Irradiation par microfaisceau de particules alpha : Implication des espèces réactives de l'oxygène dans l'effet de voisinage.

Ionizing radiation-induced bystander effects arise in bystander cells that receive signals from directly irradiated cells. To date, free radicals are believed to play an active role in the bystander response, but this is incompletely characterized. To mark temporal and spatial impacts of bystander effect, we employed a precise -particle microbeam to target a small fraction of sub-confluent osteoblastic cell cultures (MC3T3-E1). We identified the cellular membrane and mitochondira like two distinct places generating reactive oxygen species. The global oxidative stress observed after irradiation was significantly attenuated after filipin treatment, evidencing the pivotal role of membrane in MC3T3-E1 cells bystander response. To determine impact of bystander effect at a cell level, cellular consequences of this membrane-dependant bystander effect were then investigated. A variable fraction of the cell population (10 to 100%) was individually targeted. In this case, mitotic death and micronuclei yield both increased in bystander cells as well as in targeted cells demonstrating a role of bystander signals between irradiated cells in an autocrine or paracrine manner. Our results indicate a complex interaction of direct irradiation and bystander signals that lead to a membrane-dependant amplification of cell responses.L'effet de voisinage radio-induit s'observe dans les cellules voisines de cellules irradiées mais non directement touchées par l'irradiation. A ce jour, les espèces réactives de l'oxygène (EROs) sont considérées comme ayant un rôle actif dans la survenue de réponse au voisinage, mais leur implication n'est pas encore totalement définie. Afin de déterminer leur impact dans la réponse au voisinage, à la fois temporellement et spatialement, des irradiations par microfaisceau de particules sont mises au point afin de cibler une fraction définie de cellules au niveau du noyau dans une culture cellulaire. Les irradiations sont pratiquées sur des cellules normales ostéoblastiques, à sous-confluence, nommées MC3T3-E1. L'observation directe de la génération d'EROs cellulaires et mitochondriales révèle que la réponse bystander est caractérisée par un stress cellulaire d'origine double et temporellement distinct. A court terme, la signalisation issue de la membrane induit une importante production d'EROs impliquée dans l'apparition de cassures double brin de l'ADN retardées. Les mitochondries produisant des EROs jusque 6 heures après l'irradiation semblent être impliquées dans un processus à long terme différent. L'étude indépendante de la réponse des cellules ciblées et voisines met en évidence un phénomène nouveau. L'effet de voisinage induit une réponse des cellules voisines mais est aussi impliqué dans une amplification de la réponse des cellules ciblées par l'irradiation. Ainsi toute cellule, irradiée ou non, peut recevoir ces signaux bystander et répondre. L'ensemble de cette étude mène à considérer les cultures cellulaires comme des réseaux complexes de communication; leurs réponses à l'irradiation indiquent une relation mêlée entre signaux relatifs à l'irradiation elle-même et réponse au voisinage. Cette complexité du phénomène d'effet de voisinage peut justifier que son incidence sur l'établissement de règles de radioprotection et sur la courbe linéaire sans seuil n'est pas encore clairement déterminée

Irradiation par microfaisceau de particules alpha (implication des espèces réactives de l'oxygène dans l'effet de voisinage)

