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.

S1263ESTRO 37Meanwhile, recurrent patients tended to exhibit increased post-RT peripheral neutrophil count (4.2×10 9 /L vs. 3.6×10 9 /L, p=0.09) and NLR (8.5 vs. 6.5, p=0.08) in comparison with non-recurrent patients.Multivariate analysis (MVA) identified patient age >50 years old (HR=3.4,p=0.02), weight loss during RT> 7.5% (HR=3.2,p=0.03), and post-RT peripheral NLR >7.05 (HR=2.5, p=0.04, 5-year OS 71.4% vs. 87.8%)as unfavorable prognostic factors for OS.There was also a nonsignificant trend in the MVA that patients with post-RT peripheral NLR >7.05 showed worse PFS (HR=1.9,p=0.06, 5-year PFS 64.1% vs. 81.8%). ConclusionPost-RT NLR predicted OS and PFS in LANPC patients treated with sequential chemoradiotherapy.The dynamic change of the routinely tested immune factors could help make appropriate treatment options and follow-up strategies.

Concurrent cetuximab based radio-chemotherapy is a validated scheme in head and neck squamous cell carcinoma (HNSCC). Cancer Stem Cells (CSCs) are highly resistant to treatment, have large migratory abilities, and are hypothesized to be responsible for a significant part of recurrences. Apoptotic signaling in CSCs is a major way of treatment escape, through Bcl-2 proteins family. The aim of the present study was to explore the synergic effect between cetuximab and an anti-Bcl-2 antibody (ABT-199), when combined or not with photon radiation. HNSCC chemo and radio resistant human cell line (SQ20B) and its corresponding stem cell line (SQ20B/CSCs) were used to test the treatment combinations. HaCaT cell line was used to assess toxicity on healthy cell population. SQ20B/CSCs subpopulation was isolated through double cell sorting: side population (SP) (Hoechst exclusion) and CD44 staining: SP/CD44High. SQ20B and SQ20B/CSCs proliferation, invasion/migration, and apoptosis were studied after an exposition to cetuximab 5nM + ABT-199 10mM treatment +/- 10Gy photon irradiation. Invasion and migration were assessed based on scratch wound assay with or without matrigel. Apoptosis was measured using caspases 3/7. 3D spheroid assay was performed to validate the results in a 3D culture approach. EGFR, phospho-EGFR (Tyr1068), Bcl-2 and Bcl-xl protein expression were studied. Cetuximab strongly inhibited SQ20B proliferation, migration and invasion whereas it had little effect on SQ20B/CSCs. Conversely, ABT-199 significantly inhibited these properties on SQ20B/CSCs, without showing any effect on SQ20B parental cell line. Cetuximab-ABT-199 combined with radiation had a significant inhibitory effect on both SQ20B and SQ20B/CSCs proliferation, migration and invasion. Although exclusive cetuximab had no pro-apoptotic effect, activation of caspases 3/7 was induced by ABT-199 and enhanced by the cetuximab+ABT-199 combination in both populations. Cetuximab was a strong inhibitor of 3D-spheroid proliferation in SQ20B, whereas ABT-199 strongly decreased spheroid size in CSCs. EGFR was overexpressed in SQ20B, and under-expressed in SQ20B/CSCs. Bcl2 was overexpressed in SQ20B/CSCs. Although cetuximab moderately inhibited HaCaT cell proliferation, the drug combination did not significantly enhanced toxicity. Cetuximab+ABT-199 combined with photon radiation significantly inhibited proliferation, invasion and migration of SQ20B HNSCC cell line and its CSCs subpopulation, with an acceptable toxicity profile on healthy cell lines. Apoptotic cell death was enhanced by this drug combination.

