[Stereotactic body radiation therapy for hepatocellular carcinoma: Results from a retrospective multicentre study]. / Radiothérapie en conditions stéréotaxiques du carcinome hépatocellulaire : résultats d'une étude rétrospective multicentrique.

PURPOSE: The purpose of this paper was to describe local control, overall survival, progression-free survival and toxicity of CyberKnife®-based stereotactic body radiation therapy of hepatocellular carcinoma. MATERIAL AND METHODS: Records of all the patients treated for hepatocellular carcinoma at the Eugene-Marquis cancer centre, Rennes and the Bretonneau hospital, Tours (France), between November 2010 and December 2016, were reviewed. Radiation therapy was performed as a salvage treatment, while awaiting liver transplantation or if no other treatment was possible. RESULTS: One hundred and thirty-six patients were consecutively included in the study. The median follow-up was 13months. Median total dose prescribed, fractionation and overall treatment time were respectively 45Gy, three fractions and 5 days. Overall survival, progression-free survival and local control rates at 1year and 2years were 79.8 % and 63.5 %, 61.3 % and 39.4 %; 94.5 % and 91 %. Two grade 3 acute toxicity events and two grade 4 late toxicity events corresponding to a duodenal ulcer have been reported. Seven patients underwent classic radiation-induced hepatitis and 13 patients showed non-classical radiation-induced hepatitis. Barcelona Clinic Liver Cancer stage, World Health Organisation grade and planning target volume were correlated with overall survival in univariate Cox analysis. CONCLUSION: Stereotactic body radiation therapy is effective and well-tolerated for inoperable hepatocellular carcinoma or as a bridge to liver transplantation. Toxicity is mainly related to cirrhotic background and requires a selection of patients and strict dose constraints.

[Documentation management: from theory to practice]. / La gestion documentaire: de la théorie à la pratique.

The management of the documentation is one of the key points regarding the efficacy and the performance of the quality management of health centres. It offers to all professionals the possibility to be informed on the procedures in use, leading to a pool of documents for improvement of organisations and for securing the critical steps of the patient management. In this paper, we will describe the optimal organisation of the documentation according to Haute autorité de santé (HAS) and ISO recommendations, then we will discuss in concrete terms the potential methods usable for the production of a tool well adapted to our routine practice, in order to achieve the objectives for security.

[The French preliminary experience of the use of a seed-projector for exclusive iodine 125 prostate brachytherapy: feasibility and acute toxicity]. / L'expérience française de l'utilisation d'un projecteur de source pour la curiethérapie prostatique exclusive par implants permanents d'iode radioactif: faisabilité et données de toxicité aiguë.

PURPOSE: To analyse a new technique for prostate brachytherapy with permanent Iodine implants characterized by the use of a seed projector after a 3D dosimetric peroperative treatment planning (FIRST technique). PATIENTS AND METHOD: 395 patients have been treated in France with this technique in six radiotherapy centres between November 2002 and December 2005 for a localized prostate cancer. RESULTS: Thirteen patients (3.3%) developped a urinary retention, and respectively 7.8 and 26.5% an acute RTOG grade 3 and 2 toxicity. The 6-weeks IPSS score was equal or lower to 15 in 73% with a 11 median IPSS value. A failure of the loading with the seed-projector, leading to a manual loading of the seeds, occurred in 9 patients (2.3%) in two centres, directly related to the loading procedure with the seed-projector in 5 cases. The median duration of the procedure was reduced by 30 minutes for the patients treated in 2005. CONCLUSIONS: This multicenter study establishes the feasibility of the routine use of a seed projector for permanent iodine 125 prostate implants with an initial tolerance similar to the best results published for other implants techniques.

[Study of a new commercial film for high energy photon dosimetry]. / Etude d'un film récemment commercialisé pour la dosimétrie des faisceaux de photons de haute énergie.

Dosimetric properties of a new film, the Extended Dose Range-EDR2, manufactured by Kodak, have been studied. We have established the response of the film versus dose and compared it with that of X-OMAT V films. We found a linear response with dose, for the range from 0.5 to 4 Gy. No dependence of this curve with beam quality and with depth is observed. EDR2 films are useful for dosimetric study of high-energy photon beam, especially when high dose gradient occurs such as for Intensity Modulated Radiotherapy Treatment.

Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to gamma-radiation but activation of this sequence is defective in radioresistant SQ20B cells.

PURPOSE: To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to 7-rays. MATERIALS AND METHODS: Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (delta(psi)m), reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry. RESULTS: In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in delta(psi)m and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, gamma-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway. CONCLUSIONS: Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.

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.

Palmitic and stearic acids bind Ca2+ with high affinity and form nonspecific channels in black-lipid membranes. Possible relation to Ca2+-activated mitochondrial pores.

