The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression.

In response to different environmental stresses, eIF2α phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2α-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2α-ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.

Computational identification of transcriptionally co-regulated genes, validation with the four ANT isoform genes.

BACKGROUND: The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. RESULTS: From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases. CONCLUSIONS: Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.

Computational analysis of the transcriptional regulation of the adenine nucleotide translocator isoform 4 gene and its role in spermatozoid glycolytic metabolism.

Computational phylogenetic analysis coupled to promoter sequence alignment was used to understand mechanisms of transcriptional regulation and to identify potentially coregulated genes. Our strategy was validated on the human ANT4 gene which encodes the fourth isoform of the mitochondrial adenine nucleotide translocator specifically expressed during spermatogenesis. The movement of sperm flagella is driven mainly by ATP generated by glycolytic pathways, and the specific induction of the mitochondrial ANT4 protein presented an interesting puzzle. We analysed the sequences of the promoters, introns and exons of 30 mammalian ANT4 genes and constructed regulatory models. The whole human genome and promoter database were screened for genes that were potentially regulated by the generated models. 80% of the identified co-regulated genes encoded proteins with specific roles in spermatogenesis and with functions linked to male reproduction. Our in silico study enabled us to precise the specific role of the ANT4 isoform in spermatozoid bioenergetics.

Adenine nucleotide translocase 2 is a key mitochondrial protein in cancer metabolism.

Adenine nucleotide translocase (ANT), a mitochondrial protein that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, plays an essential role in cellular energy metabolism. Human ANT presents four isoforms (ANT1-4), each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proliferation. Thus, ANT1 is specific to muscle and brain tissues; ANT2 occurs mainly in proliferative, undifferentiated cells; ANT3 is ubiquitous; and ANT4 is found in germ cells. ANT1 and ANT3 export the ATP produced by oxidative phosphorylation (OxPhos) from the mitochondria into the cytosol while importing ADP. In contrast, the expression of ANT2, which is linked to the rate of glycolytic metabolism, is an important indicator of carcinogenesis. In fact, cancers are characterized by major metabolic changes that switch cells from the normally dual oxidative and glycolytic metabolisms to an almost exclusively glycolytic metabolism. When OxPhos activity is impaired, ANT2 imports glycolytically produced ATP into the mitochondria. In the mitochondrial matrix, the F1F0-ATPase complex hydrolyzes the ATP, pumping out a proton into the intermembrane space. The reverse operations of ANT2 and F1F0-ATPase under glycolytic conditions contribute to maintaining the mitochondrial membrane potential, ensuring cell survival and proliferation. Unlike the ANT1 and ANT3 isoforms, ANT2 is not pro-apoptotic and may therefore contribute to carcinogenesis. Since the expression of ANT2 is closely linked to the mitochondrial bioenergetics of tumors, it should be taken into account for individualizing cancer treatments and for the development of anticancer strategies.

Cell cycle-dependent conjugation of endogenous BRCA1 protein with SUMO-2/3.

BACKGROUND: BRCA1, the main breast and ovarian cancer susceptibility gene, has a key role in maintenance of genome stability, cell cycle and transcription regulation. Interestingly, some of the numerous proteins which interact with BRCA1 protein undergo conjugation with small ubiquitin-like modifiers (SUMO). This post-translational modification is related to transcription, DNA repair, nuclear transport, signal transduction, and to cell cycle stress response. METHODS AND RESULTS: Protein sequence analysis suggests that sumoylation target sites belong to the RING finger and BRCT domains (BRCA1 C-terminus), two crucial regions for BRCA1 function. Moreover putative SUMO interacting motifs are present in the sequence of many proteins of BRCA1 network. Using immunoprecipitations and western blotting, we show the conjugation of endogenous nuclear BRCA1 protein with SUMO-2/3. BRCA1 conjugation with SUMO-2/3 is linked to the cell cycle in a cell line dependent manner since no cell cycle dependence of sumoylation is observed in MCF7 breast cancer cells. In contrast, BRCA1 conjugation with SUMO-2/3 is linked to the oxidative stress independently to the cell line, in DU145, MCF7 and 293 T cells. CONCLUSION AND GENERAL SIGNIFICANCE: Our data reveal a new BRCA1 regulation pathway implying sumoylation in response to cell cycle progression and oxidative stress, providing a possible mechanism for the involvement of BRCA1 gene in tumorigenesis.

Quantitative two-dimensional HRMAS 1H-NMR spectroscopy-based metabolite profiling of human cancer cell lines and response to chemotherapy.

