Gadolinium-Based Nanoparticles Can Overcome the Radioresistance of Head and Neck Squamous Cell Carcinoma Through the Induction of Autophagy.

Radiation therapy is a mainstay in the therapeutic management of Head and Neck Squamous Cell Carcinoma (HNSCC). Despite significant progress in this field, radioresistance still accounts for most treatment failures. Gadolinium-based nanoparticles (GBNs) have shown great promises as radiosensitizers but the underlying sensitizing mechanism is still largely unknown with regards to the disparities obtained in in vitro studies. In this study, we show that a new formulation of GBNs, AGuIX®, can radiosensitize HNSCC after cell uptake and further accumulation in lysosomes. Although radiation alone triggered late apoptosis and mitochondrial impairment, the pre-treatment with GBNs led to complex DNA damage and a specific increase of autophagic cell death. In addition, a significant radio-enhancement effect was obtained after the pre-conditioning of cells with a glutathione inhibitor before GBNs treatment and radiation exposure. Overall, our results provide additional information on the radio-enhancing properties of GBNs in the management of radioresistant HNSCC.

Cellular and molecular portrait of eleven human glioblastoma cell lines under photon and carbon ion irradiation.

This study aimed to examine the cellular and molecular long-term responses of glioblastomas to radiotherapy and hadrontherapy in order to better understand the biological effects of carbon beams in cancer treatment. Eleven human glioblastoma cell lines, displaying gradual radiosensitivity, were irradiated with photons or carbon ions. Independently of p53 or O(6)-methylguanine-DNA methyltransferase(1) status, all cell lines responded to irradiation by a G2/M phase arrest followed by the appearance of mitotic catastrophe, which was concluded by a ceramide-dependent-apoptotic cell death. Statistical analysis demonstrated that: (i) the SF2(2) and the D10(3) values for photon are correlated with that obtained in response to carbon ions; (ii) regardless of the p53, MGMT status, and radiosensitivity, the release of ceramide is associated with the induction of late apoptosis; and (iii) the appearance of polyploid cells after photon irradiation could predict the Relative Biological Efficiency(4) to carbon ions. This large collection of data should increase our knowledge in glioblastoma radiobiology in order to better understand, and to later individualize, appropriate radiotherapy treatment for patients who are good candidates.

[Cardiac troponin I and C: analytical comparison and clinical-biological interpretation of three troponins assays]. / Troponines I et T cardiaques: comparaison analytique et étude clinicobiologique de trois trousses de troponine.

Many assays 1(st), 2(nd) even 3(rd) generation are at present available to determine the concentration of cardiac troponin I and T. With the redefinition of upper reference value in the acute coronary syndromes, the aim of this study was to evaluate the clinical and analytical performance of 2 troponins assays: Troponin Ic 2(nd) generation (AccuTnI) on Access 2 of Beckman Coulter and Troponin Tc 3(rd)generation (Troponin T STAT) on Elecsys 2010 of Roche Diagnostics. The analytical performance observed with these 2 assays are accurate (analytical and functional sensitivity, repetability and reproductibility). Comparing each method with Dade Behring assay (Flex Troponine-I Cardiaque, TROP) on Dimension RxL, the correlation observed with AccuTnI kit on Access 2 can be put into the equation: AccuTnI = 1.08 (TnIc TROP) - 0.34, r = 0.99. On the contrary, it's more difficult to compare cTnI and cTnT. The study of decisonnal values indicated by Beckman Coulter for cTnI (0.04 microg/L at the 99 degrees percentil, 0.06 microg/L for a CV < or =10%) show a better specificity (76%) and predictive positive value (89%) with a sensitivity at 100% at 0.1 microg/L, fixed and used in the laboratory for its better agreement between sensibility / specificity and its imprecision below 10 %. For the cTnT values published by Roche Diagnostics (0.01 microg/L), at the 99 degrees percentil and 0.03 microg/L for a CV < or = 10%, the specificity is lower, so the decisionnal value 0.1 microg/L seems to be more suitable. During this study, few false positive and negative cTnT values have been observed, in patients with complex pathologies; this eventuality must be taken in consideration if clinical findings are not in good accordance with laboratory results.

