Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells

Purpose : To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. Materials and methods : HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm -2) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. Results : The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. Conclusion : The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.     

Mesenchymal stem cells are sensitive to treatment with kinase inhibitors and ionizing radiation

Introduction

Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance for normal tissue repair after cancer treatment. Small molecule receptor kinase inhibitors (RKIs) have recently been introduced into cancer treatment. However, the influence of these drugs—particularly in combination with radiotherapy—on the survival of MSCs is largely unknown.

Methods

The sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells to small molecule kinase inhibitors of the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ) receptors, as well to inhibitors of c-Kit, was examined in combination with ionizing radiation (IR); cell survival and proliferation were assessed. Expression patterns of different kinase receptors and ligands were investigated using gene arrays.

Results

MSCs were highly sensitive to the tyrosine kinase inhibitors SU14816 (imatinib) and SU11657 (sunitinib), but showed only moderate sensitivity to the selective TGFβ receptor 1 inhibitor LY2109761. Primary adult human fibroblasts were comparably resistant to all three inhibitors. The addition of IR had an additive or supra-additive effect in the MSCs, but this was not the case for differentiated fibroblasts. Proliferation was markedly reduced in MSCs following kinase inhibition, both with and without IR. Gene expression analysis revealed high levels of the PDGF α and β receptors, and lower levels of the TGFβ receptor 2 and Abl kinase. IR did not alter the expression of kinase receptors or their respective ligands in either MSCs or adult fibroblasts.

Conclusion

These data show that MSCs are highly sensitive to RKIs and combination treatments incorporating IR. Expression analyses suggest that high levels of PDGF receptors may contribute to this effect.     

电离辐射对松果腺细胞功能的影响

目的研究电离辐射对松果腺细胞凋亡、cAMP和褪黑素(MLT)含量的变化规律及特点。方法采用流式细胞术检测松果腺细胞凋亡。采用放免分析法检测松果腺细胞中cAMP含量。采用高效液相色谱分析法检测松果腺细胞中MLT含量。结果小鼠受0．5～6Cy X射线全身照射12h，随剂量增加松果腺细胞凋亡百分率增加；照射后24h，随剂量增加松果腺细胞中cAMP含量降低。小鼠受1～6Cy照射后12h，随剂量增加松果腺细胞合成和分泌MLT功能减弱。结论高剂量电离辐射对松果腺细胞功能有抑制作用．     

Effects of multiple doses of ionizing radiation on cytokine expression in rat and human cells

Purpose: To determine the effect of daily fractionated irradiation on the expression of growth factors and cytokines in different cardiac and vascular cell types.

Materials and methods: Cell cultures of rat cardiac myocytes, fibroblasts, a rat cardiac microvascular endothelial cell line and human artery endothelial cells were irradiated with doses of 2?Gy, given daily during 5 consecutive days. Twenty‐four hours after each fraction, gene expression was determined by competitive or semiquantitative polymerase chain reaction. Protein secretion into culture media was determined by enzyme‐linked immunoabsorbant assay.

Results: Of all investigated mRNA levels, transforming growth factor (TGF)‐ß1 and fibroblast growth factor (FGF)‐2 were slightly upregulated in the rat cardiac endothelial cell line after irradiation. TGF‐ß1 protein secretion by these cells was slightly, but non‐significantly, elevated. Interleukin 1ß protein levels in myocyte culture media were decreased in control cultures at days 3 and 4 compared with day 2. No significant changes were observed in expression of FGF‐2 in either of the four cell types. Moreover, no changes were observed in gene expression of platelet‐derived growth factors A, B and interleukin 8 in the human artery endothelial cells.

Conclusions: Fractionated irradiation leads to minor changes in the expression of specific cytokines in cardiac myocytes, fibroblasts and endothelial cells.     

