Regulation of FE65 nuclear translocation and function by amyloid beta-protein precursor in osmotically stressed cells

FE65, a neural adaptor protein, interacts with amyloid beta-protein precursor (APP) and is known to regulate amyloid beta generation from APP. FE65 also associates with nuclear proteins; however, its physiological function in the nucleus remains unclear. A fixed population of cytoplasmic FE65 is tethered to membranes by binding APP. This membrane-tethered FE65 is liberated from membranes by APP phosphorylation, which is facilitated by a stress-activated protein kinase in sorbitol-treated cells. Here we show that liberated FE65, which is distinct from "virgin" FE65 in the cytoplasm, translocates into the nucleus and accumulates in the nuclear matrix forming a patched structure. Targeting of FE65 into the nuclear matrix was suppressed by the APP intracellular domain fragment, which is generated by consecutive cleavages of APP. Thus, nuclear translocation of FE65 is under the regulation of APP. In the nucleus, FE65 induced gammaH2AX, which plays an important role in DNA repair as a cellular response by stress-damaged cells. These observations suggest that APP-regulated FE65 plays an important role in the early stress response of cells and that FE65 deregulated from APP induces apoptosis.     

Cell-killing efficiency and number of platinum atoms binding to DNA,RNA, and protein molecules of hela cells treated with combinations of hyperthermia and cisdiamine (glycolato)platinum(II)

HeLa S-3 cells were treated with^195mPt-radiolabeledcisdiamine(glylato)platinum(II) (254-S) for 60 min at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentration (Do) of 254-S for a 60-min treatment at 0‡C, 25‡C, 37‡C, 40‡C, 42‡C, and 44‡C was 233,132, 61.1, 42.7, 25.6, and 9.9 ΜM, respectively. By using identically treated cells, the numbers of Pt atoms combined with DNA, RNA, and protein molecules were determined in the subcellular fractions. Thus, the D₀ values given as drug concentrations were replaced with the number of Pt atoms combined in each fraction. Then, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt atoms combined and was calculated for each molecule. The efficiency for the DNA molecule was 0.61X10⁴, 1.09xl0⁴, 1.88xl0⁴, 1.90xl0⁴, 2.66xl0⁴, and 5.88xl0⁴ nucleotides, respectively, for the conditions described. From 0‡C to 44‡C, the cell-killing efficiency of Pt atoms increased by a factor of 9.6.     

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Proteinase-activated receptors (PARs; PAR_1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR₄, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR₄ stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.

Methods

EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).

Results

Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR₄ agonist peptide (AYPGKF-NH₂, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR₄ stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR₄-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR₄-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR₄-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR₄ stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.

Conclusion

PAR₄ stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.     

Studies on Microbial Metabolism of Sugar Nucleotides

The fermentative production of uridine diphosphate N-acetylglucosamine (UDPAG) from 5′-UMP and glucosamine by dried cells of baker’s yeast was studied. UDPAG was found to accumulate in a reaction system containing 5′-UMP, glucosamine, fructose, inorganic phosphate and magnesium ions with dried baker’s yeast as an enzyme source. UDPAG was separated from the reaction mixture by means of anion exchange column chromatography and was identified by several biochemical methods.

The reaction conditions for the fermentative production of UDPAG were examined. The yield of UDPAG was about 40~66% based on 5′-UMP added when fermentation conditions were optimized. The concentration of glucosamine and potassium phosphate buffer, and pH as well as the water content of dried cells greatly affected the formation of UDPAG.     

Muc4 is required for activation of ErbB2 in signet ring carcinoma cell lines

Signet-ring cell carcinoma is one of the most malignant tumors, classified histologically as a poorly differentiated adenocarcinoma. The ErbB2/ErbB3 complex is often constitutively activated, which suggests that the ErbB2/ErbB3 signaling pathway may be important for malignancy of this tumor. However, the mechanism underlying this activation has not been understood. Here, we show that ErbB2 and Muc4 bind in signet ring carcinoma cells, which was not seen in highly differentiated adenocarcinoma cell lines. ErbB3 was suggested to be a substrate of ErbB2 because knockdown of ErbB2 resulted in less phosphorylation of ErbB3. Inhibition of expression of Muc4 at the cell surface by the treatment of the cells with benzyl-GalNac, an inhibitor of mucin secretion, blocked phosphorylation of ErbB3, suggesting that activity of ErbB2 depends on the expression of Muc4. These results supply the biochemical backgrounds in recent studies suggesting the contribution of Muc4 in the tumorigenesis.     

Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biological properties of mutant TK15 and mutant-induced transformants

A mutant derived from a temperature-sensitive mutant of Rous sarcoma virus ( tsNY68 ) which showed extremely low infectivity was characterized. Infection of chicken embryo fibroblast cells with the mutant, TK15 , induced two types of transformants, mutant-producing 15c (+) and nonvirus -producing 15c (-) transformants. 15c (+) cells expressed all four viral genes normally and produced a normal level of virus particles. No complementation was observed between the mutant and avian leukosis viruses. However, when 15c (+) cells were cocultured with nonvirus -producing cells transformed by Y73, a replication-defective avian sarcoma virus, a high titer of Y73 virus was recovered. From its biological properties, the mutant seemed to have a defect(s) outside the viral genes. Biochemical analysis of the TK15 mutant (T. Koyama , F. Harada, and S. Kawai , J. Virol. 51:154-162, 1984) revealed that it had a defect in packaging its own genomic RNA. During replication of TK15 virus, the TK15 mutant appeared to segregate at high frequency more defective variants that induced 15c (-) transformants, in most of which only the src gene was expressed. The mechanism for the segregation of 15c (-) transformants is discussed with respect to the defect of the mutant.     

Establishment and characterization of novel patient-derived osteosarcoma xenograft and cell line

Osteosarcoma is an aggressive mesenchymal malignancy of the bone. Patient-derived models are essential tools for elucidating the molecular mechanisms associated with poor prognosis and the development of novel anticancer drugs. This study described the establishment of a patient-derived cancer model of osteosarcoma. Primary osteosarcoma tumor tissues were obtained from an osteosarcoma patient and inoculated in the skin of immunodeficient mice, followed by transplantation to other mice upon growth. Cells were maintained in monolayer cultures, and the capability of spheroid formation was assessed by seeding the cells on culture dishes. The invasion ability of cells was monitored by Matrigel assay, and genomic and proteomic backgrounds were examined by mass spectrometry. A cell line was established from patient-derived tumors and showed similar histology to that of the primary tumor tissue. Additionally, these cells formed spheroids on low-attachment tissue-culture dishes and exhibited invasive capabilities, and we confirmed that the genomic backgrounds were similar between patient-derived xenograft tumors and the cell line. Furthermore, the proteome of the patient-derived tumors and the cells exhibited similar, but not identical, patterns to that of the original tumor tissue. Our results indicated that this patient-derived xenograft model and cell line would be useful resources for osteosarcoma research.     

beta-Adrenergic receptors in rabbit liver plasma membranes. Predominance of beta 2-receptors and mediation of adrenergic regulation of hepatic glycogenolysis

[3H]Dihydroalprenolol was used to study beta-adrenergic binding sites in plasma membranes isolated from rabbit liver. Specific binding was measured at 25 degrees C as the difference between total binding and binding in the presence of 2 microM dl-propranolol or 10 microM l-isoproterenol. Binding was saturable and stereoselective. The maximum number of binding sites (Bmax) was 434 +/- 41 fmol/mg of protein. The Kd for this binding as determined by Scatchard analysis was 1.39 +/- 0.09 nM. This value agreed well with the Kd value (1.27 +/- 0.12 nM) determined by kinetic analysis. The potency order for the displacement of bound [3H]dihydroalprenolol was isoproterenol greater than epinephrine greater than norepinephrine, indicative of beta 2-receptors. Use of beta 1- and beta 2-subtype-selective inhibitors also supported the interpretation that the binding characteristics are those of beta 2-receptors. Computer-aided analysis of this inhibition indicated that the beta-receptors in this membrane are predominantly, if not exclusively, of the beta 2-subtype. That these receptors are responsible for mediating catecholamine stimulation of hepatic glycogenolysis was deduced from the inhibition of agonist-stimulated glycogenolysis, in isolated hepatocytes, by beta-receptor subtype-selective antagonists. Thus, the hydrochloride of (t-butylamino-3-ol-2-propyl)oximino-9 fluorene, a beta-antagonist which has higher affinity at beta 2-sites than at beta 1-sites, was 3 orders of magnitude more potent in inhibiting isoproterenol-stimulated glycogenolysis than either atenolol or practolol, both of which are beta 1-selective antagonists. These results resemble the inhibition of [3H]dihydroalprenolol binding in plasma membranes. The glycogenolytic effects of catecholamines occurred with the potency order isoproterenol greater than epinephrine greater than norepinephrine. Thus, both by radioligand binding studies and by metabolic studies, the functional adrenergic receptor in the rabbit liver is shown to be of the beta 2-subtype.