Subcellular distribution and metabolic fate of exogenous ceramides taken up by HL-60 cells.

Ceramides (Cer) are key intermediates in the metabolism of sphingomyelin and are also important second messengers. We report that natural long-chain ceramides added to the incubation medium in microgram amounts are internalized in HL-60 cells as well as the short-chain analogue C2-Cer and targeted to various subcellular compartments. No significant difference was detected in the ability of HL-60 cells to metabolize exogenous Cer containing a short (acetyl) versus long (palmitoyl or oleoyl) acyl chain. After a 2-h incubation time with [14C]-C16 ceramides, most of the cell-bound radioactivity was found in free ceramides. Sphingomyelin was the major metabolized sphingolipid containing labeled ceramides and only a small proportion of exogenous ceramides were converted to neutral glycolipids and gangliosides. Up to 20% of the exogenous ceramides taken up by the cells were recovered in mitochondria, mostly as authentic C16 ceramides and C16 sphingomyelin, along with a trace amount of labeled GM3 ganglioside. These results are consistent with the notion that exogenous natural ceramides enter cells, can be further metabolized in situ and partly targeted to mitochondria, which are known to be involved in the control of programmed cell death.

Phospholipids reacylation and palmitoylcoa control tumour necrosis factor-alpha sensitivity.

From the hypothesis that in TNF-alpha-resistant cells the activity of mitochondrial phospholipase A2 could be reversed by a lysophospholipid acyltransferase, we report that the mitochondrial reacylation of phosphatidylcholine as phosphatidylethanolamine was considerably higher in C6 (TNF-alpha-resistant) than in WEHI-164 (TNF-alpha-sensitive) cells. TNF-alpha did not modify the phospholipids' reacylation in C6, while in WEHI-164 it was increased several-fold. These results suggest that TNF-alpha is not sufficient to restore the barrier permeability in sensitive cells, but may be enough to explain the absence of permeability change in resistant cells. AcylCoA esters, depending on whether the acyl group is unsaturated or saturated (palmitic acid), could control membrane permeability either by participating in the reacylation of phospholipids or keeping the pore in a closed state. The analysis of the endogenous acylCoA ester pools of both cell lines show that the amount of palmitoylCoA is higher in resistant than sensitive cell lines. TNF-alpha treatment does not change these results.

Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria.

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three-fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long-chain bases found in free ceramides were d18:1 sphingosine, d18:0 3-ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3-ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long-chain base of glucosylceramide of outer membranes.

Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion.

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.

Kinetics of internalization and subcellular binding sites for T3 in mouse liver.

The intracellular fate of radiolabeled T3 taken up by mice hepatocytes in vivo was determined at specific time intervals (2-120 min) after injection by quantitative electron microscopic radioautography. Injection of a 200-fold excess of unlabeled T3 together with [125I]-T3 resulted in a more than 90% inhibition of radioactivity detected in hepatocytes. A simple grain density (GD) analysis of radioautograms revealed that a specific labeling (GD > 1) was displayed by only five cell compartments: the plasma membrane, lipid droplets, mitochondria, nuclear envelope and nuclear matrix whereas other compartments were not labeled. Labeled compartments showed distinct changes in the pattern of labeling over time: the plasma membrane was labeled only 2 min after T3 injection, whereas labeling of the nuclear envelope was high at 2 min, decreased at 15 min and progressively increased to maximal measured levels at 120 min. After a lag time of 30 min, nuclear matrix labeling increased progressively with time. Mitochondrial labeling was found to be specific at any time point studied but showed no change over time. These ultrastructural data have been confirmed in vitro by the interaction of T3 with plasma membrane, nuclear membrane, nuclear matrix and mitochondria by real-time biospecific interaction analysis in a BIAcore system. These results demonstrate that T3 binds to hepatocytes before internalization, is transported both to mitochondria and to the nuclear envelope and translocated into the nuclear matrix.

