Masahiko Takahagi

Effect of Atm Disruption on Spontaneously Arising and Radiation-Induced Deletion Mutations in Mouse Liver

Abstract Furuno-Fukushi, I., Masumura, K., Furuse, T., Noda, Y., Takahagi, M., Saito, T., Hoki, Y., Suzuki, H., Wynshaw-Boris, A., Nohmi, T. and Tatsumi, K. Effect of Atm Disruption on Spontaneously Arising and Radiation-Induced Deletion Mutations in Mouse Liver. Radiat. Res. 160, 549–558 (2003). Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse “gpt-delta” system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi− (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi− mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8 × 10−6. The livers from Atm−/− mice yielded a virtually identical dose–response curve for X rays with a background fraction of 2.4 × 10−6. Structural analyses revealed no significant difference in the proportion of −1 frameshifts and larger deletions between Atm+/+ and Atm−/− mice, although larger deletions prevailed in X-ray-induced Spi− mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.

Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP951

Abstract Muto, M., Fujimori, A., Nenoi, M., Daino, K., Matsuda, Y., Kuroiwa, A., Kubo, E., Kanari, Y., Utsuno, M., Tsuji, H., Ukai, H., Mita, K., Takahagi, M. and Tatsumi, K. Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP95. Radiat. Res. 166, 723–733 (2006). The murine nuclear protein Np95 has been shown to underlie resistance to ionizing radiation and other DNA insults or replication arrests in embryonic stem (ES) cells. Using the databases for expressed sequenced tags and a two-step PCR procedure, we isolated human NP95, the full-length human homologue of the murine Np95 cDNA, which consists of 4,327 bp with a single open reading frame (ORF) encoding a polypeptide of 793 amino acids and 73.3% homology to Np95. The ORF of human NP95 cDNA is identical to the UHRF1 (ubiquitin-like protein containing PHD and RING domain 1). The NP95 gene, assigned to 19p13.3, consists of 18 exons, spanning 60 kb. Several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense NP95 cDNA were, like the murine Np95-knockout ES cells, more sensitive to X rays, UV light and hydroxyurea than the corresponding parental cells. In HEK293 cells, the lack of NP95 did not affect the activities of topoisomerase IIα, whose expression had been demonstrated to be regulated by the inverted CCAAT box binding protein of 90 kDa (ICBP90) that closely resembles NP95 in amino acid sequence and in cDNA but differs greatly in genomic organization. These findings collectively indicate that the human NP95 gene is the functional orthologue of the murine Np95 gene.

Aggregative organization enhances the DNA end-joining process that is mediated by DNA-dependent protein kinase.

The occurrence of DNA double‐strand breaks in the nucleus provokes in its structural organization a large‐scale alteration whose molecular basis is still mostly unclear. Here, we show that double‐strand breaks trigger preferential assembly of nucleoproteins in human cellular fractions and that they mediate the separation of large protein–DNA aggregates from aqueous solution. The interaction among the aggregative nucleoproteins presents a dynamic condition that allows the effective interaction of nucleoproteins with external molecules like free ATP and facilitates intrinsic DNA end‐joining activity. This aggregative organization is functionally coacervate‐like. The key component is DNA‐dependent protein kinase (DNA‐PK), which can be characterized as a DNA‐specific aggregation factor as well as a nuclear scaffold/matrix‐interactive factor. In the context of aggregation, the kinase activity of DNA‐PK is essential for efficient DNA end‐joining. The massive and functional concentration of nucleoproteins on DNA in vitro may represent a possible status of nuclear dynamics in vivo, which probably includes the DNA‐PK‐dependent response to multiple double‐strand breaks.

Mutagenic substances in pyrolysate obtained by burning polyvinylchloride-product at 1000°C

Abstract In order to detect possible mutagenic substances in pyrolysate obtained by burning polyvinyl chloride product (PVC-P) at approximately 1000°C, mutagenicity of rough extracts obtained by extraction with various solvents for the products was investigated by means of reversion mutation assay using Salmonella typhimurium TA98 and TA100 with or without microsomal metabolic activation (S9 mix). Strong mutagenicity in TA98 without S9 mix was observed in acetone-extract of PVC-P. The extract was fractionated into acidic, neutral and basic by liquid–liquid distribution and the mutagenicity in TA98 without S9 mix was found in the neutral fraction. Identification of mutagenic substances in the neutral fraction from acetone extract, which showed the strongest mutagenicity, was attempted by means of thin layer chromatography and capillary gas chromatography. The results suggest that mutagenic substances from pyrolysate of PVC-P are benzanthrone and an isomer of benzo( c )cinnoline. The results also suggest that burning wastes containing plastic products is not always safe even if at 1000°C and further research on the problem is necessary.