Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cells

Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o-, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% +/- 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development.     

Cigarette smoke decreases the expression of secretory component in human bronchial epithelial cells, in vitro

Epithelial secretory component (SC) is thought to be essential for immunologic protection of the respiratory tract from viral and bacterial infection, since it transports polymeric IgA from the basolateral to the luminal surface of epithelial cells. We have hypothesized that recurrent infection in airways of cigarette smokers is at least partly a consequence of cigarette smoke-induced downregulation of the expression and/or release of SC from airway epithelial cells, subsequently resulting in decreased transcytosis of secretory IgA to the airway lumen. To test this hypothesis, we have cultured human bronchial epithelial cells (HBEC) from surgical tissues and exposed these for 20 minutes to either air or cigarette smoke. Following exposure to cigarette smoke the HBEC cultures were incubated for a further period of up to 24 h, during which time separate cultures were processed by immunocytochemistry for the presence of SC, in a time-dependent manner. The stained HBEC cultures were evaluated by colour image analysis for the percentage of total cells staining for SC. Exposure to cigarette smoke significantly decreased the percentage of total HBEC staining for secretory component from a baseline value (median and interquartile[IQ]1, IQ3) of 35.9% (26.5, 41.6) to 15.7% (8.2, 25.4; p < 0.05) 1 h after exposure, compared with exposure to air. The percentage of cells staining for secretory component were further reduced to 5.3% (3.3, 6.4; p < 0.01), 6 h after exposure, compared to exposure to air. After incubation for 24 h following exposure to cigarette smoke, there was gross cell damage and the cells were not suitable for immunocytochemical analysis. These results suggest that short-term exposure to cigarette smoke may compromise the immune barrier function of the airway mucosa by decreasing the expression and/or release of epithelial SC, thereby decreasing the transcytosis of IgA necessary for inactivating the microbial pathogens in the airway lumen.     

Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.     

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background

We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods

Both the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results

Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion

The current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.     

免疫活性细胞中的非神经元型胆碱能系统

本文对淋巴细胞、巨噬细胞、粒细胞、肥大细胞及胶质细胞等免疫活性细胞中非神经元型胆碱能系统的特点、药理学和病理生理学意义进行了综述。免疫活性细胞,尤其是淋巴细胞中存在完整、独立的胆碱能系统。与神经元型胆碱能系统相比,该系统具有不同特点,如乙酰胆碱(ACh)的合成存在于整个细胞内;ACh可能储存于细胞中某种储存结构,也可能不需储存,而是按需合成并直接释放。免疫活性细胞中的非神经元型胆碱能系统对机体免疫功能及炎症过程可能发挥重要的调控作用。对该系统的深入研究将有助于进一步阐明免疫性疾病、炎症/感染性疾病和神经退行性疾病等的病理生理和发现药物治疗的新靶标。     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Hepatic growth factor (HGF) inhibits cigarette smoke extract induced apoptosis in human bronchial epithelial cells

Low concentrations of cigarette smoke induced DNA damage and repair without leading to apoptosis in human bronchial epithelial cells. Higher concentrations of cigarette smoke, however, could induce either apoptosis or necrosis. The current study demonstrated that 15% cigarette smoke extract (CSE) induced apoptosis as evidenced by DNA content profiling (17.8 ± 2.1% vs 10.2 ± 1.6% of control, p < 0.05), LIVE/DEAD staining (60.2 ± 2.1% viable cells in CSE-treated vs 86.5 ± 2.3% in control cells, p < 0.05), and COMET assay (24.3 ± 0.6% of Apoptotic Index in the cells treated with CSE vs 4.7 ± 0.6% of control, P < 0.05). Hepatocyte growth factor (HGF) significantly blocked the cigarette smoke-induced apoptosis as shown by DNA profiling (10.8 ± 1.5% of CSE + HGF, p < 0.05), LIVE/DEAD staining (78.5 ± 1.2% in CSE + HGF treated cells, p < 0.05), and COMET assay (Apoptotic Index: 10.0 ± 0.8% in CSE + HGF treated cells, P < 0.05). This protective effect of HGF on CSE-induced apoptosis was abolished by PI3K inhibitors, wortmannin and LY294002, and by introduction of the dominant negative AKT into the cells. Furthermore, CSE plus HGF could induce phosphorylation of AKT Thr 308 and the pro-apoptotic protein, BAD. These results suggest that HGF modulates cell survival in response to cigarette smoke exposure through the PI3K/AKT signaling pathway.     

The non-neuronal cholinergic system of human skin.

