, we developed a high-resolution contrast-enhanced magnetic resonance imaging (MRI) method that allows rapid and noninvasive monitoring of apoptosis in intact organs. Instead of employing superparamagnetic iron oxide particles linked to annexin V, a new T1 contrast agent was used. To this effect, annexin V was linked to gadolinium diethylenetriamine pentaacetate (Gd-DTPA)-coated liposomes.The left coronary artery of perfused isolated rat hearts was ligated for 30 min followed by reperfusion. T(1) and T(2)* images were acquired by using an 11.7-T magnet before and after intracoronary injection of Gd-DTP-labeled annexin V to visualize apoptotic cells. A significant increase in signal intensity was visible in those regions containing cardiomyocytes in the early stage of apoptosis. Because labeling of early apoptotic cell death in intact organs by histological and immunohistochemical methods remains challenging, the use of Gd-DTPA-labeled annexin V in MRI is clearly an improvement in rapid targeting of apoptotic cells in the ischemic and reperfused myocardium.     

Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons through TNF-alpha-mediated induction of Bax and phosphorylated c-Jun NH(2)-terminal kinase

Cocaine exposure results in aberrant outgrowth and decreased survival for locus coeruleus (LC), a noradrenergic population of neurons that putatively regulates attentional function; however, the underlying mechanisms for these events are not known. We previously showed that cocaine exposure in vitro activates pro-apoptotic Bax, caspase-9, and caspase-3 in LC neurons dissected from embryonic day 14 rats, implicating that apoptosis may be orchestrated via signal transduction events. In the current study in vitro, we examined upstream events to determine the role of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on LC signal transduction, because cocaine exposure to LC neurons triggered TNF-alpha expression at 30 min as measured by ELISA. Exposure of LC neurons to recombinant-TNF-alpha resulted in decreased metabolic activity, an indicator of reduced neuron viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], and increased apoptosis (terminal deoxynucleotidyl transferase-mediated DNA nick end labeling assay). Pro-apoptotic caspase-3 was induced by cocaine starting at 30 min. Recombinant-TNF-alpha induced caspase-3 activity earlier than cocaine (15 and 20 min). The caspase-3 levels were significantly reduced when cocaine and TNF-alpha were combined with neutralizing-TNF-alpha (nTNF-alpha), respectively. Further, cocaine alone elevated phospho-p38-mitogen-activated protein kinases that persisted when combined with nTNF-alpha. However, both cocaine and TNF-alpha independently increased phospho-c-Jun NH(2)-terminal kinase and Bax levels at concurrent time periods (30 min and 1 h), and this elevation was attenuated in the presence of nTNF-alpha. These simultaneous molecular events triggered by cocaine and TNF-alpha implicate a potential apoptotic signal transduction pathway via induction of phospho-c-Jun NH(2)-terminal kinase and Bax that may lead to caspase-3 activation and apoptosis in cocaine-exposed fetal LC neurons.     

Non-fusion expression in Escherichia coli: Single-step purification of recombinant human annexin A5 for detection of apoptosis

Recombinant human annexin A5 (rh-annexin A5) was originally used to detect early stages of apoptosis in vitro. With the development of radioactive labeling and imaging techniques, annexin A5 labeled with radioactive markers can play a more important role in monitoring apoptotic cells in vivo. To obtain highly pure rh-annexin A5 with an easy and inexpensive purification approach, we constructed a pJLA503-annexin A5 expression plasmid, which could overexpress human annexin A5 in a soluble form in Escherichia coli. Then a novel purification method based on Ca2+-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of rh-annexin A5 was increased to 98%. To confirm the PS affinity of rh-annexin A5 produced by this purification protocol, a simple and reliable lipid membrane model was prepared and used in the binding test. As a probe to detect apoptosis, the fluorescein isothiocyanate-labeled rh-annexin A5 was incubated with apoptotic cells. The results showed that the labeled rh-annexin A5 possessed high affinity for PS molecule and was able to indicate different apoptotic states.     

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.     

Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.     

A new peptidic vector for molecular imaging of apoptosis, identified by phage display technology

Phosphatidylserine (PS) exposure on the cell surface is an early marker of apoptosis. To select PS binding peptides as vectors of contrast agents to image apoptosis, a phage library has been exposed to perfused mouse livers. Phages not retained on control livers during the first perfusions were used for selections on apoptotic livers in a second series of perfusions. Four selected phages were further evaluated for binding to PS-coated enzyme-linked immunosorbent assay (ELISA) plates. They presented an apparent affinity constant (Ka app) for PS ranging from 6.08x10(10) M to 1.62x10(11)M. These phages did not bind to phosphatidylcholine, and competition with annexin V confirmed their specific interaction with PS. The phage with the highest affinity-bound PS in ELISA with a Ka app=(1.6+/-0.2)x10(11)M. It carried the TLVSSL peptide that was synthesized. Specific competition with annexin V and with the synthetic peptide was performed and confirms the specificity of the interaction.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.     

Site-specific labeling of annexin V with F-18 for apoptosis imaging

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.     

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.     

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca²⁺, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[³⁵S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.     

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.     

