Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells

Functional responses of the spontaneously transformed human endothelial cell line ECV304 were studied in order to asses its applicability as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of proliferation activity of this line on the presence of growth factor was shown. The absent serum in culture medium resulted in blocking of cells in G1-phase of a cell cycle which is not typical for tumor cell lines. Low doses of beta particles emitted during [3H]thymidine decay resulted in blocking the proliferation of these cells in G2M-phase in a dose-dependent manner. Incubation of the cells with another source of beta particles, 3H2O, under condition of equal specific activities of tritium resulted in preferable accumulation of the cells in S-phase. The different efficiency of beta particles of tritium as a part of 3H2O molecule or thymidine demonstrates that various mechanisms are responsible for various check points. The check point of G1/S is absent and that complies with the presence of deletion of chromosome 9 in locus p21. The level of NO produced by constitutive form of NO-synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that ECV304 line cells retained the properties of the initial spontaneously transformed cell line obtained from human umbilical vein (HUVEC) as well as they can be used as a model system for further studies of the properties of vascular endothelial.     

Characterization of the spontaneously transformed human endothelial cell line ECV304: 1. Multiple chromosomal rearrangements in ECV304 cells

Using differential G-staining of chromosomes, the karyotype of the endothelial cell line ECV304 obtained from endotheliocytes of the human umbilical vein was studied. The cells have been shown to have a polyploid karyotype with a number of chromosomes ranging from 96 to 112, as well as multiple numerical and structural clonal chromosome abnormalities. The structural rearrangements involve almost all chromosomes of the karyotype. Several paired chromosomal rearrangements have been revealed and include del(9)(p21), as well as two derivates of chromosome 3 with a breakpoint at the p25 locus, i.e., der(3)t(3;12)(3p25;12q11～12;12q21～24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endotheliocytes and in angiogenesis is discussed. A comparison of the karyotypes of the cell line ECV304 and of the bladder carcinoma T24 cell line has shown that these karyotypes differ in all of the main cytogenetic characteristics. No identical structural chromosomal rearrangements, nor rearrangements characteristic of bladder carcinoma cells have been revealed. The studied endothelial cell line ECV304 is not identical to the T24 cell line.     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Characteristics of the spontaneously transformed human endothelial cell line ECV304. I. Multiple chromosomal rearrangements in endothelial cells ECV304

Karyotype of endothelial line ECV304 cells obtained from human umbilicus vein endothelial cells was studied using G-banding chromosome staining. It has been revealed that the cells have a polyploidy karyotype with 96-112 chromosomes and multiple numerical and structural clonal rearrangements. Almost all the chromosomes of the karyotype are involved in structural rearrangements. There are several double chromosome rearrangements revealed including del(9)(p21) as well as two derivatives of chromosome 3 with the breakpoint in the locus p25 - der(3)t(3;12)(3p25;12q11- 12q24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endothelial cells and sighs of transformation are discussed. In connection with the information received about the fact that the cells of ECV304 line are not endothelial cells but T24, urinary bladder cancer cells (which karyotype was studied by Hurst et al., 2000), the comparative analysis of the karyotypes of these two lines was carried out. It has been revealed that these two lines differ by all cytogenetic characteristics. Neither identical structural chromosomal rearrangements nor cell characteristic of urinary bladder cancer cells were detected. Our line ECV304 is not identical to the line T24.     

Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics

Endothelial cells separate the intra- and extravascular space and regulate transport processes between these compartments. Since intercellular junctions are required for these specific cell functions, the cell-cell contacts in the permanent cell line ECV304 were systematically analyzed and compared with human umbilical vein endothelial cells (HUVECs) in primary culture and with the epithelial Madin Darby Canine Kidney (MDCK) cell line. Filter-grown ECV304 cells generate a distinct electrical resistance and a permeability barrier between cell culture compartments. Electron microscopy of ECV304 cells revealed lateral membrane interdigitations, typically found in endothelial cells in vivo, with direct membrane contact sites, which prevented the diffusion of lanthanum. By immunoblot and immunofluorescence analysis, the expression and cellular localization of the tight junction and adherens-type junction proteins occludin, ZO-1, symplekin, beta-catenin, and plakoglobin were analyzed. ECV304 cells display further characteristics of endothelial cells, including the expresssion of thrombomodulin and of the vitronectin receptor CD51, as well as the secretion of plasminogen activator inhibitor 1 (PAI-1) and endothelin. However, ECV304 cells also express proteins characteristically found in epithelial cells, including E-cadherin and the desmosomal proteins desmoplakin, desmocollin, and desmoglein; occasionally desmosomal structures can be identified by electron microscopy. In conclusion, ECV304 cells express many endothelial markers and form specialized intercellular junctions that display some epithelial features. Thus this reportedly endothelial-derived permanent human cell line may be dedifferentiated toward an epithelial phenotype.     

Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: Endothelial versus epithelial features

ECV304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1-2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy. Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement. transmembrane electrical resistance, and activity of gamma-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as in T24 tells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.     

Okadaic acid induces the release of Ca from intracellular stores in ECV304 endothelial cells

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca²⁺ ([Ca²⁺]_c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca²⁺]_c both in the presence and absence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ stores with either ATP (1 μM) or thapsigargin (100 nM) prevented any further release of Ca²⁺ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca²⁺), a prior release of Ca²⁺ induced by okadaic acid reduced the magnitude of the response to ATP (1 μM). Taken together these observations indicate that okadaic acid induces Ca²⁺ release from the agonist-sensitive pool. The okadaic acid-induced Ca²⁺ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca²⁺]_c oscillations, which at their peak resulted in 40–100% cells, at any one time, having an elevated [Ca²⁺]_c. The response appeared to propagate between adjacent cells and the elevation of [Ca²⁺]_c. appeared initially in the cell periphery. In adherent cells, the release of Ca²⁺ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca²⁺ from the agonist- and thapsigargin-sensitive intracellular stores.     

The role of TG2 in ECV304-related vasculogenic mimicry

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.     

核酶抑制ECV304细胞TXS mRNA的表达

目的 从设计针对人TXSmRNA的核酶中,筛选能显著抑制TXSmRNA表达的核酶。方法 将核酶DNA亚克隆到真核pEGFP,经脂质体转染入ECV304细胞后,用RT-PCR方法测定细胞内TXSmRNA的相对含量。结果 核酶R416,R441能显著抑制ECV304细胞TXSmRNA的表达。结论 核酶可望成为人TXS有效的抑制剂。在有关TXS的基因治疗中发挥作用。     

Cytotoxic effect of achatininH (lectin) from Achatina fulica against a human mammary carcinoma cell line (MCF7)

The hemolymph-derived achatinin_H (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF7, a human mammary carcinoma cell line. IC₅₀ values as measured by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay for achatinin_H ranged from 6 to 10 μg/ml in the MCF7 cells. MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed after 48 h of treatment with 8 μg/ml when compared to untreated cells. Alterations in the tumor marker enzymes, as well as in antioxidant enzymes, were observed after achatinin_H treatment. The specificity and purity of the achatinin_H was confirmed by the Western blot assay. Achatinin_H binding to MCF7 cells was detected by anti-achatinin_H, and visualization of the achatinin_H binding sites on confluent MCF7 cells was confirmed by flourescein isothiocyanate conjugated secondary antibody. MCF7-treated cells fluoresced, indicating the presence of achatinin_H binding sites. Fluorescence-activated cell sorting analysis of the cell cycle showed a significant increase in S-phase in MCF7 cells after 48 h of achatinin_H treatment. The cells were arrested in G₂/M phase of the cell cycle after 48 h with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis. An erratum to this article can be found at     

MAPK pathway mediates the protective effects of onychin on oxidative stress-induced apoptosis in ECV304 endothelial cells