Further characterization of mitochondrial outer membrane: evidence for the presence of two endogenous sialylated glycoproteins.

The distribution of sialic acid-containing glycoproteins was investigated in highly purified mitochondrial membranes using labeled Sambucus nigra agglutinin as a detection system. Two sialylated glycoproteins were shown to be true components of the mitochondrial outer membrane. Relative to monoamine oxidase activity, these glycoproteins were found to be preferentially located in the "free" outer membrane fraction. As sialic acid is thought to be involved in molecular recognition, a role for these glycoproteins in mediating the interactions between mitochondria and other sub-cellular organelles is considered.

Involvement of mitochondrial contact sites in the subcellular compartmentalization of phospholipid biosynthetic enzymes.

The concerted synthesis of phospholipids derived from serine involving two microsomal enzymes (phosphatidylserine synthase and phosphatidylethanolamine N-methyltransferase) and a mitochondrial one (phosphatidylserine decarboxylase) occurs in reconstituted cell-free systems. Subfractionation of crude mitochondria after swelling and separating on a sucrose density gradient resulted in the isolation of two contact site-enriched fractions from total outer membranes and inner membranes, respectively. Estimation of marker enzyme activities shows a high recovery of glucose-6-phosphate phosphatase (a marker for the endoplasmic reticulum) associated with contact site-enriched fractions. Accordingly, the linked synthesis of phosphatidylserine, phosphatidylethanolamine, and at a lesser extent phosphatidylcholine can occur. This biosynthetic pathway was absent from purified contact site-enriched fractions correlative with the absence of glucose-6-phosphate phosphatase activity. Reconstitution experiments, including contact site-enriched fractions incubated with endoplasmic reticulum-rich fraction, led to the restoration of the linked synthesis of phospholipids, thereby demonstrating that a reversible association between these two fractions can occur. These functional interactions between the endoplasmic reticulum and mitochondria are confirmed at the ultrastructural level using either chemical or physical fixation before resin embedding. These results show that the interorganelle trafficking of lipids may involve only highly specialized microdomains of both membranes, thereby allowing the maintenance of a specific lipid composition and distribution within membranes.

Evidence for the presence of alpha and beta-related T3 receptors in rat liver mitochondria.

Although the dependence of mitochondrial structure and function on thyroid hormone status is well established, several attempts to demonstrate a direct pathway of T3 action on mitochondria have been made during the last decade without being firmly conclusive. In this study, we present evidence firstly for the presence of specific binding sites for [125I]-T3 in rat liver mitochondria 5 min after injection, as assessed by ultrastructural autoradiography. In the same way, using immunocytological techniques and protein immunoblotting, T3 receptor-like immunoreactivity was revealed mainly in the nucleus and mitochondria of hepatocytes. Whereas the colloidal gold labeling over mitochondria was found to be specific at the ultrastructural level, these results were confirmed biochemically by Western blotting experiments which revealed the presence of two protein bands in mitochondria: a stronger one of 55 kDa and a weaker one of 48 kDa. At the opposite, receptor T3 mRNAs were not detected in mitochondria by ultrastructural in situ hybridization thus confirming that the synthesis of receptor T3 occurs in the cytoplasm and that nuclear-encoded T3 receptors may belong to the bulk of cytosolic precursor polypeptides which are targeted to and imported into mitochondria. These results confirm that a direct pathway of T3 action on mitochondria occurs in situ which could now explain how the rapid activation of several mitochondrial functions can take place within minutes after thyroid hormone injection.

Further evidence for both functional and structural microcompartmentation within the membranes of two associated organelles, mitochondrion and endoplasmic reticulum.