L effet de voisinage radio-induit s observe dans les cellules voisines de cellules irradiées mais non directement touchée par l irradiation. A ce jour, les radicaux libres sont considérés comme ayant un rôle actif dans la survenue de réponse au voisinage, mais leur implication n est pas encore totalement définie. Afin de déterminer leur impact dans la réponse au voisinage, à la fois temporellement et spatialement, des irradiations par microfaisceau de particules alpha sont mises au point afin de cibler une fraction définie de cellules au niveau du noyau dans une culture cellulaire avec un nombre prédéterminé d ions. Les irradiations sont pratiquées sur des cellules normales ostéoblastiques à sous-confluence nommée MC3T3-E1. Deux sources distinctes sont ici identifiées dans les cellules comme productrices d espèces réactives de l oxygène après irradiation ciblée : la membrane cytoplasmique et les mitochondries. L inhibition du stress oxydatif général observé après irradiation par la filipin (déstructurant les radeaux lipidiques) met en évidence le rôle primordial de la membrane dans la survenue d effet de voisinage. Les conséquences cellulaires de l effet de voisinage sont aussi étudiées à l échelle de la cellule en termes de mort cellulaire et induction de micronoyaux. Une fraction de 10 à 100% de cellules est individuellement ciblée dans la population cellulaire. Dans ce cas, l induction de mort mitotique et de micronoyaux augmentent dans les cellules voisines, ainsi que dans les cellules directement irradiées. Ces observations démontrent un impact de l effet de voisinage entre cellules irradiées selon un mode autocrine ou paracrine. Ce mémoire indique une interaction complexe entre signaux relatifs à l irradiation et signaux relatifs à l effet de voisinage (dépendant de la membrane) conduisant à une amplification.de la réponse cellulaire originelleIonizing radiation-induced bystander effects arise in bystander cells that receive signals from directly irradiated cells. To date, free radicals are believed to play an active role in the bystander response, but this is incompletely characterized. To mark temporal and spatial impacts of bystander effect, we employed a precise alpha-particle microbeam to target a small fraction of sub-confluent osteoblastic cell cultures (MC3T3-E1). We identified the cellular membrane and mitochondira like two distinct places generating reactive oxygen species. The global oxidative stress observed after irradiation was significantly attenuated after filipin treatment, evidencing the pivotal role of membrane in MC3T3-E1 cells bystander response. To determine impact of bystander effect at a cell level, cellular consequences of this membrane-dependant bystander effect were then investigated. A variable fraction of the cell population (10% to 100%) was individually targeted. In this case, mitotic death and micronuclei yield both increased in bystander cells as well as in targeted cells demonstrating a role of bystander signals between irradiated cells in an autocrine or paracrine manner. Our results indicate a complex interaction of direct irradiation and bystander signals that lead to a membrane-dependant amplification of cell responses.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

International audienceFree radicals are believed to play an active role in the bystander response. This study investigated their origin as well as their temporal and spatial impacts in the bystander effect.We employed a precise alpha-particle microbeam to target a small fraction of subconfluent osteoblastic cells (MC3T3-E1). γH2AX-53BP1 foci, oxidative metabolism changes, and micronuclei induction in targeted and bystander cells were assessed.Cellular membranes and mitochondria were identified as two distinct reactive oxygen species producers. The global oxidative stress observed after irradiation was significantly attenuated after cells were treated with filipin, evidence for the primal role of membrane in the bystander effect. To determine the membrane's impact at a cellular level, micronuclei yield was measured when various fractions of the cell population were individually targeted while the dose per cell remained constant. Induction of micronuclei increased in bystander cells as well as in targeted cells and was attenuated by filipin treatment, demonstrating a role for bystander signals between irradiated cells in an autocrine/paracrine manner.A complex interaction of direct irradiation and bystander signals leads to a membrane-dependent amplification of cell responses that could influence therapeutic outcomes in tissues exposed to low doses or to environmental exposure

Glutathione Depletion and Carbon Ion Radiation Potentiate Clustered DNA Lesions, Cell Death and Prevent Chromosomal Changes in Cancer Cells Progeny

