Maria D'alessio

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.

Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells

Abstract:  Among the non‐neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin‐specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT1, MT2); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonins antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca2+ influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.

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.

Melatonin as an apoptosis antagonist

Abstract:  The pineal hormone melatonin (Mel), in addition to having a well‐established role as a regulator of circadian rhythms, modulates nonneural compartments by acting on specific plasma membrane receptors (MT1/MT2) present in many different cell types. Mel plays immunomodulatory roles and is an oncostatic and antiproliferative agent; this led to the widespread belief that Mel may induce or potentiate apoptosis on tumor cells, even though no clear indications have been presented so far. Here we report that Mel is not apoptogenic on U937 human monocytic cells, which are known to possess MT1 receptors at the times (up to 48 h) and doses (up to 1 mM) tested. Mel does not even potentiate apoptosis, but instead, significantly reduces apoptosis induced by both cell‐damaging agents (intrinsic pathway) and physiological means (extrinsic pathway). The doses required for the antiapoptotic effect (≥100 μM) are apparently not compatible with receptor stimulation (receptor affinity <1 nM). However, receptor involvement cannot be ruled out, because we discovered that the actual Mel concentration active on cells was lower than the nominal one because of sequestration by fetal calf serum (FCS). Accordingly, in FCS‐free conditions, Mel doses required for a significant antiapoptotic effect are much lower.

Magnetic fields protect from apoptosis via redox alteration.

Abstract:  Magnetic fields (MFs) are receiving much attention in basic research due to their emerging ability to alter intracellular signaling. We show here that static MFs with intensity of 6 mT significantly alter the intracellular redox balance of U937 cells. A strong increase of reactive oxygen species (ROS) and a decrease of glutathione (GSH) intracellular levels were found after 2 h of MF exposure and maintained thereafter. We found that also other types of MFs, such as extremely‐low‐frequency (ELF) MFs affect intracellular GSH starting from a threshold at 0.09 mT. We previously reported that static MFs in the intensity range of 0.3–60 mT reduce apoptosis induced by damaging agents (Fanelli et al., 1998). Here, we show that ELF‐MFs are also able to protect U937 from apoptosis. Interestingly, this ability is limited to the ELF intensities able to alter redox equilibrium, indicating a link between MFs antiapoptotic effect and the MF alteration of intracellular redox balance. This suggests that MF‐produced redox alterations may be part of the signaling pathway leading to apoptosis antagonism. Thus, we tested whether MFs may still exert an antiapoptotic action in cells where the redox state was artificially altered in both directions, that is, by creating an oxidative (via GSH depletion with BSO) or a reducing (with DTT) cellular environment. In both instances, MFs fail to affect apoptosis. Thus, a correct intracellular redox state is required in order for MFs to exert their antiapoptotic effect.

Intracellular Pro-oxidant Activity of Melatonin Deprives U937 Cells of Reduced Glutathione without Affecting Glutathione Peroxidase Activity

Abstract:  It was long believed that melatonin might counteract intracellular oxidative stress because it was shown to potentiate antioxidant endogenous defences, and to increase the activity of many antioxidant enzymes. However, it is now becoming evident that when radicals are measured within cells, melatonin increases, rather than decreasing, radical production. Herein we demonstrate a pro‐oxidant effect of melatonin in U937 cells by showing an increase of intracellular oxidative species and a depletion of glutathione (GSH). The activity of glutathione peroxidase is not modified by melatonin treatment as it does occur in other experimental models.

Rescue of Cells from Apoptosis by Antioxidants Occurs Downstream from GSH Extrusion

Abstract: Antioxidants‐that is, scavengers of free radicals and anaerobic conditions (5%CO2, 95% N2)‐protect monocytic U937 cells from damage‐induced apoptosis. Antioxidants rescue the cells acting on the apoptotic pathway at a step downstream from gluthatione extrusion. Reducing agents, such as DTT, also reduce stress‐induced apoptosis. Thus, apoptotic GSH extrusion triggers the downstream events of apoptosis by leaving cells unprotected against thiols oxidation and radical production.

Non‐apoptogenic Ca2+‐Related Extrusion of Mitochondria in Anoxia/Reoxygenation Stress

Abstract:  Tumor cells often develop molecular strategies for survival to anoxia/reoxygenation stress as part of tumor progression. Here we describe that the B lymphoma Epstein–Barr‐positive cells E2r survive reoxygenation in spite of a very high and long‐lasting increase in cytosolic Ca2+ and the loss of about half of their mitochondria due to specific extrusion of the organelles from the cells. The extrusion typically occurs 3 days after reoxygenation, and a regular mitochondrial asset is regained after further 24 h.

Analysis of calcium changes in endoplasmic reticulum during apoptosis by the fluorescent indicator chlortetracycline

Abstract:  Many studies suggest that endoplasmic reticulum (ER) Ca2+ pool rather than cytosolic Ca2+ may play a crucial role in triggering apoptosis. In this study, we performed an image analysis of cells loaded with the fluorescent dye chlortetracycline (CTC) to in situ analyze Ca2+ changes within the ER in apoptosing promonocytic U937 cells. The results, validated through the use of thapsigargin (THG) as ER Ca2+ depletor, confirm the findings that apoptotic cells have a Ca2+‐depleted ER, in contrast with treated but still viable cells.

Oxidative Upregulation of Bcl‐2 in Healthy Lymphocytes

Abstract:  In many cell systems, pharmacological glutathione (GSH) depletion with the GSH neosynthesis inhibitor buthionine sulfoximine (BSO) leads to cell death and highly sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents. However, some tumor cells upregulate Bcl‐2 in response to BSO, thus surviving the treatment and failing to be chemosensitized. Cell lines of monocytic and lymphocytic origins respond to BSO treatment in an opposite way, lymphocytes being chemosensitized and unable to transactivate Bcl‐2. In this article we investigate the response to BSO of lymphocytes freshly isolated from peripheral blood of healthy donors. After ensuring that standard separation procedures do not alter per se lymphocytes redox equilibrium nor Bcl‐2 levels in the first 24 h of culture, we show that BSO treatment promotes the upregulation of Bcl‐2, with a mechanism involving the increased radical production consequent to GSH depletion. Thus, BSO treatment may increase the differential cytocidal effect of cytotoxic drugs in tumor versus normal lymphocytes.