Antioxidants delay clinical signs and systemic effects of ENU induced brain tumors in rats.

According to our previous study suggesting that antioxidant properties of phytochemicals in the diet decrease glioma aggressiveness, we used a SUVIMAX-like diet ("Supplementation en VItamines et Minéraux AntioXydants") (enriched with alpha-tocopherol, beta carotene, vitamin C, zinc, and sodium selenite), adapted to rats. The present results showed that each of the antioxidants inhibited growth of glioma cells in vitro. When used in combination for in vivo studies, we showed a highly significant delay in the clinical signs of the disease, but not a statistical significant difference in the incidence of glioma in an Ethyl-nitrosourea (ENU)-model. The SUVIMAX-like diet decreased candidate markers of tumoral aggressiveness and gliomagenesis progression. The mRNA expressions of 2 common markers in human glioma: Mn-SOD (Manganese Superoxide Dismutase) and IGFBP5 (insulin growth factor binding protein) were reduced in the tumors of rats fed the antioxidant diet. In addition, the transcripts of two markers linked to brain tumor proliferation, PDGFRb (platelet-derived growth factor receptor beta) and Ki-67, were also significantly decreased. On the whole, our results suggest a protective role for antioxidants to limit aggressiveness and to some extent, progression of gliomas, in a rat model.

Increase in PGE2 biosynthesis induces a Bax dependent apoptosis correlated to patients' survival in glioblastoma multiforme.

Prostaglandin E(2) plays multiple roles both in the physiology and the physiopathology of human brain, which are not completely understood. We have identified in a subset of human glioblastoma multiforme (GBM) tumors, the most common form of adult brain cancer, an increased expression of mPGES-1, the enzyme which catalyses the isomerization of PGH(2) into PGE(2) downstream of cyclooxygenase 2 (COX-2). The sensitivity of primary cultures of GBM to apoptosis was augmented by the overexpression of mPGES-1, whereas the knockdown of its expression by shRNA decreased the apoptotic threshold in vitro and stimulated tumor growth in vivo. Adding extracellular PGE(2) in the culture medium failed to reproduce mPGES-1 effect on the cell viability in vitro. However, the intracellular injection of PGE(2) induced a dose-dependent apoptosis in GBM cultures, which was dependent on the presence of Bax, a pro-apoptotic protein. We show that PGE(2) physically associates with Bax, triggering its apoptotic-like change in conformation and its subsequent association with mitochondria. Our results raise questions about the role of PGE(2) in the control of apoptosis and in its potential impact in central nervous system pathologies.

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax.

The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway.

Mitochondrial membrane permeabilization produced by PTP, Bax and apoptosis: a 1H-NMR relaxation study.

To analyze the involvement of structured water (bound to macromolecules) in apoptosis-induced mitochondrial outer-membrane permeability, we compared the dynamics of water protons from nuclear magnetic resonance (NMR) data in apoptotic liver mitochondria with that of control mitochondria incubated in vitro with free Ca(2+) (opening of the permeability transition pore, PTP) or with Bax alpha. Our results demonstrate that water molecules in apoptotic mitochondria exhibit an accelerated translational motion of structured water common with that induced by the opening of the PTP, but limited in amplitude. On the other hand, no significant quantitative change in structured water was observed in apoptotic mitochondria, a phenomenon also observed with Bax alpha-induced permeability. We conclude that the changes observed in the different water phases differ both quantitatively and qualitatively during the opening of the PTP and the Bax alpha-induced permeability, and that the apoptotic mitochondria exhibit mixed properties between these model situations.

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast.

Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.

Induction of chemoresistance in HL-60 cells concomitantly causes a resistance to apoptosis and the synthesis of P-glycoprotein.

