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.     

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.     

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

The functional peculiarities of spontaneously transformed human endothelial ECV304 cell line were studied to estimate its adequacy as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of the proliferative activity of this line on the presence of growth factors was shown. The absence of serum in the nutrition medium leads to the blockage of cells in the G₁ phase of cell cycle, which is not characteristic of tumor cell lines. Low doses of beta particles emitted during the decay of the [³H]-thymidine blocked, dose-dependent proliferation of these cells in the G₂/M phase. The incubation of the cells in medium with another source of β particles, ³H₂O, resulted in the predominant accumulation of cells in the S phase under conditions of equal specific tritium activities. The different efficiency of β particles of tritium as a part of the H₂O molecule or thymidine demonstrates that different mechanisms are responsible for different checkpoints. The checkpoint of G₁/S is absent, which agrees with the presence of the deletion of chromosome 9 at locus p21. The level of NO produced by the constitutive form of NO synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that the ECV304 line cells retained properties of the initial spontaneously transformed cell line obtained from the human umbilical vein (HUVEC) and can be used as a model system for further studies of properties of the vascular endothelium.     

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.     

The most frequent chromosomal abnormalities in karyotypes of patients with reproductive disorders

Cytogenetic and molecular cytogenetic characteristics have been studied in 210 couples with fertility problems. The patients’ karyotypes contained various chromosomal rearrangements in 46 cases (10.95%). The structural chromosomal rearrangements such as pericentric inversions, Robertsonian translocations, balanced reciprocal translocations, and marker chromosomes were more frequent than numerical chromosome aberrations (89.13 and 10.87% of cases, respectively). We have found 19 (4.52%) karyotypes with “hidden’ low mosaicism in X and Y chromosomes. We believe that the patients with chromosomal anomalies in the karyotype need differentiated treatment.     

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.     

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.     

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。     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.     

Cytogenetic investigations in three cell types of a Saudi family with ataxia telangiectasia

Summary Chromosomal analyses were performed on lymphocytes, fibroblasts and lymphoblastoid cell lines derived from a Saudi family with ataxia telangiectasia (AT). The three siblings of a consanguineous marriage were all affected. The lymphocytes of the AT homozygotes (probands) showed an increase of 2- to 6-fold and 4- to 8-fold respectively, in the frequency of spontaneous and X-ray-induced chromosomal aberrations compared with controls, while the parents (obligate heterozygotes) of the patients showed no notable difference. The unirradiated lymphocytes from the oldest AT sibling, an 11-year-old boy (AT1), showed specific rearrangements involving chromosomes 7 and 14 [t(7;14)(q35;q12)] and 12 and 14 [t(12;14)(q23;q12)] in two different clones. The most severely affected sibling was a 9-year-old girl (AT2) who presented with a clone showing a novel rearrangement involving chromosomes 14 and 17, namely: del(14) (q31q32) and dup(17)(q21–q24). The lymphocytes from the third sibling, a 2-year-old boy (AT3), showed a t(2;14)(p24;q12). In addition, an inv(14)(q12q32) was observed in all three AT patients, while inv(7)(p14q35) was found only in patients 2 and 3. The lymphocytes from the AT parents and controls showed normal karyotypes. The breakpoints involving chromosomes 2,12 and 17, observed in our studies, have rarely been reported in other series of AT patients. No non-random chromosomal rearrangements were observed either in the skin fibroblasts or in the lymphoblastoid cell lines derived from the AT patients, although all cell lines showed an increase in both spontaneous and radiation-induced chromosomal breaks per cell. The present study constitutes the first report on a cytogenetic analysis of a Saudi family with three AT siblings.     

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11-->5q15, 7pter-->q22, 11, 13q14-->qter, 20pter-->p12, X) and losses (8pter-->p2, 18q12-->qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter-->5q11, 12q12-->q23, 15p13-->p11, and 16q21-->q24 and losses of 2pter-->2p24, 4q28-->qter, and 6q25-->qter) can be viewed as changes that occurred in a putative metastatic founder cell.     

利用相互染色体涂色技术分析两株人肺腺癌细胞系(A549和GLC-82)的核型特征

肺癌是全世界癌症死亡中的一个主要的原因。除吸烟外,一些肺癌患者的发病与氡气污染相关。该研究采用包括染色体分选、正向和反向染色体涂色技术,分析了两株肺腺癌细胞系A549和GLC-82的核型特征。A549和GLC-82细胞系都属于非小细胞肺癌细胞系,但诱因不同,后者来源于一个长期生活在氡气污染环境肺癌病人的癌组织。染色体涂色结果表明,这两株肺癌细胞系发生了复杂的染色体重排。在A549和 GLC-82细胞系中,除正常染色体拷贝数变化外,还分别存在13条和24条畸变染色体。约一半的畸变染色体是通过非相互易位形成的,其余的畸变染色体则是通过一些正常染色体的片段缺失或重复而产生的。尽管这两株肺癌细胞系都没有共同的畸变染色体, 但它们似共享两个染色体易位断裂点：HSA8q24和12q14。     

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.     

