Glutathione peroxidase-1 expression enhances recovery of human breast carcinoma cells from hyperoxic cell cycle arrest

We previously reported that hyperoxia (95% O(2)) induces an S-phase cell cycle arrest in glutathione peroxidase-deficient human carcinoma cells T47D-H3 (Exp. Cell Res. 256:347-357; 2000). Here, we investigated whether increasing the peroxide scavenging capacity via glutathione peroxidase-1 (GPx1) expression can prevent cell cycle alterations induced by oxidative stress. We show that GPx1-proficient T47D-GPx-2 transfectant cells, in which GPx1 concentration is most elevated in mitochondria (Biochem. Biophys. Res. Commun. 272:416-422; 2000), are partially resistant to cell cycle inhibition induced by hyperoxia or menadione exposure. Transient cell growth resistance was observed at the level of cell cycle phase distribution, Cdk2 activity, and DNA synthesis after 40 h hyperoxia. This differential resistance was associated with an inhibition of ROS production and lipid peroxidation induced by hyperoxia. After 64 h hyperoxic exposure, cell growth was completely abolished in both cell lines, despite elevated glutathione levels. However, in contrast to the GPx1-deficient cells, T47D-GPx-2 cells showed an increased capacity to recover from a cell cycle arrest mediated by a 64 h hyperoxic stress. Differential recovery was also observed at the ultrastructural level between Gpx1-proficient and -deficient cells. These data indicate that GPx1 played an important role in the cell capacity to recover from hyperoxic insults. The limited protection conferred by GPx1 during hyperoxia suggests that the deleterious effects were partially mediated by peroxide-derived free radicals, but also involved the action of nonperoxide-derived reactive species.     

Hypergravity affects cell cycle progression and caveolin-1 expression of in vitro cultured human primary endothelial cells

In hypogravity conditions unloading of skeletal muscle fibres causes alterations in skeletal muscle structure and functions including growth, gene expression, cell differentiation, cytoskeletal organization, contractility and plasticity. Recent studies have identified sphingosine I -phosphate (SPP) as a lipid mediator capable of eliciting intracellular Ca2+ transients, cell proliferation, differentiation, suppression of apoptosis, as well as cell injury repair. The aim of this research is to evaluate a possible involvement of SPP in skeletal muscle cells differentiation and repair from space-flight damage. Particularly, we investigated the Ca2+ sources and the changes on the cytoskeletal rearrangement induced by SPP in a mouse skeletal (C2C12) myoblastic cell line. Confocal fluorescence imaging revealed that SPP elicited Ca2+ transients which propagated throughout the cytosol and nucleus. This response required extracellular and intracellular Ca2+ mobilization. SPP also induced cell contraction through a Ca2(+)- independent/Rho-dependent pathway. The nuclear Ca2+ transients are suggestive for an action of SPP in the differentiation program and damage repair.     

Introduction of the human AVPR1A gene substantially alters brain receptor expression patterns and enhances aspects of social behavior in transgenic mice

Central arginine vasopressin receptor 1A (AVPR1A) modulates a wide range of behaviors, including stress management and territorial aggression, as well as social bonding and recognition. Inter- and intra-species variations in the expression pattern of AVPR1A in the brain and downstream differential behavioral phenotypes have been attributed to differences in the non-coding regions of the AVPR1A gene, including polymorphic elements within upstream regulatory areas. Gene association studies have suggested a link between AVPR1A polymorphisms and autism, and AVPR1A has emerged as a potential pharmacological target for treatment of social cognitive impairments and mood and anxiety disorders. To further investigate the genetic mechanism giving rise to species differences in AVPR1A expression patterns and associated social behaviors, and to create a preclinical mouse model useful for screening drugs targeting AVPR1A, we engineered and extensively characterized bacterial artificial chromosome (BAC) transgenic mice harboring the entire human AVPR1A locus with the surrounding regulatory elements. Compared with wild-type animals, the humanized mice displayed a more widely distributed ligand-AVPR1A binding pattern, which overlapped with that of primates. Furthermore, humanized AVPR1A mice displayed increased reciprocal social interactions compared with wild-type animals, but no differences in social approach and preference for social novelty were observed. Aspects of learning and memory, specifically novel object recognition and spatial relocation recognition, were unaffected. The biological alterations in humanized AVPR1A mice resulted in the rescue of the prepulse inhibition impairments that were observed in knockout mice, indicating conserved functionality. Although further behavioral paradigms and additional cohorts need to be examined in humanized AVPR1A mice, the results demonstrate that species-specific variations in the genomic content of regulatory regions surrounding the AVPR1A locus are responsible for differential receptor protein expression patterns across species and that they are likely to contribute to species-specific behavioral variation. The humanized AVPR1A mouse is a potential preclinical model for further understanding the regulation of receptor gene expression and the impact of variation in receptor expression on behaviors, and should be useful for screening drugs targeting human AVPR1A, taking advantage of the expression of human AVPR1A in human-relevant brain regions.KEY WORDS: AVPR1A, Humanized mouse, Social behavior, Species-specific, Microsatellite, Autism     