A mitochondrial hydrophobic component that forms Ca2+-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids--mainly palmitic and stearic. These fatty acids, similar to the mitochondrial hydrophobic component, bind Ca2+ with high affinity in comparison with unsaturated fatty acids, saturated fatty acids with shorter aliphatic chains, phospholipids, and other lipids. Ca2+-binding is inhibited by Mg2+ but not by K+. For palmitic acid, the Kd for Ca2+ was 5 microM at pH 8.5 and 15 microM at pH 7.5, with the Bmax of 0.48 +/- 0.08 mmol/g. This corresponds to one Ca2+ ion for eight palmitic acid molecules. The data of IR spectroscopy confirm that Ca2+ does not form ionic bonds with palmitic and stearic acids under hydrophobic conditions. It has been found that in the presence of Ca2+, palmitic and stearic acids, but not unsaturated FFA induce a nonspecific permeability in black-lipid membranes. Addition of Ca2+ in order to induce the permeability transition, increases the extractable amount of palmitic and stearic acids, the effect being prevented by a phospholipase A2 inhibitor. The possible involvement of palmitic and stearic acids in the mitochondrial nonspecific permeability is discussed.

[Gross tumor volume and clinical target volume: esophageal tumors]. / Volume tumoral macroscopique et volume-cible anatomoclinique en radiothérapie: tumeurs de l'aesophage.

The oesophagus is divided into four regions: cervical oesophagus, and intrathoracic oesophagus with an upper, mid- and lower thoracic portion. Cancer may occur on each of these regions. Computed tomography of the thorax and superior abdomen and endoscopic ultrasound are necessary for reliable staging. CT simulation allows accurate definition of tumour volume. GTV includes tumour volume and regional lymph nodes. CTV encompasses GTV plus a safety margin and lymph node areas considered to harbour potential microscopic disease. The extent of prophylactic lymph node irradiation depends on the anatomic location of the primary tumour.

Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis.

To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2 h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Delta(psi)m), between 4 and 48 h after treatment. Ceramide levels began to increase 2 h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48 h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations.

Dietary spermidine and spermine participate in the maturation of galactosyltransferase activity and glycoprotein galactosylation in rat small intestine.

This study considered the role of dietary polyamines in the maturation of intestinal glycoprotein galactosylation during postnatal development. In the rat small intestine, O-glycan: beta-1,3-galactosyltransferase and N-glycan: beta-1,4-galactosyltransferase are, respectively, involved in the glycan chain biosynthesis of mucins and of glycoproteins in the brush border membranes. Their activities increase significantly at weaning, in parallel with a rise in the intestinal content of spermidine and spermine (as determined by high performance liquid chromatography) and in proportion to the polyamine increase in food intake. The oral ingestion of spermidine or spermine (at 0.4 micromol/g body) by immature suckling rats for 4 d reproduced the levels of spermine and spermidine in their intestines at the time of weaning and induced precocious and significant rises in O-glycan: and N-glycan: galactosyltransferase activities to those normally found after weaning. In parallel, more galactose residues (detected in the complex oligosaccharide chains of glycoproteins by specific lectins after electrophoresis and transfer to nitrocellulose membranes) were observed in the brush border membranes of spermidine- and spermine-treated rats. In contrast, the ingestion of putrescine or ornithine had no effect. Diets with different levels of polyamines (milks and commercial diet), when given at weaning, induced variable evolutions of the galactosylation process, partly in relation to the amounts of polyamines ingested. These results indicate that spermidine and spermine are maturation factors that can reproduce, in immature rats, the same increase in intestinal glycoprotein galactosylation that is normally observed during weaning. They also suggest that the maturation of glycoprotein galactosylation may be a multifactorial event in which spermidine and spermine are both involved.

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.

[Adenocarcinomas of the distal esophagus and cardia. Anatomy and volume of irradiation]. / Adénocarcinome du bas oesophage et du cardia. Anatomie et volumes à irradier.

The esophagus is divided into four regions: cervical esophagus, intrathoracic esophagus with upper, mid and lower thoracic portion. Cancer may occur on each of these regions. Computed tomography of the thorax and superior abdomen and endoscopic ultrasound are necessary for reliable staging. CT simulation allows accurate definition of tumor volume. GTV includes tumor volume and regional lymph nodes. CTV encompasses GTV plus safety margin and lymph nodes areas considered to harbor potential microscopic disease. The extent of prophylactic lymph node irradiation depends on the anatomic location of the primary tumor.

Comparison of methods used to study cell death in an adherent tumoral cell line with moderate clonogenic radiosensitivity.

Our objective was to compare different methods for studying programmed cell death in adherent H460 non-small lung cancer cells of moderate clonogenic radiosensitivity. The major effect of gamma-radiation was found to be the release of cells from the substratum. The different methods gave complementary and unexpected information: a) with the TUNEL method, a few non-apoptotic cells were found in the culture medium; b) with the flow cytometry after propidium iodide labeling, some hypodiploid cells which remained attached to the substratum were apoptotic, as demonstrated by the effect of a caspase inhibitor; c) with the annexin V labeling, the detached cells were demonstrated either necrotic or very late apoptotic; d) the mitochondria transmembrane potential (deltapsim), measurements demonstrated that the mitochondria were implicated in cell death induced by gamma-radiation. These data illustrate the need to use several complementary methods in the study of apoptosis in adherent cells exposed to gamma-radiation.