Effects of multiple doses of ionizing radiation on cytokine expression in rat and human cells

PURPOSE: To determine the effect of daily fractionated irradiation on the expression of growth factors and cytokines in different cardiac and vascular cell types. MATERIALS AND METHODS: Cell cultures of rat cardiac myocytes, fibroblasts, a rat cardiac microvascular endothelial cell line and human artery endothelial cells were irradiated with doses of 2 Gy, given daily during 5 consecutive days. Twenty-four hours after each fraction, gene expression was determined by competitive or semiquantitative polymerase chain reaction. Protein secretion into culture media was determined by enzyme-linked immunoabsorbant assay. RESULTS: Of all investigated mRNA levels, transforming growth factor (TGF)-ss1 and fibroblast growth factor (FGF)-2 were slightly upregulated in the rat cardiac endothelial cell line after irradiation. TGF-ss1 protein secretion by these cells was slightly, but non-significantly, elevated. Interleukin 1ss protein levels in myocyte culture media were decreased in control cultures at days 3 and 4 compared with day 2. No significant changes were observed in expression of FGF-2 in either of the four cell types. Moreover, no changes were observed in gene expression of platelet-derived growth factors A, B and interleukin 8 in the human artery endothelial cells. CONCLUSIONS: Fractionated irradiation leads to minor changes in the expression of specific cytokines in cardiac myocytes, fibroblasts and endothelial cells.     

pETNF-P16质粒的构建及其在食管癌细胞中的辐射诱导表达特性

目的　构建重组质粒pETNF P16并研究其在食管鳞状细胞癌细胞系EC970 6中X射线照射诱导表达特性和基因 放射治疗食管癌的可行性。方法　构建重组质粒pETNF P16 ,通过脂质体介导转染EC970 6细胞。利用ELISA ,Westernblot和免疫细胞化学的方法检测pETNF P16在被转染的EC970 6细胞中的X射线照射诱导表达特性。结果　成功构建真核载体pETNF P16并转染EC970 6细胞。在不同剂量X射线照射后被转染细胞中TNFα的表达量明显高于 0Gy组 (P <0 0 5或P <0 0 1)。 2GyX射线照射后 2～ 4 8hTNFα和P16的表达量明显高于对照组 (P <0 0 5或P <0 0 0 1)。在正常的EC970 6细胞中没有P16的表达 ,在转染组和辐射诱导组中P16有较强的表达。结论　在pETNF P16质粒转染的EC970 6细胞中X射线可诱导TNFα和P16的表达增强。本研究结果为临床食管癌基因 放射治疗进一步研究提供实验基础     

siRNA干扰明胶酶对人肾小管细胞ICAM-1表达的影响

目的 利用小干扰RNA(siRNA)抑制人肾小管上皮细胞(HKC)的基质金属蛋白酶-2(MMP-2)和MMP-9,即明胶酶A和B的表达,观察其对胞间黏附分子1(ICAM-1)表达的影响.方法 培养人肾小管上皮细胞,以100nmol/L佛波醇酯(PMA)刺激18h,脂质体转染抑制MMP-2或MMP-9表达的siRNA或无关siRNA.提取细胞总RNA或胞浆蛋白,利用Real-Time PCR、Western blot或免疫荧光结合激光共聚焦显微镜技术检测MMP-2、MMP-9或ICAM-1的表达情况.结果 特异性的siRNA能有效抑制HKC中MMP-2、MMP-9的mRNA和蛋白表达;免疫荧光结果显示经PMA刺激后HKC的ICAM-1表达上调,同时MMP-9-siRNA转染组人肾小管细胞的ICAM-1蛋白表达水比其他实验组高(P＜0.05).结论 抑制肾小管上皮细胞MMP-9的表达可使ICAM-1表达上调,提示除了细胞外基质降解途径外,MMP-9还可通过炎症途径影响肾脏纤维化进程.     

Differences in ICAM-1 and TNF-alpha expression between large single fraction and fractionated irradiation in mouse brain

