Early molecular changes in irradiated aortic endothelium.
Irradiated aortic endothelial cells (EC) exhibit distinct morphological, functional, and physiological responses to ionizing radiation (IR). However, the molecular basis for these responses has not been fully characterized. Cultured bovine and rat aortic endothelial cells were exposed to single fraction doses (0-30 Gy) of gamma radiation. IR caused dose-dependent DNA strand breaks which were repaired to near baseline levels within 30 min. A dose-dependent inhibition of cell growth was noted for IR greater than 1 Gy. At doses greater than 2.5 Gy, morphologic changes consistent with apoptosis and loss of cell viability were present beginning 12-16 h after radiation, with subsequent detachment of EC from the cell monolayer. By Western blot analysis, expression of p53, gadd45, p21, and bax protein increased in a time-and dose-dependent manner; p53 expression was maximal at 3 h after IR, and gadd45, bax and p21 levels peaked at 6 h. By Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), levels of p53 mRNA were not significantly increased after IR, whereas gadd45 exhibited time- and dose-dependent increase in mRNA synthesis after IR. Activation of intracellular caspases, manifest by proteolytic poly (ADP-ribose) polymerase (PARP) and lamin B cleavage, was maximal at 15 h after IR, concident with other indices of EC apoptosis, including oligonucleosomal DNA degradation, TUNEL immunostaining, and morphologic changes. The tripeptide protease inhibitor z-Val-Ala-Asp (zVAD) prevented PARP and lamin cleavage, DNA fragmentation, morphological changes, and cell detachment in irradiated EC. The combined data suggested that gamma radiation induces a dose- and time-dependent sequence of early events in cultured EC with modulate growth arrest, apoptosis, and possibly premature senescence in surviving cells.
Gajdusek
C
C
Department of Neurological Surgery, University of Washington, Seattle, WA, USA.
Onoda
K
K
London
S
S
Johnson
M
M
Morrison
R
R
Mayberg
M
M
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000502:physiology / Q000528:radiation effects; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D024161:Collagen Type XI; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004249:DNA Damage / Q000502:physiology; D004307:Dose-Response Relationship, Radiation; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D005720:Gamma Rays; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000097642:GADD45 Proteins
D000590:Amino Acid Chloromethyl Ketones; D020169:Caspases; D024161:Collagen Type XI; D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D020741:rho GTP-Binding Proteins; D000097642:GADD45 Proteins; C494828:Bax protein, rat; C066455:PARP protein, Bos taurus; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C499322:Parp1 protein, rat