title: Chronic hypoxia protects against gamma-irradiation-induced apoptosis by inducing bcl-2 up-regulation and inhibiting mitochondrial translocation and conformational change of bax protein.
abstract: Malignant tumours contain zones of chronic or acute hypoxia, which influence their prognosis and progression. The goal of our study was to understand the role of hypoxia in radio-resistance in a squamous cell carcinoma cell line of the head and neck (KB-3-1 cells). Cell growth was evaluated by Trypan blue exclusion under chronic hypoxia (3-5% O2) for 4 weeks or under normal conditions (21% O2). Cells were then gamma-irradiated either by X-ray (2-6 Gy) or UV-C radiation (0.001-10 J/cm(2)). Apoptosis was estimated by double staining with orange acridine and ethydium bromide and fluorescence microscopy. DNA content was estimated by FACS analysis. Expression of Bax, Bcl-2 and P53 was assessed by immunofluorescence and Western blotting. ROS production was measured by dichlorofluorescein fluorescence. Cell growth depends on oxygen tension. It decreased by 42 and 70% at 5 and 3% O2 compared to control with a significant cell cycle arrest rather than increased mortality. Hypoxic cells are more radio-resistant (x2.5) than normoxic cells. Under chronic hypoxia, Bcl-2 increased considerably in cells compared to control, while Bax and P53 did not change. After irradiation, in hypoxic cells very weak expression of the pro-apoptotic Bax protein and no translocation of Bax to the mitochondria were observed. In addition, irradiation of control KB-3-1 cells demonstrated a large increase in ROS production (x2) compared to cells irradiated identically under hypoxia. In conclusion, chronic hypoxia: i) seems to slow-down cell growth of KB-3-1 cells without inducing apoptosis, ii) induces Bcl-2 overexpression and prevents radiation-induced apoptosis by inhibiting ROS production and altering Bax subcellular redistribution and conformational changes.
authors.affiliation: Fundamental and Applied Bioenergetics Laboratory, INSERM E02-21, Joseph Fourier University, BP 53X, 38041 Grenoble cedex 9, France. catherine.riva-lavieille@ujf-grenoble.fr
authors.forename: Olivier
authors.initials: O
authors.lastname: Cuisnier
authors.forename: Raphael
authors.initials: R
authors.lastname: Serduc
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Lavieille
authors.forename: Michel
authors.initials: M
authors.lastname: Longuet
authors.forename: Emile
authors.initials: E
authors.lastname: Reyt
authors.forename: Catherine
authors.initials: C
authors.lastname: Riva
mesh_terms: D017209:Apoptosis; D001692:Biological Transport; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D006801:Humans; D000860:Hypoxia; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000473:pathology; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species; D013997:Time Factors; D014343:Trypan Blue / Q000494:pharmacology; D014466:Ultraviolet Rays; D015854:Up-Regulation; D014965:X-Rays; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D004396:Coloring Agents; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D014343:Trypan Blue; D010100:Oxygen