In human skin both resident and transiently residing cells are part of the extra- or non-neuronal cholinergic system, creating a highly complex and interconnected cosmos in which acetylcholine (ACh) and choline are the natural ligands of nicotinic and muscarinic receptors with regulatory function in both physiology and pathophysiology. ACh is produced in keratinocytes, endothelial cells and most notably in immune competent cells invading the skin at sites of inflammation. The cholinergic system is involved in basic functions of the skin through autocrine, paracrine, and endocrine mechanisms, like keratinocyte proliferation, differentiation, adhesion and migration, epidermal barrier formation, pigment-, sweat- and sebum production, blood circulation, angiogenesis, and a variety of immune reactions. The pathophysiological consequences of this complex cholinergic "concert" are only beginning to be understood. The present review aims at providing insight into basic mechanisms of this highly complex system.     

Cigarette smoke irreversibly modifies glutathione in airway epithelial cells

In patients with chronic obstructive pulmonary disease (COPD), an imbalance between oxidants and antioxidants is acknowledged to result in disease development and progression. Cigarette smoke (CS) is known to deplete total glutathione (GSH + GSSG) in the airways. We hypothesized that components in the gaseous phase of CS may irreversibly react with GSH to form GSH derivatives that cannot be reduced (GSX), thereby causing this depletion. To understand this phenomenon, we investigated the effect of CS on GSH metabolism and identified the actual GSX compounds. CS and H(2)O(2) (control) deplete reduced GSH in solution [Delta = -54.1 +/- 1.7 microM (P < 0.01) and -39.8 +/- 0.9 microM (P < 0.01), respectively]. However, a significant decrease of GSH + GSSG was observed after CS (Delta = -75.1 +/- 7.6 microM, P < 0.01), but not after H(2)O(2). Exposure of A549 cells and primary bronchial epithelial cells to CS decreased free sulfhydryl (-SH) groups (Delta = -64.2 +/- 14.6 microM/mg protein, P < 0.05) and irreversibly modified GSH + GSSG (Delta = -17.7 +/- 1.9 microM, P < 0.01) compared with nonexposed cells or H(2)O(2) control. Mass spectrometry (MS) showed that GSH was modified to glutathione-aldehyde derivatives. Further MS identification showed that GSH was bound to acrolein and crotonaldehyde and another, yet to be identified, structure. Our data show that CS does not oxidize GSH to GSSG but, rather, reacts to nonreducible glutathione-aldehyde derivatives, thereby depleting the total available GSH pool.     

Cigarette smoke extract increases albumin flux across pulmonary endothelium in vitro

Cigarette smoking causes lung inflammation, and a characteristic of inflammation is an increase in vascular permeability. To determine if cigarette smoke could alter endothelial permeability, we studied flux of radiolabeled albumin across monolayers of porcine pulmonary artery endothelium grown in culture on microporous membranes. Extracts (in either dimethylsulfoxide or phosphate-buffered saline) of cigarette smoke in a range estimate of concentrations simulating cigarette smoke exposure to the lungs in vivo caused a dose-dependent increase in albumin flux that was dependent on extracellular divalent cations and associated with polymerization of cellular actin. The effect was reversible, independent of the surface of endothelial cells exposed (either luminal or abluminal), and due primarily to components of the vapor phase of smoke. The effects occurred without evidence of cell damage, but subtle morphological changes were produced by exposure to the smoke extracts. These findings suggest that cigarette smoke can alter permeability of the lung endothelium through effects on cytoskeletal elements.     

Cigarette smoke induces the release of CXCL-8 from human bronchial epithelial cells via TLRs and induction of the inflammasome

COPD is a chronic airway disease associated with inflammation and cigarette smoking. Airway epithelial cells are the first cells exposed to cigarette smoke (CS) and can release CXCL-8 and IL-1β. These cytokines are involved in acute and chronic inflammatory processes in COPD. The aim of this study was to investigate whether toll-like receptors (TLRs) located in/on epithelial cells were involved in cigarette smoke-induced cytokine production. Here we demonstrate that CS induces the release of CXCL-8 and IL-1β from human bronchial epithelial cells (HBE-14o). CS-induced CXCL-8 production was inhibited by an antibody against TLR4 and by inhibitory ODN suggesting the involvement of TLR4 and TLR9. In addition, exposure of HBE-14o cells to TLR4 or TLR9 ligands resulted in the release of CXCL-8 and IL1β. TLR4 and also TLR9 were present on the cell surface and the expression of both receptors decreased after CS exposure. The molecular mechanism of the CS-induced CXCL-8 production by the epithelial cells was further investigated. It was found that P2X7 receptors and reactive oxygen species were involved. Interestingly, the inflammasome activator monosodium urate crystals (MSU) induced the release of CXCL-8 and IL-1β and the caspase-1 inhibitor Z-VADDCB suppressed the CS-induced release of CXCL-8. In addition, CS, CpGODN, lipopolysaccharide and MSU all increased the expression of caspase-1 and IL-1β. In conclusion, our results demonstrate that CS releases CXCL-8 from HBE-14o cells via TLR4 and TLR9 and inflammasome activation. Therefore, inflammasome signaling in airway epithelial cells may play an important role in pathogenesis of diseases like COPD.     

Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells

Background

To demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC) — the particulate fraction of tobacco smoke — were examined.

Methods

The human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs) were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and <5% apoptosis. Changes in gene expression and signaling responses were determined by RT-PCR, western blotting and immunocytofluorescence.

Results

NHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK) kinase (MEK), and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2), demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml) or TNF-α (50 ng/ml) had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls.

Conclusion

The acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.     

Cigarette smoke extract alters genome‐wide profiles of circular RNAs and mRNAs in primary human small airway epithelial cells

As a novel kind of non‐coding RNA, circular RNAs (circRNAs) were involved in various biological processes. However, the role of circRNAs in the developmental process of chronic obstructive pulmonary disease (COPD) is still unclear. In the present study, by using a cell model of COPD in primary human small airway epithelial cells (HSAECs) treated with or without cigarette smoke extract (CSE), we uncovered 4,379 previously unknown circRNAs in human cells and 903 smoke‐specific circRNAs, with the help of RNA‐sequencing and bioinformatic analysis. Moreover, 3,872 up‐ and 4,425 down‐regulated mRNAs were also identified under CSE stimulation. Furthermore, a putative circRNA‐microRNA‐mRNA network was constructed for in‐depth mechanism exploration, which indicated that differentially expressed circRNAs could influence expression of some key genes that participate in response to pentose phosphate pathway, ATP‐binding cassette (ABC) transporters, glycosaminoglycan biosynthesis pathway and cancer‐related pathways. Our research indicated that cigarette smoke had an influence on the biogenesis of circRNAs and mRNAs. CircRNAs might be involved in the response to CSE in COPD through the circRNA‐mediated ceRNA networks.     

Cigarette smoke alters the secretome of lung epithelial cells

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label‐free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.     

香烟提取物活化大鼠支气管平滑肌细胞蛋白激酶C亚型及下调钾通道BKCa、Kv1.5表达

大电导的钙活化钾通道（large—conductance calcium—activated potassium channel,BKCa）和电压依赖性钾通道Kv1．5在气道高反应性的发生机制中具有重要作用。已知吸烟可致气道高反应,但钾通道的变化在其发病中的作用尚需进一步阐明。本文旨在研究香烟提取物（cigarette smoke extract,CSE）对培养的大鼠支气管平滑肌细胞（bronchial smooth muscle cells,BSMCs）钾通道BKCa和Kv1．5表达的直接作用,以及蛋白激酶C（protein kinaseC,PKC）在其中的作用。实验采用原代培养大鼠BSMCs,用5％CSE刺激,免疫印迹检测PKC亚型的表达和转位,半定量RT—PCR、免疫印迹实验检测BKCa和Kv1．5的mRNA和蛋白表达,然后用PKC抑制剂BIM和G6e6983与CSE共作用,检测其对BKCa和Kv1．5的mRNA和蛋白表达的影响。结果显示,5％CSE使PKCε、η、θ发生明显的膜转位,并使BKCa。和Kv1．5的蛋白和mRNA表达明显降低;选择性PKC抑制剂BIM或G6e6983与CSE共同作用,均可使BKCa和Kv1．5的蛋白和mRNA表达部分恢复。上述结果提示,CSE可引起BSMCs的BKCa和Kv1．5表达下调,PKCε、η、θ参与其信号转导。     

Expression and function of the non-neuronal cholinergic system in endothelial cells

Increasing evidence has shown the expression of the non-neuronal cholinergic system in endothelial cells. In the present experiments the expression of choline acetyltransferase (ChAT) was investigated in human endothelial cells by anti-ChAT immunohistochemistry and anti-ChAT immunofluorescence. Positive ChAT immunoreactivity was found in cultures of human umbilical endothelial cells (HUVEC) and a human angiosarcoma cell line (HAEND). In HUVEC and HAEND choline acetyltransferase activity and small amounts of acetylcholine were also detected. Positive ChAT-immunoreactivity was demonstrated in situ in endothelial cells of the human umbilical cord. In addition, in experiments with confocal laser scanning microscopy positive anti-ChAT immunoreactivity was found in situ in endothelial cells of human skin blood vessels. In the first functional experiments with HUVEC acetylcholine appeared to mediate a small facilitatory effect on the expression of intracellular adhesion molecule-1. The present experiments demonstrate the wide existence of ChAT in human endothelial cells. Further experiments are addressed to elucidate the biological role of acetylcholine in the endothelium and possible differences between the different subtypes of endothelial cells.     

Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

Background

Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Cigarette smoking is the leading risk factor for the development of COPD and 25% of asthmatics smoke. Smoking asthmatics have worse symptoms and more frequent hospitalizations compared to non-smoking asthmatics. The degree of neutrophil recruitment to the airways correlates with disease severity in COPD and during viral exacerbations of asthma. We have previously shown that HRV and cigarette smoke, in the form of cigarette smoke extract (CSE), each induce expression of the neutrophil chemoattractant and activator, CXCL8, in human airway epithelial cells. Additionally, we demonstrated that the combination of HRV and CSE induces expression of levels of CXCL8 that are at least additive relative to induction by each stimulus alone, and that enhancement of CXCL8 expression by HRV+CSE is regulated, at least in part, via mRNA stabilization. Here we further investigate the mechanisms by which HRV+CSE enhances CXCL8 expression.

Methods

Primary human bronchial epithelial cells were cultured and treated with CSE alone, HRV alone or the combination of the two stimuli. Stabilizing/destabilizing proteins adenine/uridine-rich factor-1 (AUF-1), KH-type splicing regulatory protein (KHSRP) and human antigen R (HuR) were measured in cell lysates to determine expression levels following treatment. siRNA knockdown of each protein was used to assess their contribution to the induction of CXCL8 expression following treatment of cells with HRV and CSE.

Results

We show that total expression of stabilizing/de-stabilizing proteins linked to CXCL8 regulation, including AUF-1, KHSRP and HuR, are not altered by CSE, HRV or the combination of the two stimuli. Importantly, however, siRNA-mediated knock-down of HuR, but not AUF-1 or KHSRP, abolishes the enhancement of CXCL8 by HRV+CSE. Data were analyzed using one-way ANOVA with student Newman-Keuls post hoc analysis and values of p≤ 0.05 were considered significant.

Conclusions

Induction of CXCL8 by the combination of HRV and CSE is regulated by mRNA stabilization involving HuR. Thus, targeting the HuR pathway may be an effective method of dampening CXCL8 production during HRV-induced exacerbations of lower airway disease, particularly in COPD patients and asthmatic patients who smoke.     

Cigarette smoke extract increases S-adenosylmethionine and cystathionine in human lung epithelial-like (A549) cells

The effect of cigarette smoke extract (CSE) on S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and sulfur amino acid metabolism was examined in human lung epithelial-like (A549) cells exposed to various CSE concentrations (2.5-100%) for 24 or 48 h. Intracellular SAM and SAM/SAH ratio were elevated after exposure to CSE for 48 h. Cell SAH content decreased, but the effect was not consistent. Cellular cystathionine, cysteine, and methionine levels were increased after CSE exposure for 48h. Sub-acute exposure to CSE induced increases in cellular SAM and SAM/SAH ratio. The transsulfuration pathway was likely activated by CSE since cystathionine increased, potentially contributing to the increased total intracellular GSH content.     

Transformed human bronchial epithelial cells (beas-2b) alter the growth and morphology of normal human bronchial epithelial cells in vitro

Normal human bronchial epithelial cells (BE) and adenovirus-12 SV40 hybrid virus transformed, non-tumorigenic human bronchial epithelial cells (BEAS-2B) were cultured for 7 days in a serum free hormone supplemented medium. BE cells after 3 days in culture were exposed to conditioned medium (CM_t) from confluent BEAS-2B cells. By day 7, CM_t-treated BE cells exhibited a lower colony forming efficiency (CFE), fewer cells per colony, and a reduced mitotic index (MI) and BrdU (bromodeoxyuridine) labeling index. CM_t also enhanced the expression of a terminally differentiated squamous phenotype in BE cells. Cell free lysates from BEAS-2B cells (CFL_t) had effects similar to CM_t on the MI and morphology of BE cells. In contrast, CM_t and CFL_t did not inhibit the growth, or alter the morphology of BEAS-2B cells. Conditioned medium from BE cells (CM_n) did not reduce the growth of BEAS-2B cells, and had little effect on the morphology of BE cells. In co-culturesAbbreviations BE normal bronchial epithelial cells - BEAS-2B adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells - CM_n conditioned medium from BE cells - CM_t conditioned medium from BEAS-2B cells - CF_n cell free lysate from BE cells - CFL_t cell free lysate from BEAS-2B cells - BrdU bromodeoxyuridine - KGM keratinocyte growth medium - TGF- transforming growth factor type - NCI-LHC National Cancer Institute-Laboratory of Human Carcinogenesis Contribution No. 2801 from the Pathobiology Laboratory, University of Maryland.