Constitutional mismatch repair deficiency (CMMRD) is an autosomal recessive condition associated with a high risk of pediatric cancers. It is caused by homozygous or compound heterozygous pathogenic germline variants in one of four mismatch repair (MMR) genes (i.e., MLH1, MSH2, MSH6 and PMS2) [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF]. Mono-allelic (heterozygous) MMR gene variants lead to autosomal dominant Lynch syndrome [START_REF] Kohlmann | Lynch Syndrome[END_REF].

While Lynch syndrome is one of the most common cancer predisposition syndromes and leads to an increased risk of colorectal cancer, endometrial cancer and other malignancies in adults [START_REF] Kohlmann | Lynch Syndrome[END_REF], CMMRD is rare and leads to an increased probability to develop brain tumors, hematological malignancies, colorectal cancer and a wide range of other cancers in children, adolescents and young adults [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF]. In addition, most patients with CMMRD have nonneoplastic features with multiple café-au-lait maculae (CALM) being the most prevalent [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF][START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF]. This report summarizes the 5 th meeting held by the 'Care for CMMRD' (C4CMMRD) consortium in Leiden, the Netherlands, on July 6 th 2019. The consortium was established in 2013 and aims at improving the care of patients with CMMRD and their families by increasing knowledge and awareness of the syndrome through developing guidelines for diagnosis and clinical care, establishing a database to record clinical details of known patients with CMMRD and conducting collaborative studies. Meetings are held every one to two years and aim at updating the members with the latest results and developments of ongoing research and at initiating new study proposals. Thirty-five participants from nine countries and various medical fields (including basic and translational researchers, pediatric oncologists, clinical geneticists, gastroenterologists and molecular geneticists) attended the meeting in Leiden.

CMMRD-database in Paris

For research purposes a database with CMMRD patients was established in the Gustave Roussy Cancer Campus in Villejuif, France. To start the meeting an update on its current status was given by Chrystelle Colas. At the time of the meeting, 87 patients with CMMRD from 66 families were included, 27 of whom are still alive (age range 3 -48 years). PMS2 is the gene which most often carries pathogenic variants (n=34), followed by MSH6 (n=19), MSH2 (n=8) and MLH1 (n=4). Molecular results are lacking for one patient. All but one patient developed at least one malignancy. There is a total of 154 tumor diagnoses in 86 patients with tumors of the central nervous system being the most frequent (n=64, 41%), followed by hematological malignancies (n=45, 28%), mainly of T lymphoblastic lymphomas, and Lynch syndrome-related malignancies (n=43, 27%). This database was used as a starting point for several studies of which (preliminary) results were discussed during the meeting.

Role of functional assays

The previously developed C4CMMRD criteria for the clinical suspicion of a CMMRD diagnosis in young cancer patients [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF] were designed to have high diagnostic sensitivity at the cost of specificity. Detection of pathogenic variants in both alleles of an MMR gene is required to confirm the diagnosis. A definite molecular diagnosis or, equally important, a rejection of this diagnosis is also needed when testing for CMMRD as a differential diagnosis to neurofibromatosis type 1 (NF1)/Legius syndrome in a malignancy-free child with NF1-signs without a causative NF1 or SPRED1 mutation [START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF]. Unfortunately, molecular genetic testing is not always definitive, and the diagnosis of CMMRD is frequently confounded by MMR variants of unknown significance (VUS) and PMS2 pseudogenes.

The need to resolve diagnostic ambiguities has led to the development of functional CMMRD assays and highly sensitive microsatellite instability (MSI) assays that detect lowfrequency microsatellite length variants in non-neoplastic tissues, a diagnostic hallmark of CMMRD.

Current functional approaches include assessing methylation tolerance in combination with MSI in primary lymphoblastoid cell lines [START_REF] Bodo | Care for C (2015) Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents[END_REF] and assessment of the MMR capacity of protein extracts from patient cells [START_REF] Shuen | Functional Repair Assay for the Diagnosis of Constitutional Mismatch Repair Deficiency From Non-Neoplastic Tissue[END_REF]. Martine Muleris presented data of the methylation tolerance test that was performed on 85 patients with a CMMRD-like phenotype and 92 controls.

Previously it was shown, in a smaller cohort, that this test can discern CMMRD patients and healthy controls and may therefore be a useful diagnostic tool in CMMRD-like patients [START_REF] Bodo | Care for C (2015) Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents[END_REF].

The results of the methylation tolerance test in this larger cohort will be published elsewhere.

While being reliable, these functional assays performed in specialized laboratories may not easily be scalable. Furthermore, they require fresh patient material [START_REF] Bodo | Care for C (2015) Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents[END_REF][START_REF] Shuen | Functional Repair Assay for the Diagnosis of Constitutional Mismatch Repair Deficiency From Non-Neoplastic Tissue[END_REF]. MSI assays for CMMRD detection may be applied also on ascertained patient DNA in retrospective studies, and are likely to need less specialized laboratories. The first MSI assay assessing low-level MSI in three dinucleotide repeat markers in patient peripheral blood leukocytes (PBLs) is simple, fast and scalable, but has the disadvantage of being insensitive to MSH6 deficiency due to the type of microsatellite analyzed [START_REF] Ingham | Simple detection of germline microsatellite instability for diagnosis of constitutional mismatch repair cancer syndrome[END_REF].

At the meeting, Richard Gallon presented a sensitive and scalable MSI assay that detects low-level MSI in patient PBLs using 24 mononucleotide repeat markers. The assay method was developed in a pilot cohort of 5 CMMRD patients and 40 controls, and was validated by analyzing an additional 27 CMMRD patients, and 54 controls, blind to sample status, as well as 40 Lynch syndrome patients. The assay achieved 97% sensitivity and 100% specificity, including the detection of MSH6 deficient patients, and patients with hypomorphic PMS2 variants [START_REF] Gallon | A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes[END_REF]. The single false negative result was attributed to the patient's chemotherapyinduced aplasia when this sample was collected, as additional samples of this patient collected after recovery from aplasia were correctly classified [START_REF] Gallon | A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes[END_REF].

Marta Pineda presented a high sensitivity MSI (hs-MSI) assessment that can be used in nonneoplastic tissues of Lynch syndrome and CMMRD carriers using a panel of 186 mononucleotide repeat markers. This approach was applied to a training cohort including 15 blood samples from negative controls, 48 from Lynch syndrome individuals and 12 from CMMRD patients. MSI score was significantly higher in blood DNA samples from CMMRD patients compared to healthy controls, without overlapping. This finding was confirmed in a validation set including 36 blinded samples (18 controls and 18 CMMRD provided by the C4CMMRD consortium) reaching 100% specificity and sensitivity, also including the detection of MSH6 deficient patients. Moreover, high hs-MSI scores were not detected in blood from germline TP53, POLE, POLD1 and NF1 mutation carriers and early-onset Lynch syndrome, showing that the assay discriminates between CMMRD and other hereditary syndromes with overlapping phenotypes. The results of this approach had good correlation with the MSI assay presented by Richard Gallon. The results of this work have been recently published by González-Acosta et al. [8].

Patrick Benusiglio presented a proof-of-concept study of another assay detecting ultra-low MSI in leukocytes enabling rapid and accurate diagnosis of CMMRD, which will be published elsewhere.

