Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53(+/+)) and Trp53 heterozygous (Trp53(+/-)) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53(+/+) embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53(+/+) embryonic mice, while no such effect was found in the Trp53(+/-) embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.     

Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress

Therapeutic irradiation of the brain can cause a progressive cognitive dysfunction that may involve defects in neurogenesis. In an effort to understand the mechanisms underlying radiation-induced stem cell dysfunction, neural precursor cells isolated from the adult rat hippocampus were analyzed for acute (0-24 h) and chronic (3-33 days) changes in apoptosis and reactive oxygen species (ROS) after exposure to X rays. Irradiated neural precursor cells exhibited an acute dose-dependent apoptosis accompanied by an increase in ROS that persisted over a 3-4-week period. The radiation effects included the activation of cell cycle checkpoints that were associated with increased Trp53 phosphorylation and Trp53 and p21 (Cdkn1a) protein levels. In vivo, neural precursor cells within the hippocampal dentate subgranular zone exhibited significant sensitivity to radiation. Proliferating precursor cells and their progeny (i.e. immature neurons) exhibited dose-dependent reductions in cell number. These reductions were less severe in Trp53-null mice, possibly due to the disruption of apoptosis. These data suggest that the apoptotic and ROS responses may be tied to Trp53-dependent regulation of cell cycle control and stress-activated pathways. The temporal coincidence between in vitro and in vivo measurements of apoptosis suggests that oxidative stress may provide a mechanistic explanation for radiation-induced inhibition of neurogenesis in the development of cognitive impairment.     

Radiation-induced teratogenic effects in fetal mice with varying Trp53 function: influence of prior heat stress

Teratogenesis induced by radiation in fetal mice has been closely linked to Trp53-dependent apoptosis. This study examined teratogenesis in tails and limb digits of fetal mice with varying Trp53 status after a 4-Gy radiation exposure, with and without a prior 40.5 degrees C, 60-min heat stress. Irradiation earlier in gestation (day 11) produced greater effects than later (day 12) exposure, but in both cases the maximum teratogenic effect of radiation occurred in Trp53 normal fetuses, the minimum in Trp53 null fetuses, and intermediate effects in Trp53 heterozygotes, indicating dominance of Trp53-dependent apoptosis. Heat stress 24 h prior to irradiation on day 11 did not alter the teratogenic effects in Trp53 normal or heterozygous fetuses, but it reduced effects in the Trp53 null fetuses. Conversely, heat stress immediately before irradiation on day 11 amplified teratogenesis in Trp53 null fetuses, still with only a small or no effect on fetuses with full or partial Trp53 function, respectively. These results indicate little effect of mild heat on Trp53-dependent apoptosis after irradiation, but they also suggest heat-induced amplification of Trp53-independent processes that led to apoptosis when heat was delivered near the time of radiation exposure, and heat-induced protection of that process when sufficient expression time was allowed. However, Trp53-dependent apoptosis, when functional, acted as the ultimate determinant of radiation-induced teratogenic effects during early organogenesis. On gestation day 12, radiation effects were diminished, but heat stress 24 h prior to radiation exposure had a large amplifying effect in Trp53 normal or heterozygous fetuses. In the absence of functional Trp53, the sensitizing effect of the heat was diminished. The results may suggest that at later times in organ development, DNA repair is more active, allowing some cells to escape radiation-induced Trp53-dependent apoptosis. However, heat may be able to significantly inhibit this active repair and increase the teratogenic effect of radiation. A diminished effect in the absence of functional Trp53 is consistent with an influence of heat on inhibiting DNA repair, but with a diminished probability of apoptosis.     

Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.     

