Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.     

Antioncogenic effect of adenovirus E1A in human tumor cells.

Stable expression of the adenovirus 5 E1A gene reduced anchorage-independent growth and tumorigenic potential, caused cytoskeletal reorganization, induced flat morphology, and restored contact inhibition in three human tumor cell lines. By these criteria, E1A appears to be functionally indistinguishable from a tumor suppressor gene in this context. The apparent paradox accorded by the observations of the ability of E1A to transform rodent cells in cooperation with other oncogenes suggests that E1A may be the prototype of a class of growth-regulatory proteins having context-specific transforming and antioncogenic activities.     

Recombinant E1-deleted adenovirus vector induces apoptosis in two lung cancer cell lines.

Although replication-defective adenoviruses (Ads) are used as vectors for delivering therapeutic genes to cancer cells, various effects of the viruses on the proliferation of lung cancer cells have been reported. Experiments were carried out to determine whether or not E1-deleted Ad vectors (Ad5-CMV-lacZ) affected cell kinetics in two different types of lung cancer cell line in vitro. A dose-dependent relationship was measured between the vector multiplicity of infection (MOI) and the efficiency of lacZ gene transfer to lung cancer cells. The growth curves of vector-infected cells were shifted to the right compared with those of vehicle-exposed cells in a vector MOI-dependent fashion. The slowed cell proliferation resulted from both increased cell death and slower cell cycle progression of the vector-infected cells. The morphology of vector-exposed cells revealed apoptotic features including nuclear condensation and fragmented nuclei. These results indicate that using a higher vector MOI causes a higher gene transfer rate, but may induce apoptosis of infected cells. Although vector-induced apoptosis may be advantageous in inhibiting tumour growth, apoptosis of vector-infected cells may also reduce transgene expression in cancer cells. Minimization of the induction of apoptosis of vector-infected cells is important for the prolongation of the transduction efficiency of Ad vectors.     

腺病毒EIA基因对细菌脂多糖所致肺泡上皮细胞MMP-9FFIMP-1失衡的影响

目的：明确腺病毒E1A基因对细菌脂多糖（lipopolysaccharide,LPS）所致肺泡上皮细胞基质金属蛋白酶9（MMP-9）／基质金属蛋白酶组织抑制剂1（TIMP-1）失衡是否存在影响。方法：将人Ⅱ型肺泡上皮细胞系A549细胞分为正常对照组、转染腺病毒E1A质粒组（E1A＋组）和转染不含有腺病毒E1A的空白质粒组（EIA一组）,分别以不同浓度的LPS刺激．刺激后12、24h用反转录聚合酶链反应技术检测各组细胞MMP-9mRNA和TIMP．1mRNA的表达,用酶联免疫吸附分析（ELISA）检测MMP．9和TIMP-1蛋白的表达。结果：在LPS刺激12h后E1A＋组的MMP-9／TIMP-1mRNA比值高于其他2组（P=0．045）：在刺激24h后3组之间的差别更加显著（P=0．032）。MMP-9/TIMP-1蛋白比值在LPS刺激12h后E1A＋组高于其他2组．但无统计学意义（P=0．069）,在刺激24h后3组蛋白比值的差别比较显著（P=0．039）。结论：腺病毒EIA基因可导致Ⅱ型肺泡上皮细胞在LPS刺激下大量释放MMP-9．使MMP-9／TIMP-1的失衡进一步加重。     

Induction of ICAM-1 expression on alveolar epithelial cells during lung development in rats and humans.

