Akira Ootsuyama

One hundred percent tumor induction in mouse skin after repeated beta irradiation in a limited dose range

Repeated beta irradiation of the backs of mice three times a week with radiation doses of 250 to 1180 cGy per exposure induced 100% incidence of tumors. The incidence of skin tumors appeared to be determined by the total number of repeated exposures in this dose range. An abrupt delay in tumor emergence was observed when the radiation dose was reduced from 250 to 150 cGy per exposure, indicating the existence of a critical threshold. Mouse skin resembles human skin rather than rat skin in its response to radiation.

Threshold-like dose of local beta irradiation repeated throughout the life span of mice for induction of skin and bone tumors.

The backs of female ICR mice were irradiated with beta rays from 90Sr-90Y three times a week throughout life. Previously we observed 100% tumor incidence at five different dose levels ranging from 1.5 to 11.8 Gy per exposure, but no tumor on repeated irradiation with 1.35 Gy for 300 days (Radiat. Res. 115, 488, 1988). In the present study, delay of tumor development was again seen at a dose of 1.5 Gy per exposure, with further delay at 1.0 Gy. The final tumor incidence was 100% with these two doses. At 0.75 Gy per exposure, no tumor appeared within 790 days after the start of irradiation, but one osteosarcoma and one squamous cell carcinoma did finally appear. These findings indicate a threshold-like response of tumor induction in this repeated irradiation system and further suggest that the apparent threshold may be somewhat less than 0.75 Gy per exposure.

Threshold effect for teratogenic risk of radiation depends on dose-rate and p53-dependent apoptosis.

Purpose : To obtain evidence that the p53 gene is indispensable for reduction of high teratogenic risk of radiation at a high dose-rate to zero risk by lowering the dose-rate. Materials and methods : Wild-type p53 (+/+), heterozygous p53 (+/-) and null p53 (-/-) mice were exposed to γ-rays at high or low dose-rates during days 9.5-10.5 of gestation. The incidence of malformations and prenatal deaths was studied. Frequencies of cells dying by apoptosis were measured during or after protracted irradiation. Results : After irradiation with 2 Gy, the frequency of apoptotic cells increased to 20% for p53 (+/+) mice and did not increase at all for p53 (-/-) mice. For p53 (+/+) mice, 2 Gy γ-rays induced 70% malformations when given at 1.06 Gy/min, but no malformations above the control when given at 1.2m Gy/min. In contrast, after irradiation of p53 (-/-) foetuses with 2 Gy at 1.2m Gy/min, the incidence of malformations increased 12% above control levels. Conclusion : Foetal irradiation with 2 Gy at 1.2m Gy/min was not teratogenic for p53 (+/+) mice but teratogenic for p53 (-/-) mice. This indicates that the p53 gene is indispensable for a threshold effect in the risk of radiation at low doses or dose-rates.

Sequential changes in transforming growth factor (TGF)-β1 concentration in synovial fluid and mRNA expression of TGF-β1 receptors in chondrocytes after immobilization of rabbit knees

Abstract We have previously reported that a combination of transforming growth factor (TGF)-β1 and basic fibroblast growth factor (bFGF) synergistically increases the proliferation of chondrocytes obtained from knee joint immobilized for 7–14 days in male Japanese white rabbits. In the present study, we performed experiments with chondrocytes and synovial fluid obtained from rabbit knees immobilized for 0–42 days, to clarify the sequential changes in TGF-β1 and bFGF concentrations in synovial fluid and the mRNA expression of TGF-β1 receptor type I (RI) and II (RII) in chondrocytes after immobilization. The combination of TGF-β1 and bFGF had a synergistic effect on the proliferation of chondrocytes obtained from knee joints immobilized for 7–14 days. The concentration of TGF-β1 in synovial fluid was significantly higher (up to 3.6-fold) at 7–28 days after immobilization compared with that at 2 days. The mRNA for RI and RII was expressed during the whole immobilization period. The concentration of bFGF was kept at the same level at 2–7 days after immobilization, and gradually decreased thereafter. In the early stages of degenerated cartilage, up to 14 days after immobilization, the concentrations of both TGF-β1 and bFGF were higher in the synovial fluid and mRNA expression of TGF-β1 receptors in chondrocytes was kept.