Our recent studies have shown that onychin could protect rabbit aortic rings from lysophosphatidylcholine-induced injury by preserving endothelium-dependent relaxation and alleviating acute endothelial damage mediated by oxidative stress. However, the effect of onychin on apoptosis of endothelial cells induced by oxidative stress was not evaluated. In the present study, we investigated the effect of onychin on Hydrogen Peroxide (H2O2) induced apoptosis of ECV304 endothelial cells. Cultured human umbilical vein endothelial cell line (ECV304) was pretreated with vehicle (DMSO), genistein, or different concentrations of onychin (0.1, 0.3, 1, 3, and 10 micromol/L) for 30 minutes and then exposed to 1 mmol/L H2O2 for 24 hours. Cell apoptosis was determined by TUNEL and flow cytometric analysis. Meanwhile, Western-blot was used to measure the expression of phospho-ERK1/2, phospho-p38 and caspase-3. Our data showed that onychin treatment exhibited a protective effect on ECV304 endothelial cells from H2O2-induced apoptosis in a concentration-dependent manner. Moreover, onychin attenuated H2O2-induced phosphorylation of p38MAPK and increased H2O2-induced phosphorylation of ERK1/2. Furthermore, onychin decreased the activation of caspase-3. The opposing effects of onychin on phosphorylation levels of p38MAPK and ERK1/2, and its caspase-3 inhibition might play a role in the beneficial effect of onychin on endothelial injury.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against -light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively G _M3, substituted with C₂₄₁ and C₁₆₀ fatty acid and C₁₈₁ sphingosine, was found in this B cell derived cell line. A G _M3 (NeuGc) to G _M3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute G _M3 amounts of about 0.3 mg 10^–9 viable cells were determined. Exogenous application of G _M3, which has been isolated from large cell preparations, to 187.1 cells showed growth inhibition in a concentration dependent manner. Using the MTT-assay and the [³H]thymidine incorporation assay, the cells exhibited a strong reduction in metabolic and proliferative activity, respectively, after exposure of cells to G _M3. G _M3 was applied in concentrations between 3M and 30M, giving evidence for strong inhibitory effects at 30M G _M3 and less but significant suppression after application of G _M3 concentrations lower than 20M. No cellular response was observed at the lowest concentration (3M) used in this study. Hybridoma cells as well as other cell types like fibroblasts, muscle cells and endothelial cells, are in general characterized by high expression of the G _M3 ganglioside, which is known to act as a modulator of cellular growth in monolayer cultures of adherent cells. Since gangliosides are released to the culture medium by cell lysis, i.e. cell death, and/or by active membrane shedding, the results obtained in this study suggest a growth regulatory role of G _M3 in high density hybridoma cell cultures.Abbreviations DMB 1,2-diamino-4,5-methylenedioxybenzene - FAB-MS fast atom bombardment-mass spectrometry - GSL(s) glycosphingolipid(s) - HPLC high performance liquid chromatography - HPTLC high performance thin layer chromatography - MTT 3,(4,5 dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - PBS phosphate buffered saline The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Galß1–4Glcß1-1Cer; gangliotriaosylceramide or GgOse₃Cer; GalNAcß1–4Galß1–4Glcß1-1 Cer; gangliotetraosylceramide or GgOse₄Cer, Galß1–3GalNAcß1–4Galß1–4Glcß1-1Cer; G _M3(NeuAc), II³NeuAc-LacCer; G _M3(NeuGc), II³NeuGc-LacCer; G _M2(NeuGc), II³NeuGc-GgOse₃Cer; G _M1 or G _M1a, II³NeuAc-GgOse₄Cer; G _M1b, IV³NeuAc-GgOse₄Cer.     