Up-regulation of mitochondrial peripheral benzodiazepine receptor expression by tumor necrosis factor alpha in testicular leydig cells. Possible involvement in cell survival.

Porcine Leydig cells in primary cultures are resistant to tumor necrosis factor alpha (TNFalpha) cytotoxicity. Here we report that these cells can be rendered sensitive to TNFalpha killing by treatment with the translational inhibitor cycloheximide, suggesting the existence of proteins that can suppress the death stimulus induced by the cytokine. In search of these cytoprotective proteins, we focused on the constituents of the mitochondrial permeability transition pore (PT pore), whose opening has been shown to play a critical role in the TNFalpha-mediated death pathway. We found that TNFalpha up-regulated mRNA and protein expression of the mitochondrial peripheral benzodiazepine receptor (PBR), an outer membrane-derived constituent of the pore. A strong correlation was established between the resistance of the cells to TNFalpha killing and the density of PBR-binding sites. Concomitantly, TNFalpha down-regulated Bcl-2 mRNA and protein expression. As Bcl-2 has been shown to be an endogenous inhibitor of the PT pore, we hypothesize that the TNFalpha-induced up-regulation of PBR expression may compensate for the decrease in Bcl-2 levels to prevent the opening of the PT pore.

Implication of insulin and nutritional factors in the regulation of intestinal galactosyltransferase activity during postnatal development.

In the rat small intestine, galactosyltransferases are the enzymes implicated in the biosynthesis of glycoproteins of the brush-border membranes and mucins. During postnatal development, the circulating insulin level increased at weaning in parallel with the activities of intestinal galactosyltransferases on O-glycans and N-glycans. This study deals with the role of insulin in the regulation of galactosyltransferase activities during postnatal development. The treatment of immature suckling rats with insulin induced a precocious increase in the activities of the O-glycan and N-glycan galactosyltransferases, partly reproducing the increase in galactosyltransferase activity normally found at weaning, since the O-glycan galactosyltransferase activity increased more quickly than the N-glycan galactosyltransferase activity. The sensitivity of the two galactosyltransferase activities to insulin disappeared after weaning, a period when drastic diet changes occur. In 22-day-old rats submitted to prolonged nursing (high-fat diet), the activities of the O-glycan and N-glycan galactosyltransferases were lower than those found in age-matched normally weaned rats (high-carbohydrate diet), indicating a delay in the maturation of the intestine of prolonged-nursing rats. The circulating insulin level of these animals stayed lower than that of the age-matched weaned rats. When the prolonged-nursing animals were treated with insulin, the O-glycan and N-glycan galactosyltransferase activities reached levels similar to those of the weaned rats. These observations suggest that insulin is one of the maturation factors for intestinal glycoprotein galactosylation and may be partly responsible for the natural enhancement of intestinal galactosyltransferase activities observed during postnatal development in relation to the dietary changes at weaning.

Calcium-binding properties of the mitochondrial channel-forming hydrophobic component.

A hydrophobic, low-molecular weight component extracted from mitochondria forms a Ca2+-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3-8.5, the component has a high-affinity binding site for Ca2+ with a Kd of 8 x 10(-6) M, while at pH 7.5 this Kd was decreased to 9 x 10(-5) M. Bmax for the Ca2+-binding site did not change significantly with pH. In the range studied, 0.2 +/- 0.06 mmol Ca2+/g component were bound or one calcium ion to eight molecules of the component. The Ca2+ binding was strongly decreased by 50-100 mM Na+, but not by K+. Treatment of mitochondria with CaCl2 prior to ethanolic extraction resulted in a high level of Ca2+-binding capacity of the partially purified component. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition, when added to the mitochondrial suspension, decreased the Ca2+-binding activity of the purified extract severalfold. The calcium-binding capability of the partially purified component correlates with its calcium-channel activity. This indicates that the channel-forming component might be involved in the permeability transition that stimulates its formation.

Influence of spermine on intestinal maturation of the glycoprotein glycosylation process in neonatal rats.

Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an alpha-1, 2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in alpha-1,2-fucosyltransferase level and of the decrease in alpha-l-fucosidase level after treatment with spermine, several alpha-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development.

Spermidine-induced glycoprotein fucosylation in immature rat intestine.

In rat small intestine, during postnatal development, the glycoprotein fucosylation is markedly increased at weaning. At the same time, a rise in the intestinal spermidine level was observed, partly due to the increase in the spermidine content of solid food given to animals at this period as compared to the spermidine content of milk. In order to mimic the spermidine increase observed in weanling rat intestines, we had treated suckling rats with spermidine by oral ingestion to study its role as maturation factor of the small intestine. In spermidine-treated suckling rats, the spermidine and N-acetyl-spermidine contents were highly increased. Spermidine treatment induced the rise in alpha-1,2-fucosyltransferase activity and the precocious appearance in the brush-border membrane of some alpha-1,2-fucoproteins in weaned rats. Such results indicate that spermidine could be a maturation factor implicated in the appearance of alpha-1,2-fucoproteins naturally observed at weaning time.