DNA methylation and chromosomal rearrangements in reconstructed karyotypes of Hordeum vulgare L.

Summary. One standard and two reconstructed barley karyotypes were used to study the influence of chromosomal rearrangements on the distribution pattern of DNA methylation detectable at the chromosome level. Data obtained were also compared with Giemsa N-bands and high gene density regions that had been previously described. The effect of chromosomal reconstruction in barley seems to be decidedly prominent in the repositioning of genomic DNA methylation along metaphase chromosomes. In comparison to the standard karyotype, the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complements not subjected to structural alterations. Moreover, differences may occur between corresponding regions of homologues. Some specific chromosomal bands, including the nucleolus-organizing regions, showed a relative constancy in the methylation pattern, but this was not the case when the two satellites were combined by translocation in chromosome 6H^5H of line T-30. Our results suggest that epigenetic changes like DNA methylation may play an important role in the overall genome reorganization following chromosome reconstruction. Correspondence: R. Cremonini, Dipartimento di Biologia, Università di Pisa, Via L. Ghini 5, 56126 Pisa, Italy.     

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.     

p38L12 TAT融合肽对山梨糖醇引起的 ECV304细胞ATF2磷酸化的抑制作用

构建p38 Loop-12(L12)的TAT融合表达载体,纯化原核表达的p38L12融合蛋白并鉴定其在真核细胞内的功能.利用PCR方法分别扩增出p38L12及其“T-X-Y”双磷酸化位点的AF突变体p38L12（AF）片段,克隆入His标记的TAT-EGFP融合蛋白原核表达载体pHTE（pET14b-His-TAT-EGFP）,经酶切、测序鉴定正确后,将重组质粒转化原核表达菌,诱导表达纯化融合蛋白;将融合蛋白加入ECV304细胞后于荧光显微镜下观察并行Western印迹分析,检测融合蛋白的细胞内转导活性;通过检测内源性ATF2磷酸化水平,鉴定高渗刺激下p38L12对内源性p38活性的影响.成功构建了p38L12和p38L12(AF)片段与TAT的融合表达载体,并获得相应的融合蛋白.在ECV304细胞中可见导入的HTE-p38L12和THE-p38L12(AF)融合蛋白具有较高的细胞内转导活性和转导效率,并可竞争性抑制高渗刺激对内源性p38的活化.基于HIV-1 TAT细胞转导系统证实p38L12可竞争性抑制高渗刺激诱导的内源性p38对ATF2的活化,从而发挥对p38激活特异性抑制的功能.     

46,XX, der(15),t(Y;15)(q12;p11) karyotype in an azoospermic male

We report on a Yq/15p translocation in a 23-year-old infertile male referred for Klinefelter Syndrome testing, who had azoospermia and bilateral small testes. Hormonal studies revealed hypergonadotropic hypogonadism. Conventional cytogenetic procedures giemsa trypsin giemsa (GTG) and high resolution banding (HRB) and molecular cytogenetic techniques Fluorescence In Situ Hybridization (FISH) performed on high-resolution lymphocyte chromosomes revealed the karyotype 46,XX, t(Y;15)(q12;p11). SRY-gene was confirmed to be present by classical Polymerase Chain Reaction (PCR) methods. His father carried de novo derivative chromosome 15 [45,X, t(Y;15)(q12;p11)] and was fertile; the karyotype of the father using G-band technique confirmed a reciprocal balanced translocation between chromosome Y and 15. In the proband, the der (15) has been inherited from the father because the mother had a normal karyotype (46,XX). In the proband, the der (15) could have produced genetic imbalance leading to unbalanced robertson translocation between chromosome Y and 15, which might have resulted in azoospermia and infertility in the proband. The paternal translocation might have lead to formation of imbalanced ova, which might be resulted infertility in the proband. Sister''s karyotypes was normal (46,XX) while his brother was not analyzed.     

Prenatal diagnosis and molecular cytogenetic characterization of an unusual complex structural rearrangement in a pregnancy following intracytoplasmic sperm injection (ICSI).

We report on a balanced complex chromosomal aberration detected in a fetus after amniocentesis. The pregnancy was achieved after intracytoplasmic sperm injection. GTG-banding revealed a complex structurally rearranged karyotype with a translocation between chromosomes 5 and 15 and an additional paracentric inversion in the der(15) between bands 5q11.2 and 5q15. Ag-NOR staining showed an interstitial active nuclear organizer region in the der(15). Molecular cytogenetic analyses using whole-chromosome-painting probes, comparative genomic hybridization, and multicolor banding did not point to further structural aberrations or imbalances. Therefore, a complex rearrangement with three breakpoints has occurred, and the karyotype can be described as 46,XX,der(5)t(5;15) (q11.2;p12),der(15)t(5;15)(q11.2;p12)inv(5)(q11.2q15).