In conclusion, several reliable MSI assays for a rapid CMMRD diagnosis are developed with the support of the C4CMMRD consortium and at least one of them is suitable for scalable screening of at-risk populations (see proposal for the assessment of Prevalence of CMMRD in patients with T-cell acute lymphoblastic lymphoma).

CMMRD-like phenotypes

Differential diagnoses in patients with a "CMMRD-like" phenotype in whom neither identification of bi-allelic germline MMR mutations nor functional or MSI assays could confirm this diagnosis were another topic of the 5 th C4CMMRD meeting.

Clara Ruiz-Ponte presented the case of a boy who fulfilled the C4CMMRD criteria for suspecting CMMRD. This boy, who had a maternal family history of Lynch syndrome, developed colorectal cancer at 12 years of age and had a skin nodule suspected of being a neurofibroma. However, he only carried the maternally inherited pathogenic MSH2 variant and was negative for CMMRD in all functional and MSI assays that were applied. Therefore, other possible scenarios were explored that could explain the early age of tumour onset.

Interestingly paternally-inherited low/moderate-penetrance variants in other cancer predisposing genes or genes described as genetic modifiers of Lynch syndrome were identified. Data of this patient suggest that the combination of several low-risk modifier alleles in addition to the pathogenic MSH2 variant may be responsible for the CMMRD-like phenotype in this patient [START_REF] Fernandez-Rozadilla | Early Colorectal Cancers Provide New Evidence for a Lynch Syndrome-to-CMMRD Phenotypic Continuum[END_REF]. Katharina Wimmer presented three cases with a "CMMRD-like" phenotype likely explained by germline POLE pathogenic variants. These included a previously published case of a 14 year old boy with colorectal cancer, colon adenomas, a pilomatricoma and multiple CALM [START_REF] Wimmer | A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency[END_REF] and two unpublished cases, one being a 31 year old male with colorectal cancer, adenomatous polyposis, glioblastoma, CALM and pilomatricomas, and the other a 4 year old girl with a malignant central nervous system tumor and CALM. As it was the case for a POLE pathogenic variant found in a medulloblastoma patient with a "CMMRD-like" phenotype who was published after our meeting [11], the POLE mutations found in all three patients presented at the meeting were de novo and were previously seen as somatic but never as germline mutations. Taken together, these cases support the evolving notion that specific POLE exonuclease domain variants, typically seen as somatic mutations in hyper-mutated tumors, confer a phenotype reminiscent of CMMRD when present as a germline mutation. Katharina Wimmer also presented two siblings, diagnosed with bowel cancer as teenagers, who both had a maternally inherited, heterozygous PMS2 pathogenic variant and a paternally inherited POLD1 variant likely to affect POLδ exonuclease activity. This suggests that this "CMMRD-like" phenotype could be caused by digenic inheritance of MMR and polymerase proofreading inactivating mutations.

Marine Le Mentec and Chrystelle Colas presented a patient with duodenal cancer at age 17 with a maternally inherited heterozygous PMS2 pathogenic variant as well as a paternally inherited heterozygous POLE variant of unknown significance.

Taken together, these cases demonstrate that sequencing of POLE and POLD1 should be considered in patients with a "CMMRD-like" phenotype in whom CMMRD can't be confirmed (neither molecularly nor functionally).

CMMRD and early onset systemic lupus erythematosus

As listed in the C4CMMRD consensus guidelines, there are a number of non-neoplastic features, such as pilomatricomas, that are indicative of CMMRD in the (young) cancer patient or in a patient with suspected NF1 but without an NF1 or SPRED1 pathogenic variant [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF][START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF]. At the meeting, Yael Goldberg introduced a new non-neoplastic feature by presenting two cases of young children with CMMRD and pediatric systemic lupus erythematosus (SLE). Age of onset was five years in both children. One of them did not have any cancer at the time of diagnosis [START_REF] Toledano | Paediatric systemic lupus erythematosus as a manifestation of constitutional mismatch repair deficiency[END_REF]. Taken together with three previously published children with CMMRD and SLE [START_REF] Rahner | Nine novel pathogenic germline mutations in MLH1, MSH2, MSH6 and PMS2 in families with Lynch syndrome[END_REF][START_REF] Plaschke | Compound heterozygosity for two MSH6 mutations in a patient with early onset of HNPCC-associated cancers, but without hematological malignancy and brain tumor[END_REF][START_REF] Lindsay | Simultaneous colonic adenocarcinoma and medulloblastoma in a 12-year-old with biallelic deletions in PMS2[END_REF], these patients indicate that pediatric onset SLE should be considered a diagnostic criterion of CMMRD and CMMRD testing should be offered if additional features are present. This may alert early diagnosis, but the treatment of SLE in those patients may be challenging. Immune checkpoint inhibitors are under investigation as a treatment for CMMRD-related cancers, but may cause SLE to flare, while steroid treatment for SLE may mitigate the effect of immune checkpoint inhibitors.

Psychological impact

Eveline Bleiker was invited to the meeting to give a presentation on her experience with the psychological impact of another severe cancer predisposition syndrome, namely Li Fraumeni syndrome (LFS), and see what can be learned from this in the context of CMMRD. Based on experiences and literature on LFS it is expected that the uptake of genetic testing in those who are aware of their hereditary factor will be high, particularly for siblings of affected children. It is expected that in 20-30% of patients with a confirmed diagnosis the levels of distress will be high. The large majority of these patients might experience specific worries related to CMMRD and to coping with cancer in their family. Professional psychosocial support should be offered to all. Worries about the cancer risk in children are expected to be high and deserve attention from a counselor and, if needed and desired, from a professional psychosocial worker. The Psychological Aspects of Hereditary Cancer questionnaire can be used as a tool to identify and discuss the specific problems experienced [START_REF] Eijzenga | Effect of routine assessment of specific psychosocial problems on personalized communication, counselors' awareness, and distress levels in cancer genetic counseling practice: a randomized controlled trial[END_REF]. These expectations are based on studies with LFS. To learn more about the psychosocial issues that come with CMMRD, qualitative and quantitative studies on this topic in this population are recommended.

Experience with colonoscopic surveillance

James Hardwick outlined his experience of performing colonoscopic surveillance in a CMMRD patient in Leiden. Surveillance was commenced at 26 years of age and performed yearly for four years until the patient developed a glioblastoma. At the first colonoscopy a 2cm villous adenoma with high grade dysplasia was successfully removed by piecemeal Endoscopic Mucosal Resection. A 1cm sessile serrated polyp with low grade dysplasia and 2 sub-centimeter adenomas were also removed. Subsequent colonoscopies were performed using chromoendoscopy due to the subtle flat morphology of several of the polyps, with the removal of several more sub-centimeter sessile serrated polyps and adenomas over the four year period. In conclusion the colon is at high risk in CMMRD. Both serrated polyps and classical adenomas are found. Advanced polyps can be removed successfully endoscopically.

Intensive surveillance seems justified.

Proposals for collaborative studies and recent results

Selection criteria for CMMRD testing in children without malignancy with an NF1-like phenotype

CMMRD is a valid differential diagnosis in children without cancer who are suspected of sporadic NF1 but in whom no causative NF1 or SPRED1 variant is identified. In 2019 a consensus guideline was published by the C4CMMRD consortium, advocating testing of CMMRD in preselected patients with a higher a priori chance, rather than reflex testing of all suspected sporadic NF1 children lacking causative NF1/SPRED1 variants [START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF]. Manon Suerink and Katharina Wimmer presented the design of a prospective multicenter study to validate the specificity of the criteria by prospectively recording cases to whom CMMRD testing is offered.