NMR spectroscopy-based metabolomics still needs development in quantification procedures. A method was designed for quantitative two-dimensional high resolution magic angle spinning (HRMAS) proton-NMR spectroscopy-based metabolite profiling of intact cells. It uses referencing of metabolite-related NMR signals to protein-related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two-dimensionally visible metabolites and combination of one- and two-dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping.

A model of phospholipid biosynthesis in tumor in response to an anticancer agent in vivo.

We study, in this paper, a model for the core of the system of the Glycerophospholipid metabolism in the murine cells. It comprises the simple and enzymatic reactions of PhosphatidylEthanolamine and the PhosphatidylCholine. The model's general structure is taken from a number of books and articles. We translate this model into a set of ordinary differential equations (ODEs), to propose a quantitative explanation of the experimental experiences and the observed results. In order to make it usable as a basis for simulations and mathematical analysis we need to make precise the various constants present in the equations but which are usually not directly accessible in the literature. In a first step we considered experimental data of rat's liver cells obtained by NMR spectroscopy: given the values of metabolite concentrations we find appropriate parameter values which allow us to describe the system with ODEs. We have then performed several analyses using the developed model such as stability analysis. A first interesting result is the global stability of the system which was observed by simulation and then proved by mathematical arguments. A second important result is that we observe on the diagrams that the steady state for normal cells is precisely a singular point of order two, whereas tumoral cells present different characteristics; this fact has been proved for PhosphatidylEthanolamine N-Methyl transferase (PEMT), an enzyme which seems to be identified for the first time as a crucial element in the tumoral process. In a second step we applied our model to experimental data of proton HRMAS NMR spectroscopy for solid B16 melanoma and Lewis lung (3LL) 3LL carcinoma cells treated by Chloroethyl Nitrosourea (CENU). We performed a complete comparative analysis of parameters in order to learn the predictive statements to explain increases and decreases which one can observe in concentrations.

Combined methionine deprivation and chloroethylnitrosourea have time-dependent therapeutic synergy on melanoma tumors that NMR spectroscopy-based metabolomics explains by methionine and phospholipid metabolism reprogramming.

Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met-sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.

Mitochondrial bioenergetic background confers a survival advantage to HepG2 cells in response to chemotherapy.

Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (DeltaPsim). It thus may be predicted that DeltaPsim regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O(2) consumption/ATP production coupling, DeltaPsim maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered DeltaPsim, leading to an increased energy request to regenerate DeltaPsim in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization.

ANT2 isoform required for cancer cell glycolysis.

The three adenine nucleotide translocator (ANT1 to ANT3) isoforms, differentially expressed in human cells, play a crucial role in cell bioenergetics by catalyzing ADP and ATP exchange across the mitochondrial inner membrane. In contrast to differentiated tissue cells, transformed cells, and their rho(0) derivatives, i.e. cells deprived of mitochondrial DNA, sustain a high rate of glycolysis. We compared the expression pattern of ANT isoforms in several transformed human cell lines at different stages of the cell cycle. The level of ANT2 expression and glycolytic ATP production in these cell lines were in keeping with their metabolic background and their state of differentiation. The sensitivity of the mitochondrial inner membrane potential (Deltapsi) to several inhibitors of glycolysis and oxidative phosphorylation confirmed this relationship. We propose a new model for ATP uptake in cancer cells implicating the ANT2 isoform, in conjunction with hexokinase II and the beta subunit of mitochondrial ATP synthase, in the Deltapsi maintenance and in the aggressiveness of cancer cells.

Random mtDNA deletions and functional consequence in aged human skeletal muscle.

Mitochondrial respiratory chain deteriorates with age, mostly in tissues with high energy requirements. Damage to mitochondrial DNA (mtDNA) by reactive oxygen species is thought to contribute primarily to this impairment. However, the overall extent of random mtDNA mutations has still not been evaluated. We carried out molecular and biochemical analyses in muscle biopsies from healthy young and aged subjects. Deleted mtDNA accumulation was followed by both quantitative PCR analysis to quantify total mtDNA, and Southern-blotting, to determine deleted to full length mtDNA ratio. Enzymatic activities of the mitochondrial respiratory chain were measured in all subjects. Randomly deleted mtDNA appeared mainly in the oldest subjects (beyond 80 years old), affecting up to 70% of mtDNA molecules. The activities of complexes III and IV of the respiratory chain, complexes with mtDNA encoded subunits, are lower in the aged subjects. Physical activity could be one major parameter modulating the mitochondrial respiratory chain activity in aged muscle.

[What is the specific role of ANT2 in cancer cells?]. / Quel rôle spécifique pour ANT2 dans une cellule cancéreuse?