Teratogenic effects of mild heat stress during mouse embryogenesis: effect of Trp53

Hyperthermia can be teratogenic in fetal mice exposed during organogenesis, an effect considered to be due to heat-induced apoptosis of cells in the developing organs. We exposed pregnant mice carrying Trp53(+/+), Trp53(+/-) and Trp53(-/-) fetuses to mild whole-body hyperthermia that raised their core temperature to 40.5 degrees C for 60 min on either day 10 or 11 of gestation. On day 18 of gestation, the fetuses were removed from control and hyperthermia-treated mice and genotyped, and tail length was measured. Limb digits were examined for abnormalities. Tail length in unheated control fetuses was influenced by Trp53 status. A complete lack of functional Trp53 (Trp53(-/-)) but not partial lack of function (Trp53(+/-)) resulted in shorter tails compared to Trp53(+/+) fetuses, indicating a role for Trp53 in the regulation of tail lengthening in mouse fetuses. In all three genotypes, hyperthermia on gestation day 10 resulted in tails shorter than unheated controls, and hyperthermia on day 11 resulted in tails longer than controls. There was no effect on limb digit abnormalities. The data suggest that Trp53-dependent or independent apoptosis may not be directly involved in heat-induced teratogenesis, but that the primary teratogenic effect of heat results from the disruption of another tail length-regulating process that is independent of Trp53. However, the nature of the teratogenic outcome of that disruption depends on the gestation time. The ability of Trp53 to additionally regulate the tail lengthening process was also sensitive to the effects of heat, but that sensitivity again depended on the time of the heat stress during gestation.     

Cell cycle progression and apoptosis after irradiation in an acidic environment

We investigated the effect of an acidic environment on the radiation-induced G2/M arrest and apoptosis using RKO.C human colorectal cancer cells expressing wild-type p53 and RC10.1 cells, a subline of RKO.C cells deficient in p53 as well as p53+/+ MEFs and p53-/- MEFs (mouse embryonic fibroblasts). The cells were irradiated with 4 Gy or 12 Gy of gamma-rays in pH 7.5 medium or pH 6.6 medium. p53 accentuated the progression of cells from radiation-induced G2/M arrest to apoptosis and the pH 6.6 environment suppressed the progression of cells through G2/M-phase to apoptosis after irradiation. Further analysis indicated that the radiation-induced G2/M arrest was due mainly to G2 arrest in both pH 7.5 and pH 6.6. Therefore, it was concluded that p53 enhances, and an acidic environment suppresses, the exit of cells from radiation-induced G2 arrest by altering cyclin B1-Cdc2 kinase activity.     

Cytogenetic analysis of radiation-induced leukemia in Trp53-deficient C3H/He mice

C3H/He mice develop acute myeloid leukemia (AML) after whole-body irradiation, but the strain becomes highly susceptible to stem cell leukemia (SCL) when a null mutation is introduced into the Trp53 gene. To examine the etiology of SCL and the influence of chromosomal instability on leukemogenesis, 12 SCLs and two AMLs arising from Trp53-deficient C3H/He mice were investigated cytogenetically. Each SCL demonstrated cell-to-cell variation in the number and structural integrity of their chromosomes, indicating chromosomal instability. Typical deletion of chromosome 2 was observed in the two AML cases, while most SCL cells did not display this aberration. Deletions and rearrangements of chromosome 11 were noticeable in SCLs from Trp53 heterozygotes but not in AMLs. Analysis of loss of heterozygosity revealed that aberrations involving chromosome 11 in SCLs resulted in loss of the wild-type Trp53 allele. These results suggest that loss of Trp53 function triggers the tumorigenic process leading toward SCL through the induction of chromosomal instability, and that SCL and AML are distinct varieties of leukemia.     

