与细胞周期G2/M期进程相关的H2AX磷酸化

γH2AX焦点(foci)被普遍当做DNA双链断裂（DSB）损伤的分子标志物.为探 讨细胞周期进程相关的H2AX磷酸化规律特征,采用胸腺嘧啶双阻滞结合噻氨酯哒唑(nocodazole)的后续处理,将HeLa细胞同步于有丝分裂的前中期．然后,用流式细胞仪检测细胞周期、Western印迹和免疫荧光法,观察γH2AX表达和γH2AX焦点的形成．结果显示,细胞进入G2/M期和有丝分裂过程中,γH2AX水平显著增加 ;在无DNA DSB发生的情况下,部分M期细胞中也存在大量的γH2AX焦点．随着细 胞完成有丝分裂从M期退出再进入G1期,γH2AX的表达水平逐渐降低．这种 γH2AX表达变化特征与G2/M期密切关联的PLK1和Cyclin B1的表达规律相类似． 在4 Gy大剂量照射下,HeLa细胞于照后8 到12 h出现明显的G2/M期阻滞．γH2AX 焦点数在照后1 h达高峰,随后降低,照后8 h又上升,出现了第2个峰值．与之不同的是,在1 Gy低剂量照射下,细胞的G2/M期阻滞微弱,γH2AX焦点数在照后 0.5 h最高,随后下降,且无反弹,符合DNA DSB的修复动力学特征．因此,将γ H2AX当做DNA DSB分子标志物时,还需要考虑细胞周期变化的影响．γH2AX适合 作为1 Gy以下照射的DNA双链断裂损伤的分子标志．     

Dynamics of γH2AX formation and elimination in mammalian cells after X-irradiation

Phosphorylation of the replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γH2AX can be used as an effective marker for DSB repair and DNA damage response. In this study, we examined a bystander effect (BE) in locally irradiated embryonic human fibroblasts. Using fluorescence microscopy, we found that BE could be observed 1 h after X-ray irradiation (IR) and was completely eliminated 24 h after IR. Using immunohistochemistry and immunoblotting, we also studied kinetics of γH2AX formation and elimination in Syrian hamster and mouse tissues after whole body IR of animals. Analysis of hamster tissues at different times after IR at the dose 5 Gy showed that γH2AX-associated fluorescence in heart was decreased slowly with about a half level remaining 24 h after IR; at the same time, in brain, the level of γH2AX was about 3 times increased over the control level, and in liver, γH2AX level decreased to control values. We also report that in mouse heart the level of γH2AX measured by immunoblotting is lower than in brain, kidney and liver at different times after IR at the dose 3 Gy. Our observations indicate that there are significant variations in dynamics of γH2AX formation and elimination between non-proliferating mammalian tissues. These variations in γH2AX dynamics in indicated organs partially correlated with the expression level of the major kinase genes involved in H2AX phosphorylation (ATM and DNA-PK).     

Variability in E-selectin expression, mRNA levels and sE-selectin release between endothelial cell lines and primary endothelial cells

Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), median (range) percentage of E-selectin-positive HUVECs increased from 1.6(0.9-6. 2)% to 91.4(83.0-96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-alpha/IL-1beta. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of adhesion.     

Aurora-A controls cancer cell radio- and chemoresistance via ATM/Chk2-mediated DNA repair networks

