Protocol for rapid manipulation of mitochondrial morphology in living cells using inducible counter mitochondrial morphology (iCMM).

Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-by-step protocol for a chemically inducible dimerization system-based synthetic protein device, termed inducible counter mitochondrial morphology. This system allows artificial manipulation of mitochondrial morphology on a timescale of minutes in living mammalian cells. We also describe an AI-assisted imaging processing approach. For complete details on the use and execution of this protocol, please refer to Miyamoto et al., 2021.

CypD-mPTP axis regulates mitochondrial functions contributing to osteogenic dysfunction of MC3T3-E1 cells in inflammation.

Bone is a dynamic organ, the bone-forming osteoblasts and bone-resorbing osteoclasts form the physiological basis of bone remodeling process. During pathological process of numerous inflammatory diseases, these two aspects are uncoupled and the balance is usually tipped in favor of bone destruction. Evidence suggests that the inflammatory destruction of bone is mainly attributed to oxidative stress and is closely related to mitochondrial dysfunction. The mechanisms underlying osteogenic dysfunction in inflammation still need further investigation. Reactive oxygen species (ROS) is associated with mitochondrial dysfunction and cellular damage. Here, we reported an unexplored role of cyclophilin D (CypD), the major modulator of mitochondrial permeability transition pore (mPTP), and the CypD-mPTP axis in inflammation-induced mitochondrial dysfunction and bone damage. And the protective effects of knocking down CypD by siRNA interference or the addition of cyclosporin A (CsA), an inhibitor of CypD, were evidenced by rescued mitochondrial function and osteogenic function of osteoblast under tumor necrosis factor-α (TNF-α) treatment. These findings provide new insights into the role of CypD-mPTP-dependent mitochondrial pathway in the inflammatory bone injury. The protective effect of CsA or other moleculars affecting the mPTP formation may hold promise as a potential novel therapeutic strategy for inflammation-induced bone damage via mitochondrial pathways.

The physiological determinants of drug-induced lysosomal stress resistance.

Many weakly basic, lipophilic drugs accumulate in lysosomes and exert complex, pleiotropic effects on organelle structure and function. Thus, modeling how perturbations of lysosomal physiology affect the maintenance of lysosomal ion homeostasis is necessary to elucidate the key factors which determine the toxicological effects of lysosomotropic agents, in a cell-type dependent manner. Accordingly, a physiologically-based mathematical modeling and simulation approach was used to explore the dynamic, multi-parameter phenomenon of lysosomal stress. With this approach, parameters that are either directly involved in lysosomal ion transportation or lysosomal morphology were transiently altered to investigate their downstream effects on lysosomal physiology reflected by the changes they induce in lysosomal pH, chloride, and membrane potential. In addition, combinations of parameters were simultaneously altered to assess which parameter was most critical for recovery of normal lysosomal physiology. Lastly, to explore the relationship between organelle morphology and induced stress, we investigated the effects of parameters controlling organelle geometry on the restoration of normal lysosomal physiology following a transient perturbation. Collectively, our results indicate a key, interdependent role of V-ATPase number and membrane proton permeability in lysosomal stress tolerance. This suggests that the cell-type dependent regulation of V-ATPase subunit expression and turnover, together with the proton permeability properties of the lysosomal membrane, is critical to understand the differential sensitivity or resistance of different cell types to the toxic effects of lysosomotropic drugs.

Editor's Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (ß-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.

Sar1 GTPase Activity Is Regulated by Membrane Curvature.

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5'-[(ß,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.

DNA Gyrase Is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana.

The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides.

Downregulation of OPA3 is responsible for transforming growth factor-ß-induced mitochondrial elongation and F-actin rearrangement in retinal pigment epithelial ARPE-19 cells.

Transforming growth factor-ß signaling is known to be a key signaling pathway in the induction of epithelial-mesenchymal transition. However, the mechanism of TGF-ß signaling in the modulation of EMT remains unclear. In this study, we found that TGF-ß treatment resulted in elongation of mitochondria accompanied by induction of N-cadherin, vimentin, and F-actin in retinal pigment epithelial cells. Moreover, OPA3, which plays a crucial role in mitochondrial dynamics, was downregulated following TGF-ß treatment. Suppression of TGF-ß signaling using Smad2 siRNA prevented loss of OPA3 induced by TGF-ß. Knockdown of OPA3 by siRNA and inducible shRNA significantly increased stress fiber levels, cell length, cell migration and mitochondrial elongation. In contrast, forced expression of OPA3 in ARPE-19 cells inhibited F-actin rearrangement and induced mitochondrial fragmentation. We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin. Depletion of Mfn1 blocked the increase in cell length during TGF-ß-mediated EMT. These results collectively substantiate the involvement of mitochondrial dynamics in TGF-ß-induced EMT.

Carvacrol and 1,8-cineole alone or in combination at sublethal concentrations induce changes in the cell morphology and membrane permeability of Pseudomonas fluorescens in a vegetable-based broth.

