NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2-7 of the Np95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light, N-methyl-N -nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95 ؉/؉ ) or heterozygously inactivated (Np95 ؉/؊ ) cells. Expression of transfected Np95 cDNA in Np95-null cells restored the resistance to xrays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95 ؉/؊ cells, although slightly below that of wild type (Np95 ؉/؉ ) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher for Np95-null cells than for Np95 ؉/؉ cells or Np95 ؉/؊ cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability.Entry into and progression through the mammalian cell cycle are highly regulated processes that involve a number of positively and negatively acting proteins at the molecular level. When growing cells are exposed to DNA damage or DNA replication blocks, they arrest their cell cycle processes, apparently to allow time for repair to be completed satisfactorily. This arrest is part of a "checkpoint" function that monitors the physical state of DNA at different stages of the cycle. Cells that have lost a checkpoint control may be as DNA damage-sensitive as cells that have lost DNA repair capability. Many genes are involved in controlling the cell cycle and determining checkpoints (1-3). Furthermore, a possible link between checkpoint failure, hypersensitivity to DNA damage or replication blockage, and genomic instability has provided an important insight into processes contributing to the cellular dysfunction that leads to cancer (4).We previously produced a monoclonal antibody, Th-10a mAb, 1 that recognizes a 95-kDa mouse nuclear protein (NP95). NP95 was detected by the Th-10a mAb, specifically in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells showed a constantly high level for NP95 accumulation irrespective of cell stages during the cell cycle (5). By immunoscreening a gt-11 cDNA expression library with the Th-10a mAb, we isolated the cDNA encoding NP95 (6). Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame consisting of 782 amino acids. The open reading frame contains an unusual N-terminal domain that bears a striking resemblance to ubiqutin, a leucine zipper motif, a zinc ...

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Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a human cytidine deaminase, is a potent inhibitor of HIV replication. To explore a possible role of this protein in modulating in vivo susceptibility to HIV infection, we analyzed APOBEC3G expression in HIV-exposed seronegative individuals, HIV-seropositive patients, and healthy control subjects. The results showed that the expression of APOBEC3G is significantly increased in peripheral blood mononuclear cells (PBMCs)--mainly CD14(+) cells--and in cervical tissues of HIV-exposed seronegative individuals. Higher APOBEC3G expression correlated with a reduced susceptibility of PBMCs to in vitro infection with the HIV-1(Ba-L) R5 strain. APOBEC3G could be important in modulating in vivo susceptibility to sexually transmitted HIV infection.

ABSTRACT. Expression of novel NP95 (nuclear protein, 95 kDa), which contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-cyclin-dependent protein kinase 2 phosphorylation site, and retinoblastoma protein-binding motifs, is associated with S-phase progression of mouse cells. It is suppressed during G1 and G2/M phases in normal thymocytes but expressed at a constantly high level irrespective of cell stage in mouse T cell lymphoma cells. NP95 was shown previously to be expressed strongly only in proliferative tissues and cells. In this immunohistochemical study, we demonstrate that NP95 is localized in S-phase nuclei as dot-like foci. Double immunostaining of NP95 and proliferating cell nuclear antigen (PCNA) showed that NP95 was co-localized with PCNA. Construction of three-dimensional images indicated that NP95 was localized with PCNA in replication sites in a somewhat distinct temporal manner. During meiosis, NP95 was present not only in proliferating spermatogonia but also in meiotic spermatocytes and differentiating spermatids which were not proliferating. The possible role of NP95 in mitotic and meiotic cells is discussed.Key words: PCNA/foci/cell cycle/DNA replication/mitotic cell/meiotic cell The transition from G1 phase to S phase in the eukaryotic cell cycle involves the synthesis of both regulatory proteins and proteins directly associated with the DNA replication machinery. Oncogenic processes exert their greatest effect by targeting particular regulators of G1 or G2 phase progression (Hunter, 1997;Sherr, 1996). Novel NP95 (nuclear protein, 95 kDa) is expressed during S phase progression but is suppressed during G1 and G2/M phases in normal mouse thymocytes. In mouse T cell lymphoma, however, NP95 is expressed at high levels irrespective of cell stage (Muto et al., 1995). NP95 is expressed strongly in the testis, spleen, thymus, and lung but not in the brain, liver, or skeletal muscle which suggests a role in normal cell-cycle progression. Cloning and sequencing revealed that NP95 is a novel nuclear protein of 782 amino acids and contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-CDK2 phosphorylation site, and RB-binding motifs (Fujimori et al., 1998). In lymphoma cells, aberrant expression during the cell cycle indicates that NP95 is also associated with uncontrolled cell proliferation, but its function is as yet unknown (Muto et al., 1995). In this study, we examined the intracellular distribution of NP95 and found that it localizes in S-phase nuclei as dot-like foci. To elucidate the possible role of NP95 in both cultured somatic cells and spermatogenic cells in testis, spatial and temporal expression patterns were examined by immunohistochemical techniques including threedimensional imaging. Materials and MethodsCell culture, synchronization, and cell cycle analysisThe mouse m5S embryonic fibroblast and Balb 3T3 cell lines were kindly provided by Dr. M. S. Sasaki

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