We previously demonstrated that the translocation of phospholipids between the mitochondrion and the endoplasmic reticulum occurs via highly specialized membrane microdomains of both organelles that are in situ closely associated. As understanding of the interactions between both organelles requires characterization of the translocation sites organization, we first analysed the amino acid compositions of these sites. Using principal component analysis, we have shown that the translocation sites exhibit characteristic patterns when compared with the membranes from which they are derived. The results are discussed in terms of both functional and structural microcompartmentation within the membranes of mitochondria and endoplasmic reticulum.

Dacron vascular biomaterial triggers macrophage ectoenzyme activity without change in cell membrane fluidity.

Biomaterials induce an inflammatory reaction characterized by a rapid recruitment at the implantation site of polymorphonuclear cells and macrophages. In the course of the inflammatory response, the cellular activation triggers expression of a number of enzymes, such as 5'-nucleotidase, which is widely distributed in animal cell membranes as an ectoenzyme. It is now well established that 5'-nucleotidase activity decreases following the contact of inflammatory cells with foreign particles. In this paper we investigate a possible correlation between the enzymatic activities and the dynamic properties of the cell membrane bilayer. Dacron pieces were introduced into rats' peritoneal cavities for a period of 6 h, after which the peritoneal cells were harvested, and various enzyme assays performed, including those for cytoplasmic, lysosomal, and ectoenzymes. In parallel, we studied cell membrane fluidity, using fluorescence polarization of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cellular ultrastructural alteration resulting from the cell-biomaterial interactions using scanning and transmission electron microscopy. Our results show that: 1) macrophages spread around the Dacron fibers with cytoplasmic finger-like projections, but no phagolysosomes, 2) 5'-nucleotidase levels decrease with surgical trauma in comparison with the resident cell exudate, 3) implantation of biomaterials slightly modify the 5'-nucleotidase levels observed in the sham animal, 4) no differences in the anisotropy values indicating that membrane lipid order within the cells could not account for the observed decrease of 5'-nucleotidase activity. Thus, we can suggest that 5'-nucleotidase expression may reflect a particular feature of cell activation without a phagocytic process.

Phospholipid import into mitochondria: possible regulation mediated through lipid polymorphism.

We previously demonstrated that the translocation of microsomal phosphatidylserine to the inner mitochondrial membrane occurs via contact sites before decarboxylation. According to the specific lipid composition of contact sites, we investigated lipid polymorphism as a possible regulation mechanism of phospholipid import into mitochondria. Phosphatidylserine import into mitochondria is increased in the presence of calcium, under conditions where non bilayer lipid-structures can be induced in cardiolipin-containing membranes. The results are discussed in terms of structural as well as functional domains heterogeneity within contact sites.

Testosterone inhibits the immunostimulant effect of thymosin fraction 5 on secondary immune response in mice.

The purpose of the present investigation was to examine the in vivo influence of testosterone on the immune properties of a thymic factor (thymosin fraction 5, TF5) a partially purified thymic preparation in male Swiss IOPS/OF1 mice (5-10 weeks old). Testosterone administration (100 micrograms/ml) significantly inhibited the enhanced anti-sheep red blood cell antibody response induced by TF5 (100 micrograms/ml); this inhibition was only observed on the secondary antibody response and not on the primary. These results suggest that gonadal steroids can affect the immune response by modulating the activity of thymic factors.

Involvement of contact sites in phosphatidylserine import into liver mitochondria.

The synthesis, translocation, and decarboxylation of phosphatidylserine can occur in a cell-free system (Voelker, D. R. (1989) J. Biol. Chem. 264, 8019-8025). We made use of the spatial separation of the site of biosynthesis and the site of decarboxylation of phosphatidylserine to demonstrate that mitochondrial contact sites are intimately involved in the translocation of phosphatidylserine prior to decarboxylation. In that sense, the inhibition of phosphatidylserine decarboxylase leads to an accumulation of this phospholipid in the contact site-enriched fractions without mixing the inner membrane phospholipid pool. On the other hand, newly synthesized phosphatidylethanolamine can be exported very rapidly to the mitochondrial surface in the same way, i.e. via contact sites. These data provide further evidence for the existence of a structural and functional microcompartmentation at the inner mitochondrial membrane surface.

