Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated ß-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-ß-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

Enhanced expression of hepatocyte growth factor activator inhibitor type 2-related small peptide at the invasive front of colon cancers.

BACKGROUND: Hepatocyte growth factor activator inhibitor type 2-related small peptide (H2RSP) is a small nuclear protein abundantly expressed in the gastrointestinal epithelium. However, its functions remain unknown. AIMS: To investigate the expression and localisation of H2RSP in normal, injured and neoplastic human intestinal tissue. METHODS: Immunohistochemical examination and in situ hybridisation for H2RSP were performed using normal and diseased intestinal specimens. Its subcellular localisation and effects on the cellular proliferation and invasiveness were examined using cultured cells. RESULTS: In the normal intestine, H2RSP was observed in the nuclei of surface epithelial cells and this nuclear localisation was impaired in regenerating epithelium. In vitro, the nuclear translocation of H2RSP was observed along with increasing cellular density, and an overexpression of H2RSP resulted in a reduced growth rate and enhanced invasiveness. H2RSP expression was down regulated in well-differentiated colorectal adenocarcinomas. However, a marked up regulation of the cytoplasmic H2RSP immunoreactivity was observed in cancer cells at the invasive front. These cells showed low MIB-1 labelling, an enhanced p16 expression and nuclear beta-catenin. The number of H2RSP-positive cells in the invasive front of well-differentiated adenocarcinomas was considerably higher in the cases with lymph node metastases than in node-negative ones. CONCLUSION: In the normal intestine, the nuclear accumulation of H2RSP is a marker of differentiated epithelial cells. Although H2RSP was down regulated in colorectal adenocarcinomas, a paradoxical up regulation was observed in actively invading carcinoma cells. H2RSP immunoreactivity at the invasive front may serve as a marker of invasive phenotype of well-differentiated colon cancers.

Sonographic findings of anomalous position of the gallbladder.

Anomalous position of the gallbladder is relatively rare and has been reported only in isolated case reports. We tried to determine its ultrasound (US) findings on the basis of 18 such patients. In the left-side gallbladder group (nine patients), the gallbladder was imaged as an oval cystic mass in front of the pancreas. In all patients, the narrow neck of the gallbladder was clearly detected by US in the usual location before the main portal vein. Four of nine patients had small gallbladder stones. The retrohepatic gallbladder group (four patients) showed marked atrophy of the right lobe of the liver. Two patients had multiple gallstones in the bile ducts of the right lobe. All patients in the suprahepatic retrohepatic gallbladder group (four patients) were cirrhotic, and the anterior segment of the right lobe was markedly atrophied. In the intercostal scan, the gallbladder mimicked a perihepatic fluid. In the floating gallbladder group (one patient), the gallbladder was imaged as a cystic mass in the anterior abdominal wall. Surgical intervention showed a severely inflamed gallbladder, with small stones adhered into the anterior abdominal wall and partially ruptured. Knowledge of the wide range of US findings of malposition of the gallbladder helps in avoiding misdiagnosis.

Effects of exhaustive endurance exercise and its one-week daily repetition on neutrophil count and functional status in untrained men.

Whereas endurance exercise is known to induce marked neutrophilia, it remains to be fully understood as to whether the cell functions are altered as well as whether the adaptability of the responses to training occurs. To address both of these issues, we undertook the present longitudinal investigation in ten healthy untrained men (20-24 years, VO2max 39.1 +/- 4.2 ml/kg/min). The exercise protocol was 7 consecutive sessions of the same workload performed each day for 1.5 h at an intensity of 70% of VO2max. Peripheral blood samples were obtained before, immediately after, and 1 h after exercise on Days 1, 4, and 7, and served for determination of total and differential leukocyte counts, chemotaxis and chemiluminescence of neutrophils. Acute endurance exercise caused marked peripheral neutrophilia with significant increase in both absolute number and relative proportion of band neutrophils (p < 0.01, respectively), indicating partial recruitment of bone marrow neutrophils. While chemotaxis remained unaltered following exercise, reactive oxygen species generation of neutrophils, measured by luminol-dependent chemiluminescence upon stimulation with opsonized zymosan, was not only significantly enhanced following exercise (p < 0.01), but also associated with the proportional increase in band neutrophils (r = 0.727, p < 0.05), suggesting that neutrophils mobilized from the bone marrow following endurance exercise may possess higher responsiveness. On the other hand, the magnitude of the exercise-induced changes was reduced gradually by daily repeated exposure to endurance exercise, but none of the trends were significant except the decline in resting segmented neutrophil counts (p < 0.05) at least during a 1-wk period of repeated exercise sessions.

Gas exchange efficiency of a membrane oxygenator with use of a vibrating flow pump.

We have developed a newly designed blood pump, named the vibrating flow pump (VFP), which can generate high frequency oscillated flow. The driving frequency is 10-50 Hz, and flow volume is linearly controlled electric power (current and voltage). The VFP was applied as the pump for extracorporeal circulation (ECC) in acute animal experiments. The gas exchange efficiency of the membrane oxygenator with the VFP and a roller pump (RP) was evaluated. Under general anesthesia with halothane, 7 adult goats underwent ECC; an inflow cannula was inserted into the right atrium, an outflow cannula was sutured to the descending thoratic aorta, and total ECC was performed with a flow of about 80 ml/min/kg. The ECC system with the VFP showed excellent gas exchange efficiency compared with that of the RP. The hemodynamics of ECC using the VFP were easily maintained within normal limits. These results suggest that the VFP is very useful as a pump for ECC; thus, a compact-sized ECC system may be achieved.

