The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease.

Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation in TRIP4 encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted in TRIP4 mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and in Trip4 knocked-down C2C12 led to a significant reduction in myotube diameter ex vivo and in vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the first TRIP4 mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism.

The p.R482W substitution in A-type lamins deregulates SREBP1 activity in Dunnigan-type familial partial lipodystrophy.

Nuclear lamins are involved in many cellular functions due to their ability to bind numerous partners including chromatin and transcription factors, and affect their properties. Dunnigan type familial partial lipodystrophy (FPLD2; OMIM#151660) is caused in most cases by the A-type lamin R482W mutation. We report here that the R482W mutation affects the regulatory activity of sterol response element binding protein 1 (SREBP1), a transcription factor that regulates hundreds of genes involved in lipid metabolism and adipocyte differentiation. Using in situ proximity ligation assays (PLA), reporter assays and biochemical and transcriptomic approaches, we show that interactions of SREBP1 with lamin A and lamin C occur at the nuclear periphery and in the nucleoplasm. These interactions involve the Ig-fold of A-type lamins and are favored upon SREBP1 binding to its DNA target sequences. We show that SREBP1, LMNA and sterol response DNA elements form ternary complexes in vitro. In addition, overexpression of A-type lamins reduces transcriptional activity of SREBP1. In contrast, both overexpression of LMNA R482W in primary human preadipocytes and endogenous expression of A-type lamins R482W in FPLD2 patient fibroblasts, reduce A-type lamins-SREBP1 in situ interactions and upregulate a large number of SREBP1 target genes. As this LMNA mutant was previously shown to inhibit adipogenic differentiation, we propose that deregulation of SREBP1 by mutated A-type lamins constitutes one underlying mechanism of the physiopathology of FPLD2. Our data suggest that SREBP1 targeting molecules could be considered in a therapeutic context.

Gene- and exon-expression profiling reveals an extensive LPS-induced response in immune cells in patients with cirrhosis.

BACKGROUND & AIMS: Lipopolysaccharide (LPS)-expressing bacteria cause severe inflammation in cirrhotic patients. The global gene response to LPS is unknown in cirrhotic immune cells. METHODS: Gene-expression profiling using Affymetrix Human Exon Array analyzed the expression of 14,851 genes in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from 4 patients with cirrhosis and 4 healthy subjects. We performed validation studies using RT-qPCR in LPS-stimulated PBMCs from 52 patients and 9 healthy subjects and investigated the association of gene induction with mortality in 26 patients. RESULTS: Gene-expression profiling of LPS-stimulated cirrhotic cells showed 509 upregulated genes and 1588 downregulated genes. In LPS-stimulated "healthy" cells, 952 genes were upregulated and 838 genes downregulated. The 741 LPS-regulated genes shared by cirrhotic and "healthy" cells were involved in cytokine production/activity and induction of "immune paralysis". Comparison of functions associated with the 1356 genes, specifically regulated by LPS in cirrhotic cells, to functions of the 1049 genes, specifically regulated in "healthy" cells, allowed to define a cirrhosis-specific phenotype. Unlike in "healthy" cells, LPS failed to induce an interferon-mediated program in cirrhotic cells. In cirrhotic PBMCs, LPS specifically induced certain molecules involved in apoptosis and downregulated molecules involved in endocytic trafficking. RT-qPCR experiments showed that LPS-stimulated cirrhotic PBMCs had an enhanced induction of certain proinflammatory cytokines and chemokines. In the prognosis study, higher ex vivo LPS-induction of the inflammatory genes IL6 and CXCL5 was a significant predictor of mortality. CONCLUSIONS: Our results show that LPS-stimulated cirrhotic PBMCs exhibit an extensive and often unexpected transcriptional response.

Sex-Specific Patterns of Diaphragm Phospholipid Content and Remodeling during Aging and in a Model of SELENON-Related Myopathy.