PD-1 blockade as treatment in CMMRD

Laurence Brugieres gave a presentation on the potential of PD-1 blockade as a treatment for CMMRD-related cancer. MMR deficient cancers have been shown to respond well to this treatment modality [START_REF] Le | Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade[END_REF]. To evaluate the proportion of patients who could benefit from treatment with PD-1 inhibitors and to analyze indications and efficacy of immunotherapy in this first set of patients, an analysis of patients included in the C4CMMRD database was undertaken. In addition, collaborating researchers were contacted to include more patients who received immunotherapy.

Overall, 18 CMMRD patients treated with PD-1 inhibitors were identified. Indication for immunotherapy was high-grade glioma in 13 patients (2 for front-line treatment, 11 at relapse). The type of treatment was known for 17/18 patients: pembrolizumab for 5 and nivolumab for 12 patients (3 of which are included in a trial combining ipililumab and nivolumab). Ten patients had progressive disease, whereas 8 patients had a stabilization and/or a response. After the initiation of immunotherapy, 11 patients died with a median survival of 5 months (9 high-grade gliomas and 2 digestive tract cancers) and 7 patients were still alive with median follow-up of 20 months (4 high-grade glioma, 2 digestive tract cancers and one non-Hodgkin lymphoma).

It appears from this short series of patients that, despite a high mutation burden, not all CMMRD patients benefit from immunotherapy. It was proposed to include this series of patients in the SIGN'it project, an on-going project aiming to analyze biomarkers associated with response to PD-1 inhibitors (B. Geoerger, France). To collect more data, a specific data sheet will be sent to all investigators who have included a CMMRD patient treated with immunotherapy in the C4CMMRD database.

Guidelines for genetic counselling

Tim Ripperger followed with a presentation drawing attention to the need for guidelines regarding counselling issues faced by genetic counsellors, clinical geneticists, and oncologists taking care of CMMRD families. Following the consortiums' focus on the development of surveillance guidelines [START_REF] Vasen | Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D)[END_REF] and clinical criteria indicating when to test for CMMRD in cancer patients [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF] as well as their recent refinement regarding individuals with suspected neurofibromatosis type 1 but without an identifiable NF1 or SPRED1 mutation [START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF], we discussed and consented the crucial need of recommendations for genetic counselling of families with suspected and/or diagnosed CMMRD. Although, there is a growing body of literature dealing with CMMRD, none of the papers specifically addressed counselling issues (e.g., ethical and legal issues of predictive testing in minor siblings, or the chance of inadequate surveillance or even risk reducing surgery in parents with formal molecular diagnosis of Lynch syndrome in the absence of a family history of Lynch syndromeassociated malignancies). Moreover, we need to address the question of whether, and if so, when and how, CMMRD has to be implemented in the counselling of Lynch syndrome patients.

Vaccination

Matthias Kloor gave an update on the role of vaccinations in the prevention of cancer in Lynch syndrome and posed questions that need to be answered about a similar vaccination for CMMRD: 1) What are the neoantigen profiles of CMMRD-associated tumors? 2) Is there a pre-existing systemic immune response in CMMRD? 3) What immune response pathways are active in CMMRD, and can auto-immune symptoms be expected? and 4) What are the mechanisms of immune evasion in CMMRD tumors?.

Prevalence of CMMRD in patients with T-cell acute lymphoblastic lymphoma

Richard Gallon proposed to use a newly developed scalable MSI assay (see above) to study the prevalence of CMMRD in children with T-cell acute lymphoblastic lymphoma (T-LBL) and high-grade gliomas in PBLs from retrospective cohorts of patients with these types of cancer.

Preliminary results of surveillance according to the C4CMMRD guidelines

Zeinab Ghorbanoghli presented the preliminary results of surveillance according to the protocol as proposed by the C4CMMRD consortium in 2014 [START_REF] Vasen | Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D)[END_REF]. Data were collected from 22 patients including 12 females. Fifteen of these patients (68%) had bi-allelic PMS2variants. Seventy-seven percent of the patients had developed a previous cancer; mainly colorectal and hematological tumors. During a follow-up time of up to 5 years, 15 malignancies developed divided over 12 patients. These malignancies were most frequently located in the digestive tract, followed by brain tumors. Twelve patients were diagnosed with (multiple) adenomas in the colon. The investigators concluded that the yield of screening was very high. The preliminary results suggest that surveillance of the digestive tract is effective because many polyps were endoscopically removed and early cancers were detected. However, the benefit of screening of the brain is still uncertain.

Following this presentation, it was discussed whether surveillance guidelines should be adjusted in view of these findings. One of the participants suggested to recommend MRIscreening of the brain with intervals of 6 months instead of 6-12 months which is currently advised. In addition, the question arose whether a lower starting age for colonic surveillance (currently 8 years) should be considered given that patients have been reported with adenomatous polyps before this age. After the final analysis of the data, adjustment of the protocol will be re-discussed.

Acknowledgements

We would like to thank all meeting participants for their attendance and valuable input: Felipe Andreiuolo, Amedeo Azizi, Birgit Burkhardt, Bianca Desouza, Demetra Georgiou, Lea Guerrini-Rousseau, Yvette van Ierland, Hanna IJspeert, Danuta Januszkiewicz-Lewandowska, Maria Kurnikova, Monika Morak, Enrico Opocher, Daniel Rueda, Pauline van Schouwenburg, Irene Slavc, Helen Toledano, Anja Wagner, Liudmila Yasko


Christophe Fleury

Bernard Mignotte
email: mignotte@genetique.uvsq.fr

Jean-Luc Vayssière

Mitochondrial reactive oxygen species in cell death signaling

Keywords: mitochondria, reactive oxygen species, apoptosis, signaling

During apoptosis, mitochondrial membranes permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase independent factors as AIF) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs.

However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by antiapoptotic Bcl-2 and Bcl-xL is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggest the existence of an ancestral redox-sensitive death signaling pathway that have been independent of caspases and Bcl-2.

