Mesothelioma: Scientific clues for prevention, diagnosis, and therapy.

Mesothelioma affects mostly older individuals who have been occupationally exposed to asbestos. The global mesothelioma incidence and mortality rates are unknown, because data are not available from developing countries that continue to use large amounts of asbestos. The incidence rate of mesothelioma has decreased in Australia, the United States, and Western Europe, where the use of asbestos was banned or strictly regulated in the 1970s and 1980s, demonstrating the value of these preventive measures. However, in these same countries, the overall number of deaths from mesothelioma has not decreased as the size of the population and the percentage of old people have increased. Moreover, hotspots of mesothelioma may occur when carcinogenic fibers that are present in the environment are disturbed as rural areas are being developed. Novel immunohistochemical and molecular markers have improved the accuracy of diagnosis; however, about 14% (high-resource countries) to 50% (developing countries) of mesothelioma diagnoses are incorrect, resulting in inadequate treatment and complicating epidemiological studies. The discovery that germline BRCA1-asssociated protein 1 (BAP1) mutations cause mesothelioma and other cancers (BAP1 cancer syndrome) elucidated some of the key pathogenic mechanisms, and treatments targeting these molecular mechanisms and/or modulating the immune response are being tested. The role of surgery in pleural mesothelioma is controversial as it is difficult to predict who will benefit from aggressive management, even when local therapies are added to existing or novel systemic treatments. Treatment outcomes are improving, however, for peritoneal mesothelioma. Multidisciplinary international collaboration will be necessary to improve prevention, early detection, and treatment.

BAP1 is a novel regulator of HIF-1α.

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos.

Carriers of heterozygous germline BAP1 mutations (BAP1+/-) are affected by the "BAP1 cancer syndrome." Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/- cells secrete increased amounts of HMGB1, and that BAP1+/- carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.

BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation.

BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1+/-) developed one and often several BAP1-/- malignancies in their lifetime, mostly malignant mesothelioma, uveal melanoma, and so on. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1+/- carriers cause reduction both of IP3R3 levels and of Ca2+ flux, preventing BAP1+/- cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1+/- carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene-environment interaction in human carcinogenesis.

Asbestos induces mesothelial cell transformation via HMGB1-driven autophagy.

Asbestos causes malignant transformation of primary human mesothelial cells (HM), leading to mesothelioma. The mechanisms of asbestos carcinogenesis remain enigmatic, as exposure to asbestos induces HM death. However, some asbestos-exposed HM escape cell death, accumulate DNA damage, and may become transformed. We previously demonstrated that, upon asbestos exposure, HM and reactive macrophages releases the high mobility group box 1 (HMGB1) protein that becomes detectable in the tissues near asbestos deposits where HMGB1 triggers chronic inflammation. HMGB1 is also detectable in the sera of asbestos-exposed individuals and mice. Searching for additional biomarkers, we found higher levels of the autophagy marker ATG5 in sera from asbestos-exposed individuals compared to unexposed controls. As we investigated the mechanisms underlying this finding, we discovered that the release of HMGB1 upon asbestos exposure promoted autophagy, allowing a higher fraction of HM to survive asbestos exposure. HMGB1 silencing inhibited autophagy and increased asbestos-induced HM death, thereby decreasing asbestos-induced HM transformation. We demonstrate that autophagy was induced by the cytoplasmic and extracellular fractions of HMGB1 via the engagement of the RAGE receptor and Beclin 1 pathway, while nuclear HMGB1 did not participate in this process. We validated our findings in a novel unique mesothelial conditional HMGB1-knockout (HMGB1-cKO) mouse model. Compared to HMGB1 wild-type mice, mesothelial cells from HMGB1-cKO mice showed significantly reduced autophagy and increased cell death. Autophagy inhibitors chloroquine and desmethylclomipramine increased cell death and reduced asbestos-driven foci formation. In summary, HMGB1 released upon asbestos exposure induces autophagy, promoting HM survival and malignant transformation.

Heterozygous germline BLM mutations increase susceptibility to asbestos and mesothelioma.

Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM+/-) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM+/- mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM+/- mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM+/- mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm+/- mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm+/- mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1ß, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm+/- mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM+/- mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma.

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

Study of PTEN subcellular localization.

The tumor suppressor PTEN is a key regulator of a plethora of cellular processes that are crucial in cancer development. Through its lipid phosphatase activity PTEN suppresses the PI3K/AKT pathway to govern cell proliferation, growth, migration, energy metabolism and death. The repertoire of roles fulfilled by PTEN has recently been expanded to include crucial functions in the nucleus, where it favors genomic stability and restrains cell cycle progression, as well as protein phosphatase dependent activity at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), where PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and regulates Ca(2+) release from the ER and sensitivity to apoptosis. Indeed, PTEN is present in definite subcellular locations where it performs distinct functions acting on specific effectors. In this review, we summarize recent advantages in methods to study PTEN subcellular localization and the distinct biological functions of PTEN in different cellular compartments. A deeper understanding of PTEN's compartmentalized-functions will guide the rational design of novel therapies.