Growing evidence shows that the lipid bilayer is a key site for membrane interactions and signal transduction. Surprisingly, phospholipids have not been widely studied in skeletal muscles, although mutations in genes involved in their biosynthesis have been associated with muscular diseases. Using mass spectrometry, we performed a phospholipidomic profiling in the diaphragm of male and female, young and aged, wild type and SelenoN knock-out mice, the murine model of an early-onset inherited myopathy with severe diaphragmatic dysfunction. We identified 191 phospholipid (PL) species and revealed an important sexual dimorphism in PLs in the diaphragm, with almost 60% of them being significantly different between male and female animals. In addition, 40% of phospholipids presented significant age-related differences. Interestingly, SELENON protein absence was responsible for remodeling of 10% PL content, completely different in males and in females. Expression of genes encoding enzymes involved in PL remodeling was higher in males compared to females. These results establish the diaphragm PL map and highlight an important PL remodeling pattern depending on sex, aging and partly on genotype. These differences in PL profile may contribute to the identification of biomarkers associated with muscular diseases and muscle aging.

Abnormal Cellular Phenotypes Induced by Three TMPO/LAP2 Variants Identified in Men with Cardiomyopathies.

A single missense variant of the TMPO/LAP2α gene, encoding LAP2 proteins, has been associated with cardiomyopathy in two brothers. To further evaluate its role in cardiac muscle, we included TMPO in our cardiomyopathy diagnostic gene panel. A screening of ~5000 patients revealed three novel rare TMPO heterozygous variants in six males diagnosed with hypertrophic or dilated cardiomypathy. We identified in different cellular models that (1) the frameshift variant LAP2α p.(Gly395Glufs*11) induced haploinsufficiency, impeding cell proliferation and/or producing a truncated protein mislocalized in the cytoplasm; (2) the C-ter missense variant LAP2α p.(Ala240Thr) led to a reduced proximity events between LAP2α and the nucleosome binding protein HMGN5; and (3) the LEM-domain missense variant p.(Leu124Phe) decreased both associations of LAP2α/ß with the chromatin-associated protein BAF and inhibition of the E2F1 transcription factor activity which is known to be dependent on Rb, partner of LAP2α. Additionally, the LAP2α expression was lower in the left ventricles of male mice compared to females. In conclusion, our study reveals distinct altered properties of LAP2 induced by these TMPO/LAP2 variants, leading to altered cell proliferation, chromatin structure or gene expression-regulation pathways, and suggests a potential sex-dependent role of LAP2 in myocardial function and disease.

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins.

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (∆607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

Chronic ethanol consumption lessens the gain of body weight, liver triglycerides, and diabetes in obese ob/ob mice.

Clinical studies suggest that moderate alcohol consumption can have beneficial effects, in particular regarding cardiovascular events, insulin resistance, and type 2 diabetes. In this study, lean and obese diabetic ob/ob mice were submitted or not to chronic ethanol intake via the drinking water for 6 months, which was associated with moderate levels of plasma ethanol. Plasma levels of alanine aminotransferase and aspartate aminotransferase were not increased by alcohol intake. Ethanol consumption progressively reduced the gain of body weight in ob/ob mice, but not in lean mice, and this was observed despite higher calorie intake. Increased plasma free fatty acids and glycerol in ethanol-treated ob/ob mice suggested peripheral lipolysis. Glycemia and insulinemia were significantly reduced, whereas adiponectinemia was increased in ethanol-treated ob/ob mice. Liver weight and triglycerides were significantly decreased in ethanol-treated ob/ob mice, and this was associated with less microvesicular steatosis. Hepatic levels of AMP-activated protein kinase and the phosphorylated form of acetyl-CoA carboxylase were higher in ethanol-treated ob/ob mice, suggesting better fatty acid oxidation. However, hepatic mRNA expression of several lipogenic genes was not reduced by ethanol consumption. Finally, mild oxidative stress was noticed in the liver of ethanol-treated mice, regardless of their genotype. Hence, our data are in keeping with clinical studies suggesting that moderate ethanol intake can have beneficial effects on type 2 diabetes and insulin sensitivity, at least in part through increased levels of plasma adiponectin. However, further studies are needed to determine whether long-term drinking of light-to-moderate amounts of ethanol is safe for the liver.