Introduction

The Bcl-2/Ced-9 family proteins are involved in positive and negative regulation of apoptotic cell death (review in [START_REF] Gross | BCL-2 family members and the mitochondria in apoptosis[END_REF]). Among the anti-apoptotic members, Bcl-2 and Bcl-xL are negative regulators of cell death, preventing cells from undergoing apoptosis induced by various stimuli in a wide variety of cell types [START_REF] Korsmeyer | Bcl-2 initiates a new category of oncogenes: regulators of cell death[END_REF][START_REF] Zhong | bcl-2 inhibits death of central neural cells induced by multiple agents[END_REF], whereas others, such as Bax and Bid promote or accelerate cell death. In mammals, most of our understanding of Bcl-2 regulation of apoptosis involves its mitochondrial membrane association. Although for a long time apoptosis was considered to be under the control of nuclear events, mitochondria appear today as the central control point of programmed cell death [START_REF] Mignotte | Mitochondria and apoptosis[END_REF][START_REF] Desagher | Mitochondria as the central control point of apoptosis[END_REF][START_REF] Zamzami | The mitochondrion in apoptosis: how Pandora's box opens[END_REF][START_REF] Ferri | Mitochondria: the suicide organelles[END_REF]. It is now clear that mitochondrial membraneassociated Bcl-2 acts by regulating the release in the cytosol of pro-apoptotic factors usually sequestered in the mitochondrial intermembrane space (reviewed in other articles of this special issue of "Biochimie"). Moreover, inhibition of apoptosis by antiapoptotic Bcl-2 and Bcl-xL is associated with a protection against oxidants and/or a shift of the cellular redox potential to a more reduced state. These results have suggested that reactive oxygen species (ROS) could be mediators of apoptosis. However, others data have shown that apoptosis can occur in very low oxygen environments [START_REF] Jacobson | Programmed cell death and Bcl-2 protection in very low oxygen[END_REF][START_REF] Shimizu | Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-X L[END_REF], which has cast doubt on this interpretation. A growing number of data show now that ROS, mainly produced by the mitochondria, can be involved in cell death. On the one hand, they can produce an oxidative stress leading to cell destruction, as observed during necrosis or the so-called "post-mitochondrial" phase of apoptosis. On the other hand, in several instances, ROS derived from mitochondria are also involved in the initiation phase of apoptosis contributing to cell death signaling. The present review will focus on the role of mitochondrial ROS in cell death signaling and the possible molecular mechanisms.

Mitochondria are the major site of ROS production

ROS, such as superoxide anion, radicals, hydrogen and organic peroxides are generated by all aerobic cells as by-products of a number of metabolite reactions and in response to various stimuli [START_REF] Fridovich | The biology of oxygen radicals[END_REF]. Mitochondria are believed to be a major site of ROS production, according to an endogenous and continuous physiological process under aerobic conditions (figure 1). However, endoplasmic reticulum and nuclear membranes also contain e _ transport chains that can lose e _ and generate superoxide radical. Some fatty acid metabolites, such as those derived from arachidonic acid by the lipoxygenase pathway, are also ROS.

The physiological activity of the respiratory chain leads to the production of semi quinones, a potential source of ROS [START_REF] Papa | Reactive oxygen species, mitochondria, apoptosis and aging[END_REF]. Indeed, the respiratory chain produces ROS at Complex I (NADH/Ubiquinone oxidoreductase) and Complex III (ubiquinol/cytochrome c oxido-reductase [START_REF] Takeshige | NADH-and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH-ubiquinone reductase preparation[END_REF][START_REF] Boveris | Role of ubiquinone in the mitochondrial generation of hydrogen peroxide[END_REF][START_REF] Turrens | Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria[END_REF]. The ubiquinone site in complex III appears as the major site of mitochondrial ROS production: this site catalyzes the conversion of molecular oxygen to superoxide anion radical (O2 -) by a single electron transfer to molecular oxygen (figure 1). Moreover, the inhibition of the respiratory chain, due to a lack of oxygen or to an inhibitor, such as cyanide or antimycin A, increases the ubisemiquinone free radical level in the normal catalytic mechanism of complex III [START_REF] Turrens | Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria[END_REF].

In aqueous solutions, O2 -is moderately reactive but can generate other potent ROS such as H2O2 by dismutation. About 1-5% of mitochondrial O2 consumption leads to H2O2 production [START_REF] Chance | Hydroperoxide metabolism in mammalian organs[END_REF]. A reductive homolytic cleavage of H2O2 produces the highly oxidative and cytotoxic hydroxyl radical (OH . ) according a metal-dependent breakdown. This highly reactive radical is assumed to be directly responsible for most of the oxidative damages leading to the non-physiological necrosis [START_REF] Halliwell | Role of free radicals and catalytic metal ions in human disease: an overview[END_REF]. Alternatively, mitochondria superoxide may react with nitric oxide to produce peroxynitrite [START_REF] Groves | Peroxynitrite: reactive, invasive and enigmatic[END_REF], a potent oxidant, which causes irreversible inhibition of mitochondria respiration and damage to mitochondria components (complexes I, II, IV and V, creatine kinase, aconitase, membranes, DNA, SOD…). It induces cell death in several cell types [START_REF] Szabo | DNA damage induced by peroxynitrite: subsequent biological effects[END_REF][START_REF] O'connor | Role of peroxynitrite in the protein oxidation and apoptotic DNA fragmentation in vascular smooth muscle cells stimulated with bacterial lipopolysaccharide and interferon-gamma[END_REF][START_REF] Lin | Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease[END_REF][START_REF] Liu | Peroxyacetyl nitrate-induced apoptosis through generation of reactive oxygen species in HL-60 cells[END_REF]. Nitric oxide (NO) can also promote or inhibit apoptosis, according the cell type, the rate of production and the interaction with other intracellular molecules [START_REF] Chung | Nitric oxide as a bioregulator of apoptosis[END_REF].

Oxidative stress

An excess of ROS in mitochondria causes an oxidative stress, with an enhanced activity of the antioxidant defense system and mitochondrial damage. Several situations, such as dysfunctional complex I [START_REF] Robinson | Human complex I deficiency: clinical spectrum and involvement of oxygen free radicals in the pathogenicity of the defect[END_REF], chemical poisoning and ischaemia followed by reperfusion, may subject tissues and cells to oxidative stress The main targets of ROS in mitochondria are the protein components of the membranes and the polyunsaturated fatty acids. In membranes, ROS and lipids affect cysteine residues (sulfhydryl groups), causing intramolecular cross-linkings and formation of proteins aggregates. OH

. radicals can initiate lipid peroxidation and generate peroxyl-and alkoxyl radical intermediates. Oxidants increase the release of calcium from mitochondria, thus stimulating calcium-dependent enzymes, such as proteases, nucleases and phospholipases. Because of the absence of mitochondria DNA-protecting proteins, the low efficiency reparation mechanism and the proximity of the respiration chain, mtDNA is a privileged target for ROS [START_REF] Shoji | DNA damage induced by tumour necrosis factor-alpha in L929 cells is mediated by mitochondrial oxygen radical formation[END_REF].

Protection of the cell

To prevent oxidative damage, mammalian cells have developed a complex antioxidant defense system that includes non-enzymatic antioxidants (e.g. glutathione, thioredoxine) as well as enzymatic activities (e.g. catalase, superoxide dismutase (SOD)) [START_REF] Sies | Oxidative stress: from basic research to clinical application[END_REF].

Mitochondria possess an antioxidant system with the superoxide dismutase (SOD), NADH and a complete glutathione redox system, which is formed of glutathione reductase, reduced glutathione (GSH), and glutathione peroxidase (figure 1). This system allows the reduction of oxidants, such as hydroperoxide. As mitochondria are generally devoid of catalase, the hydroperoxide detoxification mainly relies on GSH peroxidase. Various chemicals are known to induce oxidative stress, either as the result of an enhanced free radical production (e.g. xenobiotics utilizing cytochrome P450 for detoxification) or by impairment of their destruction provoked by glutathione depletion. Indeed, irreversible injury may occur following a depletion of the mitochondrial pool of glutathione [START_REF] Reed | Glutathione: toxicological implications[END_REF]. Mitochondrial GSH plays an important role in preserving mitochondria membrane integrity and in assuring the reduced state of the intramitochondrial protein thiol groups. Moreover, although coenzyme Q10 can also act as a pro-oxidant [START_REF] Yamamura | Dual involvement of coenzyme Q10 in redox signaling and inhibition of death signaling in the rat heart mitochondria[END_REF], it can prevent ROS accumulation occurring under unfavorable condition [START_REF] Teranishi | Effects of coenzyme Q10 on changes in the membrane potential and rate of generation of reactive oxygen species in hydrazine-and chloramphenicol-treated rat liver mitochondria[END_REF]. Last, macrophages of uncoupling protein-2 deficient mice generate more ROS [START_REF] Arsenijevic | Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production[END_REF], which is in agreement with the idea that a mitochondrial mild uncoupling and non-coupled oxidations could safe guard cells from high oxygen level and ROS generation [START_REF] Skulachev | Uncoupling: new approaches to an old problem of bioenergetics[END_REF].

