Mitochondria can orchestrate sex differences in cell fate of vascular smooth muscle cells from rats.

BACKGROUND: In basal conditions, vascular smooth muscle cells freshly isolated from aortas of male and female rats display marked sex differences in terms of redox balance and susceptibility to ultraviolet radiation-induced cell death. In particular, in the same experimental conditions, cells from male rats are more susceptible to oxidative stress and underwent apoptosis, while cells from female rats underwent premature senescence. In the present work, the mechanism involved in cell fate after ultraviolet radiation exposure is investigated. METHODS: Vascular smooth muscle cells, isolated from the descending aortas of both female and male Sprague-Dawley young rats, were exposed to a single sub-cytotoxic dose of ultraviolet radiation (200 mJ/cm(2)). The distribution and the expression of molecules involved in cell survival and mitochondrial physiology were evaluated by static and flow cytometry using commercial kits and antibodies. Statistical analyses were performed by using Student's t test and two-way ANOVA. RESULTS: After exposure to ultraviolet radiation, an upregulation of survival proteins such as BclxL, survivin and the presence in the nucleus of NF-κB were found in cells from females. Conversely, pro-apoptotic proteins such as Bax, caspase-3, and caspase-9 as well as loss of mitochondrial membrane potential were found in cells from male rats. CONCLUSIONS: Our results suggest that (i) mitochondria, being producers of ROS, can orchestrate sex differences in cell fate of VSMC and (ii) mitochondrial dysfunction may be a significant mechanism by which cardiovascular risk factors lead to the formation of vascular lesions in a sex-specific way.

Increased frequency of immunoglobulin (Ig)A-secreting cells following Toll-like receptor (TLR)-9 engagement in patients with Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.

Botulinum neurotoxin type A counteracts neuropathic pain and facilitates functional recovery after peripheral nerve injury in animal models.

A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100ß and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain.

The Rac-activating toxin cytotoxic necrotizing factor 1 oversees NK cell-mediated activity by regulating the actin/microtubule interplay.

The cell cytoskeleton is widely acknowledged as a master for NK cell function. Specifically, actin filaments guide the NK cell binding to target cells, engendering the formation of the so-called immunological synapse, while microtubules direct the killer behavior. All these cytoskeleton-dependent activities are competently governed by the Rho GTPases, a family of regulatory molecules encompassing the three different subfamilies, Rho, Rac, and Cdc42. By using a Rac GTPase-activating bacterial protein toxin from Escherichia coli named cytotoxic necrotizing factor 1 (CNF1), we obtained results supporting the activation of Rac GTPase as a booster for effector cell-binding efficiency, recruitment ability, and, consequently, cytotoxicity. In particular, the augmented killer capacity of CNF1-treated NK cells was associated with the increased expression of certain cell adhesion or activation-associated molecules and the reshaping of the actin and microtubule networks. Importantly, CNF1 counteracted the activity exerted by toxins disrupting the cytoskeletal architecture. Hence, the activation of Rho GTPases, particularly Rac, induced by CNF1, appears to orchestrate a dynamic cross talk between microtubules and actin filaments, leading to a fruitful NK cell activity and polarization state. Our findings suggest that protein toxins might be viewed as modulators of NK cell cytotoxic activity and could possibly be regarded as useful pharmacological tools for certain Rho-linked immune diseases in the near future.

Cultured cells as a model system for the study of UV-induced cytotoxicity.

In vivo, UV radiation induces a series of morphological and ultrastructural alterations in human epidermis. These and other changes eventually lead to well described pathological modifications including erythema and cancer. Morphological alterations are easier to detect in cultured cells, such as human keratinocytes or other epithelial cells. One can use different intensities of different radiation types (UV-A, -B and -C) and expose cell monolayers to different doses. In these experimental conditions it is possible to evaluate radiation risks and to provide additional information thanks to the reproducibility and the enormous amplification of the phenomena normally occurring in vivo. Alterations observed in structural studies can be summarized as the succession of the following events: (i) cell retraction with loss of cell-cell interactions; (ii) surface blebbing; and eventually (iii) cell death. Cytoskeletal components play a key role in this cascade. Morphogenesis of these changes can be ascribed to oxidative modifications due to reactive oxygen species formation following radiation that can modify both cell membrane and cytoskeleton. The use of in vitro systems can be of great relevance in the understanding of the pathogenetic mechanisms of UV radiation changes and to determine possible drugs capable of counteracting UV-mediated subcellular pathology.

