Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures

Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.     

Hexokinase II detachment from the mitochondria potentiates cisplatin induced cytotoxicity through a caspase-2 dependent mechanism

Cancer cells are frequently glycolytic and over-express hexokinase II (HXK II). In cancer cells, the majority of hexokinase II is localized to the mitochondria through interaction with the voltage dependent anion channel (VDAC). Disruption in the binding of hexokinase II to the mitochondria has been shown to promote mitochondrial injury provoked by pro-apoptotic proteins.

The present study demonstrates that cisplatin induces the PIDD (P53 induced protein with a death domain) dependent activation of caspase-2. In turn, caspase-2 cleaves and activates Bid, resulting in the oligomerization of Bak and the release of cytochrome c. Notably, the detachment of hexokinase II from the mitochondria markedly potentiates the onset of caspase-2 induced mitochondrial damage, thus resulting in a synergistic induction of cisplatin induced cytotoxicity.     

Mitochondrial involvement in tracheary element programmed cell death

The mitochondria pathway is regarded as a central component of some types of programmed cell death (PCD) in animal cells where specific signals cause the release of cytochrome c from mitochondria to trigger a proteolytic cascade involving caspases. However, plant cells lack canonical caspases, therefore a role for the mitochondria in programmed cell death in plant cells is not obvious. Using plant cells which terminally differentiate, we provide evidence supporting the involvement of mitochondria in PCD, however the release of cytochrome c is insufficient to trigger the PCD. Prior to execution of cellular autolysis initiated by the rupture of the large central vacuole to release sequestered hydrolases, mitochondria adopt a definable morphology, the inner membrane depolarizes prior to death, and cytochrome c is released from mitochondria. However, PCD can be blocked despite translocation of cytochrome c. These results suggest a role for the mitochondria in this PCD but do not support the current animal model for a causative role of cytochrome c in triggering PCD.     

The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana

Plant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DeltaPsim) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DeltaPsim loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DeltaPsim and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DeltaPsim loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DeltaPsim changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DeltaPsim alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DeltaPsim loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.     

Actin-based cellular framework for glucose signaling by Arabidopsis hexokinase1

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.Key words: Arabidopsis, F-actin, glucose signaling, hexokinase, hTalin, mitochondria, polysomes, protoplasts, transient expression assay, fluorescence microscopy     

A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed.     

Vesicle-associated membrane protein of Arabidopsis suppresses Bax-induced apoptosis in yeast downstream of oxidative burst

Programmed cell death (PCD) in many systems is controlled by relative amounts of the apoptosis-regulating proteins Bax and Bcl-2 through homo- or heterodimerization. Here we show that Bax-induced PCD of yeast was suppressed by transformation with a vesicle-associated membrane protein from Arabidopsis (AtVAMP), which was isolated by screening a cDNA expression library against sugar-induced cell death in yeast. AtVAMP expression blocked Bax-induced PCD downstream of oxidative burst. AtVAMP also prevented H(2)O(2)-induced apoptosis in yeast and in Arabidopsis cells. Reduced oxidation of lipids and plasma membrane proteins was detected in the AtVAMP-transformed yeast, suggesting improved membrane repair. Inhibition of intracellular vesicle trafficking by brefeldin A induced apoptosis from a sublethal concentration of H(2)O(2). No protection occurred by overexpression of the yeast homolog SCN2. However, efficient suppression of yeast PCD occurred by expression of a chimeric gene, composed of the conserved domains from yeast, fused to the variable N-terminal domain from Arabidopsis, resulting in exchange of the proline-rich N-terminal domain of SCN2 with a proline-poor Arabidopsis sequence. Our results suggest that intracellular vesicle traffic can regulate execution of apoptosis by affecting the rate of membrane recycling and that the proline-rich N-terminal domain of VAMP inhibited this process.     

Dimerization and processing of procaspase-9 by redox stress in mitochondria

We studied the mechanism of intra-mitochondrial death initiator caspase-9 activation by a redox response, in which hydrogen peroxide (H(2)O(2)) caused a subtle decrease in the inner membrane potential (Deltapsim) with little evidence of cytochrome c release. Initiation of the intra-mitochondrial autocleavage of procaspase-9 preceded the onset of caspase cascade induction in the cytosol. Purified mitochondria demonstrated procaspase-9 processing and releasing abilities when exposed to H(2)O(2). Bcl-2 overexpression caused accumulation of the active form caspase-9 in the mitochondria, rendering the cells resistant to the redox stress. Intriguingly, disulfide-bonded dimers of autoprocessed caspase-9 were generated in the mitochondria in the pre-apoptotic phase. Using a substrate-analog inhibitor, dimer formation of procaspase-9 was also detectable inside the mitochondria. Furthermore, thiol reductant thioredoxin blocked the caspase-9 activation step and the cell death induction. Thus, redox stress-responsive thiol-disulfide converting reactions in the mitochondrion seemed to mediate procaspase-9 assembly that allows autoprocessing. This study offers an explanation for the recent observation that Apaf-1-null cells can execute apoptosis, which can be blocked by Bcl-2, and supports the proposition that the cytochrome c-Apaf-1-procaspase-9 complex functions in the caspase amplification rather than in its initiation.     

Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes

Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.     

