p53-dependent delayed effects of radiation vary according to time of irradiation of p53 + / - mice

Abstract

We previously reported that in p53 (+ / -) mice that had been given a whole-body dose of 3 Gy at 8 weeks of age, p53-dependent delayed effects of radiation, as manifested in T-cell receptor (TCR) variant fractions (VF) instability in mouse splenocytes, were biphasic, namely, induction of TCR-VF mutation reappeared at 44 weeks. The manifestation of the delayed effects and the measures of biological markers varied according to the timing of irradiation. We also reported that the decrease in function of the p53 gene was related to the effects of a delayed mutation. In the present study, we investigated the functions and mutations of the p53 gene in old age for p53 (+ / -) mice following irradiation at various ages. p53 (+ / -) mice were given a whole-body dose of 3 Gy at 8, 28 or 40 weeks of age. There were significant differences for all variables tested at 8 weeks of age. This was similarly the case for mice irradiated at 28 weeks of age, in which there were also significant differences in TCR VF and the percentage of apoptosis. In mice irradiated at 40 weeks of age, there were significant differences for all considered variables except for the p53 allele. We demonstrated that the different patterns of delayed mutation of the p53 gene at 56 weeks of age depended on the age at which mice had undergone 3-Gy whole-body irradiation. Our conclusions are limited to variation in p53-dependent delayed effects according to the time of irradiation.

Keywords: age; delayed effects; irradiation; p53.

Fig. 1.

Time-course of the TCR VF of splenic T lymphocytes in p53 ^{+ / −} mice following irradiation with 3 Gy at 8, 28 or 40 weeks of age. Open circles: non-irradiated (sham-irradiated) control mice. Filled circles: irradiated mice given a whole-body dose of 3 Gy at 8 weeks of age [14]. Filled triangles: irradiated mice given a whole-body dose of 3 Gy at 28 weeks of age. Filled squares: irradiated mice given a whole-body dose of 3 Gy at 40 weeks of age. Each point indicates the mean ± SD of 4–16 mice.

Fig. 2.

Frequency of apoptotic cells in the spleens of p53 ^{+ / −} mice at 56 weeks of age 4 h after irradiation with 3 Gy. All mice were exposed to 3 Gy (after previous earlier irradiation) again at 56 weeks of age to evaluate the activity of radiation-induced apoptosis. The frequency of apoptosis was evaluated at 4 h after the irradiation with the modified TUNEL methods. Unfilled: non-irradiated (sham-irradiated) control mice. Striped: irradiated mice previously given a whole-body dose of 3 Gy at 8 weeks of age [14]. Black: irradiated mice previously given a whole-body dose of 3 Gy at 28 weeks of age. Gray: irradiated mice previously given a whole-body dose of 3 Gy at 40 weeks of age. Each bar indicates the mean ± SD of 5–8 mice. In the time-course of the apoptosis assay, a Student's t-test was utilized to determine the differences between irradiated mice and non-irradiated mice at each time-point. *P < 0.05, ***P < 0.001.

Fig. 3.

A: Protein expression of p53, phospho-p53 (p53-ser15/18) and p21 in p53 ^{+ / −} mice at 56 weeks of age after 3-Gy whole-body irradiation either at 28 or at 40 weeks of age. Western blot analysis was performed. Representative data are shown. The expression levels of p53, phospho-p53 and p21 were normalized to actin. Unfilled: non-irradiated (sham-irradiated) control mice. Black: irradiated mice given a whole-body dose of 3 Gy at 28 weeks of age. Gray: irradiated mice given a whole-body dose of 3 Gy at 40 weeks of age. Each group contained 3–8 mice. Data are presented as the means ± SD. *P < 0.05. **P < 0.01. B: Protein expression of phospho-p53 (p53-ser15/18) in p53 ^{+ / −} mice at 56 weeks of age after 3-Gy whole-body irradiation at 8, 28 or 40 weeks of age. The data for 8 weeks of age was from previous data [15]. The previous data and the present data were combined. Unfilled: non-irradiated (sham-irradiated) control mice. Striped: irradiated mice given a whole-body dose of 3 Gy at 8 weeks of age [15]. Black: irradiated mice given a whole-body dose of 3 Gy at 28 weeks of age. Gray: irradiated mice given a whole-body dose of 3 Gy at 40 weeks of age. Each group contained 3–8 mice. Data are presented as the means ± SD. *P < 0.05.

Fig. 4.

The loss of the p53 allele in p53 ^{+ / −} mice at 56 weeks of age after 3-Gy whole-body irradiation at 8, 28 or 40 weeks of age. Each group contained 3–9 mice. Data are presented as the means ± SD. *P < 0.05 compared with the non-irradiated control p53 ^{+ / −} mice.

Fig. 5.

MSP (methylation-specific PCR) assay for p53 in p53 ^{+ / −} mice at 56 weeks of age after 3-Gy whole-body irradiation at 8, 28 or 40 weeks of age. Each group contained 3–5 mice.