Redox imbalance and immune functions: opposite effects of oxidized low-density lipoproteins and N-acetylcysteine.

This study investigates the in vitro effects of oxidized low-density lipoproteins (ox-LDL), 'physiological' pro-oxidants, N-acetylcysteine (NAC), a free radical scavenger and glutathione precursor, and their combination on human peripheral blood mononuclear cell functions. We found that treatment with ox-LDL induced a significant down-regulation of proliferative response to mitogens, antigens and interleukin-2. Lipid extracts from ox-LDL were able to reproduce the same effect as the lipoprotein. On the other hand, NAC exposure induced a significant up-regulation of proliferative responses to all the stimuli used. Moreover, we showed that natural killer (NK) cell-mediated cytotoxic activity was significantly down-regulated by ox-LDL while treatment with NAC induced a significant up-regulation of NK-cell activity. Finally, we found that ox-LDL and NAC exerted opposite effects on the cytokine network, interfering both at the protein secretion level and the messenger RNA synthesis level. More importantly, when NAC was used in combination with ox-LDL the proliferative responses, NK-cell-mediated cytotoxic activity and cytokine production were restored to values comparable to controls. These data indicate that ox-LDL and NAC modulate immune functions, exerting opposite effects reflecting their pro-oxidant and antioxidant behaviours. Our results add new insights to the key role played by redox imbalance as a modulator of immune system homeostasis and suggest that an antioxidant drug such as NAC could be useful against pathologies associated with an increase in lipid peroxidation.

N-Acetylcysteine counteracts erythrocyte alterations occurring in chronic obstructive pulmonary disease.

A key role has been proposed for reactive oxygen species (ROS) in chronic obstructive pulmonary disease (COPD). Aim of the present work was to evaluate possible implications of ROS in the integrity and function of the cell type mainly involved in oxygen uptake and delivery to the peripheral tissues: the erythrocyte. Red blood cells (RBCs) were thus collected from blood samples from COPD patients. Furthermore, blood samples from the same patients treated with the antioxidizing drug of widespread use in such disease i.e., N-acetylcysteine (NAC), were also considered. Morphometric and analytical cytology studies were then conducted. We report herein that: (i) alterations of RBC ultrastructure were detectable in RBCs from COPD patients, that (ii) relevant changes of spectrin cytoskeleton and glycophorin expression were also found and that (iii) NAC treatment was capable of significantly counteracting these changes. These results are consistent with a reappraisal of the role of RBCs in this disease.

Carboxyfullerenes protect human keratinocytes from ultraviolet-B-induced apoptosis.

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.

C60 carboxyfullerene exerts a protective activity against oxidative stress-induced apoptosis in human peripheral blood mononuclear cells.

C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a free-radical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-d-ribose (dRib) or TNF-alpha plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-d-ribose or TNF-alpha plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria.

Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis.

In mice, the Fas/Fas ligand (FasL) system has been shown to be involved in germ cell apoptosis. In the present study we evaluated the expression of Fas and Fas ligand (FasL) in fetal and adult human testis. Semiquantitative RT-PCR demonstrated the expression of Fas and FasL messenger ribonucleic acids in adult testis, but not in fetal testis (20-22 weeks gestation). In situ RT-PCR and immunohistochemistry experiments on adult human testis demonstrated the expression of FasL messenger ribonucleic acid and protein in Sertoli and Leydig cells, whereas the expression of Fas was confined to the Leydig cells and sporadic degenerating spermatocytes. The number of Fas-positive germ cells per 100 Sertoli cell nuclei was increased in 10 biopsies with postmeiotic germ cell arrest compared to 10 normal testis biopsies (mean, 3.82 +/- 0.45 vs. 2.02 +/- 0.29; P = 0.0001), but not in 10 biopsies with meiotic germ cell arrest (mean, 1.56 +/- 1.07). Fas and FasL proteins were not expressed in cases of idiopathic hypogonadotropic hypogonadism. Together, these findings may suggest that Fas/FasL expression in the human testis is developmentally regulated and under gonadotropin control. The increased germ cell expression of Fas in patients with postmeiotic germ cell arrest suggests that the Fas/FasL system may be involved in the quality control mechanism of the produced gametes.

