Arnaud Bruneel

Anticancer drugs induce necrosis of human endothelial cells involving both oncosis and apoptosis

The endothelium is the first physiological barrier between blood and tissues and can be injured by physical or chemical stress, particularly by the drugs used in cancer therapy. We found that four anticancer agents: etoposide, doxorubicin, bleomycin and paclitaxel induced apoptosis in human umbilical vein endothelial cells (HUVECs) (as judged by DNA fragmentation) with a time- and concentration-dependent decrease in bcl-2 protein but without the involvement of p53. As revealed by immunoblotting, bax protein was expressed in HUVECs treated with 1 mg/ml etoposide whereas bcl-2 protein disappeared. Oncosis occurred parallel to apoptosis with the release of lactate dehydrogenase into the supernatant, and, for doxorubicin and etoposide with the inversion of the distribution of angiotensin I-converting enzyme between supernatant and cells. Among the four tested anticancer drugs, only doxorubicin induced an oxidative stress, with significative malondialdehyde production. Thus, human endothelial cells in confluent cultures seem to be in an equilibrium of resistance to apoptosis related to bcl-2 expression; this equilibrium can be disrupted by a chemical stress, such as the antiproliferative drugs known as pro-apoptotic for tumour cells. For doxorubicin and bleomycin, this cellular toxicity can be related to their unwanted effects in human cancer therapy. Low doses of doxorubicin, paclitaxel or etoposide, however, could induce apoptosis of endothelial cells of new vessels surrounding the tumour, thus leading to specific vessel regression with minimal toxic effects for the endothelium of the other vessels. These findings provide evidence of relationships between endothelial toxicity of anticancer drugs and the key role of bcl-2 for resistance of endothelium cells toward apoptosis; moreover lack of p53 and bax in quiescent cells contributes to resistance of endothelial cells to DNA-damaging agents.

Reactive Oxygen Species, AMP-activated Protein Kinase, and the Transcription Cofactor p300 Regulate α-Tubulin Acetyltransferase-1 (αTAT-1/MEC-17)-dependent Microtubule Hyperacetylation during Cell Stress

Background: Tubulin acetylation is a hallmark of microtubule stabilization, which may modulate the binding of microtubule-associated proteins. Results: Microtubules are hyperacetylated because of stress-induced cellular signaling upstream of the tubulin acetyltransferase MEC-17/αTAT1. Conclusion: MEC-17/αTAT1 is regulated by p300, reactive oxygen species, and AMP-activated protein kinase. Significance: Microtubule hyperacetylation is important for cell adaptation to stress through autophagy induction and for cell survival. Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.

Identification of Rat Targets of Anti-Soluble Liver Antigen Autoantibodies by Serologic Proteome Analysis

BACKGROUND Anti-soluble liver antigen (SLA) autoantibodies are specific for autoimmune hepatitis type 1 and are the only immunologic marker found in 15-20% of hepatitis cases previously considered cryptogenic. Anti-SLA antibodies react with the 100 000g supernatant from rat liver homogenate, but the molecular targets remain controversial. METHODS We characterized anti-SLA targets by one- and two-dimensional immunoblotting analysis. The recognized proteins were identified by peptide mass fingerprint analysis after matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. RESULTS Three proteins of 35 kDa and pI 6.0, 50 kDa and pI between 6.0 and 6.5, and 58 kDa and pI between 6.5 and 7.0 were stained more intensely by anti-SLA positive-sera than by control sera. After in-gel tryptic digestion, MALDI-TOF analysis of the generated peptides enabled the clear identification of N-hydroxyarylamine sulfotransferase, isoforms of alpha-enolase, and isoforms of catalase. CONCLUSIONS Possible antigens for anti-SLA antibodies include a sulfotransferase, alpha-enolase(s), and catalase(s). Two-dimensional electrophoresis combined with mass spectrometry offers a versatile tool to identify molecular targets of autoantibodies and thus to improve diagnostic tools and the understanding of the immune process.

Red blood cell Thomsen-Friedenreich antigen expression and galectin-3 plasma concentrations in Streptococcus pneumoniae–associated hemolytic uremic syndrome and hemolytic anemia

Pneumococcal hemolytic uremic syndrome (P‐HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking.

MALDI-TOF MS applied to apoC-III glycoforms of patients with congenital disorders affecting O-glycosylation. Comparison with two-dimensional electrophoresis

The O‐glycan abnormalities accompanying some congenital disorders of glycosylation, namely conserved oligomeric Golgi‐congenital disorders of glycosylation (COG‐CDGs) and ATP6V0A2‐CDGs, are mainly detected using electrophoresis methods applied to circulating apolipoprotein C‐III. The objective of this study was to evaluate the reliability of MALDI‐TOF MS of apoC‐III for the detection and characterization of CDG‐associated O‐glycan defects.

