Establishment of Methylation-Specific PCR for the Mouse p53 Gene.

Methylation-specific PCR (MSP) of the mouse p53 gene has not yet been reported. We searched the CpG islands, sequenced the bisulfited DNA, and designed PCR primers for methylation and unmethylation sites. DNA from a young mouse produced a strong PCR product with the unmethylated primer and a weaker band with the methylated primer. DNA from an old mouse produced bands of similar intensities with both primers. In radiation-induced tumors, DNA from an old mouse yielded similar bands with both types of primers. We suggest that MSP is a valuable technique for the epigenetic study of the mouse p53 gene.

Dynamics of delayed p53 mutations in mice given whole-body irradiation at 8 weeks.

PURPOSE: Ionizing irradiation might induce delayed genotoxic effects in a p53-dependent manner. However, a few reports have shown a p53 mutation as a delayed effect of radiation. In this study, we investigated the p53 gene mutation by the translocation frequency in chromosome 11, loss of p53 alleles, p53 gene methylation, p53 nucleotide sequence, and p53 protein expression/phosphorylation in p53(+/+) and p53(+/-) mice after irradiation at a young age. METHODS AND MATERIALS: p53(+/+) and p53(+/-) mice were exposed to 3 Gy of whole-body irradiation at 8 weeks of age. Chromosome instability was evaluated by fluorescence in situ hybridization analysis. p53 allele loss was evaluated by polymerase chain reaction, and p53 methylation was evaluated by methylation-specific polymerase chain reaction. p53 sequence analysis was performed. p53 protein expression was evaluated by Western blotting. RESULTS: The translocation frequency in chromosome 11 showed a delayed increase after irradiation. In old irradiated mice, the number of mice that showed p53 allele loss and p53 methylation increased compared to these numbers in old non-irradiated mice. In two old irradiated p53(+/-) mice, the p53 sequence showed heteromutation. In old irradiated mice, the p53 and phospho-p53 protein expressions decreased compared to old non-irradiated mice. CONCLUSION: We concluded that irradiation at a young age induced delayed p53 mutations and p53 protein suppression.

The late effects of radiation on lifespan, lymphocyte proliferation and p53 haplodeficiency in mice.

PURPOSE: We investigated the effect of irradiation on the lifespan of eight-week-old mice, the number of lymphocytes in bone marrow and the levels of p53 protein expression in the splenocytes. METHODS AND MATERIALS: Eight-week-old mice, wild-type p53 (p53(+/+)) and heterozygous p53 (p53(+/-)), were irradiated with 3 Gy. The cell numbers and cell cycle phases of bone marrow cells were determined by flow cytometry. The splenocyte proliferation was evaluated by a fluorescent cell viability assay. The p53 expression was evaluated by Western blotting. RESULTS: The lifespan of the irradiated mice was shorter than that of the non-irradiated mice. In irradiated 72-week-old p53(+/+) mice and 56-week-old p53(+/-) mice, the number of lymphocytes in bone marrow decreased as compared to that in the non-irradiated mice. In 56-week-old p53(+/-) mice, the S- and G2/M-phases of lymphocytes in the irradiated mice were increased compared to that in the non-irradiated mice. The splenocyte proliferation in p53(+/+) mice decreased with age, and the proliferation in the irradiated mice was much lower than that in the non-irradiated mice. In 72-week-old p53(+/+) mice after re-irradiation, the p53 protein expression in the splenocytes of the irradiated mice was delayed as compared to those from the non-irradiated mice. CONCLUSION: We suggest that the decrease in the number of lymphocytes in bone marrow and the delayed p53 expression in splenocytes from the irradiated mice are related to the shortened lifespan after irradiation at a young age.

Radiation dose-rate effect on mutation induction in spleen and liver of gpt delta mice.