Influence of prior exposure to low-dose adapting radiation on radiation-induced teratogenic effects in fetal mice with varying Trp53 function

Teratogenesis in tails and limb digits of fetal mice with varying Trp53 status was examined after exposure of pregnant females to 4 Gy gamma radiation with and without a prior 30-cGy exposure. Prior low-dose exposure modified the teratogenic effects of radiation in a manner dependent upon Trp53 status and gestation time. A 4-Gy exposure on gestation day 11 resulted in tail shortening and digit abnormalities. A 30-cGy exposure 24 h prior to a 4-Gy radiation exposure on day 11 reduced the extent of both digit abnormalities and the tail-shortening effects in Trp53(+/+) fetuses and also reduced tail shortening in Trp53(+/-) fetuses, but to a lesser extent. However, the pre-exposure enhanced the tail-shortening effects of 4 Gy in Trp53(-/-) fetuses. In contrast, a 30-cGy exposure given 24 h prior to a 4-Gy exposure on gestation day 12 had no effect on the reduced tail length resulting from the 4-Gy exposure of Trp53(+/+) or Trp53(+/-) fetuses, but it partly protected Trp53(-/-) fetuses against reduced tail length. A 4-Gy exposure alone on day 12 did not result in any increase in the frequency of digit abnormalities in Trp53(-/-) fetuses so any protective effect of the preirradiation could not be detected. However, the preirradiation did result in protection against in digit abnormalities in Trp53(+/-) fetuses. We conclude that radiation-induced teratogenesis reflects both Trp53-dependent and independent processes that lead to apoptosis, and these respond differently to prior adapting doses.     

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.     

Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice

We have studied the effects of a defect in the p53 gene on spontaneous and radiation-induced somatic mutation frequencies in vivo by measuring T-cell receptor (TCR) and hypoxanthine phosphoribosyltransferase (HPRT) mutant frequencies (MFs) in p53 deficient mice both before and after exposure to X-irradiation. In the absence of irradiation, the TCR and HPRT mutant frequencies were roughly two-fold higher in p53 null (-/-) mice than in wild-type (+/+) mice. Unexpectedly, the TCR and HPRT MFs were slightly lower in heterozygote p53 (+/-) than in wild-type (+/+) mice, however. After 2 weeks 2Gy whole body irradiation the TCR and HPRT MFs were about two-fold higher in the p53 null (-/-) and p53 (+/-) mice than in the wild-type. Taken together, these findings suggest that a defect in the p53 gene may lead to TCR and HPRT mutants being recovered at higher frequencies in both irradiated and unirradiated mice, but it should be emphasized that the effects we have observed are not particularly strong, albeit that they are statistically significant. Interestingly, several of the highest TCR MF values that we observed in the course of our experiments were recorded in p53 (-/-) animals that had developed thymomas and hence appeared to be cancer prone.     

Friend leukemia virus infection enhances DNA damage-induced apoptosis of hematopoietic cells,causing lethal anemia in C3H hosts

Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53(-/-)) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53(-/-) --> C3H p53(+/+) bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.     

Radiation-induced apoptosis in the adult central nervous system is p53-dependent

Oligodendrocytes and subependymal cells in the adult CNS have been shown to undergo radiation-induced apoptosis. Here, we examined the role of p53 in radiation-induced apoptosis in the adult mouse CNS. In the spinal cord of p53+/+ mice, apoptotic glial cells were observed within 24 h after irradiation, and the apoptotic response peaked at 8 h. These apoptotic cells demonstrated the immunohistochemical phenotype of oligodendrocytes, and decreased oligodendrocyte density was observed at 24 h after 22 Gy. A similar time course of radiation-induced apoptosis was seen in subependymal cells in the adult mouse brain. Radiation-induced apoptosis was preceded by an increase in nuclear p53 expression in glial cells of the spinal cord and subependymal cells of the brain. There was no evidence of radiation-induced apoptosis in the spinal cord and subependymal region of p53-/- animals. We conclude that the p53 pathway may be a mechanism through which DNA damage induces apoptosis in the adult CNS.     