Ibuprofen administration attenuates serum TNF-alpha levels, hepatic glutathione depletion, hepatic apoptosis and mouse mortality after Fas stimulation.

Fas stimulation recruits neutrophils and activates macrophages that secrete tumor necrosis factor-alpha (TNF-alpha), which aggravates Fas-mediated liver injury. To determine whether nonsteroidal anti-inflammatory drugs modify these processes, we challenged 24-hour-fasted mice with the agonistic Jo2 anti-Fas antibody (4 microg/mouse), and treated the animals 1 h later with saline or ibuprofen (250 mg/kg), a dual cyclooxygenase (COX)-1 and COX-2 inhibitor. Ibuprofen attenuated the Jo2-mediated recruitment/activation of myeloperoxidase-secreting neutrophils/macrophages in the liver, and attenuated the surge in serum TNF-alpha. Ibuprofen also minimized hepatic glutathione depletion, Bid truncation, caspase activation, outer mitochondrial membrane rupture, hepatocyte apoptosis and the increase in serum alanine aminotransferase (ALT) activity 5 h after Jo2 administration, to finally decrease mouse mortality at later times. The concomitant administration of pentoxifylline (decreasing TNF-alpha secretion) and infliximab (trapping TNF-alpha) likewise attenuated the Jo2-mediated increase in TNF-alpha, the decrease in hepatic glutathione, and the increase in serum ALT activity 5 h after Jo2 administration. The concomitant administration of the COX-1 inhibitor, SC-560 (10 mg/kg) and the COX-2 inhibitor, celecoxib (40 mg/kg) 1 h after Jo2 administration, also decreased liver injury 5 h after Jo2 administration. In contrast, SC-560 (10 mg/kg) or celecoxib (40 or 160 mg/kg) given alone had no significant protective effects. In conclusion, secondary TNF-alpha secretion plays an important role in Jo2-mediated glutathione depletion and liver injury. The combined inhibition of COX-1 and COX-2 by ibuprofen attenuates TNF-alpha secretion, glutathione depletion, mitochondrial alterations, hepatic apoptosis and mortality in Jo2-treated fasted mice.

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient.

Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.

A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus.

A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient's skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient's mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor.

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.

Inducible nitric oxide synthase (iNOS) activity could be responsible for resistance or sensitivity to IFN-gamma-induced apoptosis in several human hepatoma cell lines.

Response to interferon-gamma (IFN-gamma)-induced apoptosis of human hepatoma cell lines (HHCLs) is variable. We analyzed this different behavior in Hep3B, Chang-liver, HepG2, and HuH7 cells. We studied (1) IFN-gamma-induced apoptosis, (2) protein expression of Stat1, (3) binding of nuclear proteins to IFN-gamma activated sequence (GAS), (4) mRNA and expression of proteins acting in apoptosis, and (5) HuH7 sensitivity after inducible nitric oxide synthase (iNOS) siRNA transfection. IFN-gamma induced apoptosis in Hep3B and Chang-liver cells only. In all HHCLs, Stat1 protein increased. Binding of proteins and transactivation activity of GAS increased much more in HuH7. In all HHCLs, caspase activity and apoptotic proteins were not implicated in resistance or sensitivity. iNOS mRNA and protein expression increased in HuH7, disappeared in Hep3B, and remained unchanged in Chang-liver and HepG2. We compared the role of iNOS in Hep3B and HuH7. The iNOS inhibitor, L-NAME, sensitized HuH7 to IFN-gamma, Hep3B/HuH7 coculture partially inhibited Hep3B apoptosis, and HuH7 transfection with iNOS siRNA induced a 50% inhibition of iNOS protein and cell apoptosis. GAS activity and overexpression of iNOS in HuH7, but not in the other HHCLs, suggest that this enzyme could play an important role in the resistance of HuH7 to IFN-gamma-induced apoptosis, perhaps by the antiapoptotic action of NO.

