Simona Coppola

Oxidative Bax dimerization promotes its translocation to mitochondria independently of apoptosis

Bax is a cytosolic protein, which in response to stressing apoptotic stimuli, is activated and translocates to mitochondria, thus initiating the intrinsic apoptotic pathway. In spite of many studies and the importance of the issue, the molecular mechanisms that trigger Bax translocation are still obscure. We show by computer simulation that the two cysteine residues of Bax may form disulfide bridges, producing conformational changes that favor Bax translocation. Oxidative, nonapoptogenic treatments produce an up‐shift of Bax migration compatible with homodimerization, which is reverted by reducing agents; this is accompanied by translocation to mitochondria. Dimers also appear in pure cytosolic fractions of cell lysates treated with H2O2, showing that Bax dimerization may take place in the cytosol. Bax dimer‐enriched lysates support Bax translocation to isolated mitochondria much more efficiently than untreated lysates, indicating that dimerization may promote Bax translocation. The absence of apoptosis in our system allows the demonstration that Bax moves because of oxidations, even in the absence of apoptosis. This provides the first evidence that Bax dimerization and translocation respond to oxidative stimuli, suggesting a novel role for Bax as a sensor of redox imbalance.

Heart infarct in NOD-SCID mice: Therapeutic vasculogenesis by transplantation of human CD34+ cells and low dose CD34+KDR+ cells

Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast: specifically, the human cord blood (CB) CD34+KDR+ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. Particularly, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in the immunocompromised NOD‐SCID mouse model, and monitored the effects of myocardial transplantation of human CB CD34+ cells on cardiac function. Specifically, we compared the therapeutic effect of unseparated CD34+ cells vs. PBS and mononuclear cells (MNCs); moreover, we compared the action of the CD34+KDR+ cell subfraction vs. the CD34+KDR– subset. CD34+ cells significantly improve cardiac function after MI, as compared with PBS/MNCs. Similar beneficial actions were obtained using a 2‐log lower number of CD34+KDR+ cells, while the same number of CD34+KDR– cells did not have any effects. The beneficial effect of CD34+KDR+ cells may mostly be ascribed to their notable resistance to apoptosis and to their angiogenic action, since cardiomyogenesis was limited. Altogether, our results indicate that, within the CD34+ cell population, the CD34+KDR+ fraction is responsible for the improvement in cardiac hemodynamics and hence represents the candidate active CD34+ cell subset.

White cell apoptosis in packed red cells

BACKGROUND: After the removal of the buffy coat, packed red cell (RBC) transfusion units still contain white cells that may undergo apoptosis as a result of storage conditions (1‐6 degrees C). The aim of the present study was the evaluation of this phenomenon in view of the possible influence it may have on febrile nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: Three independent methods (microscopy, DNA electrophoresis, and cytometry) were used to evaluate apoptosis in white cells present in 13 RBC units. Of these units, 10 had been collected into CPD/saline‐adenine‐glucose‐mannitol and 3 into CPDA‐1; each bag was split in two parts, one of which was irradiated. RBCs were stored at 1 to 6 degrees C, and samples were periodically withdrawn for study. The proliferative capacity of stored lymphocytes was evaluated after phytohemagglutinin stimulation and tritiated thymidine incorporation. RESULTS: Apoptosis was found to occur in both granulocytes and lymphocytes, starting from the first 48 to 72 hours of storage. The choice of the anticoagulant‐preservative solution and the effect of irradiation did not influence the amount and the timing of the apoptotic phenomenon. Lymphocyte proliferative capacity was found to decrease sharply with storage time. CONCLUSION: Conditions of storage in RBCs induce consistent apoptosis in residual white cells. The possible clinical implications of the relationships between apoptosis and the induction of biologic response modifiers (that may cause interleukin‐mediated febrile non‐hemolytic transfusion reactions) and between apoptosis and immune reactions remain to be elucidated.

Glutathione depletion up-regulates Bcl-2 in BSO-resistant cells

Glutathione depletion by inhibition of its synthesis with buthionine sulfoximine (BSO) is a focus of the current research in antitumor therapy, BSO being used as chemosensitizer. We had previously shown that two human tumor cell lines (U937 and HepG2) survive to treatment with BSO: BSO can elicit an apoptotic response, but the apoptotic process is aborted after cytochrome c release and before caspase activation, suggesting the development of an adaptive response (FASEB J., 1999, 13, 2031–2036). Here, we investigate the mechanisms of such an adaptation. We found that following BSO, U937 up‐regulate Bcl‐2 mRNA and protein levels, by a mechanism possibly involving NF‐κB transcription factor; the increase in protein level is limited by a rapid decay of Bcl‐2 in BSO‐treated cells, suggesting that redox imbalance speeds up Bcl‐2 turnover. BSO‐dependent Bcl‐2 up‐regulation is associated with the ability to survive to BSO. Indeed, 1) its abrogation by CAPE or protein synthesis inhibition sensitizes U937 to BSO; 2) in a panel of four tumor lines, BSO‐resistant (U937, HepG2, and HGB1) but not BSO‐sensitive (BL41) cells can up‐regulate Bcl‐2 following GSH depletion; remarkably, only the latter are chemosensitized by BSO.

GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells

The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes.

Anti-apoptotic effect of HIV protease inhibitors via direct inhibition of calpain

Treatment with drugs designed to inhibit the HIV protease ameliorates immune functions in AIDS patients, reducing cell deletion by apoptosis even in the absence of inhibition of viral spread. This suggests that they interact with the intrinsic apoptotic signaling. We found that caspases, the main executioner of the apoptotic process, are not directly inhibited. In search for the mechanism responsible for their anti-apoptotic effect, we have found that indinavir and ritonavir are able to inhibit apoptosis only in those cell systems where apoptosis involves the activation of calpains. They directly inhibit a calpain-like activity expressed in lysates from apoptotic cells, to the same extent as commercially available calpain inhibitor 1. In in vitro assays with purified calpains, indinavir and ritonavir strongly inhibit m-calpain, and moderately mu-calpain. These results have great therapeutic implications, going beyond AIDS treatment, since many degenerative disorders involve abnormal calpain activation, indicating calpain as an ideal pharmacological target. Indinavir and ritonavir, potent m-calpain inhibitors, largely used since several years on humans without important negative side effects, may become powerful tools against those pathologies.

Multiple mechanisms for hydrogen peroxide-induced apoptosis.

The mechanisms of cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet well clarified. Here, we show that during recovery after H2O2 treatment, apoptosis occurs in two different waves, peaking at 8 h (early) and 18 h (late) of recovery from oxidative stress. The two peaks are differentially modulated by a set of inhibitors of metabolic processes, which suggests that the first peak depends on DNA break formation, whereas the second may be correlated with H2O2‐induced mitochondrial alterations.

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

A mutation in PAK3 with a dual molecular effect deregulates the RAS/MAPK pathway and drives an X-linked syndromic phenotype

Loss-of-function mutations in PAK3 contribute to non-syndromic X-linked intellectual disability (NS-XLID) by affecting dendritic spine density and morphology. Linkage analysis in a three-generation family with affected males showing ID, agenesis of corpus callosum, cerebellar hypoplasia, microcephaly and ichthyosis, revealed a candidate disease locus in Xq21.33q24 encompassing over 280 genes. Subsequent to sequencing all coding exons of the X chromosome, we identified a single novel variant within the linkage region, affecting a conserved codon of PAK3. Biochemical studies showed that, similar to previous NS-XLID-associated lesions, the predicted amino acid substitution (Lys389Asn) abolished the kinase activity of PAK3. In addition, the introduced residue conferred a dominant-negative function to the protein that drives the syndromic phenotype. Using a combination of in vitro and in vivo studies in zebrafish embryos, we show that PAK3(N389) escapes its physiologic degradation and is able to perturb MAPK signaling via an uncontrolled kinase-independent function, which in turn leads to alterations of cerebral and craniofacial structures in vivo. Our data expand the spectrum of phenotypes associated with PAK3 mutations, characterize a novel mechanism resulting in a dual molecular effect of the same mutation with a complex PAK3 functional deregulation and provide evidence for a direct functional impact of aberrant PAK3 function on MAPK signaling.

Colocalization of the VEGF-R2 and the common IL-3/GM-CSF receptor beta chain to lipid rafts leads to enhanced p38 activation

Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF‐R2 and granulocyte‐macrophage colony‐stimulating factor receptor (GM‐CSFR) membrane localization and cell signalling in TF1‐KDR cells (TF1 leukaemic cells that overexpress VEGF‐R2/KDR). Activation of either GM‐CSFR or VEGF‐R2 was shown to determine the migration of both receptor elements (VEGF‐R2 and the common β‐chain of the GM‐CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM‐CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF‐R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the β‐chain of the GM‐CSFR. Co‐stimulation of TF1‐KDR cells with both GM‐CSF and VEGF‐A resulted in massive migration of both the common GM‐CSFR β‐chain and VEGF‐R2 to lipid rafts and sustained p38 mitogen‐activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM‐CSF and VEGF‐A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF‐ and GM‐CSF‐dependent proliferation of TF1‐KDR and the survival of primary acute myeloid leukaemia blasts.