This study aimed to investigate the effects of sublethal concentrations of carvacrol (CAR) and 1,8-cineole (CIN) alone and in combination on the morphology, cell viability and membrane permeability of Pseudomonas fluorescens ATCC 11253 cultivated in a vegetable-based broth. Transmission and scanning electron microscopy images of bacterial cells exposed to CAR and CIN alone or in combination showed marked ultrastructural changes after 1h of exposure. These changes included shrunken protoplasm, discontinuity of the outer and cytoplasmic membranes and leakage of the intracellular material. Confocal scanning laser microscopy images corroborated the electron microscopy data, showing a decrease in the number of SYTO-9 cells (intact cells) with a concomitant increase in the number of PI-positive cells (dead cells). All of these morphological changes are indicative of increased membrane permeability and the loss of bacterial envelope integrity, which ultimately lead to cell death. The combination of sublethal concentrations of CAR and CIN could be applied to inhibit the growth of P. fluorescens on vegetables.

Protection of exenatide for retinal ganglion cells with different glucose concentrations.

Exendin-4 is a peptide resembling glucagon-like peptide-1 (GLP-1), which has protective effects on nerve cells. However, the effects of Exendin-4 on retinal ganglion cells (RGC) are still under clear. The purpose of the present study is to demonstrate that exenatide prevents high- or low-glucose-induced retinal ganglion cell impairment. We observed the expression of GLP-1R in RGC-5 cells by immunofluorescence and Western blot. To investigate the effect of exenatide on RGC-5 cells incubated different glucose concentrations, CCK-8 measured the survival rates and electron microscopy detected cellular injury. The expression levels of Bcl-2 and Bax were analyzed by immunocytochemistry and Western blot. Exenatide protects RGC-5 from high- or low-glucose-induced cellular injury and the optimum concentration was 0.5µg/ml. Exenatide can inhibit high- or low-glucose-induced mitochondrial changes. Exenatide protects RGC-5 from high- or low-glucose-induced Bax increased and Bcl-2 decreased. Furthermore, the protective effect of exenatide could be inhibited by Exendin (9-39). These findings indicate that exenatide shows a neuroprotective effect for different glucose concentrations-induced RGC-5 cells injury. Exenatide could protect RGC-5 cells from degeneration or death, which may protect retinal function and have a potential value for patients with diabetic retinopathy.

Inhibition of glycoprotein synthesis in the endoplasmic reticulum as a novel anticancer mechanism of (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG) has been found to trigger the unfolded protein response (UPR) likely due to the inhibition of glucosidase II, a key enzyme of glycoprotein processing and quality control in the endoplasmic reticulum (ER). These findings strongly suggest that EGCG interferes with glycoprotein maturation and sorting in the ER. This hypothesis was tested in SK-Mel28 human melanoma cells by assessing the effect of EGCG and deoxynojirimycin (DNJ) on the synthesis of two endogenous glycoproteins. Both tyrosinase and vascular endothelial growth factor (VEGF) protein levels were remarkably reduced despite unaltered mRNA expression in EGCG- or DNJ-treated cells compared to control. The hindrance of tyrosinase and VEGF protein synthesis could be prevented by proteasome inhibitor, lactacystine. Collectively, our results support that glucosidase II inhibitor EGCG interferes with protein processing and quality control in the ER, which diverts tyrosinase, VEGF, and likely other glycoproteins towards proteasomal degradation. This mechanism provides a novel therapeutic approach in dermatology and might play an important role in the antitumor effect or hepatotoxicity of EGCG.

Testosterone-induced hypertrophy, fibrosis and apoptosis of cardiac cells--an ultrastructural and immunohistochemical study.

BACKGROUND: Androgen abuse is an increasing problem amongst professional and amateur athletes. Moreover, testosterone, apart from its widely accepted indications, is used for a variety of other indications such as aging and ischemia. Its actions are mainly attributed to a specific genomic mechanism through the androgen receptor, but emerging evidence reveals non-genomic effects as well. The use of androgens has been linked with several adverse effects. The purpose of this study was to examine the effects of testosterone on the morphology and the ultrastructure of the myocardium and to investigate the possible role of apoptosis. MATERIAL/METHODS: We used 12 adult male Wistar rats, separated into 2 groups. Group A consisted of 6 rats that were administered high doses of testosterone enanthate, while group B consisted of 6 male Wistar rats that received placebo (normal saline) intramuscularly. After the last day of treatment, all rats were anesthetized and sacrificed, and the hearts were removed and processed for optical and electron microscopy and immunohistochemical detection of caspase-3, an apoptosis marker. RESULTS: We found significant myocardial hypertrophy along with abundant ultrastructural alterations. The immunohistochemical staining of the myocardial cells for caspase-3 was positive in group A (experimental group), which is interpreted as an activation of apoptosis by testosterone treatment. CONCLUSIONS: Testosterone abuse has serious adverse effects, including myocardial hypertrophy, myocardial fibrosis and activation of apoptosis. These findings need to be taken into account whenever androgens are prescribed to improve performance or as hormone therapy.

Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1.

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.

Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition.

Trichoplein/mitostatin (TpMs) is a keratin-binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)-dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca(2+)-dependent stimuli that require ER-mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria-ER juxtaposition.

Dynamics of lamin-A processing following precursor accumulation.

Lamin A (LaA) is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear membrane (INM) of the nuclear envelope (NE). Newly synthesized prelamin A (PreA) undergoes extensive processing involving C-terminal farnesylation followed by proteolysis yielding non-farnesylated mature lamin A. Different inhibitors of these processing events are currently used therapeutically. Hutchinson-Gilford Progeria Syndrome (HGPS) is most commonly caused by mutations leading to an accumulation of a farnesylated LaA isoform, prompting a clinical trial using farnesyltransferase inhibitors (FTI) to reduce this modification. At therapeutic levels, HIV protease inhibitors (PI) can unexpectedly inhibit the final processing step in PreA maturation. We have examined the dynamics of LaA processing and associated cellular effects during PI or FTI treatment and following inhibitor washout. While PI reversibility was rapid, with respect to both LaA maturation and associated cellular phenotype, recovery from FTI treatment was more gradual. FTI reversibility is influenced by both cell type and rate of proliferation. These results suggest a less static lamin network than has previously been observed.

Mitochondria isolated in nearly isotonic KCl buffer: focus on cardiolipin and organelle morphology.

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation.

Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain.

Mechanical forces and geometric constraints play critical roles in determining cell functionality and tissue development. Novel experimental methods are essential to explore the underlying biological mechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patterned substrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold is coated with a cell repulsive NCO-sP(EO-stat-PO) hydrogel which in turn is covalently patterned by fibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesive micro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cell culture conditions without unspecific adhesion. The applicability of our system is demonstrated by studying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with different orientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of 0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predominantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologically, myotube formation was incomplete after 4 days of culture, but actin striations were observed exclusively on the 45 degrees line patterns subjected to CTS, the direction of maximum shear strain.

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells.

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.

HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis.

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell death in mammalian and fission yeast cells, suggesting that Vpr may affect a conserved cellular process. It is unclear, however, whether Vpr-induced yeast cell death mimics Vpr-mediated apoptosis in mammalian cells. We have recently identified a number of Vpr suppressors that not only suppress Vpr-induced cell death in fission yeast, but also block Vpr-induced apoptosis in mammalian cells. These findings suggest that Vpr-induced cell death in yeast may resemble some of the apoptotic processes of mammalian cells. The goal of this study was to develop and validate a fission yeast model system for future studies of apoptosis. Similar to Vpr-induced apoptosis in mammalian cells, we show here that Vpr in fission yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes of mitochondrial membrane potential. Moreover, Vpr triggers production of reactive oxygen species (ROS), indicating that the apoptotic-like cell death might be mediated by ROS. Interestingly, Vpr induces unique morphologic changes in mitochondria that may provide a simple marker for measuring the apoptotic-like process in fission yeast. To verify this possibility, we tested two Vpr suppressors (EF2 and Hsp16) that suppress Vpr-induced apoptosis in mammalian cells in addition to a newly identified Vpr suppressor (Skp1). All three proteins abolished cell death mediated by Vpr and restored normal mitochondrial morphology in the yeast cells. In conclusion, Vpr-induced cell death in fission yeast resembles the mammalian apoptotic process. Fission yeast may thus potentially be used as a simple model organism for the future study of the apoptotic-like process induced by Vpr and other proapoptotic agents.

Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells.

The mechanisms of sodium selenite-induced cell death in cervical carcinoma cells were studied during 24 h of exposure in the HeLa Hep-2 cell line. Selenite at the employed concentrations of 5 and 50 micromol/L produced time- and dose-dependent suppression of DNA synthesis and induced DNA damage which resulted in phosphorylation of histone H2A.X. These effects were influenced by pretreatment of cells with the SOD/catalase mimetic MnTMPyP or glutathione-depleting buthionine sulfoximine, suggesting the significant role of selenite-generated oxidative stress. Following the DNA damage, selenite activated p53-dependent pathway as evidenced by the appearance of phosphorylated p53 and accumulation of p21 in the treated cells. Concomitantly, selenite activated p38 pathway but its effect on JNK was very weak. p53- and p38-dependent signaling led to the accumulation of Bax protein, which was preventable by specific inhibitors of p38 (SB 203580) and p53 (Pifithrin-alpha). Mitochondria in selenite-treated cells changed their dynamics (shape and localization) and released AIF and Smac/Diablo, which initiated caspase-independent apoptosis as confirmed by the caspase-3 activity assay and the low effect of caspase inhibitors z-DEVD-fmk and z-VAD-fmk on cell death. We conclude that selenite induces caspase-independent apoptosis in cervical carcinoma cells mostly by oxidative stress-mediated activation of p53 and p38 pathways, but other selenite-mediated effects, in particular mitochondria-specific ones, are also involved.