Further characterization of mitochondrial contact sites: effect of short-chain alcohols on membrane fluidity and activity.

Two mitochondrial contact site-enriched fractions were further characterized by freeze-fracture electron microscopy. Examination of the replicas revealed that these two fractions are in the form of vesicles with membrane sheets attached to the vesicles. The physical state of these fractions has been investigated by means of steady-state fluorescence polarization to assess the effects of alcohols on their fluidity properties and activity. Comparison between intact membranes and their extracted lipids shows that butanol and hexanol induce a differential increase of the overall membrane fluidity in the two contact site-enriched fractions. This alteration in the membrane dynamics leads to a complex modulation of cytochrome c oxidase activity in both fractions. These results bring further evidence for the existence of morphologically and functionally distinct domains in mitochondrial membranes.

Mitochondrial contact sites. Lipid composition and dynamics.

Two membrane fractions of intermediate density between inner and outer mitochondrial membranes were isolated by density gradient centrifugation from osmotically lysed mitochondria and mitoplasts of liver. These fractions were characterized by the presence of both monamine oxidase and cytochrome c oxidase activities and bound hexokinase. 1) The content of the fractions in proteins and lipids was assessed by biochemical determination. Thin-layer and gas-liquid chromatography showed that the two contact site-enriched fractions contain predominantly phosphatidylcholine (31%), phosphatidylethanolamine (27%, half-unsaturated), and cardiolipin (27%, fully unsaturated). 2) The dynamics of the fractions were assessed by fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene as a probe and by fluorescence decay measurements. We have verified that differences in static anisotropy cannot be exclusively attributed to differences in fluorescence lifetimes. On the contrary, the results indicated an increased lipid mobility in "inner membrane contact sites," which is probably related to a lower cholesterol to phospholipid ratio, as well as a lower saturation of the fatty acyl chains when compared with "outer membrane contact sites." Taken all together, the spectroscopic measurements confirm the biochemical results, leading to the idea that the two populations of contact sites have different physicochemical properties, which are probably mainly determined by the membrane from which they are derived. They constitute microdomains enriched either in inner or outer mitochondrial membranes.

Dolichol kinase activity: a key factor in the control of N-glycosylation in inner mitochondrial membranes.

Inner mitochondrial membranes from liver contain a dolichol kinase which required CTP as a phosphoryl donor. Kinase activity was linear with protein concentration and unlike other reported kinases, activated almost equally well by Mg2+, Mn2+ or Ca2+. Thin-layer chromatography showed that the reaction product co-migrated with authentic dolichyl monophosphate. The phosphorylation of dolichol did not occur in presence of ATP, GTP or UTP but required exogenous dolichol for maximal activity. Newly synthesized [3H]dolichyl monophosphate has been shown to be glycosylated in the presence of GDP[14C]mannose or UDP[14C]glucose. The double labeled lipids formed by the sugar nucleotide-dependent reactions were identified respectively as [14C]mannosylphosphoryl[3H]dolichol and [14C]glucosylphosphoryl [3H]dolichol. These results are discussed in terms of regulation of N-glycosylation processes in inner mitochondrial membranes from liver.

Triggering of mannosyltransferase activity in inner mitochondrial membranes by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids.

The activity of GDPmannose:dolichyl monophosphate mannosyltransferase in inner mitochondrial membranes can be triggered by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids. The efficiency of this incorporation and the efficiency of the enzyme activity are not equivalent. Among a variety of amphiphiles which were tested, the highest mannosyltransferase activity was obtained with the mixture of lipids extracted from the outer mitochondrial membranes. The results presented here appear consistent only with a mechanism involving collisional contacts of the phospholipid vesicles and fusion with the membranes. ESR spectroscopy confirms that (a) the incorporation process is followed by solubilization of dolichyl monophosphate molecules in the lipid phase and (b) the general organization of the inner mitochondrial membranes is not perturbed by the addition of dolichyl monophosphate.