Analysis of aberrant methylation in DNA repair genes during malignant transformation of human bronchial epithelial cells induced by cadmium

Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood yet. Aberrant methylation was investigated in order to obtain insight into the DNA repair-related epigenetic mechanisms underlying CdCl(2)-induced malignant transformation of human bronchial epithelial cells (16HBE). Gene expression and DNA methylation were assessed in untreated control cells; 5th, 15th, and 35th passage of CdCl2-treated cells and tumorigenic cells (TCs) from nude mice by using high-performance liquid chromatography, real-time PCR, Western blot analysis, and methylation-specific PCR assay. During Cd-induced malignant transformation, global DNA methylation progressively increased and was associated with the overexpression of the DNA methyltransferase genes DNMT1 and DNMT3a but not DNMT3b. Expression of both the messenger RNA and proteins of the DNA repair genes (hMSH2, ERCC1, XRCC1, and hOGG1) progressively reduced and DNA damage increased with Cd-induced transformation. The promoter regions of hMSH2, ERCC1, XRCC1, and hOGG1 were heavily methylated in the 35th passage transformed cells and the TCs. The DNA demethylating agent 5-aza-2'-deoxycytidine could reverse the Cd-induced global DNA hypermethylation, DNMT hyperactivity, and the silencing of hMSH2, ERCC1, XRCC1, and hOGG1 in a time-dependent manner. The results indicate that DNMT1 and DNMT3a overexpression can result in global DNA hypermethylation and silencing of the hMSH2, ERCC1, XRCC1, and hOGG1 genes. They may partly explain the epigenetic mechanisms underlying the carcinogenesis due to Cd.     

X线修复交叉互补基因1对氢醌致人支气管上皮细胞DNA损伤的保护作用(英文)

目的探索氢醌对人支气管上皮细胞遗传毒性的分子机制,并研究X线修复交叉互补基因1(XRCC1)对氢醌致人支气管上皮细胞DNA损伤是否有保护作用。方法通过RNA干扰敲除XRCC1基因,通过转染重组质粒pEGFP-C1-pU6-dsRNA建立XRCC1缺陷细胞;正常人支气管上皮细胞和转染空载体pEGFP-C1的细胞分别作为正常对照组和载体对照组;3种细胞用不同浓度(10 ～100 μmol.L-1)的氢醌作用4 h,分别进行MTT实验和彗星实验来检测氢醌的毒性。结果 MTT结果显示,不同浓度(10 ～100μmol.L-1)氢醌作用的XRCC1缺陷细胞,490 nm波长处吸光度值低于对照组细胞,提示缺陷细胞的细胞存活率比正常细胞低;彗星实验结果显示,不同浓度的氢醌对XRCC1缺陷细胞DNA损伤比对照组细胞更严重,而2个对照组细胞之间没有明显差异。结论 XRCC1基因在氢醌导致的细胞损伤的修复方面起着重要的作用。     

SWCNTs induced autophagic cell death in human bronchial epithelial cells

Carbon nanotubes are being actively introduced in electronics, computer science, aerospace, and other industries. Thus, the urgent need for toxicological studies on CNTs is mounting. In this study, we investigated the alterations in cellular response with morphological changes induced by single-walled carbon nanotubes (SWCNTs) in BEAS-2B cells, a human bronchial epithelial cell line. At 24 h after exposure, SWCNTs rapidly decreased ATP production and cell viability as well a slight increase in the number of cells in the subG1 and G1 phases. In addition, SWCNTs increased the expression of superoxide dismutase (SOD)-1, but not SOD-2, and the number of cells generating ROS. The concentration of Cu and Zn ions also increased in a dose-dependent manner in cells exposed to SWCNTs. SWCNTs significantly enhanced the release of nitric oxide, interleukin (IL)-6, and IL-8 and up-regulated the expression of chemokine- and cytokine-related genes. Furthermore, the levels of autophagy-related genes, especially the DRAM1 gene, and the autophagosome formation-related proteins, were clearly up-regulated together with an increase of autophagosome-like vacuoles. Based on these results, we suggest that SWCNTs induce autophagic cell death through mitochondrial dysfunction and cytosolic damage in human bronchial epithelial cells.     

