Hdm2 and nitric oxide radicals contribute to the p53-dependent radioadaptive response.
OBJECTIVE
The aim of this work was to characterize the radioadaptive response at the molecular level.


METHODS
We used wild-type (wt) p53 and mutated (m) p53-containing cells derived from the human lung cancer H1299 cell line, which is p53-null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulations of p53, the human homolog of endogenous murine double minute 2 (Hdm2), and inducible nitric oxide synthase were analyzed with Western blotting. Quantification of chromosomal aberrations was estimated by scoring dicentrics per cell.


RESULTS
In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low-dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about two- to fourfold after challenging irradiation subsequent to a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of 5, 5'-(2, 5-Furanidiyl)bis-2-thiophenemethanol (RITA) or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an inducible nitric oxide synthase inhibitor), and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover radioresistance developed when wtp53 cells were treated with isosorbide dinitrate (an NO-generating agent) alone.


CONCLUSIONS
These findings suggest that NO radicals are initiators of the radioadaptive response, acting through the activation of Hdm2 and the depression of p53 accumulations.
Takahashi
Akihisa
A
Department of Biology, School of Medicine, Nara Medical University, Nara, Japan.
Matsumoto
Hideki
H
Ohnishi
Takeo
T
D045744:Cell Line, Tumor; D002470:Cell Survival; D002869:Chromosome Aberrations; D003497:Cyclic N-Oxides / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D016158:Genes, p53 / Q000235:genetics*; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D011836:Radiation Tolerance / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D006146:Guanidines; D007093:Imidazoles; D010879:Piperazines; D016159:Tumor Suppressor Protein p53; C079391:2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; D009569:Nitric Oxide; C482205:nutlin 3; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C004479:pimagedine