In the mitochondrial internal membrane, the adenine nucleotide translocator (ANT) carries out the ATP/ADP exchange between cytoplasm and mitochondrial matrix. Three isoforms with different kinetic properties are encoded from three different genes in Human: the muscle specific ANT1 and the ubiquitary ANT3 isoforms export ATP produced by mitochondrial oxidative phosphorylation (OXPHOS). The ANT2 isoform is specifically expressed in proliferative cells with a predominant glycolytic metabolism and is associated with cellular undifferentiation which is a major characteristic in carcinogenesis. Its role would be to import into mitochondria ATP produced by the glycolysis, energy essential to several intramitochondrial functions, particularly to maintenance of the membrane potential (Delta Psi m), conditioning cellular survival and proliferation. The mechanism of regeneration of this Delta Psi m gradient would involve at least three major proteins: the hexokinase II isoform, the ANT2 isoform and the F1 part of the mitochondrial ATP synthase complex. Taking into account this major role of ANT2 in cell proliferation and the very low expression of this isoform in differentiated tissues, this protein or its transcript could be chosen as a target for an anticancer strategy. Furthermore, previous studies showed that molecules of the cisplatin family, used as chemotherapeutic agents, led to the destruction of the mitochondrial membrane potential and thus to cell death. Does the anticancer effect of these molecules result, at least partially, from this mitochondrial aggression? If it is the case, the ANT2 isoform, mainly involved in the generation of this potential by its ATP4-/ADP3- exchange, could be considered as a more specific targeting by an RNA interference approach.

ANT2 expression under hypoxic conditions produces opposite cell-cycle behavior in 143B and HepG2 cancer cells.

Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.

Quantification of mitochondrial DNA deletion, depletion, and overreplication: application to diagnosis.

BACKGROUND: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. METHODS: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. RESULTS: Both methods quantified 5-76% of deleted mtDNA in muscle, 59-97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r(2)) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. CONCLUSIONS: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.

Oxygen consumption and expression of the adenine nucleotide translocator in cells lacking mitochondrial DNA.

It has been shown previously that human rho degrees cells, deprived of mitochondrial DNA and consequently of functional oxidative phosphorylation, maintain a mitochondrial membrane potential, which is necessary for their growth. The goal of our study was to determine the precise origin of this membrane potential in three rho degrees cell lines originating from the human HepG2, 143B, and HeLa S3 cell lines. Residual cyanide-sensitive oxygen consumption suggests the persistence of residual mitochondrial respiratory chain activity, about 8% of that of the corresponding parental cells. The fluorescence emitted by the three rho degrees cell lines in the presence of a mitochondrial specific fluorochrome was partially reduced by a protonophore, suggesting the existence of a proton gradient. The mitochondrial membrane potential is maintained both by a residual proton gradient (up to 45 to 50% of the potential) and by other ion movements such as the glycolytic ATP(4-) to mitochondrial ADP(3-) exchange. The ANT2 gene, encoding isoform 2 of the adenine nucleotide translocator, is overexpressed in rho degrees HepG2 and 143B cells strongly dependent on glycolytic ATP synthesis, as compared to the corresponding parental cells, which present a more oxidative metabolism. In rho degrees HeLa S3 cells, originating from the HeLa S3 cell line, which already displays a glycolytic energy status, ANT2 gene expression was not higher as in parental cells. Mitochondrial oxygen consumption and ANT2 gene overexpression vary in opposite ways and this suggests that these two parameters have complementary roles in the maintenance of the mitochondrial membrane potential in rho degrees cells.

Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion.

Mitochondrial DNA depletion is a well established cause of severe liver failure in infancy. The autosomal inheritance of this quantitative mitochondrial DNA defect supports the involvement of a nuclear gene in the control of mitochondrial DNA level. We previously described a case of a 28-month-old child presenting with a progressive liver fibrosis due to a mitochondrial DNA depletion (85% at 12 months of age). As this syndrome was clinically liver-restricted, a liver transplant was initially discussed. We report the clinical, biochemical and molecular follow-up of this child, now 6 years old. The patient displayed a spontaneous gradual improvement of his liver function with continuous increment of clotting factor values since 32 months of age. A marked reduction of the previous extensive fibrosis was evidenced on a liver biopsy performed at 46 months of age associated with a dramatic decrease of the mitochondrial DNA depletion (35%). Consequently, an almost complete restoration of respiratory chain activities containing mitochondrial DNA-encoded subunits was observed. This is the first report of a revertant phenotype in liver mitochondrial DNA depletion syndrome.