鼻咽癌细胞CIC-3在细胞周期中的表达

用免疫荧光、激光共聚焦显微镜图像分析及膜片钳等技术研究了鼻咽癌上皮CNE-2Z细胞容积激活性氯通道候选基因CIC-3的表达及其在细胞周期中与容积激活性氯电流及细胞容积调节性回缩(regulatory volume decrease,RVD)的关系。结果显示,CNE-2Z细胞表达CIC-3。CIC-3蛋白主要位于细胞内而不是在细胞膜上,其表达水平及其在细胞中的分布呈细胞周期依赖性。G1期细胞的CIC-3表达水平较低而S期则较高,M期细胞的表达水平中等。在细胞周期中,CIC-3表达水平与细胞RVD能力及容积激活性氯电流水平呈反比。上述观察结果提示,CIC-3可能参与细胞周期的调节,但CNE-2Z细胞中的CIC-3可能不是与RVD有关的氯通道。     

鼻咽癌细胞ClC-3在细胞周期中的表达

用免疫荧光、激光共聚焦显微镜图像分析及膜片钳等技术研究了鼻咽癌上皮CNE-2Z细胞容积激活性氯通道候选基因C1C-3的表达及其在细胞周期中与容积激活性氯电流及细胞容积调节性回缩(regulatoryvolumedecrease,RVD)的关系.结果显示,CNE-2Z细胞表达ClC-3.ClC-3蛋白主要位于细胞内而不是在细胞膜上,其表达水平及其在细胞中的分布呈细胞周期依赖性.G1期细胞的ClC-3表达水平较低而S期则较高,M期细胞的表达水平中等.在细胞周期中,ClC-3表达水平与细胞RVD能力及容积激活性氯电流水平呈反比.上述观察结果提示,ClC-3可能参与细胞周期的调节,但CNE-2Z细胞中的ClC-3可能不是与RVD有关的氯通道.     

Exercise affects the gene expression profiles of human white blood cells.

White blood cells (WBCs) express tens of thousands of genes, whose expression levels are modified by genetic and external factors. The purpose of the present study was to investigate the effects of acute exercise on gene expression profiles (GEPs) of WBCs and to identify suitable genes that may serve as surrogate markers for monitoring exercise and training load. Five male participants performed an exhaustive treadmill test (ET) at 80% of their maximal O(2) uptake (Vo(2 max)) and a moderate treadmill test (MT) at 60% Vo(2 max) for exactly the same time approximately 2 wk later. WBCs were isolated by the erythrocyte lysis method. GEPs were measured using the Affymetrix GeneChip technology. After scaling, normalization, and filtering, groupwise comparisons of gene expression intensities were performed, and several measurements were validated by real-time PCR. We found 450 genes upregulated and 150 downregulated (>1.5-fold change; ANOVA with Benjamini-Hochberg correction, P < 0.05) after ET that were closely associated with the gene ontology lists "response to stress" and "inflammatory response". Analysis of mean expression levels after MT showed that the extent of up- and downregulation was workload dependent. The genes for the stress (heat shock) proteins HSPA1A and HSPH1 and for the matrix metalloproteinase MMP-9 showed the most prominent increases, whereas the YES1 oncogene (YES1) and CD160 (BY55) were most strongly reduced. Despite different methodological approaches used, the consistency of our results with the expression data of another study (Connolly PH, Caiozzo VJ, Zaldivar F, Nemet D, Larson J, Hung SP, Heck JD, Hatfield GW, Cooper DM. J Appl Physiol 97: 1461-1469, 2004) suggests that expression fingerprints are useful tools for monitoring exercise and training loads and thereby help to avoid training-associated health risks.     

Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42.

G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues. A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K. The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus. The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product. Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K. G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S. cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.     