Assessing DNA damage and health risk using biomarkers

Increased genomic instability has been found associated with cancer and aging. The p53 tumor suppressor protein is a major determinant of genomic instability as a regulator of cell cycle control and apoptosis in response to DNA damage. To investigate the rate of age-related mutation accumulation in the absence of p53, we crossed Trp53 null mice with transgenic mice harboring a lacZ mutational target gene. In the hybrid animals, lacZ mutation frequencies at early age (i.e. at about 2 months) were found to be the same as in the control lacZ animals. However, up until about 6 months, when the Trp53-knockout mice usually die from cancer, mutations were found to accumulate with age in the spleen, and to a lesser extent in the liver, at a more rapid rate than in the control Trp53(+/+) or Trp53(+/-), lacZ hybrid mice. Treatment of 2-3-month-old Trp53(-/-), lacZ hybrid mice with the powerful mutagen ethyl nitrosourea (ENU) resulted in a higher number of mutations induced in the liver but not in the spleen, as compared to the Trp53(+/+), lacZ mice. These results suggest that p53 is not an important determinant of gene mutation induction, either spontaneously during development or after treatment with a mutagen. The accelerated age-related accumulation of mutations in normal spleen and liver could be explained by the defect in apoptosis, which would prevent severely damaged cells from being eliminated.     

Effect of exogenous wild-type P53 on melanoma cell death pathways induced by irradiation at different linear energy transfer

Summary We investigated the effect of exogenous wild-type p53 on the radiation-induced cells apoptosis and necrosis at different levels of linear energy transfer (LET) to evaluate its mechanisms. The human melanoma cell line A375, which bears wild-type p53 gene status, was used, as well as the transfectant A375 cells (A375/p53) with adenoviral vector containing the wild-type p53 gene. We exposed these cells to X-rays and to accelerated carbon-ion (C-) beams. Cellular sensitivities were determined by using clonogenic assay. Apoptotic and necrotic cell deaths were determined morphologically by dual staining (acridine orange and ethidium bromide) using fluorescence microscopy. We discovered that (1) there was no significant difference in survival fraction between A375 cells and A375/p53 cells irradiated by C-beams with greater than 32 KeV/μm LET, (2) although apoptosis in the two kinds of cells increased in an LET-dependent manner, exogenous wild-type P53 induced cell apoptosis efficiently in A375/p53 relative to A375 cells with X-rays or high-LET irradiation, and (3) by high-LET irradiation, the number of necrosis in A375 cells increased significantly (P<0.05) in comparison with A375/p53 cells. These results indicate that in high-LET irradiation apoptosis induction is p53 dependent partly and exogenous wild-type P53 plays an important role in modulating cell death type, although there was no significant difference in cellular radiosensitivities. Our observation in the study offers the potential application of high-LET radiation combined with p53 in the management of human patients with melanoma.     

Effect of low-level radiation on the death of male germ cells

Hormetic and adaptive responses induced by low-level radiation in hematopoietic and immune systems have been observed, as shown by stimulatory effects on cell growth and resistance to subsequent radiation-induced cytogenetic damage. However, in terms of cell death by apoptosis, the effects of low-level radiation are controversial: Some studies showed decreased apoptosis in response to low-level radiation while others showed increased apoptosis. This controversy may be related to the radiation doses or dose rates and also, more importantly, to the cell types. Testes are one of the most radiosensitive organs. The loss of male germ cells after exposure to ionizing radiation has been attributed to apoptosis. In the present study, the effects of low-level radiation at doses up to 200 mGy on mouse male germ cells in terms of apoptosis and the expression of apoptosis-related proteins were examined at different times after whole-body exposure of mice to low-level radiation. In addition, the effect of pre-exposure to low-level radiation on subsequent cell death induced by high doses of radiation was examined to explore the possibility of low-level radiation-induced adaptive response. The results showed that low-level radiation in the dose range of 25-200 mGy induced significant increases in apoptosis in both spermatogonia and spermatocytes, with the maximal effect at 75 mGy. The increased apoptosis is most likely associated with Trp53 protein expression. Furthermore, 75 mGy low-level radiation given pre-irradiation led to an adaptive response of seminiferous germ cells to subsequent high-level radiation-induced apoptosis. These results suggest that low-level radiation induces increased apoptosis in male germ cells but also induces a significant adaptive response that decreases cell death after a subsequent high-dose irradiation.