PURPOSE: To elucidate the brain molecular response to irradiation. The expression of the intercellular adhesion molecule (ICAM-1) and tumour necrosis factor-alpha (TNF-alpha) in the mouse brain was compared after single-dose and fractionated whole-brain irradiation. MATERIALS AND METHODS: Mice received a single dose of 2, 10 or 20 Gy or a fractionated dose (2 Gy day(-1)) of 10, 20 or 40 Gy. ICAM-1, and TNF-alpha mRNA expression were quantified by the highly sensitive real-time polymerase chain reaction technique. Expression of ICAM-1 protein was quantified by dual-labelled monoclonal antibody assay. RESULTS: After a 20-Gy single dose, there was an increase in ICAM-1 and TNF-alpha mRNA levels (14- and 11-fold, respectively) as well as a significant increase in the level of ICAM-1 protein (p=0.0243). The expression of ICAM-1 and TNF-alpha mRNA increased at the end of the 40-Gy fractionated regimen (3.55- and 2.30-fold, respectively). CONCLUSIONS: The molecular response of the brain to single-dose irradiation was rapid, while its response to fractionated irradiation was slow. This finding is consistent with clinical observations and could be of use when designing strategies to mitigate radiation sequelae.     

miR-1抑制SDF-1表达和分泌调控HMSC-bm向肝癌细胞迁移

目的 明确肿瘤抑制性micro RNA-1（miR-1）能否通过抑制SDF-1的表达和分泌,调控骨髓间充质干细胞（HMSC-bm）向肿瘤组织的转移.方法 （1）分别利用miR-1表达质粒pSuper-miR-1和miR-1反义寡核苷酸,在肝癌细胞系HepG2中过表达或下调miR-1,Western blot和酶联免疫吸附实验分别检测肝癌细胞蛋白裂解物和培养上清液中SDF-1的表达和分泌水平;（2）构建融合SDF-1 3UTR的pGL3重组萤光素酶报告基因载体,将其与pSuper-miR-1共转染HEK293细胞,利用双萤光报告基因检测系统检测重组萤光素酶的表达情况.（3）利用pSuper-miR-1或miR-1反义寡核苷酸转染接种于Transwell下层小室的HepG2细胞过表达miR-1或下调miR-1水平,检测TransweH上层小室HMSC-bm向下层小室HepG2肝癌细胞的迁移情况.结果 （1）在肝癌细胞,HepG2过表达miR-1能够显著抑制SDF-1蛋白表达和分泌,而下调miR-1水平则能够诱导SDF-1的表达和分泌.（2） miR-1能够抑制携带SDF-1 3UTR的pGL3重组萤光素酶报告基因活性.（3）在Transwell下层小室的HepG2细胞中过表达miR-1后,上层小室中HMSC-bm向下层小室迁移细胞数量显著减少,而下调下层小室HepG2细胞中miR-1表达水平,则明显增加HMSC-bm向HepG2肝癌细胞的迁移.结论 miR-1能够直接抑制肝癌细胞SDF-1的表达和分泌,并藉此降低肝癌细胞对HMSC-bm的趋化作用.     

高糖状态下内脂素对血管内皮细胞单核细胞趋化蛋白1及细胞间黏附分子1表达的影响

目的探讨内脂素在高糖状态下对血管内皮细胞单核细胞趋化蛋白1(MCP-1)和细胞间黏附分子1(ICAM-1)蛋白表达的影响及机制。方法体外培养人脐静脉内皮细胞(HUVEC)分为3组:空白对照组(0mmol/L葡萄糖处理)、生理葡萄糖组(5.5mmol/L葡萄糖处理)、高糖组(25mmol/L葡萄糖处理),分别用不同浓度内脂素(0、10、50、100ng/ml)培养24h。另取HUVEC,在p38MAPK特异性抑制剂SB203580预处理30min后,加入内脂素(100ng/ml)及葡萄糖(0、5.5、25mmol/L)培养24h。采用反转录-聚合酶链反应(RT-PCR)检测HUVEC中p38MAPKmRNA表达量,采用Westernblotting检测HUVEC中磷酸化p38MAPK蛋白表达水平,并用双抗体夹心酶联免疫吸附试验(ELISA)检测细胞培养上清液中的MCP-1、ICAM-1蛋白表达量。结果空白对照组、生理葡萄糖组中,内脂素促进HUVEC中p38MAPKmRNA转录、p38MAPK蛋白磷酸化以及MCP-1、ICAM-1蛋白的表达,并呈浓度依赖性(P<0.01);与空白对照组和生理葡萄糖组相比,高糖组内...     