塞替派诱发永生化人支气管上皮细胞的恶性转化（一）

目的 研究塞替派对人类支气管上皮细胞的致癌转化效应。方法 利用永生化人支气管上皮细胞（BEAS－2B）为靶细胞模型,以塞替派诱导BEAS－2B细胞的恶性转化,并伴随研究了塞替派转化细胞（EBAS－TE）的增殖动力学、细胞周期和锚着独立生长能力等转化表型特征。结果 经传代和软琼脂培养基筛选,BEAS－TE具有较为稳定的转化表型和细胞生物学特性。结论 塞替派对人支气管上皮细胞具有较强的恶性转化效应。     

环磷酰胺诱发永生化人支气管上皮细胞的恶性转化

研究环磷酰胺 (CP)对人类支气管上皮细胞的致癌转化效应 .实验利用永生化人支气管上皮细胞(BEAS 2B)为靶细胞模型 ,研究CP诱导的BEAS 2B细胞 (BEAS CP)的恶性转化 ,并伴随研究了BEAS CP转化细胞的增殖动力学 ,细胞周期和非贴壁依赖性生长等转化表型特征 .结果显示 ,经传代和软琼脂培养基筛选 ,BEAS CP细胞已具有较为稳定的转化表型和细胞生物学特性 ,可能是具有裸小鼠致瘤性的恶性转化细胞 .结果表明CP对BEAS 2B细胞具有恶性转化效应     

塞替派诱发人支气管上皮恶性转化成瘤细胞的染色体畸变

目的　旨在了解转化细胞在成瘤过程中的细胞遗传学改变。方法　运用染色体G显带技术研究永生化人支气管上皮细胞 (BEAS 2B)恶性转化后的裸小鼠接种成瘤细胞 (BEAS TT)的染色体畸变。结果　瘤细胞在传代早期基本以近二倍体细胞为主 ,随着细胞代龄的增加 ,各肿瘤细胞系的细胞染色体数目变化趋势不同 ,其中BEAS TTa逐渐形成以多倍体细胞为主的细胞群 ,而BEAS TTb ,BEAS TTc则以近二倍体细胞为主份额细胞。核型分析表明 3个瘤细胞系的核型与BEAS TE不同 ,在其基础上有新的染色体 (14号染色体 )丢失和标记染色体 (M4 )的增加。结论　细胞染色体数目不稳定 ,14号染色体的丢失和M4染色体的增加 ,可能与BEAS TE的裸小鼠成瘤性有关     

Procaterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells

β₂ agonists reduce the frequency of exacerbations in patients with bronchial asthma and chronic obstructive pulmonary disease caused by respiratory virus infection. β₂ agonists reduce the production of pro-inflammatory cytokines. However, the inhibitory effects of β₂ agonists on the infection of rhinovirus, the major cause of exacerbations, have not been well studied. To examine the effects of a β₂ agonist, procaterol, on rhinovirus infection and rhinovirus infection-induced airway inflammation, human tracheal epithelial cells were infected with a major group rhinovirus, type 14 rhinovirus. Rhinovirus infection increased viral titers and the content of pro-inflammatory cytokines, including interleukin-1β and interlukin-6, in supernatant fluids and rhinovirus RNA in the cells. Procaterol reduced rhinovirus titers and RNA, cytokine concentrations, and susceptibility to rhinovirus infection. Procaterol reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for type 14 rhinovirus, and the number of acidic endosomes in the cells from which rhinovirus RNA enters into the cytoplasm. Procaterol inhibited the activation of nuclear factor kappa-B (NF-κB) proteins including p50 and p65 in the nuclear extracts, while it increased the cytosolic amount of the inhibitory kappa B-α and intracellular cyclic AMP (cAMP) levels. A selective β₂-adrenergic receptor antagonist ICI 118551 [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] reversed the inhibitory effects of procaterol on rhinovirus titers and RNA, susceptibility to rhinovirus infection, pro-inflammatory cytokines production, ICAM-1 expression, acidic endosomes, and NF-κB. ICI 118551 also reversed the effects of procaterol on cAMP levels. Procaterol may inhibit rhinovirus infection by reducing ICAM-1 and acidic endosomes as well as modulate airway inflammation in rhinovirus infection.     