秦皮甲素对AGEs致损的ECV304细胞增殖及氧化应激的影响

为研究秦皮甲素对血管内皮细胞的保护作用,采用CCK-8法观察秦皮甲素对体外AGEs培养的人脐静脉内皮细胞增殖的影响。检测不同浓度AGEs以及秦皮甲素作用后对内皮细胞一氧化氮(NO)、不对称二甲基精氨酸(ADMA)水平的影响以及内皮细胞氧化应激有关指标:活性氧簇(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD);脂肪代谢相关指标:乳酸脱氢酶(lactic dehydrogenase,LDH)、总胆固醇(total cholesterol,CHO)、甘油三酯(triglyceride,TG)和低密度脂蛋白(low density lipoprotein,LDL),同时分别检测粘附相关因子:血管细胞粘附分子-1(VCAM-1)和细胞间粘附分子-1(ICAM-1)的表达水平。结果显示200 mg/L AGEs对人内皮细胞ECV304增殖有显著抑制作用,秦皮甲素可对抗AGEs导致的内皮细胞增殖抑制,并呈浓度依赖性。在25 mg/L时,保护效应达到最高。秦皮甲素可抵抗ROS生成。同时可改善细胞的脂类代谢:胆固醇、LDL以及TG含量在秦皮甲素作用后改善明显。秦皮甲素可显著抑制内皮粘附因子VCAM-1的表达。秦皮甲素还可上调NO水平,下调ADMA水平。总之,秦皮甲素可有效促进人血管内皮细胞增殖并在改善氧化应激,脂代谢,粘附因子和NO释放等方面发挥作用。     

High glucose induced human umbilical vein endothelial cell injury: involvement of protein tyrosine nitration

The dysfunction and further damage of endothelium play an important role in the development and progression of diabetic vascular complications. Protein tyrosine nitration is involved in endothelial cell injury induced by high glucose. Little is known about protein nitration in human umbilical vein endothelial cells (ECV304) induced by high glucose. In the present article, exposure of ECV304 to 30 mM high glucose (HG30) and 40 mM high glucose (HG40) or hemin–nitrite–H₂O₂ system for 72 h, the cell injury in ECV304 induced by high glucose and exogenous nitrating agent was studied. After 72 h treatment, it was found that high glucose stimulated ECV304 injury in a dose-dependent manner, including reducing cell viability, increasing malondialdehyde (MDA) content, decreasing glutathione (GSH) content, increasing intracellular reactive oxygen species (ROS), increasing the production of nitric oxygen (NO) (increased nitrite content in cell and nitrate content in medium) and generating protein tyrosine nitration. It was also found that protein tyrosine nitration could induce cell injury further. By comparison the protein tyrosine nitration induced by high glucose condition and extrinsic factors (hemin–nitrite–H₂O₂ system), it may be speculated that protein is nitrated selectively to generate nitrotyrosine in diabetic vascular complications.     

Cantharidin decreased viable cell number in human osteosarcoma U-2 OS cells through G2/M phase arrest and induction of cell apoptosis

ABSTRACT

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G₂/M phase arrest. CTD increased the production of reactive oxygen species and Ca²⁺, and elevated the activities of caspase-3 and ?9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G₂/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.     

Cxc chemokine receptor expression on human endothelial cells.

CXC chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation. However, recent evidence suggests that these molecules can also regulate endothelial cell functions such as migration, angiogenesis and proliferation. In this study we have investigated CXC chemokine receptor expression in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. We found that both cell types express mRNA for chemokine receptors CXCR1, CXCR2 and CXCR4, but not CXCR3. Flow cytometric analysis revealed low levels of CXCR1 but higher levels of CXCR4 cell surface expression. HUVECs responded to SDF-1alpha with a rapid and robust calcium flux, however no calcium flux was seen with either IL-8 or Gro-alpha. HUVECs and ECV304 cells did not proliferate in response to CXC chemokines, although ECV304 cells did migrate towards SDF-1alpha and IL-8. These data demonstrate that HUVECs and the endothelial cell line, ECV304 express functional CXC chemokine receptors.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.     

Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation

To study the changes in gene expression in endothelial cells stimulated by lipopolysaccharide (LPS) we performed subtraction hybridization on control human umbilical vein endothelial cells (HUVEC) versus HUVEC stimulated by LPS. A novel cDNA, named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), was cloned from our differentially expressed EST database of HUVEC cDNA library (GenBank Accession No. ). Computational analysis showed that EOLA1 is 1404bp long, encoding a 158aa, 17.8kDa protein, mapped to chromosome Xq27.4 with 5 exons, expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. Stable transfection of EOLA1 stimulates ECV304 cell proliferation. Our data suggest that the physical interaction of EOLA1 and MT2A may have an important role of cell protection in inflammation reaction.