Real-time in vivo imaging of p16Ink4a reveals cross talk with p53

Expression of the p16^Ink4a tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16^Ink4a expression is also induced by tissue culture stress, physiological mechanisms regulating p16^Ink4a expression remain unclear. To eliminate any potential problems arising from tissue culture–imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16^Ink4a expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16^Ink4a expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16^Ink4a response in check. These results unveil a backup tumor suppressor role for p16^Ink4a in the event of p53 inactivation, expanding our understanding of how p16^Ink4a expression is regulated in vivo.     

A Novel RNA-Binding Protein,Ossa/C9orf10, Regulates Activity of Src Kinases To Protect Cells from Oxidative Stress-Induced Apoptosis

During the process of tumor progression and clinical treatments, tumor cells are exposed to oxidative stress. Tumor cells are frequently resistant to such stress by producing antiapoptotic signaling, including activation of Src family kinases (SFKs), although the molecular mechanism is not clear. In an attempt to identify the SFK-binding proteins selectively phosphorylated in gastric scirrhous carcinoma, we identified an uncharacterized protein, C9orf10. Here we report that C9orf10 (designated Ossa for oxidative stress-associated Src activator) is a novel RNA-binding protein that guards cancer cells from oxidative stress-induced apoptosis by activation of SFKs. Exposure to oxidative stress such as UV irradiation induces the association of Ossa/C9orf10 with regulatory domains of SFKs, which activates these kinases and causes marked tyrosine phosphorylation of C9orf10 in turn. Tyrosine-phosphorylated Ossa recruits p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase) and behaves as a scaffolding protein for PI3-kinase and SFKs, which activates the Akt-mediated antiapoptotic pathway. On the other hand, the carboxyl terminus of Ossa has a distinct function that directly binds RNAs such as insulin-like growth factor II (IGF-II) mRNA and promotes the extracellular secretion of IGF-II. Our findings indicate that Ossa is a dual-functional protein and might be a novel therapeutic target which modulates the sensitivity of tumors to oxidative stress.Tumor cells are exposed to oxidative stress in various situations in vivo. Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H₂O₂) are generated by exposure of cancer cells to hypoxia, followed by reperfusion; radiotherapy; photodynamic therapy; and some chemotherapeutic agents such as cisplatin (6, 25). This production of ROS generally induces apoptosis, whereas some tumor cells become resistant to this kind of apoptosis by some mechanism such as elevated expression of antioxidant thiols in the cells (17, 27).Src family kinases (SFKs) play important roles in various cell functions such as cell proliferation, cell adhesion, and cell migration (26), and the activities of SFKs often correlate with the malignant potential of cancer and a poor prognosis (37). Activation of c-Src is observed after the cells are exposed to oxidative stress (1, 9, 11, 34), and the activation of SFKs contributes to the resistance to apoptosis induced upon cellular stress. For instance, treatment of cells with oxidative stress such as UV irradiation or H₂O₂ causes apoptotic cell death, which is rescued by expression of v-Src (28). In our attempt to identify the key molecules that promote the expansion of gastric scirrhous carcinoma in vivo by mediating signals from activated SFKs, we identified an uncharacterized protein, C9orf10.C9orf10 (Homo sapiens chromosome 9 open reading frame 10) was originally found by the human genome sequence project as an annotated protein, and the gene was mapped to chromosome 9q22.31 (12). C9orf10 was recently detected within the Purα-containing mRNA-protein complex in the brain, although no functional information about this protein is available (14). We show that C9orf10 protects cells from apoptosis through activation of SFKs in response to oxidative stress. The kinase activity of SFKs is regulated by two intramolecular interactions. The inactive form is achieved by interaction of the SH2 domain with the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the linker region between the SH2 domain and the catalytic domain (30). C9orf10 functions as a novel activator of SFKs that unfolds the inactive form of SFKs by association with both the SH2 and SH3 domains of SFKs. Tyrosine phosphorylation of C9orf10 is induced by the activated SFKs in turn, producing scaffolds to recruit phosphatidylinositol 3-kinase (PI3-kinase) and activate PI3-kinase-Akt signaling, which plays a key role in protecting cancer cells from oxidative stress-induced apoptosis. Therefore, we named C9orf10 Ossa (oxidative stress-associated Src activator).We also showed that the carboxyl terminus of Ossa directly binds to RNA, suggesting a distinct role for Ossa as an RNA-binding protein. As one of the target RNAs, Ossa directly binds to insulin-like growth factor II (IGF-II) mRNA, which subsequently enhances the extracellular secretion of IGF-II. Because an increase in IGF-II promotes cell proliferation, the RNA-binding function of Ossa may also contribute to the survival of cancer cells in vivo.Scirrhous gastric carcinoma diffusely infiltrates a broad region of the stomach and is frequently associated with metastasis to lymph nodes and peritoneal dissemination and therefore has the worst prognosis among the various types of gastric cancer (35). Blocking of the survival signaling mediated by Ossa, which sensitizes the cancer cells to stress-induced apoptosis, may be a novel therapeutic approach for gastric scirrhous carcinoma cells.     