The appearance of multidrug-resistant (MDR) proteins or the acquisition of a defective apoptotic programme are major drawbacks in the treatment of cancers since both induce a resistance to classical chemotherapy. However, a link between the two mechanisms has not, as yet, been clearly established. In this study, HL-60 cells cultured in the continual presence of a sub-lethal dose of doxorubicin (dox; HL-60/Dox) were used as a model to study acquired chemoresistance. During the induction of chemoresistance, the appearance of a functional P-glycoprotein (P-gp), in addition to the expression of anti-apoptotic Bcl-2, Bcl-XL and pro-apoptotic Bax proteins was assessed. Parental cells which are sensitive to dox, have no P-gp activity and express Bcl-2 and Bax. After 4 weeks of treatment, a functional P-gp was detected in HL-60/Dox cells. In addition, the synthesis of Bcl-2 appeared to be replaced by Bcl-XL while that of Bax remained unchanged. These cells were also resistant to apoptosis induced by both P-gp and non-P-gp substrates. This inability to induce apoptosis could have resulted from the induction of the expression of the inhibitor of apoptosis protein (XIAP). Our data show that acquired chemoresistance could involve a parallel induction of P-gp and an impairment of the apoptotic pathway.

D-2-Hydroxyglutarate does not mimic all the IDH mutation effects, in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH.

Somatic mutations in isocitrate dehydrogenase (IDH)-1 and -2 have recently been described in glioma. This mutation leads to a neomorphic enzymatic activity as the conversion of isocitrate to alpha ketoglutarate (αKG) is replaced by the conversion of αKG to D-2-hydroxyglutarate (D-2HG) with NADPH oxidation. It has been suggested that this oncometabolite D-2HG via inhibition of αKG-dioxygenases is involved in multiple functions such as epigenetic modifications or hypoxia responses. The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG. We show that the overexpression of mutant IDH in glioma cells or treatment with D-2HG triggered an increase in cell proliferation. However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis. Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate. These data show that besides D-2HG production, mutant IDH affects other crucial metabolite pools. These observations lead to a better understanding of the biology of IDH mutations in gliomas and their response to therapy.

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase.

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.

A conformation-dependent monoclonal antibody against active chicken acetylcholinesterase.

We show that the C-131 monoclonal antibody, directed against chicken AChE, recognizes active chicken AChE, but not the SDS-denatured or heat-inactivated protein. Previous results indicated that C-131 only binds to the active enzyme, and not to inactive molecules which also occur in the embryonic chicken brain. In contrast with C-131, other monoclonal antibodies obtained in the same series, such as C-6 and C-54, also recognize denatured or inactive AChE. It is noteworthy that these antibodies all seem to react with a trypsin-sensitive peptide which is present in chicken but not in mammalian or Torpedo AChE, whereas the C-131 antibody binds trypsin-modified as well as intact molecules. These results show that C-131 is highly conformation-dependent, specific for active AChE. They confirm our previous conclusion that active and inactive molecules arise from different folding processes.

The substitution of the C-terminus of bax by that of bcl-xL does not affect its subcellular localization but abrogates its pro-apoptotic properties.

The interaction of the anti-apoptotic members of the Bcl-2 family with mitochondria, through their hydrophobic C-terminus, has been proposed to play a crucial role in the execution phase of apoptosis. We report here that a substitution of the C-terminal end of pro-apoptotic bax by that of anti-apoptotic bcl-xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae. In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells. These results suggest that the C-terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.

Involvement of the N-terminus of Bax in its intracellular localization and function.

We have identified, using site-directed mutagenesis, a proline located at position 13 of Baxalpha (Bax) as crucial for the maintenance of its cytosolic conformation. The substitution of this proline by a valine results in a strong binding of Bax to mitochondria and to conformational changes monitored by a decreased sensitivity of Bax to mild proteolysis and the enhancement of its oligomerization state. Deletion of the C-terminus of Bax does not modify its intracellular localization. On the other hand, the pro-apoptotic activity of Bax is enhanced by a deletion of the C-terminus in the absence of the N-terminus but is decreased in its presence. These results suggest that both extremities functionally interact to control the activity but not the subcellular localization of Bax.

Resistance to apoptosis is increased during metastatic dissemination of colon cancer.