Les cellules souches de cancer ORL, sous-population hautement migratoire, semblent être à l’origine de la résistance aux traitements. Nous avons montré dans un précédent travail, sur des modèles de culture cellulaire en deux dimensions, que l’association de l’ABT-199 au cétuximab et à l’irradiation photonique semblait inhiber de façon synergique la prolifération, l’invasion et la migration de la lignée radiorésistante SQ20B et de leur sous-population de cellules souches cancéreuses. L’objectif de notre travail était de confirmer ces résultats dans un modèle de culture cellulaire en tridimensionnel, plus transposable aux modèles in vivo. La sous-population de cellules souches cancéreuses a été isolée par double tri. Les cellules SQ20B et leur sous-population de cellules souches cancéreuses sont ensuite été transductées par un lentivirus inactivé fluorescent dans le rouge, puis ensemencées dans des puits coniques à faible adhérence. Les sphéroïdes formés ont été exposés à de l’ABT-199 (10 μM) et/ou à du cétuximab (5 nM), avec ou sans une irradiation photonique de 4 Gy. L’analyse de la progression tumorale spontanée et de la réponse au traitement a été réalisée par le vidéomicroscope IncuCyte®, avec quantification de l’intensité de fluorescence en trois dimensions. Le cétuximab inhibe la progression tumorale des SQ20B, alors que l’ABT-199 a peu d’effet sur celles-ci. À l’inverse, le cétuximab a peu d’effet sur la sous-population de cellules souches cancéreuses, mais l’ABT-199 inhibe significativement la progression tumorale des cellules souches cancéreuses. L’ABT-199 potentialise l’effet du cétuximab dans les deux populations. L’irradiation photonique de 4 Gy majore l’effet de cette association thérapeutique, permettant de diminuer de plus de 90 % la taille tumorale après deux semaines de traitement, comparativement au bras témoin. L’association d’ABT-199, de cétuximab et d’irradiation photonique inhibe de façon synergique la progression tumorale des SQ20B et de leur sous-population de cellules souches cancéreuses. Ces résultats seront prochainement évalués avec un modèle in vivo.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy still associated with severe mortality, due to loco-regional recurrences or distant metastasis. Head and neck cancer stem cells (CSCs) are highly resistant to treatment and have large migratory abilities. These particular properties could explain treatment resistances in this location. If EGFR is strongly overexpressed in 80-100% of HNSCC, HER2 and HER3 seem to be also expressed in these lines. The aim of the present study was to explore the efficacy of HER1-2-3 blockade through cetuximab-pertuzumab association with or without photon irradiation on the proliferation and migration/invasion capabilities of a HNSCC chemo and radio resistant human cell line (SQ20B) and its corresponding stem cell line (SQ20B/CSCs). SQ20B/CSCs subpopulation was isolated through double cell sorting: side population (SP) (Hoechst exclusion) and CD44 staining: SP/CD44High. SQ20B and SQ20B/CSCs proliferation was studied after treatment with cetuximab 5nM + pertuzumab 20mg/mL treatment +/- 10Gy photon irradiation. Invasion and migration were assessed with scratch wound assay with or without matrigel. EGFR, phospho-EGFR (Tyr1068), HER2 and HER3 basal protein expression was studied. Activation or inhibition of RAS/MAPK and AKT-mTOR downstream signaling cascade was studied through phospho-AKT (Ser473), phospho-MEK1/2(Ser217/221) expression exposed to combined treatments. Cetuximab strongly inhibits SQ20B proliferation, migration and invasion when it has a small effect on SQ20B/CSCs. Cetuximab-pertuzumab treatment combined with radiation has a potent significant inhibitory effect on SQ20B and SQ20B/CSCs proliferation, migration and invasion. EGFR is overexpressed in SQ20B, and under-expressed in SQ20B/CSCs, while HER2 and HER3 are expressed equivalently in both populations in basal conditions. Phospho-AKT is strongly expressed in SQ20B, at the opposite of SQ20B/CSCs which express phospho-MEK1/2. Cetuximab-pertuzumab treatment combination with 10Gy photon irradiation switches off both phospho-AKT and phospho-MEK1/2 expression in the two populations. Cetuximab-pertuzumab couple pan-HER treatment combined with photon irradiation significantly inhibits proliferation, invasion and migration of SQ20B HNSCC cell line and its CSCs subpopulation, through both AKT-mTOR and Ras-MAPK downstream signaling blockade. HER family seems to be a promising therapeutic target in HNSCC.