环磷酰胺及塞替派诱导人支气管上皮细胞恶性转化的超微结构

为了解抗癌化疗药环磷酰胺(CP)及塞替派(TE)诱导的永生化人支气管上皮细胞恶性转化过程中的细胞超微结构的动态变化,采用透射电镜方法观察的细胞模型由对照永生化人支气管上皮细胞（BEAS-2B）,受环磷酰胺（BEAS-CP）和塞替派（BEAS-TE）诱导的恶性转化细胞组成. 发现经CP和TE暴露的细胞可出现细胞死亡,凋亡和转化,它们具有各自的超微结构特征．说明逃逸细胞死亡,凋亡的支气管上皮基细胞呈增生性改变,这种增生性基细胞经过扩增后将成为转化细胞群主体并且具有肿瘤细胞的增生特性.     

Radon-induced reduced apoptosis in human bronchial epithelial cells with knockdown of mitochondria DNA

Radon and radon progeny inhalation exposure are recognized to induce lung cancer. To explore the role of mitochondria in radon-induced carcinogenesis in humans, an in vitro partially depleted mitochondrial DNA (mtDNA) cell line (ρ-) was generated by treatment of human bronchial epithelial (HBE) cells (ρ+) with ethidium bromide (EB). The characterization of ρ- cells indicated the presence of dysfunctional mitochondria and might thus serve a reliable model to investigate the role of mitochondria. In a gas inhalation chamber, ρ- and ρ+ cells were exposed to radon gas produced by a radium source. Results showed that apoptosis was significantly increased both in ρ- and ρ+ cells irradiated by radon. Moreover, apoptosis in ρ- cells showed a lower level than in ρ+ cells. Radon was further found to depress mitochondrial membrane potential (MMP) of HBE cells with knockdown mtDNA. Production of reactive oxygen species (ROS) was markedly elevated both in ρ- and ρ+ cells exposed to radon. The distribution of phases of cell cycle was different in ρ- compared to ρ+ cells. Radon irradiation induced a rise in G2/M and decrease in S phase in ρ+ cells. In ρ- cells, G1, G2/M, and S populations remained similar to cells exposed to radon. In conclusion, radon-induced changes in ROS generation, MMP and cell cycle are all attributed to reduction of apoptosis, which may trigger and promote cell transformation, leading to carcinogenesis. Our study indicates that the use of the ρ- knockdown mtDNA HBE cells may serve as a reliable model to study the role played by mitochondria in carcinogenic diseases.     

环磷酰胺及塞替派诱导人支气管上皮细胞恶性转化的超微结构

为了解抗癌化疗药环磷酰胺 (CP)及塞替派(TE)诱导的永生化人支气管上皮细胞恶性转化过程中的细胞超微结构的动态变化 ,采用透射电镜方法观察的细胞模型由对照永生化人支气管上皮细胞(BEAS- 2 B) ,受环磷酰胺 (BEAS- CP)和塞替派(BEAS- TE)诱导的恶性转化细胞组成 .发现经 CP和 TE暴露的细胞可出现细胞死亡 ,凋亡和转化 ,它们具有各自的超微结构特征 .说明逃逸细胞死亡 ,凋亡的支气管上皮基细胞呈增生性改变 ,这种增生性基细胞经过扩增后将成为转化细胞群主体并且具有肿瘤细胞的增生特性     

Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-l-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.     

DNA repair gene deficiency does not predispose human bronchial epithelial cells to benzo(a)pyrene-induced cell transformation