The effect of dose rate on radiation-induced mutations in two somatic tissues, the spleen and liver, was examined in transgenic gpt delta mice. These mice can be used for the detection of deletion-type mutations, and these are the major type of mutation induced by radiation. The dose rates examined were 920 mGy/min, 1 mGy/min and 12.5 microGy/min. In both tissues, the number of mutations increased with increasing dose at each of the three dose rates examined. The mutation induction rate was dependent on the dose rate. The mutation induction rate was higher in the spleen than in the liver at the medium dose rate but was similar in the two tissues at the high and low dose rates. The mutation induction rate in the liver did not show much change between the medium and low dose rates. Analysis of the molecular nature of the mutations indicated that 2- to 1,000-bp deletion mutations were specifically induced by radiation in both tissues after high- and low-dose-rate irradiation. The occurrence of deletion mutation without any sequence homology at the break point was elevated in spleen after high-dose-rate irradiation. The results indicate that the mutagenic effects of radiation in somatic tissues are dependent on dose rate and that there is some variability between tissues.

A comparison of the mutagenic and apoptotic effects of tritiated water and acute or chronic caesium-137 gamma exposure on spleen T lymphocytes on normal and p53-deficient mice.

PURPOSE: This study was carried out to compare the mutagenic effects on spleen T lymphocytes of mice exposed to tritiated water (HTO) and chronic or acute (137)Cs gamma irradiation. MATERIALS AND METHODS: p53 wild type (p53(+/+)) and p53 null type (p53(-/-)) mice were exposed to a total dose of 3 Gy of HTO, chronic (137)Cs and acute (137)Cs. RESULTS: In spontaneous T-cell receptor (TCR) variant fractions and fractions following exposure to HTO, chronic (137)Cs and acute (137)Cs, TCR variant fractions in p53(+/+) mice were 5.9 x 10(-4), 9.8 x 10(-4), 6.4 x 10(-4) and 20.1 x 10(-4), respectively. In contrast, those fractions were increased in p53(-/-) mice to 11.2 x 10(-4), 18.8 x 10(-4), 15.7 x 10(-4) and 31.3 x 10(-4), respectively. The frequency of apoptotic cells of the spleen 12 h after HTO injection increased to 5.0% in p53(+/+) mice, but did not increase at all in p53(-/-) mice. CONCLUSIONS: When compared on the basis of induced TCR variant fractions in p53(-/-) mice, HTO (7.6 x 10(-4)) was 1.7 times more mutagenic than chronic (137)Cs (4.5 x 10(-4)), but 2.6 times less mutagenic than acute (137)Cs gamma irradiation (20.1 x 10(-4)).

Measurement of mutant frequency in T-cell receptor (TCR) gene by flow cytometry after X-irradiation on EL-4 mice lymphoma cells.

It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytometry. In this study, we developed an in vitro system using murine EL-4 lymphoma cells and observed frequency of cells defective in TCR gene expression after exposure to ionizing irradiation. EL-4 cells were stained with fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD3 antibodies. They were analyzed with a flow cytometer to detect mutant EL-4 cells lacking surface expression of TCR/CD3 complexes which showed CD3-, CD4+ due to a somatic mutation at the TCR genes. Mutant cells could be observed at 2 days after 3 Gy irradiation. MF of EL-4 cells was 6.7x10(-4) for 0 Gy and the value increased to the maximum level of 39x10(-4) between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic dose-response model. After the peak value the TCR MF gradually decreased with a half-life of approximately 3.2 days. We also examined the hprt mutant frequencies at seven days after irradiation and the cytokinesis-blocked micronucleus frequency at 20 hrs after irradiation. The frequencies of hprt mutation and micronuclei were found to be best fitted by a linear-quadratic dose-response model and a linear dose-response model, respectively. The method to detect mutation on TCR gene is quick and easy in comparison with other methods and is considered useful for the mutagenicity test.

Prolonged increase in T-cell receptor (TCR) variant fractions of spleen T lymphocytes in pregnant mice after gamma irradiation.

