Direct detection of p53 -/- thymocyte appearing at an early stage of radiation-induced thymic lymphomagenesis in p53 +/- heterozygous B10 mice.

PURPOSE: The appearance of tumor suppressor protein 53 (p53) -/- thymocytes at an early stage of radiation-induced lymphomagenesis was investigated in the p53 heterozygous (+/-) B10 mice following a single dose of irradiation, since most thymic lymphomas manifested the loss of the wild-type p53 allele and the loss of heterozygosity was thought to be an early event critical for radiation-induced thymic lymphomagenesis in p53 +/- mice. MATERIALS AND METHODS: The mice were exposed to a single dose (6 Gy) of irradiation to induce thymic lymphomas and, at various times after irradiation, treated with an extremely high dose (30 Gy) of whole-body irradiation to enrich p53 -/- thymocytes and, 24 h later, the remaining thymocytes were assayed for cell surface markers and p53 genotype. RESULTS: In a significant fraction of the p53 +/- mice 5 weeks after 6 Gy irradiation, there was a relative increase in the number of cluster of differentiation (CD) 4+CD8+ thymocyte subpopulation among thymocytes remaining after 30 Gy irradiation. The CD4+CD8+ double-positive (DP) thymocytes were shown to contain p53-/- cells, and the number of p53 -/- thymocytes was more than 10(5) in those individuals. CONCLUSIONS: The results clearly indicated that an extremely high dose (30 Gy) of whole-body irradiation enabled us to directly detect p53 -/- thymocytes in an abundant p53 +/- thymocyte population and that proliferative p53 -/- thymocytes develop in a CD4+CD8+ DP thymocyte subpopulation within a few weeks after a single dose (6 Gy) of irradiation.

p53 (TRP53) deficiency-mediated antiapoptosis escape after 5 Gy X irradiation still induces stem cell leukemia in C3H/He mice: comparison between whole-body assay and bone marrow transplantation (BMT) assay.

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.

Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site.

Retroviral DNA integration is mediated by the viral protein integrase. However, elements of the host DNA repair machinery such as the phosphatidylinositol 3-kinase (PI-3K)-related protein kinase family system would play a role in the integration of viral DNA into the host DNA. Here, we show that a host PI-3K-related protein kinase, DNA-dependent protein kinase (DNA-PK), plays a role in the specific integration of retroviral DNA and induction of retroviral diseases in vivo. DNA-PK-deficient scid mice inoculated with Friend leukemia virus (FLV) exhibited a random integration into their genomic DNA and expressed the viral envelope protein gp70. However, the specific integration of FLV at Spi-1 or Fli-1 sites did not occur in association with the significant resistance of scid mice to FLV-induced leukemogenesis. In contrast, the knockout of another member of the PI-3K-related protein kinase family, encoded by the ataxia telangiectasia mutated (ATM) gene, resulted in mice as sensitive to FLV-induced leukemogenesis as the wild type mice. FLV was specifically integrated into the DNA at Spi-1 and Fli-1 sites with significant expression of these transcription factors. These findings indicated that DNA-PK would be essential for controlling the in vivo integration of FLV at specific sites as well as the susceptibility to FLV-induced leukemogenesis.

DNA-dependent protein kinase enhances DNA damage-induced apoptosis in association with Friend gp70.

Friend leukemia virus (FLV) infection strongly enhances gamma-irradiation-induced apoptosis of hematopoietic cells of C3H hosts leading to a lethal anemia. Experiments using p53 knockout mice with the C3H background have clarified that the apoptosis is p53-dependent and would not be associated with changes of cell populations caused by the infection with FLV. In bone marrow cells of FLV + total body irradiation (TBI)-treated C3H mice, the p53 protein was prominently activated to overexpress p21 and bax suggesting that apoptosis-enhancing mechanisms lay upstream of p53 protein in the signaling pathway. Neither of DNA-dependent protein kinase (DNA-PK)-deficient SCID mice nor ataxia telangiectasia mutated (ATM) gene knockout mice with the C3H background exhibited a remarkable enhancement of apoptosis or p53 activation on FLV + TBI-treatment indicating that DNA-PK and ATM were both essential. ATM appeared necessary for introducing DNA damage-induced apoptosis, while DNA-PK enhanced p53-dependent apoptosis under FLV-infection. Surprisingly, viral envelope protein, gp70, was co-precipitated with DNA-PK but not with ATM in FLV + TBI-treated C3H mice. These results indicated that FLV-infection enhances DNA damage-induced apoptosis via p53 activation and that DNA-PK, in association with gp70, might play critical roles in modulating the signaling pathway.