Regulation of transforming growth factor gene expression in human endometrial adenocarcinoma cells

We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF- and TGF-β genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-, TGF-β and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF- and TGF-β. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF- mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF- mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF- stimulated proliferation of both cell lines whereas exogenous TGF-β inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-β inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.     

VEGF expression and its reguration by p53 gene transfection in endometrial carcinoma cells

Vascular endothelial growth factor (VEGF) that activates endothelial cell growth induces angiogenesis, which is indispensable to tumor igenesis and tumor progression. On the other hand, tumor suppressor gene p53 has been considered to regulate VEGF expression, but the detailed relationship between them remains unclear. In this study, we aimed to study VEGF expression in endometrial carcinoma cells and the effect of p53 gene transfection on VEGF expression using p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF protein, p53 protein and beta-actin was performed using 11 endometrial carcinoma cell lines. Levels of VEGF in the cultured media were measured by Enzyme immunoassay(EIA). Transfection of wild p53 gene was carried out by SuperFect method in HEC-50B cells, which had mutant p53 gene and did not express p53 protein. The results of immunoblotting were analyzed by NIH image and expressed as values. The results of EIA were expressed as the relative value. The VEGF value was 0.8 +/- 0.3 (n = 6) in p53-wild group, whereas in p53-mutant group it was 1.6 +/- 0.8 (n = 5). VEGF expression was correlated significantly with p53 status (P < 0.05). VEGF levels in p53 gene-transfected cells and the conditioned medium were decreased in 48 hours after p53 gene transfection. VEGF expression was down-regulated by p53 in endometrial carcinoma cells.     

鼻咽癌细胞CIC-3在细胞周期中的表达(英文)

用免疫荧光、激光共聚焦显微镜图像分析及膜片钳等技术研究了鼻咽癌上皮cNE-2Z细胞容积激活性氯通道候选基因C1C-3的表达及其在细胞周期中与容积激活性氯电流及细胞容积调节性回缩(regulatorly volume decrease,RVD)的关系。结果显示,CNE-2Z细胞表达CIC-3。C1C-3蛋白主要位于细胞内而不是在细胞膜上,其表达水平及其在细胞中的分布呈细胞周期依赖性。G1期细胞的C1C-3表达水平较低而S期则较高,M期细胞的表达水平中等。在细胞周期中,C1C-3表达水平与细胞RVD能力及容积激活性氯电流水平呈反比。上述观察结果提示,C1C-3可能参与细胞周期的调节,但CNE-2Z细胞中的C1C-3可能不是与RVD有关的氯通道。     

Ultrastructural characterization of two new human endometrial carcinoma cell lines and normal human endometrial epithelial cells cultured on extracellular matrix

Summary Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix. This research was supported by research grants CA31733, CA45727, and ES07017, from the National Institutes of Health, Bethesda, MD. G. P. S. is a Jefferson Pilot Fellow in Academic Medicine. A preliminary account of this work was presented at the 1988 U.S.-Canadian Academy of Pathology Annual Meeting (Lab. Inves. 58:12a, 1988).     

PPARdelta activation induces COX-2 gene expression and cell proliferation in human hepatocellular carcinoma cells

Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. Recently, many studies have shown increased expression of COX-2 in a variety of human malignancies, including hepatocellular carcinoma (HCC). Therefore, it becomes important to know more about what determines COX-2 expression. In this work, we have studied the effect of PPARdelta activation on COX-2 expression using a selective agonist (GW501516) in human hepatocellular carcinoma (HepG2) cells. Activation of PPARdelta resulted in increased COX-2 mRNA and protein expression. The mechanism behind the induction seems to be increased activity of the proximal promoter of the COX-2 gene, spanning nucleotides -327 to +59. The increased COX-2 protein expression and promoter activity induced by the GW501516 was also confirmed in the monocytic cell line THP-1. Induced levels of COX-2 have previously been associated with resistance to apoptosis and increased cell proliferation in many cell types. In HepG2 cells, we observed a dose-dependent increase in cell number by GW501516 treatment for 72h. The levels of PCNA, used as an indicator of cell division were induced, and the cell survival promoting complex p65 (NF-kappaB) was phosphorylated under GW501516 treatment. We conclude that PPARdelta activation in HepG2 cells results in induced COX-2 expression and increased cellular proliferation. These results may suggest that PPARdelta plays an important role in the development of HCC by modulating expression of COX-2.