Chronic hypoxia protects against gamma-irradiation-induced apoptosis by inducing bcl-2 up-regulation and inhibiting mitochondrial translocation and conformational change of bax protein.
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.
Cuisnier
Olivier
O
Fundamental and Applied Bioenergetics Laboratory, INSERM E02-21, Joseph Fourier University, BP 53X, 38041 Grenoble cedex 9, France. catherine.riva-lavieille@ujf-grenoble.fr
Serduc
Raphael
R
Lavieille
Jean-Pierre
JP
Longuet
Michel
M
Reyt
Emile
E
Riva
Catherine
C
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
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