To elucidate whether nitric oxide secreted from irradiated cells affects cellular radiosensitivity, we examined the accumulation of inducible nitric oxide synthase, TP53 and HSP72, the concentration of nitrite in the medium of cells after X irradiation, and cellular radiosensitivity using two human glioblastoma cell lines, A-172, which has a wild-type TP53 gene, and a transfectant of A-172 cells, A-172/mp53, bearing a mutated TP53 gene. Accumulation of inducible nitric oxide synthase was caused by X irradiation of the mutant TP53 cells but not of the wild-type TP53 cells. Accumulation of TP53 and HSP72 in the wild-type TP53 cells was observed by cocultivation with irradiated mutant TP53 cells, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase, aminoguanidine, to the medium. Likewise, accumulation of these proteins was observed in the wild-type TP53 cells after exposure to conditioned medium from irradiated mutant TP53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. The radiosensitivity of wild-type TP53 cells was reduced when the cells were cultured in conditioned medium from irradiated mutant TP53 cells compared to conventional fresh growth medium. Collectively, these findings indicate the potential importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to ionizing radiation.
bRibosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.
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