Apoptosis dysfunction in metastases has been suggested to participate in their poor response to conventional anticancer treatments. To address this question, we have analyzed the sensitivity to cell death induced by non-steroid anti-inflammatory drug, Sulindac, the most common drug used in colon cancer chemotherapy, 5-fluorouracil (5-FU) and the short chain fatty acid, butyrate (Bu) in cell lines derived from a primary colorectal tumor (ALT-I) as well as the liver (ALT-F) and the lymph-node (ALT-G) metastases. We have previously shown both in vitro by analyzing anchorage-independent cell proliferation and in vivo by subcutaneous injection into athymic nude mice that the ALT-F and ALT-G cells were more tumorigenic than the primary ALT-I cells. All these cell lines, derived from an untreated patient, were highly resistant to apoptosis induced by 5-FU and Sulindac but were sensitive to Bu-induced apoptosis. The resistance to apoptosis was, as quantified by the induction of caspase activity and the relative percentage of apoptotic cells, higher in the metastatic cell lines, than in the ALT cell line. When compared to the primary tumor, more anti-apoptotic bcl-2 and less pro-apoptotic bax were expressed in the liver and lymph node metastatic cell lines. Quite remarkably, the expression of bax was up-regulated during Bu-treatment, a feature that could explain its powerful pro-apoptotic activity.

Molecular forms of acetylcholinesterase in dystrophic (mdx) mouse tissues.

We analyzed the activity of acetylcholinesterase (AChE) and its molecular forms in the tissues of normal and dystrophic (mdx) mice, at different developmental stages. We studied the brain, the heart and the serum, in addition to four predominantly fast-twitch muscles (tibialis, plantaris, gastrocnemius and extensor digitorum longus (EDL)) and the slow-twitch, soleus muscle. We found no difference between mdx and control mice in the AChE activity of the brain and the heart. The skeletal muscles affected by the disease undergo active degeneration counterbalanced by regeneration between 3 and 14 weeks after birth. The distribution of AChE patches associated with neuromuscular junctions was abnormally scattered in mdx muscles, and in some cases (tibialis and soleus), the number of endplates was more than twice that of normal muscles. There were only minor differences in the concentration and pattern of AChE molecular forms during the acute phase of muscle degeneration and regeneration. After this period, however, we observed a marked deficit in the membrane-bound G4 molecular form of AChE in adult mdx tibialis, gastrocnemius and EDL but not in the plantaris or in the soleus, as compared with their normal counterparts. Whereas the amount of AChE markedly decreased in the serum of normal mice during the first weeks of life, it remained essentially unchanged in the serum of mdx mice. It is likely that this excess of AChE activity in serum originates from the muscles. A deficit in muscle G4 was also reported in other forms of muscular dystrophy in the mouse and chicken. Since it is not correlated to the acute phase of the disease in mdx and also occurs in genetically different dystrophies, it probably represents a secondary effect of the dystrophy.

Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: Expression of the molecular forms of acetylcholinesterase.

We attempted to cultivate muscle cells from chick embryos in a serum-free, defined medium similar to that proposed by Bottenstein and Sato (1979) for the growth and differentiation of a murine neuronal cell-line. (1) We found that muscle cells from the legs of 11-day old chick embryos can be cultivated in a medium containing the different components indicated by Bottenstein and Sato, with 2 g/l bovine serum albumin, without serum or chick embryo extract. Myoblasts attached to the gelatin-coated dishes without any addition of attachment factors. They differentiated into myotubes in a similar manner as in classical serum supplemented media. (2) The level of cellular AchE activity was comparable in cultures grown in the presence of fetal calf serum (FCS), of horse serum (HS) and in the defined medium. The percentage of A(12) form was however higher in the defined medium (25-30%) than in FCS supplemented medium (about 5-6%). In HS supplemented medium the A(12) form was not detectable, partly because horse serum contains immunoglobulins which bind chicken AChE. The addition of defined medium components to FCS medium cultures did not lead to an increase of A(12). In contrast, the addition of a small amount (1%) of fetal calf serum to DM cultures reduced the level of A(12) in a drastic manner. FCS components therefore seem to repress the biosynthesis of A(12) AChE, or increase its degradation. (3) We estimated intracellular and extracellular compartments of AChE. The ratio of endocellular to ectocellular AChE decreased with the age of the cultures. The G(1) form was intracellular at all stages analyzed, but the other molecular forms were located in both cellular compartment, in different proportion: A(12) and G(4) seemed to be located preferentially in the external compartment, whereas G(2) was preferentially intracellular. (4) Muscle cultures grown in the defined medium and in the presence of serum secreted globular forms of AChE in a similar manner.

[Apoptosis and pathologies of the central nervous system]. / Apoptose et pathologies du système nerveux central.