Clinical, laboratory and molecular findings and long-term follow-up data in 96 French patients with PMM2-CDG (phosphomannomutase 2-congenital disorder of glycosylation) and review of the literature

Background Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a multisystem inborn error of metabolism. Objectives To better characterise the natural history of PMM2-CDG. Methods Medical charts of 96 patients with PMM2-CDG (86 families, 41 males, 55 females) were retrospectively reviewed. Data on clinical, laboratory and molecular parameters at diagnosis were analysed. Follow-up data at last examination were reported for 25 patients. Results The patients were born between 1963 and 2011. Diagnosis of PMM2-CDG was made at a mean (SD) age of 6.8 (8.5) years. The presenting signs were mostly neurological (hypotonia, intellectual disability, cerebellar syndrome) and observed in almost all the patients. A total of 38 patients (14 males, 24 females) exhibited, in addition to neurological signs, visceral features including at least one of these: feeding difficulty requiring a nutritional support (n=23), cardiac features (n=20; pericarditis: 14, cardiac malformation: 9, cardiomyopathy: 2), hepato-gastrointestinal features (n=12; chronic diarrhoea: 7, protein-losing enteropathy: 1, ascites: 3, liver failure: 1, portal hypertension: 1), kidney features (n=4; nephrotic syndrome: 2, tubulopathy: 2) and hydrops fetalis (n=1). Twelve patients died at a mean age of 3.8 years (especially from pericarditis and other cardiac issues). Laboratory abnormalities mostly included elevated transaminases and abnormal coagulation parameters. High thyreostimulin levels, hypocholesterolemia, hypoalbuminemia and elevated transaminases were associated with the visceral phenotype. Besides the common Arg141His PMM2 variant harboured by half of the patients, 45 different variants were observed. Conclusions PMM2-CDG clinical phenotype is heterogeneous in terms of clinical course, with no clear division between neurological and visceral presentations.

PHProteomicDB: A Module for Two-dimensional Gel Electrophoresis Database Creation on Personal Web Sites

PHProteomicDB is a PHP-written module to help researchers in proteomics to share two-dimensional gel electrophoresis data using personal web sites. No technical or PHP knowledge is necessary except a few basics about web site management. PHProteomicDB has a user-friendly administration interface to enter and update data. It creates web pages on the fly displaying gel characteristics, gel pictures, and numbered gel spots with their related identifications pointing to their reference pages in protein databanks. The module is freely available at http://www.huvec.com/index.php3?rub=Download.

Cyclosporin A But Not Estradiol Can Protect Endothelial Cells Against Etoposide-Induced Apoptosis

This study was undertaken to examine the possibilities of endothelial protection toward toxicity of anticancer drugs. To test the hypothesis that estradiol (E2) and cyclosporin A (CsA) can interfere within programmed cell death in human umbilical vein endothelial cells (HUVECs), apoptosis was induced by etoposide with and without E2 or CsA. All the concentrations of E2 tested (from 10(-9) to 10(-5) M) failed to protect HUVECs. For CsA a dual effect was observed: used at 1 or 10 microg/mL in coincubation with etoposide, CsA significantly reduced etoposide-induced apoptosis but complete inhibition was not reached, whereas used at 50 microg/mL CsA did not protect HUVECs anymore and even had deleterious effects. Furthermore, a 24-h pretreatment of HUVECs by CsA at 10 microg/mL significantly protected the cells by preventing both bcl-2 level decrease and caspase-3 activation related to etoposide-induced apoptosis. Protective effects of CsA toward endothelial cells were concentration dependent; in pretreatment at 10 microg/mL, CsA was an effective protector and might contribute in vivo to inhibit obvious toxic effects caused by anticancer drugs.

Two-dimensional electrophoresis highlights haptoglobin beta chain as an additional biomarker of congenital disorders of glycosylation

Congenital disorders of glycosylation (CDGs) are rare inherited disorders affecting glycosylation of proteins and lipids and sharing very heterogeneous multivisceral symptoms. The biochemical screening of these diseases is currently limited to electrophoresis or HPLC separation/quantification of serum transferrin glycoforms and is relatively frequently hampered by genetic polymorphism. Further, it has been shown that transferrin glycosylation can be very poorly affected in confirmed CDGs. We developed a fast and simple two-dimensional (2-DE) Western-blot analysis applied to the simultaneous detection of various serum glycoproteins, i.e. haptoglobin, α1-anti-trypsin, transferrin and α1-acid glycoprotein, and applied it to a large cohort of CDGs and secondary glycosylation disorders. When separated using 2-DE, haptoglobin β glycoforms showed clear abnormalities in all interpretable CDG type I and CDG type II patterns. Although secondary glycosylation defects such as alcoholism, untreated fructosemia and bacterial neuraminidase remain to be excluded, we showed that 2-DE pattern of haptoglobin β glycoforms thus constitute a very reliable additional biomarker of all types of CDGs. Coupled with common screening techniques and glycans mass spectrometry, it can orientate and facilitate the way towards CDG molecular diagnostic.

Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography–tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1−/− mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1−/− hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.