Physiological role

Cells possess multiple sites for ROS production and a few mechanisms for their degradation. ROS are also involved in different physiological processes, as mediators in signal transduction pathway, activating proteins such as tyrosine kinase, mitogen-activated protein kinase system or small Ras proteins. Current evidence indicates that different stimuli use ROS as signaling messengers to activate transcription factors, such as AP-1 and NF-κB, and induce gene expression [START_REF] Pinkus | Role of Oxidants and Antioxidants in the Induction of AP-1, NF-kB, and Glutathione S-Transferase Gene Expression[END_REF]. Thus, they are responsible for the inducible expression of genes associated with inflammatory and immune responses. Additionally, ROS may have divergent effects according to the cell type, as they can function as mitotic stimuli [START_REF] Clement | Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: an hypothesis[END_REF], cellular senescence inducers [START_REF] Lundberg | Genes involved in senescence and immortalization[END_REF], inhibitors of druginduced tumor cell death [START_REF] Pervaiz | Superoxide anion inhibits drug-induced tumor cell death[END_REF] or cell death mediators [START_REF] Burdon | Control of cell proliferation by reactive oxygen species[END_REF][START_REF] Burdon | Superoxide and hydrogen peroxide in relation to mammalian cell proliferation[END_REF]. The large diversity of their effects on cells suggests that ROS not only cause unspecific changes in macromolecules, but also modify a number of signaling pathways.

The ability of oxidative stress, which is an excessive accumulation of ROS, to provoke necrotic cell death as a result of massive cellular damages associated to lipid peroxidation and alterations of proteins and nucleic acids, is well documented for a long time [START_REF] Halliwell | Free radicals in Biology and Medecine[END_REF]. When free radicals generation exceeds the cellular capacity to detoxify them, cellular protein and DNA damage may occur, leading to the cell death In this point of view, aerobic cells appear as being under a continual ''oxidative siege", their survival depending on a balance between ROS and antioxidants.

The possible implication of ROS as signaling molecules in more physiological deaths such as apoptosis is a more recent concept. First evidence suggesting the involvement of mitochondrial ROS in cell death arose from the study of the TNF-α-induced cytotoxicity [START_REF] Lancaster | Inhibition of target cell mitochondrial electron transfer by tumor necrosis factor[END_REF][START_REF] Schulze-Osthoff | Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation[END_REF]. The observed inhibition of the mitochondrial respiratory chain was assumed to result in the over production of ROS which would act as mediators of the death signaling pathway [START_REF] Schulze-Osthoff | Depletion of the mitochondrial electron transport abrogates the cytotoxic and Gene-Inductive effects of TNF[END_REF]. There is now mounting evidence that these compounds may be central in the cell death transduction pathways: 1) the addition of ROS or the depletion of endogenous antioxidants can promote cell death [START_REF] Carmody | Reactive oxygen species as mediators of photoreceptor apoptosis in vitro[END_REF][START_REF] Kane | Bcl-2 inhibition of neural death -decreased generation of reactive oxygen species[END_REF][START_REF] Sato | Thiol-Mediated Redox Regulation of Apoptosis. Possible roles of cellular thiols other than glutathione in T cell apoptosis[END_REF][START_REF] Guénal | Bcl-2 and Hsp27 act at different levels to suppress programmed cell death[END_REF]; 2) some antioxidants, such as thioredoxine or N-acetylcystein, act as intracellular ROS scavengers, and can inhibit activation of caspases and subsequent steps leading to the apoptotic cell death. In these models, ROS could be considered as major mediators, and not only the source of direct damage to DNA, proteins and lipids [START_REF] Iwata | Adult T cell leukemia (ATL)-derived factor/human thioredoxin prevents apoptosis of lymphoid cells induced by L-cystine and glutathione depletion: possible involvement of thiol-mediated redox regulation in apoptosis caused by pro-oxidant state[END_REF][START_REF] Mayer | N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro[END_REF][START_REF] Greenlund | Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death[END_REF][START_REF] Mehlen | Small stress proteins as novel regulators of apoptosis[END_REF][START_REF] Gottlieb | Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis[END_REF]; 3) increases in intracellular ROS are sometimes associated with PCD [START_REF] France-Lanord | Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF][START_REF] Li | Superoxide induces apoptosis in cardiomyocytes, but proliferation and expression of transforming growth factor-beta1 in cardiac fibroblasts[END_REF][START_REF] Manna | Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein-1[END_REF][START_REF] Carmody | Oxidative stress induces caspase-independent retinal apoptosis in vitro[END_REF][START_REF] Herrera | Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes[END_REF]; 4) MnSOD overexpression restores mitochondrial transmembrane potential [START_REF] Majima | Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death[END_REF] and inhibits the cell death caused by an inhibition of the respiratory chain [START_REF] Kiningham | Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death[END_REF]; 5) Cu/Zn SOD overexpression delays apoptosis by scavenging of O2-in neuronal cells deprived of nerve growth factor [START_REF] Greenlund | Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death[END_REF] and prevents early release of cytochrome c in ischemic brain [START_REF] Fujimura | The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice[END_REF]; 6) mitochondrial phospholipid hydroperoxide glutathione peroxidase overexpression inhibits increase of ROS and apoptosis [START_REF] Nomura | Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase Suppresses Apoptosis Mediated by a Mitochondrial Death Pathway[END_REF] and 7) although some PCD can occur in very low oxygen environments [START_REF] Jacobson | Programmed cell death and Bcl-2 protection in very low oxygen[END_REF][START_REF] Shimizu | Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-X L[END_REF], ROS might be produced in such condition [START_REF] Degli Esposti | Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis[END_REF]. Thus, ROS could be considered as major mediators, and not only the source of direct damage to DNA, proteins and lipids [START_REF] Tan | The regulation of reactive oxygen species production during programmed cell death[END_REF]

ROS participate in early and late steps of the regulation of apoptosis

Since the discovery of ROS involvement in TNF--induced killing, their contribution to the activation of the execution machinery was extended to PCD triggered by a wide range of apoptosis inducers [START_REF] Jacobson | Reactive oxygen species and programmed cell death[END_REF]. Notably, ROS accumulation preceded mitochondrial membrane alterations, nuclear condensation and other typical apoptotic events. This point can be illustrated by TNF- induced apoptosis. Indeed, experiments using antioxidants show that ROS act upstream of mitochondrial membrane depolarization [START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF], Bax relocalization, Cytochrome c release, executing caspases activation and nuclear fragmentation (figure 2). Similarly, an early increase in ROS levels has been found to precede mitochondrial membranes permeabilization, and in some case to be independent of caspases, in various models including apoptosis of Retinitis pigmentosa photoreceptors [START_REF] Carmody | Reactive oxygen species as mediators of photoreceptor apoptosis in vitro[END_REF][START_REF] Carmody | Oxidative stress induces caspase-independent retinal apoptosis in vitro[END_REF] and apoptosis induced by Fas [START_REF] Banki | Elevation of mitochondrial transmembrane potential and reactive oxygen intermediate levels are early events and occur independently from activation of caspases in Fas signaling[END_REF], p53 [START_REF] Li | p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2[END_REF], ischaemia [START_REF] Maulik | Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis[END_REF], DNA alkylation [START_REF] Tada-Oikawa | Generation of hydrogen peroxide precedes loss of mitochondrial membrane potential during DNA alkylation-induced apoptosis[END_REF] or etoposide (Vayssière et al. unpublished results).