The program of cell death called apoptosis is a biochemical and genetical pathway which has been well conserved throughout evolution. In the past years, the involvement of apoptosis has been shown not only during the embryonic development of the nervous system, but also in neurodegenerative diseases. Moreover, resistance to apoptosis is clearly an important factor in tumor growth and seems to be, at least partially, a major process in chemo- and radio-resistance. In this review, we briefly describe the main molecular events of the apoptotic process, known to date, and discuss possible defects in the central nervous system diseases.

Bioactive lipids and the control of Bax pro-apoptotic activity.

Lipids are key regulators of cell physiology through the control of many aspects of cellular life and survival. In particular, lipids have been implicated at different levels and through many different mechanisms in the cell death program called apoptosis. Here, we discuss the action of lipids in the regulation of the activation and the integration of Bax into the mitochondrial outer membrane, a key pro-apoptotic member of the BCL-2 family. We describe how, during apoptosis, lipids can act simultaneously or in parallel as receptors or ligands for Bax to stimulate or inhibit its pro-death activity.

Control of glioma cell death and differentiation by PKM2-Oct4 interaction.

Glioma stem cells are highly resistant to cell death and as such are supposed to contribute to tumor recurrence by eluding anticancer treatments. Here, we show that spheroids that contain rat neural stem cells (NSCs) or rat glioma stem cells (cancer stem cells, CSCs) express isoforms 1 and 2 of pyruvate kinase (PKM1 and PKM2); however, the expression of PKM2 is considerably higher in glioma spheroids. Silencing of PKM2 enhances both apoptosis and differentiation of rat and human glioma spheroids. We establish that PKM2 was implicated in glioma spheroid differentiation through its interaction with Oct4, a major regulator of self-renewal and differentiation in stem cells. The small molecule Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, increases the amount of PKM2/Oct4 complexes and thus inhibited Oct4-dependent gene expression. Taken together, our results highlight a new molecular pathway through which PKM2 can manage gliomagenesis via the control of glioma stemness by Oct4.

Prognostic impact of the expression/phosphorylation of the BH3-only proteins of the BCL-2 family in glioblastoma multiforme.

Apoptosis has a crucial role in anti-cancer treatment. The proteins of the BCL-2 family are core members of the apoptotic program. Thus, we postulated that alterations in the expression of BCL-2 protein family, and in particular in that of the Bcl-2 homology domain 3 (BH3)-only proteins (which can neutralized anti-apoptotic proteins or activate pro-apoptotic proteins) could account for differences in the overall survival (OS) of patients. To test this hypothesis, we analyzed the expression of 15 members of the BCL-2 protein family (Bax, Bak, Bok, Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Bad, Bid, Bim, Bik, Bmf, Hrk, Noxa and Puma) in glioblastoma multiforme (GBM) tumors, the most frequent brain tumor in adults. We found that none of the individual expression of these proteins is associated with a significant variation in OS of the patients. However, when all BH3 proteins were pooled to determine a BH3(score), this score was significantly correlated with OS of GBM patients. We also noted that patients with a have high level of phospho-Bad and phospho-Bim displayed a lower OS. Thus, BH3 scoring/profiling could be used as an independent prognostic factor in GBM when globally analyzed.

Prostaglandins antagonistically control Bax activation during apoptosis.

The Bax protein (Bcl-2-associated X protein) is pivotal for the apoptotic process. Bax, which resides in an inactive form in the cytosol of healthy cells, is activated during the early stages of apoptosis and becomes associated with mitochondria through poorly understood mechanisms. In this study, we show that a family of bioactive lipids, namely prostaglandins, regulates Bax-dependent apoptosis. The prostaglandin E(2) (PGE(2)) or its derivative PGA(2) binds to Bax, induces its change of conformation, and thereby triggers apoptosis. A cysteine present in the loop between the two transmembrane α-helices of Bax, Cys126 is critical for its activation. PGD(2) inhibits PGE(2) binding to Bax and PGE(2)-induced apoptosis, as well as cell death induced by staurosporine and UV-B in various cell lines. This result is consistent with the fact that apoptosis is accompanied during these treatments by an increase in PGE(2). This process is distinct, yet cooperative, from that involving the BH3-only protein Bid. Our results establish that the PGE(2)/PGD(2) balance is involved in a new early mechanism of control in the activation of Bax during apoptosis.