Spectrin changes occur in erythrocytes from patients with Fanconi's anemia and their parents.

Fanconi's anemia (FA) is a clinically and genetically heterogeneous disease which has been hypothesized to be defective in the detoxification of reactive oxygen species. In this work we report the results obtained by morphometric analyses on the red blood cells (RBCs) from FA patients and their parents. We found that a high rate of erythrocytes from both homozygous and heterozygous subjects was significantly altered. RBCs underwent in fact cytoskeleton-dependent modifications, in particular of spectrin molecule, leading to cell shrinking and blebbing. We hypothesize that these changes may be the result of an oxidative imbalance that probably lead to alterations of RBC plasticity- and deformation-associated functions. Moreover, our results also suggest the possibility to identify FA carriers by the existence of RBC abnormalities.

Induction of apoptosis in caco-2 cells by wheat gliadin peptides.

Recent experimental evidence suggests that enterocyte apoptosis is greater than hitherto assumed and may be responsible for villous atrophy in coeliac disease. We have previously demonstrated that a small peptide (M.W. 1157.5 Da), identified as the sequence H(2)N-gln-gln-pro-gln-asp-ala-val-gln-pro-phe-COOH from durum wheat gliadin, is able to prevent K 562 (S) cell agglutination induced by the peptic-tryptic digests (PT) of prolamin fractions from the cereals which are not tolerated in coeliac disease (i.e. bread wheat, rye, barley and possibly oats), and toxic A-gliadin peptides in coeliac disease. In the present study we have investigated the effects of the bread wheat gliadin digest (PT) on apoptosis of Caco-2 cells and whether the '1157.5' Da peptide may in any way interfere with them. We evaluated both earlier biochemical and later morphological nuclear apoptotic events in the human colon adenocarcinoma cell line Caco-2. After 48 h exposure to the PT gliadin digest and the '1157.5' Da peptide, apoptosis was detected both for the early-stage apoptotic cells (adherent cells) and the late-stage apoptotic ones (detached cells which were floating in the culture medium). Exposure to the PT gliadin digest resulted in a high percentage of adherent cells that underwent cell death by apoptosis (about 30%), independent of the concentration range used; while the presence in the culture medium of peptide '1157.5' Da determined complete inhibition of cell death. On the other hand, morphological nuclear modifications observed in the floating cells showed a difference in the rate of the apoptosis dependent on the PT concentration, with partial protection in the presence of the peptide. These findings show an action of bread wheat gliadin peptides leading to cell death by apoptosis in the Caco-2 cell line and that the '1157.5' Da peptide is capable of preventing such an effect.

Cytoskeleton alterations of erythrocytes from patients with Fanconi's anemia.

Fanconi's anemia (FA) is a very rare genetically heterogeneous disease which has been hypothesized to be defective in the detoxification of reactive oxygen species. In this work we report the results obtained by morphometric and biochemical analyses on the red blood cells (RBCs) from FA patients. With respect to RBCs from healthy donors the following changes have been detected: (i) a variety of ultrastructural alterations, mainly surface blebbing typical of acanthocytes and stomatocytes; (ii) a significant quantitative increase of these altered forms; (iii) modifications of spectrin cytoskeleton network; (iv) an altered redox balance, e.g. a decreased catalase activity and significant variations in the GSSG/GSH ratio. We hypothesize that remodeling of the redox state occurring in FA patients results in cytoskeleton-associated alterations of red blood cell integrity and function.

C3-fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis.

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3-fullero-tris-methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration-dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4-induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.

Subcellular alterations induced by UV-oxidized low-density lipoproteins in epithelial cells can be counteracted by alpha-tocopherol.