Poor local control and tumor escape are of major concern in head-and-neck cancers treated by conventional radiotherapy or hadrontherapy. Reduced glutathione (GSH) is suspected of playing an important role in mechanisms leading to radioresistance, and its depletion should enable oxidative stress insult, thereby modifying the nature of DNA lesions and the subsequent chromosomal changes that potentially lead to tumor escape. This study aimed to highlight the impact of a GSH-depletion strategy (dimethylfumarate, and L-buthionine sulfoximine association) combined with carbon ion or X-ray irradiation on types of DNA lesions (sparse or clustered) and the subsequent transmission of chromosomal changes to the progeny in a radioresistant cell line (SQ20B) expressing a high endogenous GSH content. Results are compared with those of a radiosensitive cell line (SCC61) displaying a low endogenous GSH level. DNA damage measurements (cH2AX/comet assay) demonstrated that a transient GSH depletion in resistant SQ20B cells potentiated the effects of irradiation by initially increasing sparse DNA breaks and oxidative lesions after X-ray irradiation, while carbon ion irradiation enhanced the complexity of clustered oxidative damage. Moreover, residual DNA double-strand breaks were measured whatever the radiation qualities. The nature of the initial DNA lesions and amount of residual DNA damage were similar to those observe

Comet assay.

<p>Mean percentage of DNA damage in SCC61, SQ20B, and DMF/BSO (TTT)-treated SQ20B cell lines after 10 Gy of X-ray or 5 Gy of carbon ion exposure. Comet assays were performed in alkaline conditions without (A) or in the presence of Fpg enzyme (B). ▵ SQ20B cells, ▴SQ20B+irradiation, • SQ20B+GSH depletion, ○ SQ20B+GSH depletion+irradiation, ▪ SCC61, □ SCC61+irradiation. *<i>P</i><0.05.</p

Cell cycle distribution.

<p>SCC61, SQ20B, and DMF/BSO (TTT)-treated SQ20B cells were exposed to X-rays or carbon ion radiation. (A) The percentage of cells in sub-G1 phase. (B) The percentage of cells in G2/M phase. ▴ SQ20B cells, ▵ SQ20B+5 Gy carbon ion radiation, ▵ SQ20B+10 Gy X-rays, • SQ20B+GSH depletion, ○ SQ20B+GSH depletion+5 Gy carbon ion radiation, ○ SQ20B+GSH-depletion+10 Gy X-rays, ▪ SCC61, □ SCC61+5 Gy carbon ion radiation, □ SCC61+10 Gy X-rays. *<i>P</i><0.05.</p

The endogenous glutathione content determined by HPLC analysis in SQ20B cells.

<p>Endogenous glutathione content was determined in SQ20B cells after 4 h of treatment with DMF (100 µM) and BSO (100 µM) (A). Panel B shows the endogenous glutathione level with and without 10 Gy of X-ray and 5 Gy of carbon ion irradiation with or without treatment with DMF/BSO. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044367#s3" target="_blank">Results</a> are expressed as mean ± S.D. for three different experiments in triplicate.</p

Kinetics of γH2AX foci.

<p>Cells were irradiated with 2 Gy of X-rays (A, C) or 1 Gy of carbon ions (B, D). ▴ SQ20B cells, ▵ SQ20B+irradiation, • SQ20B+GSH depletion, • SQ20B+GSH depletion+irradiation, □ SCC61+irradiation. In panels C and D, the reverse effect of <i>N</i>-acetyl cysteine in the presence of DMF/BSO treatment was assayed. One hundred cells were scored for each time and the measurements were made in triplicate and repeated three times. *<i>P</i><0.05.</p

Chromosomal rearrangements.

<p>Percentage of cells displaying chromosomal rearrangements including dicentric chromosome formation (A) and complex rearrangements (B). The effects were investigated in SCC61, SQ20B, and DMF/BSO (TTT)-treated SQ20B cell lines after 10 Gy of X-ray or 5 Gy of carbon ion irradiation. *<i>P</i><0.05.</p

Clonogenic cell survival assay.

<p>SQ20B, DMF/BSO (TTT)-treated SQ20B, and SCC61 cells were exposed to X-ray radiation (A) or carbon ion radiation (B). The reverse effect of NaC over the glutathione depletion treatment was evaluated by coincubating DMF/BSO (TTT)-treated SQ20B with 5 mM of <i>N</i>-acetyl cysteine (NaC) (SQ20B+TTT+NaC) before X-ray radiation exposure.</p