To investigate the relationship between the radiation-induced increase of T-cell receptor (TCR) defective variant fractions and physiological status such as pregnancy, C57BL/ 6N mice were irradiated with 3 Gy of gamma rays at various days of gestation, just before and just after pregnancy. While the highest level of variant fractions in spleen T lymphocytes appeared at 9 days postirradiation and resolved fairly rapidly for nonpregnant mice, the increased variant fractions for pregnant mice irradiated at 16.5 days of gestation reached a plateau at 14 days postirradiation and remained at high levels until 28 days after irradiation. Therefore, variant fractions 28 days postirradiation were measured to determine the overall effect of radiation on the kinetics of TCR variant fractions during gestation. There was no significant difference in the baseline TCR variant fraction between unirradiated nonpregnant and pregnant mice. TCR variant fractions after irradiation were about twofold higher in pregnant mice (from 10.5 days of gestation until delivery) than those in nonpregnant mice. Both gamma radiation and pregnancy caused a decrease in the proportion of naïve T-cell subsets and an increase in TCR variant fractions of naïve T cells. In addition, the prolonged postirradiation increase in the TCR variant fractions of pregnant mice was associated with an increase in serum progesterone level. Differences between pregnant and nonpregnant mice in the kinetics of postirradiation restoration of T-cell systems may be involved in producing the differences in residual TCR variant fractions of these mice.

TP53 and TP53-related genes associated with protection from apoptosis in the radioadaptive response.

We investigated the effect of administering priming low-dose radiation prior to high-dose radiation on the level of apoptosis and on the expression of TP53 and TP53-related genes in mouse splenocytes. The percentage of apoptotic cells was significantly lower in TP53(+/+) mice receiving priming radiation 2 to 168 h before the high-dose irradiation, compared to TP53(+/+) mice exposed to 2 Gy alone. In contrast, TP53(+/-) mice exhibited a reduced level of apoptosis only when priming was performed for 2 or 4 h prior to the high-dose irradiation. In TP53(+/+) mice, primed mice had higher TP53 expression than mice exposed to 2 Gy. Phospho-TP53 (ser15/18) expression was the highest in mice exposed to 2 Gy and intermediate in primed mice. Expression of p21 (CDKN1A) was higher in primed mice compared with mice exposed to 2 Gy. MDM2 expression remained at a high level in all mice receiving 2 Gy. Elevated phospho-ATM expression was observed only in mice exposed to 2 Gy. We conclude that TP53 plays a critical role in the radioadaptive response and that TP53 and TP53-related genes might protect cells from apoptosis through activation of the intracellular repair system.

The delayed manifestation of T-cell receptor (TCR) variants in X-irradiated mice depends on Trp53 status.

The influence of Trp53 on the radiation-induced elevation of T-cell receptor (TCR) variant fractions was examined in splenic T lymphocytes of Trp53-proficient and -deficient mice. Wild-type Trp53+/+, heterozygous Trp53+/- and null Trp53-/- mice were exposed to 3 Gy of X rays at 8 weeks of age. The fraction of TCR-defective variants was measured at various times after irradiation. Initially, the TCR variant fraction increased rapidly and reached its maximum level at 9 days after irradiation before decreasing gradually. In Trp53+/+ and Trp53+/- mice, the TCR variant fraction fell to normal background levels at 16 and 20 weeks of age, respectively. In contrast, the TCR variant fraction of Trp53-/- mice failed to decrease to background levels during the observation period. Baseline levels were then maintained for approximately 60 weeks in the Trp53+/+ mice and approximately 40 weeks in the Trp53+/- mice. After the long flat period, a significant re-increase in the fraction of TCR variants was found after 72 weeks of age in the irradiated Trp53+/+ mice and after 44 weeks of age in the irradiated Trp53+/- mice. Measurement of the fraction of apoptotic cells in the spleen and thymus 4 h after X irradiation at these ages in Trp53+/+ and Trp53+/- mice demonstrated a reduction in apoptosis in the irradiated mice compared to the nonirradiated mice. This suggests that the delayed increase in TCR variants after irradiation is due to a reduction in Trp53-dependent apoptosis.

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.

Radioadaptive response for protection against radiation-induced teratogenesis.

To clarify the characteristics of the radioadaptive response in mice, we compared the incidence of radiation-induced malformations in ICR mice. Pregnant ICR mice were exposed to a priming dose of 2 cGy (667 muGy/min) on day 9.5 of gestation and to a challenging dose of 2 Gy (1.04 Gy/min) 4 h later and were killed on day 18.5 of gestation. The incidence of malformations and prenatal death and fetal body weights were studied. The incidence of external malformations was significantly lower (by approximately 10%) in the primed (2 cGy + 2 Gy) mice compared to the unprimed (2 Gy alone) mice. However, there were no differences in the incidence of prenatal death or the skeletal malformations or the body weights between primed and unprimed mice. These results suggest that primary conditioning with low doses of radiation suppresses radiation-induced teratogenesis.