Some data raise the possibility that ROS are also required for the execution of the death program [START_REF] Kroemer | The biochemistry of programmed cell death[END_REF][START_REF] Schulz | Potassium deprivation-induced apoptosis of cerebellar granule neurons: a sequential requirement for new mRNA and protein synthesis, ICE-like protease activity, and reactive oxygen species[END_REF] This must be cautiously considered inasmuch as, in the majority of these systems, it is difficult to ascertain that the observed ROS accumulation corresponds to a causal effect and is not a side effect of the other changes accompanying the killing process [START_REF] Cai | Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss[END_REF]. For example, release of cytochrome c triggers accumulation of ROS and increases the oxidation state of the apoptotic cells. Moreover, in these cases, ROS increase most often arises during the later stage of the death program, i.e. during the destruction phase when the cell is broken down, and may be associated with a necrotic type terminal degradation of the cell. Exogenous sources of ROS such as hydrogen peroxide can induce PCD or necrosis depending upon the dose added [START_REF] Guénal | Bcl-2 and Hsp27 act at different levels to suppress programmed cell death[END_REF]. So a burst in ROS, in response to a dramatic perturbation of the physiology of the dying cell, could convert the late PCD steps into necrotic death. Therefore, it appears that at any moment the level of intracellular ROS can determine the fate of the cell: low levels of ROS can induce PCD while accumulation of high levels promotes necrosis or can lead PCD-committed cells toward necrotic-like destruction.

Origin of apoptosis-mediated ROS

Although fatty acid metabolites, such as those produced from arachidonic acid by the lipoxygenase pathway, may be mediators of apoptosis [START_REF] O'donnell | Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain[END_REF], it was established that both ROS accumulation and programmed cell death process require the presence of a functional mitochondrial respiratory chain in most ROS-dependent cell death systems [START_REF] Schulze-Osthoff | Depletion of the mitochondrial electron transport abrogates the cytotoxic and Gene-Inductive effects of TNF[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF][START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF][START_REF] Higuchi | Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function[END_REF]. Indeed, it was shown that an upstream inhibition, with chemical compounds acting on complex I [START_REF] Schulze-Osthoff | Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF], or an elimination of the electron transfer chain [START_REF] Schulze-Osthoff | Depletion of the mitochondrial electron transport abrogates the cytotoxic and Gene-Inductive effects of TNF[END_REF][START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF][START_REF] Higuchi | Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function[END_REF], by depletion of the mtDNA, prevent ROS accumulation and consequently protect cells against PCD. Another indirect argument is provided by the scavenger role of mitochondrial glutathione in the regulation of ROS-mediated PCD [START_REF] Goossens | Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity[END_REF]. The ubiquinone site in complex III appears as the major site of mitochondrial ROS production as this site catalyzes the conversion of molecular oxygen to superoxide anion which can lead to the formation of other potent ROS such as hydrogen peroxide and hydrogen radicals (figure 1). The inhibition of the respiratory chain, due to a lack of oxygen or to an inhibitor, such as cyanide or antimycin A, increases the ubisemiquinone free radical level in the normal catalytic mechanism of complex III [START_REF] Turrens | Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria[END_REF]. Such a model is supported by the observed potentiation of cell death processes in ROS-dependent PCD when electron flow was inhibited distal to the ubiquinone pool.

Mechanisms of ROS signaling

The involvement of mitochondrial ROS in some cell death transduction pathways leads to the fundamental questions concerning, on the one hand, the causal event of the increased ROS generation and, on the other hand, the molecular mechanisms underlying the ROS signaling. Two viewpoints must first be considered to address the question of the origin of ROS accumulation, which can indeed result from an increased production or from a reduced scavenging by the cellular detoxifying systems. Much of the available data converge to the hypothesis that ROS increases are the consequence of an impairment of the mitochondrial respiratory chain [START_REF] Schulze-Osthoff | Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation[END_REF][START_REF] France-Lanord | Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF][START_REF] Degli Esposti | Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis[END_REF][START_REF] Cai | Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss[END_REF][START_REF] Gudz | Direct inhibition of mitochondrial respiratory cahin complex III by cell-permeable ceramide[END_REF]. In agreement with the above considerations, the observed alterations are distal to the ubiquinone site of the complex III, but the origin of these electron flow disturbances are not clear. The only strong evidence comes from the study of ceramide-induced PCD, in which an increased H2O2 production was linked to mitochondrial Ca 2+ homeostasis perturbation as inhibition of the mitochondrial Ca 2+ uptake was shown to abolish both ROS accumulation and cell death.

Beside the question of the process of mitochondrial ROS accumulation, arises the problematic concerning the targets of these compounds or more precisely: how can they mediate PCD? Unlike fatty acid metabolites which harbor specific reactivity and are known to mediate particular signals from surface receptors, mitochondrial ROS are characterized by a lack of biological specificity or even an extreme reactivity, as for the hydroxyl radical: these are all features contrary to the requirements of a specific signaling role [START_REF] Jacobson | Reactive oxygen species and programmed cell death[END_REF]. In this way, a direct influence of ROS on PCD process would be correlated to a general damaging effect on cellular structures resulting in necrotic cell death, or perhaps to a more limited action on mitochondria, their site of production, which in turn could activate some mitochondria-dependent downstream cascades leading to PCD. Another alternative explanation would be to consider that the major effect of an increased ROS production is the subsequent imbalance of intracellular redox status, i.e. an enhancement of the cellular oxidative tonus, and that in fact the oxidative stress is the central common effector of PCD. Hence, ROS accumulation would only be one way which leads in some PCD to an oxidative status. Anti-Fas/APO-1 antibody or IL-3 withdrawal-induced PCD represent good illustrations of this model [START_REF] Van Den Dobbelsteen | Rapid and Specific Efflux of Reduced Glutathione during Apoptosis Induced by Anti-Fas/APO-1 Antibody[END_REF][START_REF] Bojes | Bcl-x L overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal[END_REF]. Indeed, no ROS accumulation can be measured in these two systems and anaerobic cultured cells deprived of IL-3 still undergo PCD [START_REF] Shimizu | Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-X L[END_REF][START_REF] Schulze-Osthoff | Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death[END_REF]. However, an oxidative stress can be shown in these models as a depletion of glutathione (GSH), a non-enzymatic cellular antioxidant, as a result of a rapid and specific efflux of glutathione, an event that takes place at the very beginning of the apoptotic process [START_REF] Van Den Dobbelsteen | Rapid and Specific Efflux of Reduced Glutathione during Apoptosis Induced by Anti-Fas/APO-1 Antibody[END_REF][START_REF] Bojes | Bcl-x L overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal[END_REF]. Moreover, it has been shown that Bcl-2 can protect cells from PCD by shifting the cellular redox potential to a more reduced state (see below). However, the observation that oxidation of thiols other than glutathione can mediate induction of PCD suggest that the intracellular thiol redox status would be the real key factor of the cell death signaling pathways [START_REF] Kane | Bcl-2 inhibition of neural death -decreased generation of reactive oxygen species[END_REF][START_REF] Sato | Thiol-Mediated Redox Regulation of Apoptosis. Possible roles of cellular thiols other than glutathione in T cell apoptosis[END_REF][START_REF] Mirkovic | Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols[END_REF][START_REF] Marchetti | Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function[END_REF]. In this model, the redox state of glutathione or other cellular antioxidants such as thioredoxine, would be in equilibrium with that of thiols resident in some redox sensitive crucial components of the execution machinery [START_REF] Kroemer | Mitochondrial control of apoptosis[END_REF]. Pro-apoptotic members of the Bcl-2 family might be targets for such a regulatory mechanism. Indeed, in healthy cells these proteins are cytosolic, whereas upon a death they redistribute to mitochondria, promoting cell death [START_REF] Wolter | Movement of Bax from the Cytosol to Mitochondria during Apoptosis[END_REF][START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF]. One could imagine that such a conformational change is induced by ROS. It has also recently been proposed that ROS could be responsible for a decrease in bcl-xL mRNA levels [START_REF] Herrera | Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes[END_REF].