TP53 and TP53-Related Genes Associated with Protection from Apoptosis in the Radioadaptive Response

Abstract Okazaki, R., Ootsuyama, A. and Norimura, T. TP53 and TP53-Related Genes Associated with Protection from Apoptosis in the Radioadaptive Response. Radiat. Res. 167, 51–57 (2007). 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 high susceptibility of heterozygous p53(+/-) mice to malformation after foetal irradiation is related to sub-competent apoptosis

PURPOSE To investigate the relationship between the incidence of radiation-induced malformations and the extent of p53-dependent apoptosis. MATERIALS AND METHODS Wild-type p53(+ / +) and heterozygous p53(+ / -) mice were exposed to X-rays at the mid-gestational period. The incidence of anomalies and prenatal deaths, the extent of apoptosis, and the levels of p53 protein were assessed. RESULTS After X-irradiation with 2 Gy, the incidence of malformation (corrected for control levels) was 0 and 30%, respectively, for p53(+ / +) and p53(+ / -). After irradiation of p53(+ / +) foetuses with 3 Gy, the frequency (F) of apoptotic cells rapidly peaked at 80% at 4 h and fell close to the control level at 48 h. The relationship between F 4h after irradiation and dose (D) (1-3Gy) is accurately expressed by a single-hit equation, F= 1 -exp ( -(a + bD)¿, where the radiation-induced apoptosis rate, b, is 0.47 for the wild type and 0.22 for the heterozygous mice. The X-irradiated foetuses showed no increase in the levels of p53 protein. CONCLUSION The higher susceptibility of irradiated p53(+ / -) foetuses to malformation is related to a twofold lower rate of apoptosis; competent removal by apoptosis of damaged cells from irradiated tissues is impaired dramatically if one of two wild-type p53 alleles is lost. The frequency of apoptotic cells in the wild type reached a maximum 4h after foetal irradiation with no measurable increase in the level of p53 protein, indicating that radiation-induced p53-mediated foetal apoptosis depends on non-transcriptional events.

A Novel ATM/TP53/p21-Mediated Checkpoint Only Activated by Chronic γ-Irradiation

Different levels or types of DNA damage activate distinct signaling pathways that elicit various cellular responses, including cell-cycle arrest, DNA repair, senescence, and apoptosis. Whereas a range of DNA-damage responses have been characterized, mechanisms underlying subsequent cell-fate decision remain elusive. Here we exposed cultured cells and mice to different doses and dose rates of γ-irradiation, which revealed cell-type-specific sensitivities to chronic, but not acute, γ-irradiation. Among tested cell types, human fibroblasts were associated with the highest levels of growth inhibition in response to chronic γ-irradiation. In this context, fibroblasts exhibited a reversible G1 cell-cycle arrest or an irreversible senescence-like growth arrest, depending on the irradiation dose rate or the rate of DNA damage. Remarkably, when the same dose of γ-irradiation was delivered chronically or acutely, chronic delivery induced considerably more cellular senescence. A similar effect was observed with primary cells isolated from irradiated mice. We demonstrate a critical role for the ataxia telangiectasia mutated (ATM)/tumor protein p53 (TP53)/p21 pathway in regulating DNA-damage-associated cell fate. Indeed, blocking the ATM/TP53/p21 pathway deregulated DNA damage responses, leading to micronucleus formation in chronically irradiated cells. Together these results provide insights into the mechanisms governing cell-fate determination in response to different rates of DNA damage.

Zero tumor incidence in mice after repeated lifetime exposures to 0.5 Gy of beta radiation

A final series of experiments on tumor induction by repeating 90Sr-90Y beta irradiation of the back of ICR mice three times weekly throughout the life span showed no tumor incidence with repeated doses of 0.5 Gy per exposure. The whole dose-response relationship for repeated irradiation with 0.5 to 11.8 Gy per exposure, summarized along with data obtained previously, showed a unique nonlinear dose response with saturation of tumor incidence at doses of over 2.5 Gy per exposure and a threshold-like value around 1.5 Gy per exposure.

Radiation Dose-Rate Effect on Mutation Induction in Spleen and Liver of gpt delta Mice

Abstract 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 µGy/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.

Effects of a Brain-Engraftable Microglial Cell Line Expressing Anti-Prion scFv Antibodies on Survival Times of Mice Infected with Scrapie Prions

We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.