Stimulation of acyl-CoA:cholesterol acyltransferase activity by brefeldin A in macrophage J774 cells.

Brefeldin A (BFA), an inhibitor of secretory pathway, enhances incorporation of radiolabeled cholesterol and oleate into cholesteryl esters in cultured cells [12]. We studied the mechanism for this effect of BFA in the macrophage J774. When incubated with 2.7 microM BFA in the absence of lipoproteins, J774 cells synthesized and accumulated 1.5- to 4-fold more cholesteryl esters than did cells which received no BFA. BFA caused neither an elevation of cholesterol synthesis, inhibition of its secretion nor changes in cholesterol transport to plasma membrane, esterification of plasma membrane cholesterol and cholesteryl ester hydrolysis. Acyl-CoA:cholesterol acyltransferase (ACAT) activity in microsomes from BFA-treated cells was 1.5- to 1.8-fold higher than that from control cells. The effect of BFA was diminished by treatment with low temperature, which is known to abolish BFA effect on Golgi formation.

Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase produced by Pseudomonas sp. A92.

Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT), was isolated from a culture broth of Pseudomonas sp. A92 by Diaion HP-20 column chromatography, solvent extraction and reverse phase HPLC. Spectroscopic analyses of the compound yielded 3-(1-hydroxyoctyl)-5-methyl-2(5H)-furanone as the proposed structure. In the presence of oxidized low density lipoprotein, acaterin inhibited the synthesis of cholesteryl ester in macrophage J774 by 50% at a concentration of 45 microM. Acaterin also inhibited ACAT activity in the rat liver microsomes by 50% at a concentration of 120 microM. Kinetic studies suggested that inhibition of ACAT by acaterin was noncompetitive with respect to oleoyl-CoA.

Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B1 and destruxin E.

Two microbial metabolites, bafilomycin B1 and destruxin E, have been found to inhibit significantly the oxidized low density lipoprotein (LDL)-induced accumulation of lipid droplets at 3 nM and 0.5 microM, respectively, in macrophage J774. The incorporation of [14C]oleate into cholesteryl esters in the cells incubated with oxidized LDL was inhibited to the same extent by the two compounds. Both compounds had no effect on the cell surface binding at 4 degrees C and the internalization of oxidized 125I-LDL as well as on the activity of acyl-CoA:cholesterol acyltransferase. However, when incubated with these compounds at 37 degrees C, receptors for oxidized LDL were partially trapped within the cell. In accordance with receptor accumulation, ATP-dependent acidification of endosomes and lysosomes was significantly inhibited by 50 nM bafilomycin B1 and 1 microM destruxin E, respectively. From these results it was concluded that the inhibition of ATP-dependent acidification of endosomes and lysosomes by bafilomycin B1 and destruxin E resulted in the reduction of oxidized LDL-induced synthesis of cholesteryl ester and thereby caused a reduced accumulation of lipid droplets in macrophage J774.

Inhibition of the uptake of oxidized low-density lipoprotein in macrophage J774 by the antibiotic ikarugamycin.

The antiprotozoal antibiotic ikarugamycin was found to significantly inhibit oxidized low-density lipoprotein(LDL)-induced accumulation of cholesteryl ester in macrophage J774 at a concentration over 1-4 microM. Cholesteryl ester synthesis from [14C]oleate in the macrophages was similarly inhibited by the antibiotic, while the synthesis of triacylglycerol and polar lipids was not affected. The internalization of oxidized [125I]LDL in macrophages was reduced to 50% by 2 microM ikarugamycin, while cell-surface binding of oxidized [125I]LDL, lysosomal hydrolysis of the internalized oxidized [125I]LDL and microsomal acyl-coenzyme A:cholesterol acyltransferase was not detectably inhibited by 5 microM ikarugamycin. The results demonstrated that ikarugamycin inhibited cholesteryl ester accumulation in macrophage J774 by specifically inhibiting the uptake of oxidized LDL.

A low CA++ level in effluent as a risk factor for the peritonitis in CAPD patients.

In vitro, some studies revealed the importance of the CA++ level in peritoneal macrophage functions in CAPD patients. We therefore retrospectively studied the relationship between the frequency of peritonitis and the concentration of Ca, Ca++, Interferon-r (IFN-gamma), Interleukin-1B (IL-1B) in the Pd effluent. Samples were taken during a peritonitis-free period. In a group of patients without peritonitis, the mean Ca++ level in the Pd effluent was 2.25 +/- 0.20 mEq/L, while in the other group with frequent episodes of peritonitis (more than one episode per 20 patient-months), the mean Ca++ level in PD effluent was 2.01 +/- 0.13 mEq/L which was significantly lower than the former (p less than 0.05). The mean Ca concentration in Pd effluent was also lower in the group with the high frequent peritonitis than the peritonitis-free group, but not significantly. The level of IFN-gamma is lower and IL-1B is higher in the group with frequent peritonitis than in the peritonitis-free group, although these differences were not significant. These evidences suggest that lower Ca++ level in the effluent of the frequent peritonitis group may impair the peritoneal macrophage function and peritoneal cell-mediated immune function and may increase a risk of the peritonitis. These results may offer a new approach for prophylaxis of peritonitis in CAPD patients.