High expression of Aurora kinase A (Aurora-A) has been found to confer cancer cell radio- and chemoresistance, however, the underlying mechanism is unclear. In this study, by using Aurora-A cDNA/shRNA or the specific inhibitor VX680, we show that Aurora-A upregulates cell proliferation, cell cycle progression, and anchorage-independent growth to enhance cell resistance to cisplatin and X-ray irradiation through dysregulation of DNA damage repair networks. Mechanistic studies showed that Aurora-A promoted the expression of ATM/Chk2, but suppressed the expression of BRCA1/2, ATR/Chk1, p53, pp53 (Ser15), H2AX, γH2AX (Ser319), and RAD51. Aurora-A inhibited the focus formation of γH2AX in response to ionizing irradiation. Treatment of cells overexpressing Aurora-A and ATM/Chk2 with the ATM specific inhibitor KU-55933 increased the cell sensitivity to cisplatin and irradiation through increasing the phosphorylation of p53 at Ser15 and inhibiting the expression of Chk2, γH2AX (Ser319), and RAD51. Further study revealed that BRCA1/2 counteracted the function of Aurora-A to suppress the expression of ATM/Chk2, but to activate the expression of ATR/Chk1, pp53, γH2AX, and RAD51, leading to the enhanced cell sensitivity to irradiation and cisplatin, which was also supported by the results from animal assays. Thus, our data provide strong evidences that Aurora-A and BRCA1/2 inversely control the sensitivity of cancer cells to radio- and chemotherapy through the ATM/Chk2-mediated DNA repair networks, indicating that the DNA repair molecules including ATM/Chk2 may be considered for the targeted therapy against cancers with overexpression of Aurora-A.     

Activation of DNA damage response signaling in mouse embryonic stem cells

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G1/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G2/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for γH2AX. Irradiation induced the formation of γH2AX foci both in G2-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G1/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to γ-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing γH2AX foci formation, which are necessary for the activation of DNA damage response.     

Autocrine regulation of γ-irradiation-induced DNA damage response via extracellular nucleotides-mediated activation of P2Y6 and P2Y12 receptors

A key component of the response to DNA damage caused by ionizing radiation is DNA repair. Release of extracellular nucleotides, such as ATP, from cells plays a role in signaling via P2 receptors. We show here that release of ATP, followed by activation of P2Y receptors, is involved in the response to γ-irradiation-induced DNA damage. Formation of phosphorylated histone variant H2AX (γH2AX) foci, which are induced in nuclei by DNA damage and contribute to accumulation of DNA-repair factors, was increased at 1-3h after γ-ray irradiation (2.0Gy) of human lung cancer A549 cells. Focus formation was suppressed by pre-treatment with the ecto-nucleotidase apyrase. Pre-treatment with ecto-nucleotidase inhibitor ARL67156 or post-treatment with ATP or UTP facilitated induction of γH2AX, indicating that extracellular nucleotides play a role in induction of γH2AX foci. Next, we examined the effect of P2 receptor inhibitors on activation of ataxia telangiectasia mutated (ATM; a protein kinase) and accumulation of 53BP1 (a DNA repair factor), both of which are important for DNA repair, at DNA damage sites. P2Y6 receptor antagonist MRS2578, P2Y12 receptor antagonist clopidogrel, and P2X7 receptor antagonists A438079 and oxATP significantly inhibited these processes. Release of ATP was detected within 2.5min after irradiation, but was blocked by A438079. Activation of ATM and accumulation of 53BP1 were decreased in P2Y6 or P2Y12 receptor-knockdown cells. We conclude that autocrine/paracrine signaling through P2X7-dependent ATP release and activation of P2Y6 and P2Y12 receptors serves to amplify the cellular response to DNA damage caused by γ-irradiation.     

Qualitative and quantitative analysis of phosphorylated ATM foci induced by low-dose ionizing radiation

We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.     

厄贝沙坦对人脐静脉内皮细胞株EA.hy926增殖、凋亡、VEGF mRNA表达的影响

目的:应用不同浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy 926的增殖、凋亡生物学效应及血管发生主要基因VEGFmRNA的表达进行体外研究,探讨厄贝沙坦对内皮细胞的血管生成效应。方法:各种浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy926共同孵育24 h。细胞增殖采用CCK8法分析,Annexin V/PI双染法检测细胞凋亡。RT-PCR验证VEGFmRNA的表达。结果:厄贝沙坦各浓度干预组细胞形态无明显变化,CCK8结果提示厄贝沙坦各干预组相比对照组细胞增殖活力增高(P<0.05),呈浓度非依赖性。流式细胞仪分析厄贝沙坦各浓度干预组细胞无明显凋亡。RT-PCR发现厄贝沙坦1×10-4,1×10-5,1×10-6mol/L浓度组VEGFmRNA表达增高(P<0.05)。结论:厄贝沙坦促进EA.hy926细胞株细胞增殖,上调VEGFmRNA的表达。这提示除了降压效应,血管紧张素受体拮抗剂在缺血性心脏病如慢性心力衰竭治疗中具有一定作用。     

Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.     