Pleiotropic effects of PI-3' kinase/Akt signaling in human hepatoma cell proliferation and drug-induced apoptosis.

IGF-II and type I-IGF receptor (IGF-IR) gene expression is increased in primary liver tumors, and transgenic mice overexpressing IGF-II in the liver develop hepatocellular carcinoma (HCC) spontaneously, suggesting that alterations of IGF-IR signaling in vivo may play a role in the auto/paracrine control of hepatocarcinogenesis. We have addressed the contribution of PI-3'K/Akt signaling on the proliferation of HepG2 human hepatoma cells and on their protection against doxorubicin-induced apoptosis. Both basal HepG2 cell DNA replication and that stimulated by IGF-IR signaling were inhibited by the specific PI-3'K inhibitor Ly294002 (Ly). In the former case, PI-3'K signaling overcame cell cycle arrest in G1 via increased cyclin D1 protein and decreased p27kip1 gene expression. Doxorubicin treatment induced apoptosis in HepG2 cells and was concomitant with the proteolytic cleavage of Akt-1 and -2. Drug-induced apoptosis was reversed by IGF-I and this effect was (i) dependent on Akt-1 and -2 phosphorylation and (ii) accompanied by the inhibition of initiator caspase-9 activity, suggesting that IGF-IR signaling interferes with mitochondria-dependent apoptosis. Accordingly, Ly enhanced doxorubicin-induced apoptosis and suppressed its reversal by IGF-I. Altogether, the data emphasize the crucial role of PI-3'K/Akt signaling (i) in basal as well as IGF-IR-stimulated HepG2 cell proliferation and (ii) in controlling both doxorubicin-induced apoptosis (e.g., drug-induced cleavage of Akt) and its reversal by IGF-I (protection against apoptosis parallels the extent of Akt phosphorylation). They suggest that targeting Akt activity or downstream Akt effectors (e.g., GSK3-beta, FOXO transcription factors) may help define novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients.

Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells.

AIM: To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment. METHODS: Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, Bcl-X(L) and cytochrome c, and the cleavage of poly (ADP-ribose) polymerase (PARP) were assayed by using Western blots. RESULTS: Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or anti-Fas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-X(L) expression. CONCLUSION: Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.

Differential responses of proliferative and non-proliferative leukemia cells to oxidative stress.

The response of three human leukemia cell lines, the proliferative promonocyte THP-1 and the promyeloid HL60 cells and the non-proliferative phorbol ester-treated HL60 cells (HL60/PMA), to oxidative stress induced by tert-butylhydroperoxide (t-BHP) treatment was analyzed by fluorescence microplate assay, anti-oxidant enzyme activity measurements, high performance liquid chromatography, yopro-1/PI incorporation, poly (ADP-ribose) polymerase and caspase 3 cleavages. After t-BHP treatment, the non-proliferative HL60/PMA cells exhibited a weak increase in reactive oxygen species (ROS) production, a better preservation of thiol content, a decrease of glutathione peroxidase activity and a high ability to undergo necrosis rather than apoptosis. Submitted to the same treatment, the proliferative HL60 and THP-1 cells exhibited a high increase of ROS production, a moderate thiol depletion and a high percentage of apoptosis. Under thiol depleting conditions, the oxidative treatment of the HL60/PMA cells resulted in a high ROS production that reached levels similar to those of the two other cell lines and in cell death mainly by necrosis. In conclusion, these results that show proliferative phenotype is essential for cell response towards oxidative stress, are of particular interest in chemotherapy involving an oxidative mechanism.