Where do ROS fit this thiol hypothesis? In this putative model, an increased production of mitochondrial ROS would result, either by a direct modification of the thiols or indirectly via a depletion of the intracellular antioxidant pool, in a shift of the redox state of the sensor SH groups to a more oxidized state. The nature of the ROS and the level of the intracellular antioxidant defenses would determine in which way regulatory components are activated to commit cells to PCD. In a hypothetical model, mitochondrial ROS modify the membrane permeability leading to the release of pro-apoptotic factors and/or activate directly executing caspases.

Inhibition of apoptosis by Bcl-2 and Bcl-xL is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state

Several lines of evidence support the idea that Bcl-2, in addition to its role on the regulation of the permeability of the outer mitochondrial membrane, might act as an antioxidant partner to block a putative ROS-mediated step in the cascade of events required for apoptosis. For example, during TNF- induced apoptosis, which involves ROS signaling [START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF], Bcl-2, as antioxidants do, prevents ROS accumulation and the subsequent events (mitochondrial membrane depolarization, Bax relocalization, Cytochrome c release, executing caspases activation and nuclear fragmentation) (compare figure 2 and3). However, the way by which Bcl-2 protects from ROS remains unclear. In some systems, Bcl-2 (or Bcl-xL) appears to influence the generation of ROS [START_REF] Kane | Bcl-2 inhibition of neural death -decreased generation of reactive oxygen species[END_REF][START_REF] Gottlieb | Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis[END_REF], while in other cases it does not affect ROS production but does prevent oxidative damage to cellular constituents [START_REF] Hockenbery | Bcl-2 functions in an antioxidant pathway to prevent apoptosis[END_REF][START_REF] Tyurina | Direct evidence for antioxidant effect of Bcl-2 in PC12 rat pheochromocytoma cells[END_REF][START_REF] Satoh | Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells[END_REF]. It has also been proposed that it functions as a pro-oxidant and influences the levels of ROS, inducing in this way endogenous cellular antioxidants [START_REF] Steinman | The Bcl-2 oncoprotein functions as a pro-oxidant[END_REF]. However, bcl-2 knock-out in mice genders perturbation of oxidant metabolism and antioxidant status in some tissues [START_REF] Hochman | Developmental changes in antioxidant enzymes and oxidative damage in kidneys, liver and brain of bcl-2 knockout mice[END_REF].

Since apoptosis might be modulated by redox sensitive proteins, the effect of Bcl-2s on cellular redox potential has been studied. Activities of antioxidant enzymes and levels of glutathione and pyridine nucleotides have been measured in pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7 cells transfected with bcl-2 [START_REF] Ellerby | Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2[END_REF] Both cell lines overexpressing bcl-2 had increased total glutathione levels and reduced ratios of oxidized glutathione to total glutathione. In addition, the NAD+/NADH ratio was two-to threefold less than that of control cell lines while they had approximately the same level of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities as control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes. Furthermore, depleting cellular thiols reversed the resistance to radiation in Bcl-2 expressing lymphoma cell lines [START_REF] Mirkovic | Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols[END_REF].

In the same way Bax and Bcl-xL were shown respectively to increase and decrease GSH levels [START_REF] Bojes | Bcl-x L overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal[END_REF] without modifying the activities of superoxide dismutase, catalase, and glutathione peroxidase or glutathione reductase. These results suggest a possible role for GSH in the mechanism by which Bax and Bcl-xL control cell death.

Thus, both Bcl-2 and Bcl-xL can protect cells from apoptosis by shifting the cellular redox potential to a more reduced state. Assuming that mitochondrial thiols constitute a critical sensor of the cellular redox potential during apoptosis [START_REF] Marchetti | Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function[END_REF], these effects could be at the mitochondrial level.

ROS, plants PCD and Yeast pseudo-apoptosis

In plants, ROS are also involved in some PCD pathways, such as the synchronous programmed death of petal cells [START_REF] Rubinstein | Regulation of cell death in flower petals[END_REF], the hypersensitive response (HR) of plants resistant to microbial pathogens [START_REF] Heath | Hypersensitive response-related death[END_REF], or the hormonally regulated cell death pathway in barley aleurone cells [START_REF] Bethke | Cell death of barley aleurone protoplasts is mediated by reactive oxygen species[END_REF]. More surprising is the occurrence of a ROS associated apoptosis-like process in unicellular eukaryotic cells like yeasts [START_REF] Madeo | A yeast mutant showing diagnostic markers of early and late apoptosis[END_REF]. Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast species do not contain endogenous caspases or Bcl-2s gene [START_REF] Greenhalf | Role of mitochondria and C-terminal membrane anchor Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae[END_REF][START_REF] Zha | Structurefunction comparisons of the proapoptotic protein Bax in yeast and mammalian cells[END_REF][START_REF] Ink | Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells[END_REF] and their cytochrome c has been shown to be ineffective to induce nuclear apoptosis in acellular assay [START_REF] Kluck | Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cellfree apoptosis system[END_REF]. However, several data obtained with S. cerevisiae suggest the existence of yet unknown PCD pathways, independent of Bcl-2s and caspases families, in which the place and the role of mitochondria are not fully understood. The cdc48 S565G mutant show typical markers of apoptosis, including exposure of phosphatidylserine, chromatin condensation and fragmentation, DNA fragmentation, and formation of minicells, which look like apoptotic bodies and it has been recently observed some markers of oxidative stress and apoptosis in aged mother cells of S. cerevisiae [START_REF] Laun | Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis[END_REF]. The heterologous expression of mammalian pro-apoptotic genes (ced-4, bax, bak) provokes mitochondrial dysfunction and the death of S. cerevisiae and S. pombe cells [START_REF] Greenhalf | Role of mitochondria and C-terminal membrane anchor Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae[END_REF][START_REF] Ink | Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells[END_REF][START_REF] Sato | Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system[END_REF][START_REF] Hanada | Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax[END_REF][100][101]. Several authors reported that this induced-cytotoxicity was characterized by typical phenotypic markers of apoptosis [START_REF] Ink | Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells[END_REF]101], identical to those observed in the pseudo-apoptotic mutant cdc48 S565G [START_REF] Madeo | A yeast mutant showing diagnostic markers of early and late apoptosis[END_REF], and other features, such as release of cytochrome c [102]. Moreover, as observed in mammals, coexpression of Bcl-2 or Bcl-xL with Bax abolishes the pseudo-apoptotic phenotype and the cell death [101][102][103][104][105][106]. Since on the one hand, Bax cytotoxicity is increased under respiratory condition [107], and, on the other hand, yeast mutants lacking superoxide dismutase were partially rescued by expression of Bcl-2 [START_REF] Kane | Bcl-2 inhibition of neural death -decreased generation of reactive oxygen species[END_REF]108], it is tempting to speculate that a mitochondrial ROS production is involved in the pseudo-apoptotic process. In agreement with this hypothesis, the apoptotic-like features observed in these yeast strains are accompanied with ROS accumulation [109]. Moreover, low concentration of H2O2 treatment or glutathione depletion could induce the pseudo-apoptosis in yeast [109]. The induced-cell death is not due to a direct cell damage, but requires an active cooperation of the cell machinery. In all these models, ROS accumulation seems to be necessary and sufficient to lead to the pseudo-apoptotic phenotype. These observations suggest the existence, in yeasts, of an ancestral redox-sensitive apoptosis pathway, which is independent of caspases and Bcl-2.