The development of cost and time-efficient in vitro assays to predict carcinogenicity of chemicals has become a very important direction for toxicological research. In this study, we generated a series of human bronchial epithelial (HBE) cells defect in DNA repair gene excision repair cross-completion 1 (ERCC1), excision repair cross-completion 2 (ERCC2), ataxia-telangiectasia mutated (ATM) and mutS homolog 2 (MSH2), respectively. The inhibition of gene expression was verified by detection of mRNA and protein levels of respective genes. The suppression of these DNA repair genes has no impact on cell proliferation or cell transformation. Although we found that the transgenic HBE cells were more sensitive in benzo(a)pyrene (BaP)-induced DNA damages measured by cytokinesis-block micronucleus (CBMN) assay and comet assay, we failed to observe enhanced effects on induction of cell transformation. HBE cells defect in DNA repair pathways did not exhibit malignantly transformed phenotype up to 20 weeks of BaP treatment, indicating that the deficiency of ERCC1, ERCC2, ATM, or MSH2 alone did not shorten the latency of cell transformation. In contrast, we found that HBE cells expressing H-Ras or c-Myc were transformed 8 or 12 weeks after BaP treatment. These findings demonstrate that silencing of a single DNA repair gene does not confer cells susceptible to chemical-induced cell transformation.     

Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair

The airway epithelium routinely undergoes damage that requires repair to restore epithelial barrier integrity. Cell migration followed by proliferation are necessary steps to achieve epithelial repair. Calcium-sensing receptor (CaSR) is implicated in cell migration and proliferation processes. Thus we hypothesized that CaSR mediates lung epithelial wound repair. We detected CaSR expression in human lung and in well-differentiated human bronchial epithelial cells (HBEC). To test the CaSR functionality, HBEC loaded with fura-2 were stimulated with extracellular Ca²⁺ ([Ca²⁺]_out) which resulted in a concentration-dependent intracellular Ca²⁺ ([Ca²⁺]_i) increase (potency ∼ 5.6 mM [Ca²⁺]_out). Furthermore, increasing [Ca²⁺]_out induced phosphorylation of the extracellular signal-regulated kinase (ERK1/2) which was blocked by siRNA-CaSR and the specific inhibitor of CaSR, NPS2390.Epithelial repair after mechanical injury of differentiated HBEC was a process dependent of [Ca²⁺]_out since it accelerated wound repair and HBEC proliferation being highest at 5 mM [Ca²⁺]_out. Furthermore, U73122 (an inhibitor of phospholipase C (PLC)) and PD 98059 (an inhibitor of ERK1/2) as well as siRNA-CaSR and NPS2390 partially inhibited wound repair and HBEC proliferation. On the other hand, mechanical injury produced an [Ca²⁺]_i wave propagation that was partially inhibited by siRNA-CaSR, NPS2390 and the extracellular Ca²⁺ chelator EGTA, which suggest a link of CaSR between cell-cell communication and wound repair in differentiated HBEC. Our data, for the first time, shows that CaSR plays an important role in airway epithelial repair, which may help to develop novel regenerative therapeutics allowing the rapid repair of lung damaged epithelium.     

Identification of genes induced by carbamazepine in human bronchial epithelial BEAS-2B cells

Some drugs are limited in their clinical application due to their propensity for inducing adverse side effects. We examined some clinical chemotherapeutic agents that have pulmonary toxic effects. Carbamazepine (CBZ) is an antiepileptic agent and its long-term use is associated with interstitial pneumonia, pulmonary fibrosis, and pulmonary infiltration with eosinophilia. CBZ is persistent in the environment and is frequently detected in water systems. A new technique in toxicity screening, “toxicogenomic technology”, represents a useful approach for evaluating the toxic properties of new drug candidates early in the drug discovery process and their potential effects on the environment. To this end, we have examined gene expression profiles in BEAS-2B cells (a human bronchial epithelial cell line) following exposure to CBZ, which induced pulmonary toxicity, by using a human oligonucleotide chip. We identified 518 up- and 496 down-regulated genes whose expression had changed by more than 1.5-fold (p<0.01) following CBZ exposure. Gene Ontology (GO) analysis showed elevation in the expression of genes involved in several key biological processes related to pulmonary toxicity, such as cholesterol metabolism, cell proliferation, and cell cycle regulation. In conclusion, the present study indicates that CBZ exerts its toxicity by modulating mRNA expression in BEAS-2B cells. We suggest that genes expressed by CBZ might serve as a molecular signature, which could be used more widely when implemented in combination with more traditional techniques, for the assessment and prediction of toxicity following CBZ-exposure.     

Differential expression of pro-inflammatory and oxidative stress mediators induced by nitrogen dioxide and ozone in primary human bronchial epithelial cells

Context: NO₂ and O₃ are ubiquitous air toxicants capable of inducing lung damage to the respiratory epithelium. Due to their oxidizing capabilities, these pollutants have been proposed to target specific biological pathways, but few publications have compared the pathways activated.

