Ionizing radiation induces a p53-dependent apoptotic mechanism in ARPE-19 cells.
OBJECTIVE
To investigate the molecular mechanisms for cell growth inhibition or apoptosis in human retinal pigment epithelium (RPE) cells after ionizing radiation.


METHODS
Cell survival studies, a TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay, and a caspase-3 immunocytochemical analysis were performed on irradiated ARPE-19 cell cultures at different time periods. Transcriptional levels of p53, p21, Bax, Fas/Fas-L, vascular endothelial growth factor ( VEGF), and pigment epithelium-derived growth factor ( PEDF) were evaluated by semiquantitative reverse transcriptional polymerase chain reaction. Mutations in the p53 gene were analyzed by DNA sequencing. Protein levels of p53, VEGF, and PEDF were evaluated by Western blot.


RESULTS
Cell viability was inversely related to radiation dose. TUNEL-positive cells were detected 6 h after radiation exposure. Caspase-3 immunocytochemical analysis revealed increased immunoreactivity in the TUNEL-positive cells. Levels of p53, p21, and Bax mRNA were greatest at the 2-h postradiation period. VEGF and PEDF mRNA and protein levels were constant. Protein levels of p53 were increased at the 4- and 6-h postradiation period.


CONCLUSIONS
Ionizing radiation induces apoptosis in normal proliferating RPE cells through p53 activation, without affecting expression of VEGF or PEDF. We documented a molecular basis for explaining the decrease in effectiveness of radiation therapy, particularly for age-related macular degeneration. In the clinical setting, selection of appropriate radiation therapy methods and the doses for specific diseases need careful evaluation.
Jiang
Yan Lin
YL
Department of Organ Therapeutics, Kobe University Graduate School of Medicine, Kobe, Japan. jiangyanlin00@hotmail.com
Escaño
Michael F T
MF
Sasaki
Ryohei
R
Fujii
Shigeki
S
Kusuhara
Sentaro
S
Matsumoto
Akira
A
Sugimura
Kazuro
K
Negi
Akira
A
D017209:Apoptosis / Q000528:radiation effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D005136:Eye Proteins; D053222:Fas Ligand Protein; D015870:Gene Expression; D006801:Humans; D020287:In Situ Nick-End Labeling; D008562:Membrane Glycoproteins / Q000235:genetics; D009414:Nerve Growth Factors; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000378:metabolism; D011839:Radiation, Ionizing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000235:genetics; D000099174:Pigment Epithelium-Derived Factor
D053148:Caspase 3; D020169:Caspases; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005136:Eye Proteins; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D015843:Serpins; D016159:Tumor Suppressor Protein p53; D042461:Vascular Endothelial Growth Factor A; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D000099174:Pigment Epithelium-Derived Factor; C494826:BAX protein, human; C493956:CDKN1A protein, human; C505661:FASLG protein, human; C505407:CASP3 protein, human