Involvement of illegitimate V(D)J recombination or microhomology-mediated nonhomologous end-joining in the formation of intragenic deletions of the Notch1 gene in mouse thymic lymphomas.

Deregulated V(D)J recombination-mediated chromosomal rearrangements are implicated in the etiology of B- and T-cell lymphomagenesis. We describe three pathways for the formation of 5'-deletions of the Notch1 gene in thymic lymphomas of wild-type or V(D)J recombination-defective severe combined immune deficiency (scid) mice. A pair of recombination signal sequence-like sequences composed of heptamer- and nonamer-like motifs separated by 12- or 23-bp spacers (12- and 23-recombination signal sequence) were present in the vicinity of the deletion breakpoints in wild-type thymic lymphomas, accompanied by palindromic or nontemplated nucleotides at the junctions. In scid thymic lymphomas, the deletions at the recombination signal sequence-like sequences occurred at a significantly lower frequency than in wild-type mice, whereas the deletions did not occur in Rag2(-/-) thymocytes. These results show that the 5'-deletions are formed by Rag-mediated V(D)J recombination machinery at cryptic recombination signal sequences in the Notch1 locus. In contrast, one third of the deletions in radiation-induced scid thymic lymphomas had microhomology at both ends, indicating that in the absence of DNA-dependent protein kinase-dependent nonhomologous end-joining, the microhomology-mediated nonhomologous end-joining pathway functions as the main mechanism to produce deletions. Furthermore, the deletions were induced via a coupled pathway between Rag-mediated cleavage at a cryptic recombination signal sequence and microhomology-mediated end-joining in radiation-induced scid thymic lymphomas. As the deletions at cryptic recombination signal sequences occur spontaneously, microhomology-mediated pathways might participate mainly in radiation-induced lymphomagenesis. Recombination signal sequence-mediated deletions were present clonally in the thymocyte population, suggesting that thymocytes with a 5'-deletion of the Notch1 gene have a growth advantage and are involved in lymphomagenesis.

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice.

Gamma ray-radiation induced significant apoptosis in peritoneal resident macrophages (PRMs) of C3H/HeJ (C3H) mice, but not in other strains of mice. To investigate the role of DNA damage in the apoptosis, DNA damage was quantified in PRMs by use of the alkaline single-cell gel electrophoresis (Comet) assay. No significant difference was found between C3H and C57Black/6 mice in either radiation-induced DNA damage or repair. Radiation induced apoptosis at the same levels in PRMs of p53 knockout mice and atm knockout mice as those of wild-type C3H mice; however radiation-induced apoptosis was significantly less extensive in the thymocytes of these mutant mice than in those of wild-type mice. Apoptosis was also induced at the same level by an irradiation in PRMs of C3H scid mice as in those of wild-type C3H mice. Therefore it was suggested that radiation-induced DNA damage and TP53, ATM, or DNA-PK-mediated cellular responses occurring downstream thereof were not involved in the radiation-induced apoptotic cell death in C3H mouse PRMs.

Adaptive response in embryogenesis: V. Existence of two efficient dose-rate ranges for 0.3 Gy of priming irradiation to adapt mouse fetuses.