Objectif de l’etude De nos jours, le pronostic des formes evoluees des carcinomes epidermoides de la tete et du cou reste defavorable. La radioresistance intrinseque de la tumeur en est en grande partie responsable. Plusieurs donnees suggerent que les deregulations du couple Keap1/Nrf2, entrainant une augmentation de l’expression de certains genes cytoprotecteurs, pourraient avoir un role cle dans les phenomenes de radioresistance. L’objectif de cette etude etait d’analyser la correlation potentielle entre la radiosensibilite et l’expression du couple Nrf2/Keap1, proposant alors ces proteines comme des biomarqueurs predictifs de radiosensibilite des carcinomes epidermoides de la tete et du cou. Materiel et methode Nous avons selectionne cinq lignees cellulaires humaines de carcinome epidermoide de la tete et du cou presentant autant de degres differents de radioresistance. La radioresistance est definie, conventionnellement, par la proportion de survie (test clonogenique de mort cellulaire) apres une irradiation de 2 Gy (survival fraction 2 : SF2). Le niveau d’expression des proteines Keap1 et Nrf2 a ete analyse par westernblot . Resultat Une nette correlation a ete etablie entre le degre de radioresistance des differentes lignees et l’expression de la proteine Keap1 (R2 = 0,94). En revanche, il n’y avait pas de correlation avec la proteine Nrf2 (R2 = 0,02). Conclusion Cette etude preliminaire, realisee in vitro, propose Keap1 comme un nouveau bio-marqueur de radioresistance et offre ainsi une nouvelle perspective dans le choix du plan de traitement des carcinomes epidermoides de la tete et du cou. En effet, connaissant les degres de radioresistance de leur tumeur, les oncologues pourraient engager les patients, des le diagnostic, vers d’autres strategies therapeutiques. Ces resultats preliminaires doivent maintenant etre confirmes par l’examen de niveau Keap1 dans les biopsies de carcinomes epidermoides de la tete et du cou afin d’evaluer leur eventuelle implication future dans la decision clinique du plan de traitement.

Les cancers épidermoïdes ORL sont associés à un fort taux de récidive loco-régionale et métastatique. Les cellules souches cancéreuses, sous-population hautement migratoire, semblent être une hypothèse majeure à l’origine de la résistance aux traitements. L’objectif du travail était de comparer l’efficacité du blocage pan-HER par une association cétuximab-pertuzumab associé à l’irradiation photonique dans les processus d’invasion et migration de la lignée SQ20B et sa sous-population souche. La sous-population de CSCs de la lignée SQ20B a été isolée par double tri selon les critères SQ20B/SP/CD44High. La prolifération des cellules SQ20B et SQ20B/CSCs a été étudiée après traitement par cétuximab 5 nM et/ou pertuzumab 20 μg/mL avec ou sans irradiation photonique à 10 Gy. L’analyse de la migration et de l’invasion a été réalisée par test de blessure avec et sans matrigel (IncuCyte). L’activation de récepteur de l’epidermal growth factor (EGFR) (Tyr1068) et des voies de signalisation intracellulaires (phospho-AKT et phospho-MEK1/2) a été étudiée en réponse aux traitements par western blot (WES). Le cétuximab inhibe la prolifération cellulaire des cellules SQ20B et non celle de la sous-population souche. L’association cétuximab-pertuzumab inhibe significativement la prolifération des cellules SQ20B et SQ20B/CSCs. La double association cétuximab-pertuzumab associée à une irradiation de 10 Gy inhibe significativement la migration et l’invasion des deux populations cellulaires. Le double traitement inhibe la phosphorylation d’EGFR dans les deux populations. Les cellules SQ20B expriment fortement phospho-AKT à l’inverse des SQ20B/CSCs qui expriment phospho-MEK1/2. Enfin, l’association cétuximab-pertuzumab-10 Gy inhibe fortement l’expression de phospho-AKT et phospho-MEK1/2. Le double traitement par cétuximab-pertuzumab associé à l’irradiation photonique inhibe significativement la prolifération, la migration et l’invasion de la lignée SQ20B et sa sous-population souche.