ATP synthesis and storage.

Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.

Mitochondria-associated membranes (MAMs) as hotspot Ca(2+) signaling units.

The tight interplay between endoplasmic reticulum (ER) and mitochondria is a key determinant of cell function and survival through the control of intracellular calcium (Ca(2+)) signaling. The specific sites of physical association between ER and mitochondria are known as mitochondria-associated membranes (MAMs). It has recently become clear that MAMs are crucial for highly efficient transmission of Ca(2+) from the ER to mitochondria, thus controlling fundamental processes involved in energy production and also determining cell fate by triggering or preventing apoptosis. In this contribution, we summarize the main features of the Ca(2+)-signaling toolkit, covering also the latest breakthroughs in the field, such as the identification of novel candidate proteins implicated in mitochondrial Ca(2+) transport and the recent direct characterization of the high-Ca(2+) microdomains between ER and mitochondria. We review the main functions of these two organelles, with special emphasis on Ca(2+) handling and on the structural and molecular foundations of the signaling contacts between them. Additionally, we provide important examples of the physiopathological role of this cross-talk, briefly describing the key role played by MAMs proteins in many diseases, and shedding light on the essential role of mitochondria-ER interactions in the maintenance of cellular homeostasis and the determination of cell fate.

Calcium signaling around Mitochondria Associated Membranes (MAMs).

Calcium (Ca2+) homeostasis is fundamental for cell metabolism, proliferation, differentiation, and cell death. Elevation in intracellular Ca2+ concentration is dependent either on Ca2+ influx from the extracellular space through the plasma membrane, or on Ca2+ release from intracellular Ca2+ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca2+ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca2+ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca2+ release and Ca2+ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca2+ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.

Ca(2+) transfer from the ER to mitochondria: when, how and why.

The heterogenous subcellular distribution of a wide array of channels, pumps and exchangers allows extracellular stimuli to induce increases in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) with highly defined spatial and temporal patterns, that in turn induce specific cellular responses (e.g. contraction, secretion, proliferation or cell death). In this extreme complexity, the role of mitochondria was considered marginal, till the direct measurement with targeted indicators allowed to appreciate that rapid and large increases of the [Ca(2+)] in the mitochondrial matrix ([Ca(2+)]m) invariably follow the cytosolic rises. Given the low affinity of the mitochondrial Ca(2+) transporters, the close proximity to the endoplasmic reticulum (ER) Ca(2+)-releasing channels was shown to be responsible for the prompt responsiveness of mitochondria. In this review, we will summarize the current knowledge of: i) the mitochondrial and ER Ca(2+) channels mediating the ion transfer, ii) the structural and molecular foundations of the signaling contacts between the two organelles, iii) the functional consequences of the [Ca(2+)]m increases, and iv) the effects of oncogene-mediated signals on mitochondrial Ca(2+) homeostasis. Despite the rapid progress carried out in the latest years, a deeper molecular understanding is still needed to unlock the secrets of Ca(2+) signaling machinery.

Biological Mechanisms and Clinical Significance of BAP1 Mutations in Human Cancer.

Among more than 200 BAP1-mutant families affected by the "BAP1 cancer syndrome," nearly all individuals inheriting a BAP1 mutant allele developed one or more malignancies during their lifetime, mostly uveal and cutaneous melanoma, mesothelioma, and clear-cell renal cell carcinoma. These cancer types are also those that, when they occur sporadically, are more likely to carry somatic biallelic BAP1 mutations. Mechanistic studies revealed that the tumor suppressor function of BAP1 is linked to its dual activity in the nucleus, where it is implicated in a variety of processes including DNA repair and transcription, and in the cytoplasm, where it regulates cell death and mitochondrial metabolism. BAP1 activity in tumor suppression is cell type- and context-dependent. BAP1 has emerged as a critical tumor suppressor across multiple cancer types, predisposing to tumor development when mutated in the germline as well as somatically. Moreover, BAP1 has emerged as a key regulator of gene-environment interaction.This article is highlighted in the In This Issue feature, p. 1079.

Tumour predisposition and cancer syndromes as models to study gene-environment interactions.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.

A Subset of Mesotheliomas With Improved Survival Occurring in Carriers of BAP1 and Other Germline Mutations.