Effect of extended exposure to low-dose radiation on autoimmune diseases of immunologically suppressed MRL/MpTn-gld/gld mice.

The purpose of this paper is to analyze the relationship between alterations of splenic T-cell subpopulations and the amelioration of autoimmune diseases of MRL/MpTn-gld/gld mice (MRL/gld mice) after extended exposure to low-dose radiation. After the onset of disease, 4-month-old MRL/gld mice were exposed to doses of 0.05, 0.2, and 0.5 Gy/day for 4 weeks (5 days/week), for total doses of 1, 4, and 10 Gy, respectively. The MRL/gld mice that were irradiated with 0.2 and 0.5 Gy/day showed an obvious decrease in the proportion of splenic CD4(-)CD8(-) T cells and remission of their autoimmune diseases. After the last irradiation, apoptotic cells were found in the white pulp of the spleen of the MRL/gld mice irradiated with 0.2 Gy/day, but not in the MRL/MpJ-+/+ mice (MRL/wild mice), which experienced a similar treatment. Before the onset of disease, 3-month-old MRL/gld mice subjected to 0.2 Gy/day showed a decrease in the proportion of splenic CD4(-)CD8(-) T cells and less remission of their autoimmune diseases than the 4-month-old mice. These results suggest that the accumulated CD4(-)CD8(-) T cells are more sensitive to radiation than other T-cell subpopulations, and that decreasing CD4(-)CD8(-) T cells with extended exposure to low-dose radiation leads to the amelioration of autoimmune disease.

Apoptosis and p53 expression in chondrocytes relate to degeneration in articular cartilage of immobilized knee joints.

OBJECTIVE: We have reported that articular cartilage showed early stage degeneration at 7 and 14 days after immobilization, moderate degeneration at 28 days, and severe degeneration at 42 days in rabbits. To test whether apoptosis occurs in association with p53 expression in chondrocytes during the process of articular cartilage degeneration, we investigated the degree of cartilage degeneration, the frequency of apoptotic cells, and the levels of p53 mRNA in rabbits and mice after knee immobilization. METHODS: Right knees of male Japanese white rabbits were immobilized in full extension with fiberglass casts for up to 42 days. Similarly, right knees of male p53 wild-type [p53 (+/+)] and p53 null [p53 (-/-)] mice were immobilized in full extension with bandage tape for up to 84 days. Apoptotic cells were confirmed by TUNEL staining on the sections of knee joints. Total RNA of articular chondrocytes obtained from Day 0 or immobilized knees was analyzed semiquantitatively by RT-PCR using specific primers for p53. RESULTS: Articular cartilage degenerated after immobilization of p53 (+/+) mouse knees, but not after immobilization of p53 (-/-) knees. Apoptotic cells were observed in articular cartilage in the femur and tibia of rabbits and p53 (+/+) mice after immobilization. However, only a few apoptotic cells were observed at the same sites in p53 (-/-) mice. In RT-PCR analysis, the levels of p53 mRNA obtained from immobilized groups were significantly higher than those of Day 0 groups in rabbit and p53 (+/+) mouse knees. CONCLUSION: Apoptosis and p53 expression in chondrocytes relate to degeneration in articular cartilage of immobilized knee joints.

Disruption of the p53 gene results in preserved trabecular bone mass and bone formation after mechanical unloading.