Early and long-term effects of radiation on intercellular adhesion molecule 1 (ICAM-1) expression in mouse urinary bladder endothelium

The aim was to assess the effect of irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells of vessels in mouse urinary bladder and to compare endothelial ICAM-1 expression with changes in bladder function (storage capacity) during the early and late radiation response phases. Female C3H/Neu mice were irradiated with doses of either 20 or 0 Gy. For assessment of ICAM-1 expression, which was measured by the intensity of the immunohistochemical staining signal in bladder endothelium, an arbitrary semiquantitative score (0 - 3) was applied. Bladder storage function was assessed by transurethral cystotonometry. A positive functional radiation response, defined as a reduction in bladder capacity by > 50%, between days 0 and 15 or 16 and 30 was found in 40 and 64% of the animals, respectively. A late functional response was observed in 71% of the animals sacrificed after day 180. Minor constitutive expression of ICAM-1 was observed in bladder endothelial cells. After irradiation, an increase in staining signal by day 2, with a maximum on day 4, and on days 16 - 28 was found, which preceded the functional radiation effects. A permanent increase in ICAM-1 staining signal was observed in the late phase on top of an age-related rise. ICAM-1 expression was significantly higher in animals with a positive late response on day 90, i.e. during the initial late phase. Irradiation induces significant early and chronic variations in ICAM-1 expression in bladder endothelium, which preceded the functional response. This suggests that endothelial ICAM-1 is involved in the pathogenesis of both the early and late phases of radiation-induced urinary bladder effects.     

细胞间黏附分子-1在颅脑损伤中的表达和炎症介导作用

介绍细胞间黏附分子-1(intercellular adhesion molecules-1,ICAM-1)的生化特性和颅脑损伤后介导损伤脑组织炎性反应的作用.另外,许多炎性因子可激活ICAM-1蛋白表达,导致炎症的级联反应.     

Early and long-term effects of radiation on intercellular adhesion molecule 1 (ICAM-1) expression in mouse urinary bladder endothelium

The aim was to assess the effect of irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells of vessels in mouse urinary bladder and to compare endothelial ICAM-1 expression with changes in bladder function (storage capacity) during the early and late radiation response phases. Female C3H/Neu mice were irradiated with doses of either 20 or 0?Gy. For assessment of ICAM-1 expression, which was measured by the intensity of the immunohistochemical staining signal in bladder endothelium, an arbitrary semiquantitative score (0?–?3) was applied. Bladder storage function was assessed by transurethral cystotonometry. A positive functional radiation response, defined as a reduction in bladder capacity by >?50%, between days 0 and 15 or 16 and 30 was found in 40 and 64% of the animals, respectively. A late functional response was observed in 71% of the animals sacrificed after day 180. Minor constitutive expression of ICAM-1 was observed in bladder endothelial cells. After irradiation, an increase in staining signal by day 2, with a maximum on day 4, and on days 16?–?28 was found, which preceded the functional radiation effects. A permanent increase in ICAM-1 staining signal was observed in the late phase on top of an age-related rise. ICAM-1 expression was significantly higher in animals with a positive late response on day 90, i.e. during the initial late phase. Irradiation induces significant early and chronic variations in ICAM-1 expression in bladder endothelium, which preceded the functional response. This suggests that endothelial ICAM-1 is involved in the pathogenesis of both the early and late phases of radiation-induced urinary bladder effects.     

细胞间黏附分子-1在颅脑损伤中的表达和炎症介导作用

介绍细胞间黏附分子-1（intercellular adhesion molecules-1，ICAM-1）的生化特性和颅脑损伤后介导损伤脑组织炎性反应的作用。另外，许多炎性因子可激活ICAM-1蛋白表达，导致炎症的级联反应。     

