Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP95.

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 IIalpha, 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.

Effect of Atm disruption on spontaneously arising and radiation-induced deletion mutations in mouse liver.

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 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-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.

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.