PURPOSE: We hypothesized that four criteria could help identify malignant mesotheliomas (MMs) most likely linked to germline mutations of BAP1 or of other genes: family history of MM, BAP1-associated cancers, or multiple malignancies; or age younger than 50 years. PATIENTS AND METHODS: Over the course of 7 years, 79 patients with MM met the four criteria; 22 of the 79 (28%) reported possible asbestos exposure. They were screened for germline BAP1 mutations by Sanger sequencing and by targeted next-generation sequencing (tNGS) for germline mutations in 55 additional cancer-linked genes. Deleterious mutations detected by tNGS were validated by Sanger sequencing. RESULTS: Of the 79 patients, 43 (16 probands and 27 relatives) had deleterious germline BAP1 mutations. The median age at diagnosis was 54 years and median survival was 5 years. Among the remaining 36 patients with no BAP1 mutation, median age at diagnosis was 45 years, median survival was 9 years, and 12 had deleterious mutations of additional genes linked to cancer. When compared with patients with MMs in the SEER cohort, median age at diagnosis (72 years), median survival for all MM stages (8 months), and stage I (11 months) were significantly different from the 79 patients with MM in the current study ( P < .0001). CONCLUSION: We provide criteria that help identify a subset of patients with MM who had significantly improved survival. Most of these patients were not aware of asbestos exposure and carried either pathogenic germline mutations of BAP1 or of additional genes linked to cancer, some of which may have targeted-therapy options. These patients and their relatives are susceptible to development of additional cancers; therefore, genetic counseling and cancer screening should be considered.

Germline BAP1 mutations induce a Warburg effect.

Carriers of heterozygous germline BAP1 mutations (BAP1+/-) develop cancer. We studied plasma from 16 BAP1+/- individuals from 2 families carrying different germline BAP1 mutations and 30 BAP1 wild-type (BAP1WT) controls from these same families. Plasma samples were analyzed by liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found a clear separation in the metabolic profile between BAP1WT and BAP1+/- individuals. We confirmed the specificity of the data in vitro using 12 cell cultures of primary fibroblasts we derived from skin punch biopsies from 12/46 of these same individuals, 6 BAP1+/- carriers and 6 controls from both families. BAP1+/- fibroblasts displayed increased aerobic glycolysis and lactate secretion, and reduced mitochondrial respiration and ATP production compared with BAP1WT. siRNA-mediated downregulation of BAP1 in primary BAP1WT fibroblasts and in primary human mesothelial cells, led to the same reduced mitochondrial respiration and increased aerobic glycolysis as we detected in primary fibroblasts from carriers of BAP1+/- mutations. The plasma and cell culture results were highly reproducible and were specifically and only linked to BAP1 status and not to gender, age or family, or cell type, and required an intact BAP1 catalytic activity. Accordingly, we were able to build a metabolomic model capable of predicting BAP1 status with 100% accuracy using data from human plasma. Our data provide the first experimental evidence supporting the hypothesis that aerobic glycolysis, also known as the 'Warburg effect', does not necessarily occur as an adaptive process that is consequence of carcinogenesis, but rather that it may also predate malignancy by many years and facilitate carcinogenesis.

Latest developments in our understanding of the pathogenesis of mesothelioma and the design of targeted therapies.

Malignant mesothelioma is an aggressive cancer whose pathogenesis is causally linked to occupational exposure to asbestos. Familial clusters of mesotheliomas have been observed in settings of genetic predisposition. Mesothelioma incidence is anticipated to increase worldwide in the next two decades. Novel treatments are needed, as current treatment modalities may improve the quality of life, but have shown modest effects in improving overall survival. Increasing knowledge on the molecular characteristics of mesothelioma has led to the development of novel potential therapeutic strategies, including: molecular targeted approaches, that is the inhibition of vascular endothelial growth factor with bevacizumab; immunotherapy with chimeric monoclonal antibody, immunotoxin, antibody drug conjugate, vaccine and viruses; inhibition of asbestos-induced inflammation, that is aspirin inhibition of HMGB1 activity may decrease or delay mesothelioma onset and/or growth. We elaborate on the rationale behind new therapeutic strategies, and summarize available preclinical and clinical results, as well as efforts still ongoing.

Oxidative stress in cardiovascular diseases and obesity: role of p66Shc and protein kinase C.

Reactive oxygen species (ROS) are a byproduct of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis. An imbalance between ROS production and the cellular antioxidant defence system leads to oxidative stress. Environmental factors and genetic interactions play key roles in oxidative stress mediated pathologies. In this paper, we focus on cardiovascular diseases and obesity, disorders strongly related to each other; in which oxidative stress plays a fundamental role. We provide evidence of the key role played by p66(Shc) protein and protein kinase C (PKC) in these pathologies by their intracellular regulation of redox balance and oxidative stress levels. Additionally, we discuss possible therapeutic strategies aimed at attenuating the oxidative damage in these diseases.

Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes.

The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca(2+)) signaling. Aequorin reacts with Ca(2+) via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca(2+) probe and how to measure Ca(2+) in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition.

The term "mitochondrial permeability transition" (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called "permeability transition pore complex" (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.