Conclusion

In conclusion, mitochondria are involved in the decision of cells to survive or not at several levels (figure 4). First, mitochondria can activate the cell death machinery by releasing in the cytosol pro-apoptotic factors such as procaspases, caspase activators (i.e. cytochrome c and Smac/Diablo) or caspase independent factors as AIF. Furthermore, the importance of mitochondria in apoptosis has been reinforced by studies showing the contribution of reactive oxygen species in the cell-death signaling. The mechanism of ROS signaling is still unknown, but it could be speculated that they act by modifying the activity of some apoptotic key regulators, such as proteins of the Bcl-2 family. If the primer function of apoptosis was possibly to clear ROS-producing cells from tissues [110,111], mitochondrial ROS production are used as active mediators in the regulation of apoptosis at different levels. Moreover, the fact that pseudo-apoptosis phenotype in yeast invariably involves ROS [109] points out these compounds as major evolution-conserved regulators of cell death. H2O2 detoxification normally occurs according two different reactions, either by the thioredoxine reductase (TR), which reduces thioredoxine (TX -> TX-H2), or by reacting with glutathione (GSH) by glutathione peroxidase (GPO). The latter reaction produces water and oxidized glutathione (GSSG), and GSSG is recycled to GSH by glutathione reductase (GR). Glutathione is mainly maintained in a reduced state, via NADPH generation in the reversible nonoxidative part of the pentose phosphate pathway [113]. When GSH level is low, H2O2 can react with Fe 2+ to produce OH -and the harmful hydroxyl anions (OH . ). Alternatively, H2O2 reacts with O2 .- producing water, molecular oxygen and OH . . Superoxide may also react with nitric oxide (NO) to produce peroxynitrite (ONOO .-), a potent oxidant, which causes irreversible inhibition of mitochondria respiration and damage to mitochondria components (complexes I, II, IV and V, creatine kinase, aconitase, membranes, DNA, SOD,…).

The complexes of the electron transfer chain in the inner mitochondrial membrane are numbered from I to IV. The electron (e-) pathway through the respiratory chain is indicated by arrows. Cyt c refers to cytochrome c. Numerous signals can lead to apoptosis and the induction pathways seem to converge to events leading to an increase in mitochondrial membranes permeability (MMP) and the release of pro-apoptotic factors from the mitochondria. Before these events, in response to numerous apoptosis inducers, among which TNF-, mitochondrial ROS production can contribute to apoptosis signaling during the induction phase. ROS accumulate as a result of a dysfunction in the mitochondrial respiratory chain. ROS also intervene in later steps in an amplifying loop, acting on mitochondrial membrane permeability, and contributing then to the activation of execution caspase.

Figure 1 :

 1 Figure 1: Schematic representation of the formation and the fate of superoxide ions (O2 .-) in mitochondria (adapted from Fleury et al. [112]).ROS can be generated by various enzymes (xanthine oxidase, lipoxygenases, NADPH oxidases,…) or during the mitochondrial oxidative phosphorylation at complex I and complex III (see text). Complex I (NADH-ubiquinone oxidoreductase) catalyzes the following reaction with electron transfer: NADH + Q + nH + (matrix) <-> NAD + + QH2 + (n-1)H + (intermembrane space), where Q is the ubiquinone Q10. The ubiquinone site (UQ) in complex III catalyzes the conversion of molecular oxygen to superoxide anion radical (O2 .-) by a single electron transfer to molecular oxygen. O2 .-are converted to hydrogen peroxide (H2O2 ) by the superoxide dismutase (SOD).H2O2 detoxification normally occurs according two different reactions, either by the thioredoxine reductase (TR), which reduces thioredoxine (TX -> TX-H2), or by reacting with glutathione (GSH) by glutathione peroxidase (GPO). The latter reaction produces water and oxidized glutathione (GSSG), and GSSG is recycled to GSH by glutathione reductase (GR). Glutathione is mainly maintained in a reduced state, via NADPH generation in the reversible nonoxidative part of the pentose phosphate pathway[113]. When GSH level is low, H2O2 can react with Fe 2+ to produce OH -and the harmful hydroxyl anions (OH . ). Alternatively, H2O2 reacts with O2 .- producing water, molecular oxygen and OH . . Superoxide may also react with nitric oxide (NO) to produce peroxynitrite (ONOO .-), a potent oxidant, which causes irreversible inhibition of mitochondria respiration and damage to mitochondria components (complexes I, II, IV and V, creatine kinase, aconitase, membranes, DNA, SOD,…).The complexes of the electron transfer chain in the inner mitochondrial membrane are numbered from I to IV. The electron (e-) pathway through the respiratory chain is indicated by arrows. Cyt c refers to cytochrome c.

Figure 3 :Figure 4 :

 34 Figure 3: Effect of Bcl-2 on mitochondrial events occurring during TNF--induced apoptosis of HeLa cells. Part a: Effect of Bcl-2 on the mitochondrial membrane potential and ROS accumulation. Panels depict typical cytograms (FSC versus fluorescence) obtained with flow cytometric analysis of Control or E/TNF treated HeLa cells that overexpress (bottom) or not bcl-2 (top). The mitochondrial membrane potential, measured by the DiOC6(3) fluorescence (left panel), and the ROS production, measured by the DCF fluorescence (right panel), are reported. Values of FSC (Forward angle Scatter) correlate to the size of the cells. Part b: Effect of Bcl-2 on Bax and cytochrome c localization in cells undergoing TNF--induced apoptosis. HeLa cells overexpressing (D-F) or not (A-C) a tetracycline-regulated inducible bcl-2 gene were treated with E/TNF for 6 h. Cells were double immunostained for cytochrome c (B, D and H) and Bax (C, F and I), and nuclei were visualized by Hoechst 33258. Arrows indicate apoptotic cells.