Ataxia telangiectasia and Rad3-related overexpressing cancer cells induce prolonged G₂ arrest and develop resistance to ionizing radiation

We investigated whether ataxia telangiectasia and rad3-related (ATR) kinases regulate prolongation of ionizing radiation (IR) induced-G? arrest and radioresistance in ataxia telangiectasia mutated-intact cancer cells. ATR overexpressing cancer cells showed prolonged-G? arrest after IR exposure and were significantly resistant to DNA damaging stresses. The phosphorylation of p-Ser1?-p53, p-Ser3??-Chk1, and p-Tyr1?-Cdk1 phosphorylation was increased until 36 h after IR exposure in ATR-overexpressing cells, whereas p-Ser1?-histone H3 decreased. ATR-overexpressing cells also showed rapid attenuation of increased γ-H2AX foci after IR exposure compared with control cells. In contrast, ATR knockdown cells had limited clearance of γ-H2AX foci after IR exposure. In conclusion, ATR overexpression seems to primarily induce prolonged G? arrest after IR exposure, which increases IR resistance by enhancing DNA damage repair. These results may provide useful clues for understanding the function of ATR in controlling IR-induced G? arrest and radiation response.     

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.     

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0. 05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.     

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.     

源自天蚕素A和爪蟾素2的杂合肽P18体外抑制内皮细胞血管生成

目的探讨杂合肽P18体外对内皮细胞EA.hy926血管生成的抑制作用.方法采用MTT法检测P18对EA.hy926细胞增殖的影响;应用Matrigel实验检测P18对内皮细胞形成管状结构的影响;利用流式细胞术分析P18对内皮细胞的损伤作用.结果 MTT结果显示P18可明显抑制EA.hy926细胞的增殖,且抑制率存在剂量依赖性;Matrigel实验表明P18具有抑制EA.hy926细胞体外分化成管状结构的作用;流式结果显示15 μM P18作用内皮细胞6 h后,所诱导的细胞坏死比例达到81.4%.结论体外实验结果表明,杂合肽P18具有体外抑制EA.hy926细胞血管生成的作用.     

Proteomic analysis by SILAC and 2D-DIGE reveals radiation-induced endothelial response: four key pathways

Epidemiological data show that ionising radiation increases the risk of cardiovascular disease. The endothelium is one of the main targets of radiation-induced damage. Rapid radiation-induced alterations in the biological processes were investigated after exposure to a clinically relevant radiation dose (2.5 Gy gamma radiation). The changes in protein expression were determined using the human endothelial cell line EA.hy926 as a model. Two complementary proteomic approaches, SILAC (Stable Isotope Labelling with Amino acids in Cell culture) and 2D-DIGE (Two Dimensional Difference-in-Gel-Electrophoresis) were used. The proteomes of the endothelial cells were analysed 4h and 24h after irradiation. Differentially expressed proteins were identified and quantified by MALDI-TOF/TOF and LTQ Orbitrap tandem mass spectrometry. The deregulated proteins were mainly categorised in four key pathways: (i) glycolysis/gluconeogenesis and synthesis/degradation of ketone bodies, (ii) oxidative phosphorylation, (iii) Rho-mediated cell motility and (iv) non-homologous end joining. We suggest that these alterations facilitate the repair processes needed to overcome the stress caused by irradiation and are indicative of the vascular damage leading to radiation-induced cardio- and cerebrovascular impairment.