Progesterone induces BRCA1 mRNA decrease, cell cycle alterations and apoptosis in the MCF7 breast cancer cell line.

BACKGROUND: Inherited mutations of the BRCA1 gene are responsible for hereditary breast and ovarian cancer syndrome. However, little is known of how disruption of BRCA1 functions preferentially increases cancer risk in hormone-dependent organs. We aimed to study whether BRCA1 was regulated by progesterone in the MCF7 breast cancer cell line. MATERIALS AND METHODS: MCF7 breast cancer cells were incubated with 10(-4) or 10(-10) M progesterone for 24 or 48 hours. BRCA1 expression, proliferation and apoptosis were analysed. RESULTS: 10(-4) M progesterone decreased cell proliferation, cell cycle progression and induced apoptosis. In addition, BRCA1 and cyclin A mRNA decreased. In contrast, none of these effects were observed in MCF7 cells incubated with 10(-10) M progesterone. CONCLUSION: The down-regulation of BRCA1 in MCF7 cells incubated with 10(-4) M progesterone seems to be a consequence of cell cycle alterations rather than a direct effect of the hormone on BRCA1.

The beneficial effect of myostatin deficiency on maximal muscle force and power is attenuated with age.

The prolonged effect of myostatin deficiency on muscle performance in knockout mice has as yet been only poorly investigated. We have demonstrated that absolute maximal force is increased in 6-month old female and male knockout mice and 2-year old female knockout mice as compared to age- and sex-matched wildtype mice. Similarly, absolute maximal power is increased by myostatin deficiency in 6-month old female and male knockout mice but not in 2-year old female knockout mice. The increases we observed were greater in 6-month old female than in male knockout mice and can primarily result from muscle hypertrophy. In contrast, fatigue resistance was decreased in 6-month old knockout mice of both sexes as compared to age- and sex-matched wildtype mice. Moreover, in contrast to 2-year old female wildtype mice, aging in 2-year old knockout mice reduced absolute maximal force and power of both sexes as compared to their younger counterparts, although muscle weight did not change. These age-related decreases were lower in 2-year old female than in 2-year old male knockout mice. Together these results suggest that the beneficial effect of myostatin deficiency on absolute maximal force and power is greater in young (versus old) mice and female (versus male) mice. Most of these effects of myostatin deficiency are related neither to changes in the concentration of myofibrillar proteins nor to the slow to fast fiber type transition.

Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3ß and p53 in response to tumor necrosis factor α.

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3ß) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3ß and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3ßS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3ß activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3ß in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3ß, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Mitochondrial CYP2E1 is sufficient to mediate oxidative stress and cytotoxicity induced by ethanol and acetaminophen.

Several cytochromes P450 (CYPs) are not only located in the endoplasmic reticulum but also within mitochondria. One such CYP is CYP2E1 which metabolizes numerous substrates and generates significant amount of reactive oxygen species. The presence of CYP2E1 in these organelles raises questions regarding its physiological role but also its possible deleterious effects in the context of drug-induced cytotoxicity. The aim of our study was to investigate the role of mitochondrial CYP2E1 in the toxicity of acetaminophen and ethanol. Hence the effects of these two compounds in cells expressing CYP2E1 in mitochondria only, or in both endoplasmic reticulum and mitochondria, were compared to those observed in mock-transfected cells. Our results indicated that when acetaminophen or ethanol were used as CYP2E1 substrates, the exclusive localization of CYP2E1 within mitochondria was sufficient to induce reactive oxygen species overproduction, depletion of reduced glutathione, increased expression of mitochondrial Hsp70, mitochondrial dysfunction and cytotoxicity. Importantly, these harmful events happened despite lower cellular level and activity of CYP2E1 when compared to cells expressing CYP2E1 in both endoplasmic reticulum and mitochondria, and this was particularly obvious with acetaminophen. Taken together, these data suggest that mitochondrial CYP2E1 could play a major role in drug-induced oxidative stress and cell demise.