电离辐射及顺铂对人肺癌细胞中TOB1基因表达的影响

目的 探讨电离辐射及其联合顺铂对人肺腺癌细胞株SPCA-1、LTEP-α-2中TOB1基因表达的影响.方法 体外培养人肺腺癌SPCA-1、LTEP-α-2细胞,用X射线一次性照射,细胞吸收剂量为2、4、6、8和10 Gy,源靶距为100 cm,吸收剂量率为200 cGy/min,在6GyX射线照射后6、12、18和24 h取样;X射线和顺铂联合对TOB1表达的研究设立单纯照射组(6 Gy)、顺铂处理组(20 mol/L顺铂)和联合处理组(6 Gy +20 mol/L顺铂);上述实验同时设立未处理的对照组.采用RT-PCR和Western blot法检测TOB1 mRNA及蛋白表达水平的变化.结果 SPCA-1、LTEP-α-2细胞在不同剂量的X射线照射后24 h,其TOB1蛋白及mRNA表达量均明显增加(t=8.25 ～24.48,P ＜0.05);不同时间检测提示,细胞在X射线照射后6h,TOB1蛋白及mRNA的表达量即开始上升,并持续到照射后24 h(t=14.23 ～15.82,P＜0.05);联合处理组TOB1的表达量均明显高于单纯照射组、顺铂处理组和对照组(t=4.67 ～ 13.67,P＜0.05).结论 TOB1基因有可能作为临床肺癌放化疗靶基因,对临床放化疗疗效的判断具有一定的应用潜力.     

低剂量电离辐射对7721细胞细胞周期及p53、Ku70和Ku80表达的影响

目的　观察电离辐射对 772 1细胞 (人肝癌细胞 )细胞周期和p53、Ku70和Ku80基因表达的影响。方法　以人肝癌细胞株 772 1为研究对象 ,通过克隆形成实验拟合出 772 1细胞的剂量存活曲线 ;用流式细胞技术检测 75mGyX射线照射后 772 1细胞周期变化 ;用原位杂交方法检测 772 1细胞p53、Ku70和Ku80基因在 75mGyX射线照射前、后的表达情况。结果　与对照组相比 ,75mGyX射线照射后 0 5～ 6h ,772 1细胞S期延长 (P <0 0 5)。p53、Ku70和Ku80基因的表达照射前与照射后比较差异无显著性。结论　 772 1细胞在 75mGyX射线照射后 ,未出现G1期阻滞 ,p53、Ku70和Ku80基因在照射前、后表达无明显变化 ,是由于这些基因自身存在缺陷或激活机制存在缺陷所致     

Sustained enhancement of liposome-mediated gene delivery and gene expression in human breast tumour cells by ionizing radiation

PURPOSE: To investigate whether irradiation improves the delivery and expression of liposome-DNA complexes in human breast tumour cells. MATERIALS AND METHODS. MDA-MB231 and MCF-7 human breast tumour cells were transfected with a liposomal SV40-luciferase complex and irradiated immediately after, at 24h after or 24h prior to transfection and in the presence or absence of serum. The amount of luciferase plasmid in the cell was evaluated after extraction by the Hirt procedure, while luciferase expression was measured using a luminescence assay. RESULTS: Ionizing radiation enhanced the liposome-mediated delivery and expression of the SV40-luciferase transgene in MDA-MB231 breast tumour cells both in the absence and presence of serum as well as in MCF-7 breast tumour cells. Improved transgene delivery and expression was observed at a clinically relevant dose of 2 Gy, and was dose-dependent over a dose range of 2-10 Gy. The effects of irradiation on transgene expression were observed with irradiation immediately prior to exposure of the cells to the liposome-transgene complex, with irradiation up to 24 h before or up to 24 h after initiation of exposure. CONCLUSIONS: Irradiation at 24 h prior to exposure of breast tumour cells to the liposome-transgene complex appears to be the optimal approach for enhancing transgene delivery and expression. These findings suggest that ionizing radiation could promote the utility of gene therapy in the treatment of breast cancer.     

Risk of delayed effects of chronic ionizing and non-ionizing irradiation in establishing hygienic standards]

The authors analyze the range and incidence of diseases in delayed periods following chronic exposure to different dose rates of ionizing radiation (IR) and high and super-high frequency electromagnetic fields (EMF) with the intensities just above the population exposure limits. The epidemiological data review lays emphasis on delayed non-tumor pathologies impacting human health and performance which have been left out of account in hygienic standards. Risks to health of personnel working in high- and super-high EMFs has been assessed in light of G. Seliet concept concerning the development of non-specific physiological reactions (general adaptation syndrome) to chronic stresses of varying nature, modeling shifts in the adaptive body reserves, and calculated radiation risks from acute and chronic IR exposures.