Involvement of the mitochondrial K(+)ATP channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures

Soybean suspension cell cultures were treated by H2O2 or nitric oxide (NO), to assess the mechanism leading to programmed cell death (PCD). Hydrogen peroxide (5 mM) induced PCD. Cells become necrotic at 20 mM H2O2, with cells exhibiting intermediate hallmarks before that (necrapoptotic cells). The level of ATP and of glucose-6-phosphate remained constant in cells undergoing PCD, while it decreased significantly in the necrotic ones. Mitochondria, isolated from 5 mM H2O2-treated (apoptotic) cells, showed that succinate-dependent oxygen consumption was slightly uncoupled, and the electrical potential difference (delta psi) weakly decreased. The addition of KCl to the delta psi formed determined a partial dissipation, which was higher than the dissipation observed in mitochondria from control cells. The addition of cyclosporin A (CsA) to de-energized mitochondria also induced delta psi formation, due to a K+ efflux from the matrix, which was decreased in mitochondria from treated cells. The same pattern of response was also observed in mitochondria isolated from 1 mM sodium nitroprusside (NO)-treated cells, exhibiting apoptotic symptoms. In mitochondria isolated from 20 mM H2O2-treated (necrotic) cells, succinate-dependent oxygen consumption was completely uncoupled, delta psi generation significantly inhibited, and CsA-dependent delta psi formation prevented. In addition, mitochondria isolated from control cells still underwent swelling, which was partially or completely prevented in mitochondria isolated from apoptotic or necrotic cells, respectively. The moderate swelling was accompanied by a slight rupture of the outer membrane and by a release of cytochrome c. These results point to the involvement of a K(+)ATP channel during the manifestation of PCD induced by H2O2 or NO in plants.     

Carbon Source-Dependent Phosphorylation of Hexokinase PII and Its Role in the Glucose-Signaling Response in Yeast

The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PII is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, and cid1/glc7. Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells with λ-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2 (S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.     

Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.

Proapoptotic proteins such as Bax, undergo translocation to the mitochondria during apoptosis, where they mediate the release of intermembrane space proteins including cytochrome c. Bax binds to the voltage-dependent anion channel (VDAC). VDAC is a beta-barrel protein located in the outer mitochondrial membrane. In planar lipid bilayers, Bax and VDAC form a channel through which cytochrome c can pass. Hexokinase II (HXK II) also binds to VDAC. HXK II catalyzes the first step of glycolysis and is highly expressed in transformed cells, where over 70% of it is bound to the mitochondria. The present study demonstrates that HXK II interferes with the ability of Bax to bind to mitochondria and release cytochrome c. Detachment of HXK II from the mitochondria-enriched fraction isolated from HeLa cells promoted the binding of recombinant Bax-Delta19 and subsequent cytochrome c release. Similarly, the addition of recombinant HXK II to the mitochondria-enriched fraction isolated from hepatocytes, cells that do not express HXK II endogenously, prevented the ability of recombinant Bax-Delta19 to bind to the mitochondria and promote cytochrome c release. Similar results were found in intact cells, in which the detachment of mitochondrial bound HXK II or its overexpression potentiated and inhibited, respectively, Bax-induced mitochondrial dysfunction and cell death.     

VDAC is a conserved element of death pathways in plant and animal systems

Programmed cell death (PCD) is very much a part of plant life, although the underlying mechanisms are not so well understood as in animals. In animal cells, the voltage-dependent anion channel (VDAC), a major mitochondrial outer membrane transporter, plays an important role in apoptosis by participating in the release of intermembrane space proteins. To characterize plant PCD pathways by investigating the function of putative components in a mammalian apoptotic context, we have overexpressed a rice VDAC (osVDAC4) in the Jurkat T-cell line. Overexpression of osVDAC4 induces apoptosis, which can be blocked by Bcl-2 and the VDAC inhibitor DIDS. Modifying endogenous VDAC function by DIDS and hexokinase II (HxKII) in Jurkat cells inhibits mitochondria-mediated apoptotic pathways. Finally, we show that DIDS also abrogates heat-induced PCD in cucumber cotyledons. Our data suggest that VDAC is a conserved mitochondrial element of the death machinery in both plant and animal cells.     

Programmed cell death during pollination-induced petal senescence in petunia

Petal senescence, one type of programmed cell death (PCD) in plants, is a genetically controlled sequence of events comprising its final developmental stage. We characterized the pollination-induced petal senescence process in Petunia inflata using a number of cell performance markers, including fresh/dry weight, protein amount, RNA amount, RNase activity, and cellular membrane leakage. Membrane disruption and DNA fragmentation with preferential oligonucleosomal cleavage, events characteristic of PCD, were found to be present in the advanced stage of petal senescence, indicating that plant and animal cell death phenomena share one of the molecular events in the execution phase. As in apoptosis in animals, both single-stranded DNase and double-stranded DNase activities are induced during petal cell death and are enhanced by Ca(2+). In contrast, the release of cytochrome c from mitochondria, one commitment step in signaling of apoptosis in animal cells, was found to be dispensable in petal cell death. Some components of the signal transduction pathway for PCD in plants are likely to differ from those in animal cells.     

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd²⁺ treatment. NO contributed caspase-3-like protease activation in Cd²⁺ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd²⁺-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd²⁺-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd²⁺-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd²⁺-induced plant PCD.     

The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.     

Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells

Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.     

植物细胞程序性死亡的分类和膜通透性调控蛋白研究进展

程序性细胞死亡是由基因调控的贯穿于真核细胞生理和发育过程的细胞自杀行为。动物细胞的程序性死亡分成3类凋亡、自噬和坏死;线粒体和溶酶体分别在前两个过程中起关键作用。关于植物细胞程序性死亡的分类还存在很多争议,焦点是植物是否有细胞凋亡这种形式,核心问题是植物细胞的线粒体外膜上没有Bcl-2家族的膜通透性调控蛋白。近年,程序性细胞死亡也在细菌中发现,LrgAB家族的膜通透性调控蛋白起着重要作用。最近的研究表明,植物叶绿体外被膜上也有LrgAB家族的同源蛋白,它们在控制叶绿体发育和程序性细胞死亡方面起重要作用。因此,叶绿体在植物细胞死亡调控中的作用应该更加受到关注。     

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.