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies. Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides. These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with UV-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell-cell and cell-substrate contacts, cell detachment and cell death by apoptosis. The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with alpha-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as alpha-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.

Decreased susceptibility to oxidative stress-induced apoptosis of peripheral blood mononuclear cells from healthy elderly and centenarians.

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma-glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells.

C3-fullero-tris-methanodicarboxylic acid protects epithelial cells from radiation-induced anoikia by influencing cell adhesion ability.

Anoikia is a type of apoptotic cell death that occurs in cells that are substrate-restricted in their growth. Buckminsterfullerenes represent a new class of chemical compounds with wide potential pharmacological antioxidant activity. In this report we provide the first demonstration that a water-soluble fullerene derivative, C3-fullero-tris-methanodicarboxylic acid, synthesized in our laboratories, is capable of inducing anoikia resistance in epithelial cells by a mechanism involving a 'trophic' effect on cell spreading-associated cytoskeletal components, i.e. on actin microfilaments.

Induction of interleukin-15 production by HIV-1 nef protein: a role in the proliferation of uninfected cells.

Several recent reports have provided evidence that Nef enhances human immunodeficiency virus HIV infectivity, and in vitro experiments with the nef gene have demonstrated the possible role of Nef in modulating immune responses. Exogenous Nef has been demonstrated to induce proliferation of normal human peripheral blood mononuclear cells (PBMC) and to enhance HIV-1 replication. The aim of this study was to evaluate the biological mechanisms by which Nef, used as exogenous protein, modulates cellular activation. We showed that exogenous Nef protein induces the proliferation of unstimulated and suboptimally stimulated normal human PBMC, while it has no effect on the proliferation of optimally stimulated PBMC. Moreover, the activating effect of exogenous Nef on PBMC proliferation was associated with an increase of IFN-gamma, TNF-alpha, and IL-6 production, while, surprisingly, IL-2 production was not affected by Nef. More importantly we showed, for the first time, that Nef exerts its activating effects on PBMC proliferation through IL-15 synthesis induction by monocyte/macrophage population. In conclusion, we found that exogenous Nef protein (i) induces activation of normal PBMC, increasing their proliferative response; (ii) modulates cytokine production; (iii) exerts its activating effects through IL-15 synthesis induction; and (iv) exerts these effects entering monocyte/macrophages. Our results might suggest that Nef enhances the rate of viral replication by a novel mechanism involving the production of IL-15.

Tyrosol, the major olive oil biophenol, protects against oxidized-LDL-induced injury in Caco-2 cells.

Experimental and clinical evidence suggest that oxidative stress causes cellular damage, leading to functional alterations of the tissue. Free radicals may thus play an important role in the pathogenesis of a number of human diseases. Among pro-oxidant agents, oxidized LDL lead to the production of cytotoxic reactive species, e.g., lipoperoxides, causing tissue injury and various subsequent pathologies including intestinal diseases. Thus, to analyze the oxidative damage induced by oxidized LDL to intestinal mucosa, we evaluated morphological and functional changes induced in the human colon adenocarcinoma cell line, Caco-2. In addition, we examined the protective effects exerted by tyrosol, 2-(4-hydroxyphenyl)ethanol, the major phenolic compound present in olive oil. Caco-2 cell treatment (24 and/or 48 h) with oxidized LDL (0.2 g/L) resulted in cytostatic and cytotoxic effects characterized by a series of morphological and functional alterations: membrane damage, modifications of cytoskeleton network, microtubular disorganization, loss of cell-cell and cell-substrate contacts, cell detachment and cell death. The oxidized LDL-induced alterations in Caco-2 cells were almost completely prevented by tyrosol which was added 2 h before and present during the treatments. Our results suggest that some biophenols, such as those contained in olive oil, may counteract the reactive oxygen metabolite-mediated cellular damage and related diseases, by improving in vivo antioxidant defenses.

Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells.

Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death. In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells. In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion. In particular, the expression of integrins such as alpha 5, alpha 6 and alpha v, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, alpha and beta catenins were significantly increased in cells exposed to CNF1. Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se. A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival. These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.