We tested the hypothesis that mechanical unloading facilitates signaling of p53, an important modulator of cell cycling and apoptosis, in bone marrow cells and thereby reduces trabecular bone volume (BV). We performed histomorphometric analyses and bone marrow cell cultures in tail-suspended (TS) p53 null (p53-/-) and wild-type (p53+/+) mice. Eight-week-old male mice were assigned to four groups after 1-week acclimatization: p53+/+ + ground control (GC), p53+/+ + TS, p53-/- + GC, and p53-/- + TS. Bilateral tibial samples were used for analysis. The histomorphometric parameters of trabecular structure, formation and resorption did not differ between the p53-/- + GC and p53+/+ + GC groups. Trabecular BV in p53+/+ + TS mice was significantly reduced to 45% of that in the p53+/+ + GC group after one week of TS. In contrast, BV in p53-/- + TS mice was preserved at the same level as that in the p53-/- + GC group. The bone formation rate (BFR) was significantly reduced in p53+/+ + TS but not in p53-/- + TS mice. Unloading significantly increased trabecular osteoclast number (Oc.N) and surface in p53+/+ + TS mice compared with the p53+/+ + GC group, but the difference was not significant between p53-/- + TS and p53-/- + GC mice. In bone marrow cell culture, the numbers of alkaline phosphatase-positive (ALP+) colony-forming units fibroblastic (CFU-f) and mineralized nodules were significantly reduced in p53+/+ + TS, but not p53-/- + TS mice. [3H]thymidine incorporation into bone marrow cells was higher in p53-/- mice than in p53+/+ mice, independent of mechanical loading or unloading. Flow cytometric cell cycle analysis revealed that unloading significantly increased the percentage of hypoploid bone marrow cells in p53+/+ mice relative to that in p53+/+ + GC mice, but there was no significant difference in ploidy between p53-/- + TS and p53-/- + GC mice. Expression levels of p53 and p21 mRNAs were enhanced after TS in bone marrow cells from p53+/+ mice. Our data show that trabecular bone mass and bone formation were preserved after tail-suspension in p53-/- mice, closely associated with ALP+ CFU-f and mineralized nodule formation in marrow cultures obtained from tibias of p53-/- mice. We speculate that bone loss due to mechanical unloading may be related to facilitation of intracellular p53-p21 signaling.

Micronuclei induced by low dose rate irradiation in early spermatids of p53 null and wild mice.

To obtain evidence of the dose-rate effect on induction of micronuclei in early spermatids, we observed frequencies in wild-type p53(+/+), heterozygous p53(+/-) and null p53(-/-) mice 14 days after gamma rays irradiation at a high (1,020 mGy/min) or a low (1.2 mGy/min) dose-rate. A dose- and dose-rate-related increase in micronuclei was seen in early spermatids with no difference between the different p53 status. These data were found to be best fitted by a linear-quadratic dose-response model at a high dose-rate, and by a linear dose-response model at a low dose-rate. The yields at 1.2 mGy/min were significantly lower than those at 1,020 mGy/min in the same manner, independent of p53 status. Testis weight declined significantly after 3 Gy irradiation, but did not depend on dose-rates. In our other studies, we observed the complete elimination both of malformation in fetuses and CD3- 4+ mutant T-lymphocytes in p53(+/+) mice, but not in p53(-/-) mice after irradiation. This indicates that concerted DNA repair and p53-dependent apoptosis are likely to completely eliminate mutagenic damage from the irradiated tissues at low doses or dose-rates in teratogenesis and lymphocytes. In the germ cell, however, irradiation at 1.2 mGy/min was mutagenic, independent of p53 status.

Role of p53 gene in apoptotic repair of genotoxic tissue damage in mice.

When DNA is damaged by exposure to a small amount of radiation, it is repaired efficiently by innate mechanisms. However, if cellular damage is more extensive, DNA repair cannot be adequately completed. To clarify the role of the p53 gene in apoptotic tissue repair, the incidence of in-vivo radiation-induced somatic mutation was evaluated by measuring the T cell receptor (TCR) gene expression in p53(+/+) and p53(-/-) mice. After gamma-irradiation with 3 Gy, the TCR mutation frequency (MF) was higher in p53(+/+) mice than in the controls. However, when the mice were exposed to 3 Gy at a low dose rate, the TCR MF did not increase in the p53(+/+) mice, whereas it increased and remained elevated in p53(-/-) mice, which are unable to induce apoptosis. In p53(+/+) mice, the TCR MF peaked 9 days after gamma-irradiation with 3 Gy at a high dose rate, and then gradually decreased with a half-life of about 13 days. However, in p53(-/-) mice, the peak level of the TCR MF did not decline significantly with time. Hence, complete repair of mutagenic damage in irradiated tissues requires the integration of DNA repair and p53-dependent apoptotic tissue repair.