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.

2012 is the penultimate year of financial support by the CPER 2007-2013 for ETOILE's research program, sustained by the PRRH at the University Claude Bernard. As with each edition we make the annual review of the research in this group, so active for over 12 years now. Over the difficulties in the decision-making process for the implementation of the ETOILE Center, towards which all our efforts are focussed, some themes (work packages) were strengthened, others have progressed, or have been dropped. This is the case of the eighth theme (technological developments), centered around the technology for rotative beam distribution heads (gantries) and, after being synchronized with the developments of ULICE's WP6, remained so by ceasing its activities, coinciding also with the retirement of its historic leader at IPNL, Marcel Bajard. Topic number 5 (In silico simulations) has suffered the departure of its leader, Benjamin Ribba, although the work has still been provided by Branka Bernard, a former postdoctoral fellow in Lyon Sud, and now back home in Croatia, still in contract with UCBL for the ULICE project. Aside from these two issues (and the fact that the theme Medico-economical simulations is now directly linked to the first one (Medical Project), the rest of the teams are growing, as evidenced by the publication statistics at the beginning of this report. This is obviously due to the financial support of our always faithful regional institutions, but also to the synergy that the previous years, the European projects, the arrival of the PRIMES LabEx, and the national France Hadron infrastructure have managed to impulse. The Rhone-Alpes hadron team, which naturally includes the researchers of LPC at Clermont, should also see its influence result in a strong presence in France Hadron's regional node, which is being organized. The future of this regional research is not yet fully guaranteed, especially in the still uncertain context of ETOILE, but the tracks are beginning to emerge to allow past and present efforts translate into a long future that we all want to see established. Each of the researchers in PRRH is aware that 2013 will be (and already is) the year of great challenge : for ETOILE, for the PRRH, for hadron therapy in France, for French hadrontherapy in Europe (after the opening and beginning of treatments in the German [HIT Heidelberg, Marburg], Italian [CNAO, Pavia] and Austrian [MedAustron, Wien Neuerstadt]) centers. Let us meet again in early 2014 for a comprehensive review of the past and a perspective for the future ...

Glutathione, a major intra-cellular antioxidant, has been reported to play a fundamental role in the resistance of some cancer cells to radiotherapy so that this molecule can be considered as a potential clinical target for their radio-sensitization.

De nombreuses trousses de 1 e, 2 e voire de 3 e generation sont actuellement disponibles pour determiner les taux seriques des troponines I ou T cardiaques. Avec la redefinition des seuils decisionnels dans le cadre des syndromes coronariens aigus, le but de cette etude a ete d’evaluer les performances analytiques et clinicobiologiques de deux trousses de troponine : Troponine Ic 2 e generation (AccuTnI™) sur Access ®2 de Beckman Coulter et Troponine Tc 3 e generation (TroponinT STAT™) sur Elecsys ®2010 de Roche Diagnostics. Les performances analytiques retrouvees pour ces deux kits sont satisfaisantes (sensibilite analytique et fonctionnelle, repetabilite et reproductibilite). En comparant chaque trousse a celle de Dade Behring (Flex™ Troponine-I Cardiaque, Trop) sur Dimension ®RxL, la correlation retrouvee avec la trousse AccuTnI™ sur Access ®2 se traduit par l’equation AccuTnI™ = 1.08 (TnIc Trop™) – 0,34 ; r = 0,99. Par contre, il est plus difficile de comparer TnIc et TnTc. L’etude des seuils decisionnels annonces par Beckman Coulter pour la TnIc (0,04 μg/L au 99 e percentil, 0,06 μg/L pour un CV ≤ 10 %) met en evidence une meilleure specificite (76 %) et valeur predictive positive (89 %), avec une sensibilite a 100 %, pour un seuil a 0,1 μg/L, defini et applique dans le laboratoire pour son meilleur compromis entre sensibilite / specificite et son imprecision inferieure a 10 %. Pour les seuils de TnTc annonces par Roche Diagnostics (0,01 μg/L au 99 e percentil et 0,03 pour un CV ≤ 10 %), la specificite est moins bonne, ainsi le seuil decisionnel a 0,1 μg/L semble mieux adapte. Lors de cette etude, quelques faux positifs et negatifs en TnTc ont ete trouves chez des patients atteints de pathologies complexes ; cette eventualite doit etre prise en compte si le resultat n’est pas en accord avec la clinique.