Objective: This work will test the premise that NO₂ and O₃ induce toxicity by activating similar cellular pathways.

Methods: Primary human bronchial epithelial cells (HBECs, n?=?3 donors) were exposed for 2?h at an air-liquid interface to 3?ppm NO₂, 0.75?ppm O₃, or filtered air and harvested 1?h post-exposure. To give an overview of pathways that may be influenced by each exposure, gene expression was measured using PCR arrays for toxicity and oxidative stress. Based on the results, genes were selected to quantify whether expression changes were changed in a dose- and time-response manner using NO₂ (1, 2, 3, or 5?ppm), O₃ (0.25, 0.50, 0.75, or 1.00?ppm), or filtered air and harvesting 0, 1, 4 and 24?h post-exposure.

Results: Using the arrays, genes related to oxidative stress were highly induced with NO₂ while expression of pro-inflammatory and vascular function genes was found subsequent to O₃. NO₂ elicited the greatest HMOX1 response, whereas O₃ more greatly induced IL-6, IL-8 and PTGS2 expression. Additionally, O₃ elicited a greater response 1?h post-exposure and NO₂ produced a maximal response after 4?h.

Conclusion: We have demonstrated that these two oxidant gases stimulate differing mechanistic responses in vitro and these responses occur at dissimilar times.     

Alteration of transcriptional profile in human bronchial epithelial cells induced by cigarette smoke condensate

Despite the significance of cigarette smoke for carcinogenesis, the molecular mechanisms that lead to increased susceptibility of human cancers are not well-understood. In our present study, the oncogenic transforming effects of cigarette smoke condensate (CSC) were examined using papillomavirus-immortalized human bronchial epithelial cells (BEP2D). Growth kinetics, saturation density, resistance to serum-induced terminal differentiation, anchorage-independent growth and tumorigenicity in nude mice were used to investigate the various stages of transformation in BEP2D cells. Illumina microarray platforms were used to explore the CSC-induced alteration of global mRNA expression profiles of the earlier period and the advanced stage of CSC-treated BEP2D cells. We showed here that a series of sequential steps arose among CSC-treated immortalized human bronchial epithelial cells, including altered growth kinetics, resistance to serum-induced terminal differentiation, and anchorage-independence growth. In the earlier period of CSC treatment, 265 genes were down-regulated and 63 genes were up-regulated, respectively, and in the advanced stage of CSC treatment, 313 genes were down-regulated and 145 genes were up-regulated, respectively. Notably, among those genes, the expression of some of imprinted genes such as IGF2, NDN, H19 and MEG3 were all silenced or down-regulated in CSC-treated cells. These genes reactivated after 5 μM 5-aza-2-deoxycytidine (5-aza-dC) treatment. These results demonstrated that long-term treatment of human bronchial epithelial cells with CSC may adversely affect their genetic and epigenetic integrity and lead to further transformation.     

烟草中甲基亚硝胺吡啶基丁酮诱发人支气管上皮细胞恶性转化的研究

目的研究吸烟致肺癌过程中人类支气管上皮细胞恶变机制,探索一种较快捷的体外研究吸烟致肺癌的途径。方法采用细胞毒性实验确定亚硝胺吡啶基丁酮(NNK)水溶液转化剂量为600μg/ml,应用NNK多次染毒法对人支气管上皮细胞系(16HBE)进行转化,对转化组织和细胞分别进行病理学检查和透射电镜与扫描电镜分析。结果16HBE细胞染毒至第23代,细胞呈恶性形态;可以在软琼脂上形成集落(锚着非依赖性实验为阳性);裸鼠成瘤实验为阳性。对裸鼠成瘤组织进行病理组织学检查,证实为低分化鳞状细胞癌,并取裸鼠成瘤组织原代培养细胞作扫描电镜和透射电镜检查,结果显示为瘤组织培养细胞具有恶性型肿瘤细胞的特征。结论NNK致16HBE细胞恶性转化能力较强,为深入研究人类支气管上皮细胞恶变机制提供了理想的生物模型。