Isolation and Characterization of Patient-derived, Toxic, High Mass Amyloid ��-Protein (A��) Assembly from Alzheimer Disease Brains

Amyloid β-protein (Aβ) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, Aβ assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10–15-nm spherical Aβ assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (>100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immunospecificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, Aβ-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other Aβ assemblies.     

Autonomous turning of cerebellar granule cells in vitro by intrinsic programs

External guidance cues play a role in controlling neuronal cell turning in the developing brain, but little is known about whether intrinsic programs are also involved in controlling the turning. In this study, we examined whether granule cells undergo autonomous changes in the direction of migration in the microexplant cultures of the early postnatal mouse cerebellum. We found that granule cells exhibit spontaneous and periodical turning without cell-cell contact and in the absence of external guidance cues. The frequency of turning was increased by stimulating the Ca²⁺ influx and the internal Ca²⁺ release, or inhibiting the cAMP signaling pathway, while the frequency was reduced by inhibiting the Ca²⁺ influx. Granule cell turning in vitro was classified into four distinct modes, which were characterized by the morphological changes in the leading process and the trailing process, such as bifurcating, turning, withdrawing, and changing the polarity. The occurrence of the 1st and 2nd modes of turning was differentially affected by altering the Ca²⁺ and cAMP signaling pathways. Collectively, the results demonstrate that intrinsic programs regulate the autonomous turning of cerebellar granule cells in vitro. Furthermore, the results suggest that extrinsic signals play a role as essential modulators of intrinsic programs.     

Membrane fusion process and assembly of cell wall during cytokinesis in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae)

During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.     

High lib mRNA expression in breast carcinomas.

Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.     

Dietary resistant starch reduces levels of glucose-dependent insulinotropic polypeptide mRNA along the jejunum-ileum in both normal and type 2 diabetic rats

It has been reported that the circulating glucose-dependent insulinotropic polypeptide (GIP) levels were reduced by an intake of some foods/drugs capable of delaying carbohydrate digestion/absorption. In this study, we revealed that feeding rats with dietary resistant starch reduced the GIP mRNA levels along the entire length of the jejunoileum in both Wistar and type 2 diabetic GK rats.     

Ciliates use both variant and universal genetic codes: evidence of omnipotent eRF1s in the class Litostomatea

Stop codon reassignments have occurred very frequently in ciliates. In some ciliate species, the universal stop codons UAA and UAG are translated into glutamine, while in some other species, the universal stop codon UGA appears to be translated into cysteine or tryptophan. The class Litostomatea has been hypothesized to be the only group of ciliates using the universal genetic code. However, the hypothesis was based on a statistical analysis of quite small sequence dataset which was insufficient to elucidate the codon usage of the class among such highly deviated phylum. In this study, together with the updated database sequence analysis for the class, we approached the problem of stop codon usage by examining the capacity of the translation termination factor eRF1 for recognizing stop codons. Using in vivo assay systems in budding yeast, we estimated the activity of eRF1 from two litostome species Didinium nasutum and Dileptus margaritifer. The results clearly showed that Didinium and Dileptus eRF1s efficiently recognize all three stop codons. This is the first experimental evidence that strongly supports the hypothesis that litostome ciliates use universal genetic code.