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.

Le NTproBNP a déjà prouvé son intérêt comme marqueur d'exclusion dans le diagnostic des insuffisances cardiaques congestives. Lors de cette étude, à partir d'une population hospitalisée à l'hôpital cardiologique, divers facteurs influençant les taux plasmatiques du NTproBNP (âge, sexe, fonction rénale et atteinte myocardique) ont été évalués à l'aide de la trousse proBNP sur Elecsys 20.10 (Roche Diagnostics, Meylan, France). Les taux moyens de NTproBNP augmentent de 695 (patients de moins de 50 ans) à 2002 ng/l (patients de plus de 80 ans), de 622 (patients sans atteinte rénale) à 6278 ng/l (insuffisants rénaux sévères). Ces divers paramètres, dont la fonction rénale sont déterminants dans l'interprétation du NTproBNP. À partir de 39 données échocardiographiques avec évaluation de la FEVG, au seuil 250 ng/l excluant une dyspnée d'origine cardiaque et 1200 ng/l à forte probabilité d'insuffisance d'origine cardiaque, les valeurs prédictives négatives du NTproBNP calculées sont respectivement de 100 et 85 %. Une corrélation inverse entre FEVG et NTproBNP (r=–0,38) est également observée. Ainsi, le NTproBNP permet rapidement de faire un screening des patients à fonction ventriculaire gauche altérée, mais pour rester discriminant dans le diagnostic de l'insuffisance cardiaque, son interprétation doit tenir compte des facteurs âge, sexe et fonction rénale.

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

To enhance the killing effects of ionizing radiation, we amplified the endogenous ceramide signal in Jurkat cell cultures using 3 different inhibitors of sphingolipid metabolism: DL‐PDMP, D‐MAPP and imipramine. Of the various possible drug combinations, only DL‐PDMP (20 μM) + imipramine (20 μM) and DL‐PDMP (20 μM) + imipramine (20 μM) + D‐MAPP (5 μM) induced a major increase in ceramide levels, reaching 240% and 340% of control values, respectively, after incubation for 48 hr. With these models, we demonstrate that endogenously formed ceramide triggers time‐ and concentration‐dependent apoptosis through induction of mitochondrial injury and activation of the caspase pathway. Cellular dysfunction includes alterations to the cellular redox potential, as assessed by the generation of ROS and total glutathione depletion, and a drop in ΔΨm. A parallel elevation of mitochondrial ceramide levels was also observed. The combination of DL‐PDMP + imipramine ± D‐MAPP with 10 Gy irradiation produced cumulative effects leading to apoptosis via mitochondrial collapse and activation of the caspase cascade. The association efficiency was confirmed in normal and acid sphingomyelinase‐deficient lymphoid cell lines. Taken together, these results suggest that increasing endogenous ceramide levels may potentially be very valuable when combined with ionizing radiation in tumor therapy. © 2002 Wiley‐Liss, Inc.

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.

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.

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.