Involvement of caspase-3-like protein in rapid cell death of Xanthomonas

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the aetiological agent of bacterial pustule disease of soybean, as well as some other strains of Xanthomonas including X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961, exhibited post-exponential rapid cell death (RCD) in Luria-Bertani (LB) medium. RCD was not displayed by Xanthomonas strains while growing in starch medium. Addition of starch to LB culture of XcgAM2 at any point of incubation during the exponential growth was found to arrest the onset of RCD. RCD in this organism was found to be associated with the synthesis of an endogenous enzyme similar to human caspase-3, a known marker of apoptosis in eukaryotes. On sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) the XcgAM2 caspase appeared to run along a 55 kDa protein molecular weight marker. The caspase-3-like protein was detected in all Xanthomonas strains tested. RCD was not detected in Escherichia coli cultures in LB medium. The caspase-3-like enzyme activity or pro-tein was also found to be absent in this bacterium. Caspase-3-like protein or Xanthomonas caspase was detected only in the cells of XcgAM2 growing in LB medium and not in those growing in starch medium. The Xanthomonas caspase protein appeared in cells at around 4 h of incubation, and peaked at around 24 h, before finally disappearing at around 54 h of incubation. However, caspase enzyme activity was detected only 12-13 h after incubation and peaked around 18-20 h. Addition of starch at the beginning or during the period of exponential growth in LB cultures of XcgAM2 terminated the synthesis of this protein. It is presumed that starch acted as the repressor of biosynthesis of the Xanthomonas caspase, thereby preventing the organism from undergoing RCD. The cells undergoing RCD also displayed the other markers of eukaryotic apoptosis. These included binding of annexin V to plasma membrane of cells undergoing RCD and the presence of nicked DNA in culture supernatant as evidenced by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay. Caspase-negative mutants of XcgAM2 did not display post-exponential RCD. The importance of RCD in Xanthomonas life cycle is not yet clear, however the phenomenon appears to have similarities with eukaryotic apoptosis.     

Preparation and bioevaluation of 99mTc-HYNIC-annexin B1 as a novel radioligand for apoptosis imaging

Annexin B1, a novel Ca²⁺-dependent PS-binding protein, has been shown to have a high affinity for PS exposed on the surface of apoptotic cells. To develop and bioevaluate an annexin B1 based PS-targeting radiotracer, annexin B1 was radiolabeled with ^99mTc using HYNIC as a bifunctional chelator. Binding assays with activated platelets and apoptotic SP2/0 cells were carried out to evaluate the in vitro biological activity of ^99mTc-HYNIC-annexin B1. Biodistribution of this radioligand was studied in normal mice. Dexamethasone-induced murine thymus apoptosis and fas-mediated murine liver apoptosis models were used to investigate the ability of radiolabeled annexin B1 to detect apoptosis in vivo. The labeling procedure yielded a compound with up to 98% radiochemical purity and good in vitro stability. The in vitro binding assays indicated that ^99mTc-HYNIC-annexin B1 retain its PS-binding activity. Biodistribution of the compound in mice showed that ^99mTc-HYNIC-annexin B1 is rapidly cleared from the blood and predominantly accumulates in the kidney. The marked increase in dexamethasone-treated murine thymus uptake and fas-mediated murine liver uptake correlated with histologic evidence of apoptosis. These data suggested that ^99mTc-HYNIC-annexin B1 retain its in vitro and in vivo biological activities. This radiotracer may therefore be useful as a novel radioligand for the noninvasive detecting of PS externalization associated with apoptosis.     

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.     

Development and characterization of annexin V mutants with endogenous chelation sites for (99m)Tc

[(99m)Tc]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the preparation and labeling of annexin V for nuclear-medicine studies, we have investigated the addition of peptide sequences that will directly form endogenous chelation sites for (99m)Tc. Three mutant molecules of annexin V, called annexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These molecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis in a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl(2) as reducing agent and glucoheptonate as exchange agent, all three proteins could be labeled with (99m)Tc to specific activities of at least 50-100 microCi/microg. The proteins retained membrane binding activity after the radiolabeling procedure, and quantitative analysis indicated a dissociation constant (K(d)) of 7 nmol/L for the annexin V-117 mutant. The labeling reaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro. Proteins labeled to a specific activity of 25-100 microCi/microg were injected intravenously in mice at a dose of 100 microg/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with previous results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [(99m)Tc]annexin V-117. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc without altering its high affinity for cell membranes. These annexin V derivatives may be useful for in vivo imaging of cell death.     

The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.     

Sarcophine-diol,a Chemopreventive Agent of Skin Cancer,Inhibits Cell Growth and Induces Apoptosis through Extrinsic Pathway in Human Epidermoid Carcinoma A431 Cells

Sarcophine-diol (SD), a structural modifications of sarcophine, has shown chemopreventive effects on 7,12-dimethylbenz(a)anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor developments in mice. Tumorigenesis is associated with uncontrolled cell growth and loss of apoptosis. In the present study, the effects of SD on cell growth and apoptosis in human epidermoid carcinoma A431 cells were determined to assess whether SD could inhibit cell growth and/or induce apoptosis, thus elucidating possible mechanism of action. MTT assay was used for cell viability; bromodeoxyuridine incorporation assay was used for cell proliferation; fluorescence-activated cell sorting analysis of annexin V/propidium iodide staining and TUNEL assay were used for determining apoptotic cells; Western blot analysis was used for determining the expression of caspase-3 and colorimetric caspase activity assays were used for determination of caspase-3, -8, and -9 activity. The results showed that SD treatment at concentration of 200 to 600 µM resulted in a concentration-dependent decrease in cell viability and cell proliferation in A431 cells, which largely inhibited cell growth. Sarcophine-diol treatment induced a strong apoptosis and significantly (P < .05) increased DNA fragmentation in A431 cells. Furthermore, SD treatment significantly (P < .05) increased the activity and expression of caspase-3 through activation of upstream caspase-8 in A431 cells rather than the activation of caspase 9. Sarcophine-diol treatment is relatively much less cytotoxic in monkey kidney normal CV-1 cells. These results suggest that SD decreases cell growth and induces apoptosis through caspase-dependent extrinsic pathway in A431 cells, and this may contribute to its overall chemopreventive effects in mouse skin cancer models.     

Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.