Kazunori Shiraishi

p53-Dependent S-Phase Damage Checkpoint and Pronuclear Cross Talk in Mouse Zygotes with X-Irradiated Sperm

ABSTRACT One difficulty in analyzing the damage response is that the effect of damage itself and that of cellular response are hard to distinguish in irradiated cells. In mouse zygotes, damage can be introduced by irradiated sperm, while damage response can be studied in the unirradiated maternal pronucleus. We have analyzed the p53-dependent damage responses in irradiated-sperm mouse zygotes and found that a p53-responsive reporter was efficiently activated in the female pronucleus. [3H]thymidine labeling experiments indicated that irradiated-sperm zygotes were devoid of G1/S arrest, but pronuclear DNA synthesis was suppressed equally in male and female pronuclei. p53−/− zygotes lacked this suppression, which was corrected by microinjection of glutathione S-transferase-p53 fusion protein. In contrast, p21−/− zygotes exhibited the same level of suppression upon fertilization by irradiated sperm. About a half of the 6-Gy-irradiated-sperm zygotes managed to synthesize a full DNA content by prolonging S phase, while the other half failed to do so. Regardless of the DNA content, all the zygotes cleaved to become two-cell-stage embryos. These results revealed the presence of p53-dependent pronuclear cross talk and a novel function of p53 in the S-phase DNA damage checkpoint of mouse zygotes.

Persistent Induction of Somatic Reversions of the Pink-Eyed Unstable Mutation in F1 Mice Born to Fathers Irradiated at the Spermatozoa Stage

Abstract Shiraishi, K., Shimura, T., Taga, M., Uematsu, N., Gondo, Y., Ohtaki, M., Kominami, R. and Niwa, O. Persistent Induction of Somatic Reversions of the Pink-Eyed Unstable Mutation in F1 Mice Born to Fathers Irradiated at the Spermatozoa Stage. Radiat. Res. 157, 661–667 (2002). Untargeted mutation and delayed mutation are features of radiation-induced genomic instability and have been studied extensively in tissue culture cells. The mouse pink-eyed unstable (pun) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts back to the wild type in germ cells as well as in somatic cells. The reversion event can be detected in the retinal pigment epithelium as a cluster of pigmented cells (eye spot). We have investigated the reversion pum in F1 mice born to irradiated males. Spermatogonia-stage irradiation did not affect the frequency of the reversion in F1 mice. However, 6 Gy irradiation at the spermatozoa stage resulted in an approximately twofold increase in the number of eye spots in the retinal pigment epithelium of F1 mice. Somatic reversion occurred for the paternally derived pun alleles. In addition, the reversion also occurred for the maternally derived, unirradiated pun alleles at a frequency equal to that for the paternally derived allele. Detailed analyses of the number of pigmented cells per eye spot indicated that the frequency of reversion was persistently elevated during the proliferation cycle of the cells in the retinal pigment epithelium when the male parents were irradiated at the spermatozoa stage. The present study demonstrates the presence of a long-lasting memory of DNA damage and the persistent up-regulation of recombinogenic activity in the retinal pigment epithelium of the developing fetus.

p21 provides stage specific DNA damage control to preimplantation embryos

The early stage embryogenesis of higher eukaryotes lacks some of the damage response pathways such as G1/S checkpoint, G2/M checkpoint and apoptosis. We examined here the damage response of preimplantation stage embryos after fertilization with 6 Gy irradiated sperm. Sperm-irradiated embryos developed normally for the first 2.5 days, but started to exhibit a developmental delay at day 3.5. p21 was activated in the delayed embryos, which carried numerous micronuclei owing to delayed chromosome instability. Apoptosis was observed predominantly in the inner cell mass of the day 4.0 embryos. Sperm-irradiated p21−/− embryos lacked the delay, but chromosome instability and apoptosis were more pronounced than the corresponding p21 wild-type embryos. We conclude from the result that damage responses come in a stage-specific manner during preimplantation stage development; p53-dependent S checkpoint at the zygote stage, p21-mediated cell cycle arrest at the morula/blastocyst stages and apoptosis after the blastocyst stage in the inner cell mass.

The Novel Surveillance Mechanism of the Trp53-Dependent S-Phase Checkpoint Ensures Chromosome Damage Repair and Preimplantation-Stage Development of Mouse Embryos Fertilized with X-Irradiated Sperm

Abstract Shimura, T., Toyoshima, M., Taga, M., Shiraishi, K., Uematsu, N., Inoue, M. and Niwa, O. The Novel Surveillance Mechanism of the Trp53-Dependent S-Phase Checkpoint Ensures Chromosome Damage Repair and Preimplantation-Stage Development of Mouse Embryos Fertilized with X-Irradiated Sperm. Radiat. Res. 158, 735–742 (2002). Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220–2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53+/+ genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53−/− embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53−/− embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.

Transcription-Independent Suppression of DNA Synthesis by p53 in Sperm-Irradiated Mouse zygotes

Cell cycle arrest in response to DNA damage is important for the maintenance of genomic integrity in higher eukaryotes. We have previously reported the novel p53-dependent S-phase checkpoint operating in mouse zygotes fertilized with irradiated sperm. In the present study, we analysed the detail of the p53 function required for this S-phase checkpoint in mouse zygotes. The results indicate that ATM kinase is likely to be indispensable for the p53-dependent S-phase checkpoint since the suppression was abrogated by inhibitors such as caffeine and wortmannin. However, ATM phosphorylation site mutant proteins were still capable of suppressing DNA synthesis when microinjected into sperm-irradiated zygotes lacking the functional p53, suggesting that the target of the phosphorylation is not p53. In addition, the suppression was not affected by α-amanitin, and p53 protein mutated at the transcriptional activation domain was also functional in the suppression of DNA synthesis. However, p53 proteins mutated at the DNA-binding domain were devoid of the suppressing activity. Taken together, the transcription-independent function of p53 associated with the DNA-binding domain is involved in the S-phase checkpoint in collaboration with yet another unidentified target protein(s).

Increased frequencies of gene and chromosome mutations after X-irradiation in mouse embryonal carcinoma cells transfected with the bcl-2 gene.

Preimplantation stage mouse embryos are known to be highly sensitive to the killing effect of DNA damaging agents such as radiation. Interestingly, however, this stage of development is well protected from radiation induction of malformation and carcinogenesis in postnatal life. In recent years, it has become clear that the stem cells of preimplantation stage embryos undergo extensive apoptosis after DNA damage. It has been postulated that this apoptosis is likely to be responsible for the resistance to malformation, by excluding cells carrying deleterious DNA damage. We have tested the possible role of apoptosis in elimination of gene and chromosome mutations in undifferentiated mouse embryonal carcinoma cell line, F9, transfected with human bcl‐2 cDNA. The colony radiosensitivity of F9 cells was not affected by overexpression of the bcl‐2 gene, but the apoptotic cell death was suppressed, as examined by DNA ladder assay and Hoechst staining. This suppression was accompanied by an increase in the frequencies of hprt mutation and micronucleus formation after X‐irradiation. These results support the idea that maintenance of genomic integrity during early development is likely to be executed by apoptotic elimination of cells at risk.