Relative biological effectiveness of high-energy iron ions for micronucleus formation at low doses

Dose-response curves for micronucleus (MN) formation were measured in Chinese hamster V79 and xrs6 (Ku80(-)) cells and in human mammary epithelial MCF10A cells in the dose range of 0.05-1 Gy. The Chinese hamster cells were exposed to 1 GeV/nucleon iron ions, 600 MeV/nucleon iron ions, and 300 MeV/nucleon iron ions (LETs of 151, 176 and 235 keV/microm, respectively) as well as with 320 kVp X rays as reference. Second-order polynomials were fitted to the induction curves, and the initial slopes (the alpha values) were used to calculate RBE. For the repair-proficient V79 cells, the RBE at these low doses increased with LET. The values obtained were 3.1 +/- 0.8 (LET = 151 keV/microm), 4.3 +/- 0.5 (LET = 176 keV/microm), and 5.7 +/- 0.6 (LET = 235 keV/microm), while the RBE was close to 1 for the repair-deficient xrs6 cells regardless of LET. For the MCF10A cells, the RBE was determined for 1 GeV/nucleon iron ions and was found to be 5.5 +/- 0.9, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/nucleon iron-ion beam was intercepted by various thicknesses of high-density polyethylene plastic absorbers, which resulted in energy loss and fragmentation. It was found that the MN yield for V79 cells placed behind the absorbers decreased in proportion to the decrease in dose both before and after the iron-ion Bragg peak, indicating that RBE did not change significantly due to shielding except in the Bragg peak region. At the Bragg peak itself with an entrance dose of 0.5 Gy, where the LET is very high from stopping low-energy iron ions, the effectiveness for MN formation per unit dose was decreased compared to non-Bragg peak areas.     

Cell cycle-related bystander responses are not increased with LET after heavy-ion irradiation

Evidence has accumulated that irradiated cells affect their unirradiated neighbors, so that they in turn display cellular responses typically associated with direct radiation exposure. These responses are generally known as bystander effects. In this study, cell cycle-related bystander responses were investigated in three strains of human fibroblasts after exposure to densely ionizing radiation. Varying the linear energy transfer (LET) from 11 to 15,000 keV microm(-1) allowed a study of the impact of the complexity of DNA damage in the inducing cells on the responses of bystander cells. Using both broad-beam and microbeam irradiation, transient bystander responses were obtained for the induction of CDKN1A (p21). The latter was also observed when the transmission of bystander signals was limited to soluble factors. Targeted irradiation of single cells in confluent cell monolayers revealed no correlation between the amount of CDKN1A protein in the bystander cells and the radial distance to the targeted cells. In line with the induction of CDKN1A in bystander cells after irradiation with different LETs, a transient delay in the first G1 phase after irradiation of G0/G1 cells was observed. However, the CDKN1A induction revealed no significant effect on premature terminal differentiation considered to underlie fibrosis in irradiated tissue. Thus the unchanged differentiation pattern in bystander cells does not indicate pronounced, long-lasting effects.     

Induction and processing of oxidative clustered DNA lesions in 56Fe-ion-irradiated human monocytes

Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0-10 Gy of (56)Fe ions (1.046 GeV/ nucleon, LET = 148 keV/microm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The (56)Fe ions produced decreased yields of DSBs (10.9 DSB Gy(-1) Gbp(-1)) and clusters (1 DSB: approximately 0.8 Fpg clusters: approximately 0.7 Endo III clusters: approximately 0.5 Endo IV clusters) compared to previous results with (137)Cs gamma rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with gamma rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with gamma rays. The enhanced levels of apoptosis observed after exposure to (56)Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or "splitting" during DNA replication.     

Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

Low-dose-rate radiation attenuates the response of the tumor suppressor TP53

Acute low-dose irradiation (0.1-1 Gy, 1.33 Gy/min) of cells of a human glioblastoma cell line, A-172, induced a dose-dependent monophasic accumulation of TP53 (formerly known as p53) and CDKN1A (formerly known as WAF1). In contrast, chronic gamma irradiation (0.001 Gy/min) produced a clear biphasic response of accumulation TP53 with the first peak at 1.5 h (0.09 Gy) and the second peak at 10 h (0.54 Gy). Significantly, when the cells were preirradiated with a chronic dose of gamma irradiation for 24 h (1.44 Gy) or 50 h (3 Gy), they no longer responded to an acute challenging dose to produce a dose-dependent response of the TP53 pathway. These findings suggest that chronic irradiation at low dose rate alters the TP53-dependent signal transduction pathway. Wearing away of the TP53 pathway by chronic exposure to radiation may have important implications for radiation protection.     

Induction of accelerated senescence by gamma radiation in human solid tumor-derived cell lines expressing wild-type TP53

Recent studies have demonstrated that p21WAF1 (now known as CDKN1A)-dependent and -independent accelerated senescence responses are a major determinant of the sensitivity of cancer cells to chemotherapeutic agents. The objective of the present study was to determine whether human solid tumor-derived cell lines that express wild-type TP53 can exhibit levels of CDKN1A induction after exposure to ionizing radiation that are sufficient to activate the accelerated senescence program. Exposure to 60Co gamma radiation (< or =8 Gy) triggered accelerated senescence in all five TP53 wild-type tumor cell lines examined, albeit to differing degrees. Three of the TP53 wild-type tumor cell lines, HCT116, A172 and SKNSH, activated the TP53 signaling pathway similarly to normal human fibroblasts, as judged by the nuclear accumulation of TP53, magnitude and duration of induction of CDKN1A mRNA and CDKN1A protein, and propensity to undergo accelerated senescence after radiation exposure. In the clonogenic survival assay, the degree of radiosensitivity of these three tumor cell lines was also in the range displayed by normal human fibroblasts. On the other hand, two other TP53 wild-type tumor cell lines, A498 and A375, did not maintain high levels of CDKN1A mRNA and CDKN1A protein at late times postirradiation and exhibited only low levels of accelerated senescence after radiation exposure. Studies with a CDKN1A knockout cell line (HCT116CDKN1A-/-) confirmed that the radiation-triggered accelerated senescence is dependent on CDKN1A function. We conclude that (1) clinically achievable doses of ionizing radiation can trigger CDKN1A-dependent accelerated senescence in some human tumor cell lines that express wild-type TP53; and (2) as previously documented for normal human fibroblasts, some TP53 wild-type tumor cell lines (e.g. HCT116, A172 and SKNSH) may lose their clonogenic potential in response to radiation-inflicted injury primarily through undergoing accelerated senescence.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

Heavy ion-induced DNA double-strand breaks in yeast

Induction of DSBs in the diploid yeast, Saccharomyces cerevisiae, was measured by pulsed-field gel electrophoresis (PFGE) after the cells had been exposed on membrane filters to a variety of energetic heavy ions with values of linear energy transfer (LET) ranging from about 2 to 11,500 keV/microm, (241)Am alpha particles, and 80 keV X rays. After irradiation, the cells were lysed, and the chromosomes were separated by PFGE. The gels were stained with ethidium bromide, placed on a UV transilluminator, and analyzed using a computer-coupled camera. The fluorescence intensities of the larger bands were found to decrease exponentially with dose or particle fluence. The slope of this line corresponds to the cross section for at least one double-strand break (DSB), but closely spaced multiple breaks cannot be discriminated. Based on the known size of the native DNA molecules, breakage cross sections per base pair were calculated. They increased with LET until they reached a transient plateau value of about 6 x 10(-7) microm(2) at about 300-2000 keV/microm; they then rose for the higher LETs, probably reflecting the influence of delta electrons. The relative biological effectiveness for DNA breakage displays a maximum of about 2.5 around 100-200 keV/microm and falls below unity for LET values above 10(3) keV/microm. For these yeast cells, comparison of the derived breakage cross sections with the corresponding cross section for inactivation derived from the terminal slope of the survival curves shows a strong linear relationship between these cross sections, extending over several orders of magnitude.     

Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells

The functionality of G(1)-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after gamma irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G(1) arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G(1) arrest.     

Heavy ion effects on cellular DNA: strand break induction and repair in cultured diploid lens epithelial cells

The relative biological effectiveness (RBE) for the induction of DNA strand breaks and the efficiency of repair of these breaks in cultured diploid bovine lens epithelial cells was measured, using accelerated heavy ions in the linear energy transfer (LET)-range up to 16,200 keV/micron. At LET values above 800 keV/micron, the number of DNA strand breaks induced per particle increases both with the atomic number of the projectile and with its kinetic energy. About 90 per cent or more of the strand breaks induced by ions with an LET of less than 10,000 keV/micron are repaired within 24 h. Repair kinetics show a dependence on the particle fluence (irradiation dose). At higher particle fluences a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At any LET value, repair is much slower after heavy-ion exposure than after X-irradiation. This is especially true for low energetic particles with a very high local density of energy deposition within the particle track. At the highest LET value (16,200 keV/micron), no significant repair is observed.     

Effects of carbon-ion radiation on the postnatal development of mice and on the yield of white spots in the mid-ventrum and tail tips

Pregnant female C57BL/10JHir mice were irradiated whole-body at 9 days of gestation with a single acute dose of carbon-ion radiation. The average linear energy transfer (LET) of the carbon ions was 50 keV/microm within a spread-out Bragg peak (SOBP). The effects were studied by scoring changes in the postnatal development of the mice as well as in the pigmentation of the cutaneous coats and tail tips of their offspring 22 days after birth. The percentage of live births was reduced in mice exposed to carbon ions at doses greater than 0.5 Gy. The survival to day 22 was also reduced in mice exposed to carbon ions at doses greater than 0.75 Gy. Moreover, the body weight at day 22 was reduced in mice exposed to carbon ions at doses greater than 0.1 Gy. A comparison of the survival to day 22 after exposure to carbon ions with our previous results for 60Co gamma rays indicated that carbon ions were twice as effective as gamma rays. White spots were found in the mid-ventrum as well as in the tail tips of offspring exposed to carbon ions in utero. The frequency and the size of the white spots in the mid-ventrum and in the tail tips increased as the dose increased. Carbon ions appear to be slightly more effective than the gamma rays used in our previous study. In the ventral white spots, no melanocytes were observed in the epidermis, dermis and hair follicles. These results indicate that prenatal exposure to carbon ions has a greater effect on the postnatal development and survival of mice than does exposure to gamma rays, and that the relative biological effectiveness is greater than that for effects on melanocyte development.     

Influence of radiation quality on the yield of DNA strand breaks in SV40 DNA irradiated in solution.

Using highly energetic particles to irradiate plasmid DNA in aerobic aqueous solution, we have compiled an extensive database on how yields of DNA single- and double-strand breaks (SSBs and DSBs) vary with radiation quality. This study was performed in a low-scavenging buffer system and covers a wide range of ion species (helium to uranium) and LETs (5 to 16,000 keV/microm). For LETs up to around 40 keV/microm for SSBs and 400 keV/microm for DSBs, the total energy deposition determines cross section. At higher LET, cross sections level off and individual plateaus for particles of different atomic numbers are observed. For each ion species this is more pronounced and occurs at lower LET for SSBs than for DSBs, leading to an increase in the DSB:SSB ratio from 1:70 for X rays to 1:6 at 500 keV/microm. At this LET, the influence of track structure becomes evident, with high local concentrations of ionization events favoring the formation of DSBs and also intratrack recombination reactions. For lower-energy ions, a saturation in production of measurable DSBs is apparent, due to correlated lesion induction within densely ionizing particle tracks. For very heavy low-energy ions, both SSB and DSB cross sections decrease with particle velocity at nearly constant LET, forming individual hooked curves when plotted as a function of LET.     

Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress

Widespread evidence indicates that exposure of cell populations to ionizing radiation results in significant biological changes in both the irradiated and nonirradiated bystander cells in the population. We investigated the role of radiation quality, or linear energy transfer (LET), and radiation dose in the propagation of stressful effects in the progeny of bystander cells. Confluent normal human cell cultures were exposed to low or high doses of 1GeV/u iron ions (LET ～ 151 keV/μm), 600 MeV/u silicon ions (LET ～ 51 keV/μm), or 1 GeV protons (LET ～ 0.2 keV/μm). Within minutes after irradiation, the cells were trypsinized and co-cultured with nonirradiated cells for 5 h. During this time, irradiated and nonirradiated cells were grown on either side of an insert with 3-μm pores. Nonirradiated cells were then harvested and allowed to grow for 20 generations. Relative to controls, the progeny of bystander cells that were co-cultured with cells irradiated with iron or silicon ions, but not protons, exhibited reduced cloning efficiency and harbored higher levels of chromosomal damage, protein oxidation and lipid peroxidation. This correlated with decreased activity of antioxidant enzymes, inactivation of the redox-sensitive metabolic enzyme aconitase, and altered translation of proteins encoded by mitochondrial DNA. Together, the results demonstrate that the long-term consequences of the induced nontargeted effects greatly depend on the quality and dose of the radiation and involve persistent oxidative stress due to induced perturbations in oxidative metabolism. They are relevant to estimates of health risks from exposures to space radiation and the emergence of second malignancies after radiotherapy.     

His+ reversions caused in Salmonella typhimurium by different types of ionizing radiation

The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co gamma rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV alpha particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/micron. The ratio of the effectiveness of this radiation to gamma rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the alpha particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames [Mutat. Res. 113, 173-215 (1983)] for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study [Roos et al., Radiat. Res. 104, 102-108 (1985)] greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating.     

The dose rate dependence of the relative biological effectiveness of 241Am versus 226Ra gamma rays

The survival of Chinese hamster cells exposed to 59.5 keV 241Am gamma rays was compared with that obtained after exposure to 226Ra gamma rays. The Fricke dosimeter in conjunction with the calculational techniques of transition-zone dosimetry was employed to determine the dose rates to the cells at the petri dish/growth medium interface. The dose rates to the cells ranged from 11 to 133 cGy/h. In all cases, cell survival versus dose was best described by a simple exponential function of dose. For both radiations, graphs of D0 versus dose rate show complex but similar patterns of peaks and valleys. As the curve for 241Am is displaced toward lower dose rates compared with that for 226Ra, the relative biological effectiveness of 241Am vs 226Ra varies considerably with dose rate, ranging from 1.7 at 20 cGy/h to 1.1 at 40 cGy/h to 1.6 at 50 cGy/h. This phenomenon may be due to the LET-dependent accumulation of cells at the G2 + M interface in the cell cycle. The mean unrestricted track-average LET of 241Am (3.7 keV/microns) is 12 times higher than that for 226Ra (0.31 keV/microns) but only one-fifth that of carbon ions (18 keV/microns) for which G2 + M pile-up is observed. Application of the in vitro data derived from this study to the clinical situation, where the dose rate decreases rapidly with distance from the source, suggests that, dose for dose, 241Am will produce results little different from those obtained with 226Ra.     