Intercellular adhesion molecule-1 (ICAM-1) is an adhesion protein involved in immune and inflammatory cell recruitment and activation. In normal, uninflamed adult rat lung, ICAM-1 is expressed at high levels on type I alveolar epithelial cells and is minimally expressed on type II cells. ICAM-1 expression by alveolar epithelial cells in vitro is a function of the state of cellular differentiation, and is regulated by factors influencing cell shape. Based upon this observation, we hypothesized that ICAM-1 expression by fetal lung epithelial cells is developmentally regulated. To investigate this hypothesis, rat and human lung tissues were obtained at time points that represent the canalicular, saccular, and alveolar stages of development. The relative expression of ICAM-1 protein and mRNA were determined in rat lungs from gestational days 18 and 21 (term = 22 days), from day 8 neonatal rats, and from adult rats. ICAM-1 protein was detectable at low level on day 18 and increased progressively during development. Relative expression of ICAM-1 protein was maximal in adult lung. Expression of ICAM-1 mRNA paralleled that of ICAM-1 protein. By immunohistochemical methods in rat and human lung, ICAM-1 was expressed at low level on cuboidal and flattening epithelial cells in the developing alveolar space at the canalicular and saccular stages; however, ICAM-1 expression was increased as epithelial cells spread and flattened during alveolarization. ICAM-1 was predominantly expressed on type I cells rather than type II cells at the alveolar stage in both the rat and human lungs. Thus, relative ICAM-1 expression progressively increased during lung development. ICAM-1 expression is correlated with the increase in surface area as alveolar structures develop and type I cell differentiation takes place. These data indicate that alveolar epithelial cell ICAM-1 expression is developmentally regulated.     

Expression of the adenovirus E1A oncogene during cell transformation is sufficient to induce susceptibility to lysis by host inflammatory cells.

Mammalian cells transformed by nononcogenic human adenoviruses exhibit high susceptibility to destruction by host mononuclear inflammatory cells. We have analyzed the viral gene regulation of the susceptibility of transformed cells to lysis by natural killer cells and activated macrophages. Comparisons of target cell lines transformed by overlapping segments of the adenovirus E1-transforming gene region revealed that isolated expression of a single oncogene, E1A, was sufficient to cause increased cytolytic susceptibility in the absence of detectable transformed cell-surface expression of viral transplantation antigens and irrespective of histocompatibility antigen identity between killer cells and target cells. These results suggest that oncogene functions that are not linked to the expression of previously recognized cell-surface target structures may actively induce neoplastic cell elimination by components of the host immune surveillance system.     

Mapping of functional domains in adenovirus E1A proteins.

We have modified the E1A gene of human subgroup C adenovirus by introducing deletions in its coding sequence. Various truncated E1A proteins were expressed in Escherichia coli, purified, and microinjected via glass capillaries into Vero cells. We monitored their movement from the cell cytoplasm to the nucleus and their ability to induce expression of H5dl312, an adenovirus E1A deletion mutant. Our results show that the carboxyl terminus of E1A contains sequences essential for rapid and efficient nuclear localization. Essential information for efficient H5dl312 complementation is contained in an internal region, comprising sequences of both exons of the E1A gene. A first exon-encoded region, however, is sufficient to induce low levels of adenovirus gene expression. Information for nuclear localization and for H5dl312 complementation are therefore encoded by distinct domains of the E1A gene. In addition, we determined that the human c-myc product was unable to complement H5dl312.     

Epithelial mesenchymal transition of non-small-cell lung cancer cells A549 induced by SPHK1

Objective:To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production of A549/vector,A549/SPHK1,A549/scramble,and A549/SPHK1/RNAi that stably expressed or silenced SPHK1.The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment.The protein and mRNA expression levels of E-cadherin,fibronectin,vimentin in A549/vector,A549/SPHK1,A549/scramble,A549/SPHK1/RNAi were detected with Western blot(WB) and quantitative PCR(QPCR) methods,respectively.Results:Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells.WB and QPCR detection results showed that,the expression of E-cadherin(a molecular marker of epithelial cells) and fibronectin,vimentin(molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1;while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells,upregulated the expression of molecular marker of epithelial cells,and downregulated the expression of molecular marker of mesenchymal cells.Conclusions:SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.     