The adaptive response is an important phenomenon in radiobiology. A study of the conditions essential for the induction of an adaptive response is of critical importance to understanding the novel biological defense mechanisms against the hazardous effects of radiation. In our previous studies, the specific dose and timing of radiation for induction of an adaptive response were studied in ICR mouse fetuses. We found that exposure of the fetuses on embryonic day 11 to a priming dose of 0.3 Gy significantly suppressed prenatal death and malformation induced by a challenging dose of radiation on embryonic day 12. Since a significant dose-rate effect has been observed in a variety of radiobiological phenomena, the effect of dose rate on the effectiveness of induction of an adaptive response by a priming dose of 0.3 Gy administered to fetuses on embryonic day 11 was investigated over the range from 0.06 to 5.0 Gy/min. The occurrence of apoptosis in limb buds, incidences of prenatal death and digital defects, and postnatal mortality induced by a challenging dose of 3.5 Gy given at 1.8 Gy/min to the fetuses on embryonic day 12 were the biological end points examined. Unexpectedly, effective induction of an adaptive response was observed within two dose-rate ranges for the same dose of priming radiation, from 0.18 to 0.98 Gy/ min and from 3.5 to 4.6 Gy/min, for reduction of the detrimental effect induced by a challenging dose of 3.5 Gy. In contrast, when the priming irradiation was delivered at a dose rate outside these two ranges, no protective effect was observed, and at some dose rates elevation of detrimental effects was observed. In general, neither a normal nor a reverse dose- rate effect was found in the dose-rate range tested. These results clearly indicated that the dose rate at which the priming irradiation was delivered played a crucial role in the induction of an adaptive response. This paper provides the first evidence for the existence of two dose-rate ranges for the same dose of priming radiation to successfully induce an adaptive response in mouse fetuses.

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

Stem-cell leukemia: p53 deficiency mediated suppression of leukemic differentiation in C3H/He myeloid leukemia.

C3H/He mice produce myeloid leukemias after whole body irradiation of 1-3Gy as compared with non-irradiated controls that produce fewer than 1% of leukemia [Radiatiton Research 127 (1991) 146]. Thus, p53-deficient C57BL/6 strain, a malignant lymphoma prone, was crossed back into C3H/He strain. Lethally irradiated wild-type mice to which p53-deficient bone marrow cells were transplanted (transplantation assay) showed dramatic change in the propensity of leukemia of myeloid lineages, the cells lacking CD3, Thy1.2, sIgM, B220, Mac-1, Gr-1, but being positive for c-Kit and CD44. Furthermore, transplanted mice subjected to 3Gy irradiation gave rise to a faster development of leukemia and a higher frequency of double-lineage leukemias than the non-irradiated control.

Putative tumor-suppressor gene regions responsible for radiation lymphomagenesis in F1 mice with different p53 status.

Regions of allelic loss on chromosomes in many tumors of human and some experimental animals are generally considered to harbor tumor-suppressor genes involved in tumorigenesis. Allelotype analyses have greatly improved our understanding of the molecular mechanism of radiation lymphomagenesis. Previously, we and others found frequent loss of heterozygosity (LOH) on chromosomes 4, 11, 12, 16 and 19 in radiation-induced lymphomas from several F1 hybrid mice. To examine possible contributions of individual tumor-suppressor genes to tumorigenesis in p53 heterozygous deficiency, we investigated the genome-wide distribution and status of LOH in radiation-induced lymphomas from F1 mice with different p53 status. In this study, we found frequent LOH (more than 20%) on chromosomes 4 and 12 and on chromosomes 11, 12, 16 and 19 in radiation-induced lymphomas from (STS/A X MSM/Ms)F1 mice and (STS/A X MSM/Ms)F1-p53KO/+ mice, respectively. Low incidences of LOH (10-20%) were also observed on chromosomes 11 in mice with wild-type p53, and chromosomes 1, 2, 9, 17 and X in p53 heterozygous-deficient mice. The frequency of LOH on chromosomes 9 and 11 increased in the (STS/A X MSM/Ms)F1-p53KO/+ mice. Preferential losses of the STS-derived allele on chromosome 9 and wild-type p53 allele on chromosome 11 were also found in the p53 heterozygous-deficient mice. Thus, the putative tumor-suppressor gene regions responsible for lymphomaganesis might considerably differ due to the p53 status.

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.

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.