Induction and rejoining of DNA double-strand breaks in normal human skin fibroblasts after exposure to radiation of different linear energy transfer: possible roles of track structure and chromatin organization

DNA double-strand breaks are nonrandomly induced by high-LET radiation. Differences in the induction and rejoining of DSBs after irradiation with ions having different LET were detected by fragment analysis. The data obtained indicate that the track structure of the traversing particle and its interaction with the different chromatin structures of the cellular DNA influence the yield as well as the distribution of the induced damage. The induction and rejoining of clustered DSBs induced by the same nitrogen ion fluence at LETs of 80-225 keV/microm were investigated by a detailed analysis of the DNA fragmentation patterns in normal human fibroblasts. The DSBs in the cells were allowed to rejoin during incubations for 0-20 h. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of fragments in the size ranges 1-6 Mbp and 5 kbp-1.5 Mbp. A strong influence of LET on the level of DSB induction was evident. The DSB yield increased from 4.5 +/- 0.2 to 10.0 +/- 0.3 DSBs per particle traversal through the cell nucleus when LET increased from 80 to 225 keV/microm. Further, the size distribution of the DNA fragments showed a significant dependence on radiation quality, with an excess of fragments at 50-200 kbp and around 1 Mbp. Differences in repair kinetics were also evident, with slower rejoining for increasing LET, and the initial nonrandom fragment distributions were still present after 1 h of repair.     

Ionizing radiation enhances the expression of the nonsteroidal anti-inflammatory drug-activated gene (NAG1) by increasing the expression of TP53 in human colon cancer cells

The induction of apoptosis in cells of human colon cancer cell lines after gamma irradiation was investigated to determine whether apoptosis was mediated by TP53 and the subsequent expression of its downstream target, the NSAID-activated gene (NAG1). HCT116 (TP53(+/+)), HCT15 (TP53 mutant) and TP53 null HCT116 (TP53(-/-)) cells were irradiated with gamma rays, and apoptosis was measured at various times after irradiation. In HCT116 TP53(+/+) cells, apoptosis was increased after irradiation; the increase was dependent on the time after treatment and the dose of gamma rays. However, in HCT15 TP53 mutant cells and HCT116 TP53(-/-) cells, there were no remarkable changes in apoptosis. The expression of TP53 protein in HCT116 cells was increased after irradiation and was followed by an increase in the expression of NAG1 protein. In contrast, the expression of NAG1 protein in TP53 mutant cells and TP53(-/-) cells was not increased by the radiation treatment, suggesting that NAG1 was required for apoptosis. The expression of NAG1 increased apoptosis in HCT116 cells, but radiation treatment did not further increase apoptosis. The transfection of a NAG1 siRNA into HCT116 cells suppressed radiation-induced apoptosis and inhibited the induction of NAG1 protein without altering the expression of TP53. a NAG1 luciferase promoter construct that included both of the TP53 binding sites, was activated by radiation in dose-dependent manner, while the promoters lacking one or both of the TP53 binding sites in the NAG1 promoter activity either was less responsive or did not respond. The findings reported here indicate that gamma radiation activates the TP53 tumor suppressor, which then increases the expression of NAG1. NAG1 mediates the induction of apoptosis in human colorectal cells.     

Dose-dependent biphasic accumulation of TP53 protein in normal human embryo cells after X irradiation

The effects of various doses of X radiation on the kinetics of accumulation of TP53 protein (formerly known as p53) were examined in normal human embryo cells. We found that the rate of accumulation of TP53 protein was biphasic at X-ray doses between 1 and 4 Gy, while monophasic accumulation was observed after X irradiation with doses higher than 6 Gy. The first phase of accumulation was detected within 1 h after irradiation, and a second phase of accumulation was detected between 6 and 12 h after irradiation. The induction of CDKN1A (formerly known as p21(WAF1/CIP1)) and MDM2 proteins was also biphasic after doses of 4 Gy or less, while monophasic accumulation was observed after 6 Gy or higher. We found that the proteasome inhibitor ALLN increased the constitutive level of TP53 protein, and no change was observed in the TP53 level after X irradiation when cells were treated with ALLN. These results indicate that the dose-dependent accumulation of TP53 is due to an inhibition of TP53 degradation, and that the induction of MDM2 might be responsible in part for the different kinetics of accumulation of TP53.