Induction of prostaglandin E synthase by gastroesophageal reflux contents in normal esophageal epithelial cells and esophageal cancer cells

The synthesis of prostaglandin E2 (PGE2) requires cyclooxygenase (COX) and prostaglandin E synthase (PGES). There are two forms of PGES: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1. In this study, we investigated the effects of gastroesophageal reflux (GER) contents on PGES and COX-2 in esophageal cells. We incubated a human normal esophageal cell line, two esophageal squamous cell carcinoma (SCC) cell lines, and two esophageal adenocarcinoma (ADC) cell lines with GER contents. The production of PGE2 by these cells was assayed with an enzyme immunoassay kit. The protein expression of COX-2, cPGES, and mPGES-1 was confirmed by immunoblot analysis. The following results were obtained: GER contents induced the expression of COX-2 in all five cell lines. In normal esophageal cells, cPGES, but not mPGES-1, was detected in the cytosolic fraction. GER contents induced the expression of cPGES in the microsomal fraction. In SCC cells, cPGES was expressed in the cytosolic fraction, and mPGES-1 was expressed in the microsomal fraction. GER contents induced the expression of mPGES-1 in the microsomal fraction. In ADC cells, cPGES was expressed in both the cytosolic and microsomal fractions. GER contents induced the expression of both cPGES and mPGES-1 in the microsomal fraction. In conclusion, our results suggest that GER contents induce PGE2 production in esophageal cells. However, there are different isoforms of PGES in normal cells, SCC cells, and ADC cells.     

Differentiation of rat intestinal epithelial cells is induced by organotypic mesenchymal cells in vitro.

Stromal-epithelial interaction is a potent driving force in the developing intestinal mucosa which ensures tissue specific cellular differentiation. The mechanisms involved are relevant to tissue renewal in adult organs yet they have not been elucidated because of the lack of appropriate in vitro models. In this study, we have investigated the interaction between intestinal mesenchymal and epithelial cells at the cellular level in vitro. Fetal rat intestinal epithelial cell colonies explanted in vitro on the 15th day of gestation, which failed to mature in plain monocultures, were reassociated in coculture with three different types of mesenchyme:fetal skin, gastric and intestinal mesenchyme. Only fetal epithelial cells cocultured with intestinal (homologous) mesenchyme acquired definite signs of differentiation within three to six days. These primitive epithelial cells were shown by electronmicroscopy to become highly polarized, connected by tight junctions and covered with a regular brush border. Three brush border enzymes were strongly expressed in homologous cocultures and their activity was sensitive to dexamethasone. In contrast, fetal epithelial cells cocultured with skin or stomach derived mesenchyme under identical conditions failed to differentiate in vitro: they remained flat, unpolarised and expressed only low enzyme activity. The unique potential of the small intestinal mesenchyme to promote intestinal epithelial differentiation is discussed.     

Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells

Adenoviruses invading the organism via normal digestive or respiratory routes require the Coxsackie-adenovirus receptor (CAR) to infect the epithelial barrier cells. Because CAR is a component of tight junctions and the basolateral membrane and is normally excluded from the apical membrane, most epithelia are resistant to adenoviruses. However, we discovered that a specialized epithelium, the retinal pigment epithelium (RPE), anomalously expressed CAR at the apical surface and was highly susceptible to adenovirus infection. These properties of RPE cells correlated with the absence of the epithelial-specific clathrin adaptor AP1B. Furthermore, knockdown of this basolateral sorting adaptor in adenovirus-resistant MDCK cells promoted apical localization of CAR and increased dramatically Adenovirus infectivity. Targeting assays showed that AP1B is required for accurate basolateral recycling of CAR after internalization. AP1B knock down MDCK cells missorted CAR from recycling endosomes to the apical surface. In summary, we have characterized the cellular machinery responsible for normal sorting of an adenovirus receptor and illustrated how tissue-specific variations in such machinery result in drastic changes in tissue-susceptibility to adenoviruses.     

Inducible nitric oxide synthase expression inhibition by adenovirus E1A

Nitric oxide (NO) is an antiviral effector of the innate immune system. Viruses that can interfere with NO synthesis may be able to replicate more rapidly than viruses that cannot limit NO synthesis. We show that the adenovirus E1A protein inhibits NO production by decreasing expression of the inducible NO synthase (NOS2). The amino-terminal portion of E1A decreases transactivation of the NOS2 5'-flanking region, limiting the DNA binding activity of NF-kappaB and inhibiting NOS2 expression. E1A is thus able to deactivate a critical component of the host defense against viral infection. Viral inhibition of NO production is a mechanism that may enable certain viruses to evade the host innate immune system.     

Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes

In the present study we have isolated and purified fractions of nonparenchymal liver cells were isolated by collagenase-pronase digestion of the biliary and connective hepatic tissue, which remained undissociated after collagenase perfusion of the liver. Fractionation of the nonparenchymal fractions was then achieved by centrifugal elutriation. Both normal rats and rats with proliferated bile duct-like structures, which were induced either by a 14-day bile duct ligation or by feeding 0.1% alpha-naphthylisothiocyanate for 28 days, were used in these studies. Using a normal rat liver, the fraction richest in biliary epithelial cells was that obtained at a pump flow rate of 36-40 ml/min. In this fraction 1.8-3.8 x 10(6) cells per liver were recovered and up to 55% of them were positive for gamma-glutamyl transpeptidase and cytokeratins 7 and 19, all of which were histochemically or immunohistochemically detected solely in the biliary structures in the intact rat liver. When the nonparenchymal cells were isolated from hyperplastic livers, the number of cells recovered in such a fraction ranged from 12 to 19 x 10(6) per liver, and as many as 60%-85% of the cells expressed phenotypes of biliary epithelial cells. These results indicate that (a) by centrifugal elutriation a fraction of nonparenchymal cells enriched in cells with biliary epithelial phenotypes can be obtained from rat liver and (b) the hepatic hyperplasia induced by biliary obstruction or alpha- naphthylisothiocyanate feeding is a useful and valid strategy for improving both the yield and the purity of the isolated biliary epithelial cells.     

NADPH氧化酶4与非小细胞肺癌上皮间质转化的相关性研究

目的探索烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)的表达与非小细胞肺癌上皮间质转化(EMT)的关系。方法收集2015~2018年在宁夏医科大学总医院经病理确诊的非小细胞肺癌患者石蜡包埋的手术标本和或纤维支气管镜取材组织标本作为肺癌组(29例),选取同期入院行肺叶或肺段切除和或纤维支气管镜取材的肺良性病变组织作为对照组(20例),采用免疫组织化学方法分别检测肺癌组及对照组肺组织标本NOX4、E-钙粘连蛋白(E-Ca)及波形蛋白(Vimentin)的表达情况。分析NOX4表达与肿瘤分期、远隔转移等肿瘤生物学行为间的相互关系。结果EMT相关标志蛋白Vimentin主要表达部位在细胞浆,在肺癌组及对照组肺组织表达的平均光密度值分别为(26.42±8.40)和(11.66±8.30),Vimentin表达高于对照组,且差异具有统计学意义(t=6.078,P<0.001);E-Ca主要表达部位在细胞膜,肺癌组及对照组E-Ca表达的平均光密度值分别为(5.15±6.67)和(14.97±7.68),E-Ca表达低于对照组,差异具有统计学意义(t=-4.819,P<0.001)。NOX4主要表达部位为细胞胞浆及胞核,NOX4在肺癌组及对照组肺组织表达的平均光密度值分别为(29.36±11.60)和(11.27±7.36),肺癌组表达较对照组明显增高,差异有统计学意义(t=6.160,P<0.001)。肺癌组NOX4蛋白的表达与E-Ca的表达呈负相关性(r=-0.612,P=0.001),NOX4的表达与Vimentin的表达呈正相关性(r=0.593,P=0.001);在对照组,NOX4蛋白的表达与E-Ca的表达无相关性(r=0.101,P=0.671),NOX4与Vimentin的表达无相关性(r=0.109,P=0.649)。结论非小细胞肺癌肿瘤组织存在NOX4以及EMT相关标志物Vimentin高表达,E-Ca低表达。NOX4表达与E-Ca表达呈负相关性,与Vimentin表达呈正相关性。肺癌组织内存在明显的EMT,NOX4可能通过氧化-抗氧化的信号途径参与EMT的过程。