Animal Cells and Systems Vol. 16, No. 2, April 2012, 95Á103

Differential expression by chrysotile in human bronchial epithelial cells Yoo-Na Seoa, Yong-Jin Leeb and Mi-Young Leea* aDepartment of Medical Biotechnology, SoonChunHyang University, Asan, Chungnam, Korea; bDepartment of Medicine, SoonChunHyung University, Cheonan, Chungnam, Korea; SoonChunHyung Environmental Health Center for Asbestos Related Disease, Cheonan, Chungnam, Korea (Received 11 April 2011; received in revised form 16 August 2011; accepted 27 September 2011)

Asbestos exposure has been known to contribute to several lung diseases named asbestosis, malignant mesothelioma OEUA & MOLECULAR

and lung cancer, but the disease-related molecular and cellular mechanisms are still largely unknown. To examine the CELLULAR effects of asbestos exposure in human bronchial epithelial cells at gene level, the global profile was BIOLOGY analyzed following chrysotile treatment. The microarray results revealed differential gene expression in response to chrysotile treatment. The up- and down-regulated by chrysotile were mainly involved in processes including metabolism, signal transduction, transport, development, transcription, immune response, and other functions. The differential gene expression profiles could provide clues that might be used to understand the pathological mechanisms and therapeutic targets involved in chrysotile-related diseases. Keywords: chrysotile; human bronchial epithelial cells; differential gene expression

Introduction profiles in human bronchial epithelial cell line BEAS- Asbestos is a family of six naturally occurring silicate 2B following chrysotile exposure through microarray fibers exploited commercially for their desirable physi- analysis. The specific analysis for those genes or classes cal properties (Frank 1993). They include serpentine of genes that change their expression during exposure class chrysotile, and an amphibole class named amo- to chrysotile could provide valuable clues in under- site, crocidolite, tremolite, anthophyllite and actinolite standing the molecular pathology of asbestos-related (Rossana et al. 2009). The inhalation of asbestos fibers disease and in searching for novel therapeutic targets. has been reported to cause serious lung diseases, including malignant lung cancer, mesothelioma and asbestosis (Park et al. 2008; Loomis et al. 2010). Materials and methods Asbestos was also reported to cause DNA double Cell culture and cell viability determination strand breaks, chromosomal aberrations, and abnor- The human bronchial epithelial cell line BEAS-2B mal chromosomal segregations (Nymark et al. 2006, (American Type Culture Collection, Rockville, MD) 2007). The molecular mechanism underlying asbestos- was maintained in bronchial epithelial cell basal associated diseases is thought to be very complex and medium (BEBM) supplemented with 2 mL bovine involves several pathways that need to be clarified. pituitary extract, 0.5 mL insulin, 0.5 mL hydrocorti- Chrysotile, also referred to as white asbestos, is the sone, 0.5 mL retinoic acid, 0.5 mL transferrin, 0.5 mL most commonly used asbestos fiber in the building triiodothyronine, 0.5 mL epinephrine, 0.5 mL human industry worldwide (Dopp et al. 2005). It is a soft, epidermal growth factor and 1% penicillin at 378Cin fibrous silicate mineral in the serpentine group of an atmosphere of 5% CO2 (Al-Wadei and Schuller phyllosilicates; as such, it is distinct from other asbesti- 2006). Chrysotile asbestos was suspended in BEBM form minerals in the amphibole group. Its idealized medium, and sonicated and triturated through a 22- chemical formula is Mg3(Si2O5)(OH)4. Chrysotile has guage needle to obtain a homogeneous suspension, and been considered to be a human carcinogen by the added directly to culture dish for 48 hr treatment. To International Agency for Research on Cancer (IARC), determine the cytotoxicity and effects of chrysotile on but the disease-related processes at the molecular and the cell growth, a trypan blue counting assay was cellular level are still unclear. performed. Recently a variety of pathological and epidemiolo- gical research on asbestos-related diseases has been reported; however, the data on the effects of chrysotile Preparation of fluorescent DNA probe and hybridization on the gene expression profile in cultured lung cells are Total RNA was extracted from the cell line using not available. We examined global gene expression the TRI reagent (MRC, OH) according to the

*Corresponding author. Email: [email protected]

ISSN 1976-8354 print/ISSN 2151-2485 online # 2012 Korean Society for Integrative Biology http://dx.doi.org/10.1080/19768354.2011.628696 http://www.tandfonline.com 96 Y.-N. Seo et al. manufacturer’s instructions (Riml et al. 2004). Each Quantitative-real-time RT-PCR total RNA sample (30 mg) was labeled with Cyanine The mRNA levels for the genes of interest in BEAS-2B (Cy5) conjugated dCTP (Amersharm, Piscataway, NJ) following exposure to 20 mg/cm2 chrysotile were ana- by a reverse transcription reaction using reverse lyzed by quantitative real-time RT-PCR using the ABI transcriptase, SuperScript ll (Invitrogen, Carlsbad, Prism 7900 Sequence Detection System (PE Applied California). The labeled cDNA mixture was then Biosystems, www.appliedbiosciences.com). Reaction concentrated using the ethanol precipitation method. mixtures contained 10 pmol/mL of each primer and The concentrated Cy5-labeled cDNA was resuspended 2SYBR Green PCR Master Mix (PE Applied Bio- in 10 mL of hybridization solution (GenoCheck, systems, www.appliedbiosciences.com), which includes Korea). Then labeled cDNA was placed on a Roche the HotStarTaqt DNA polymerase in an optimized NimbleGen Human whole genome 12-plex array buffer, the dNTP mix (with dUTP additive), the SYBRs (Roche NimbleGen, Inc., WI) and covered by a Green I fluorescent dye, and ROX dye as a passive NimbleGen H12 mixer (Roche NimbleGen, Inc., WI). reference. Thermal cycling conditions were 508C for 2 The slides were hybridized for 12 hr at 428C in a MAUI min and 958C for 10 min followed by 40 cycles of 958C system (Biomicro systems, Inc., UT). The hybridized for 30 s and 608C for 30 s, 728C for 30 s. In order to slides were washed in 2 SSC, 0.1% SDS for 2 min,  exclude the presence of unspecific products, a melting 1SSC for 3 min, and then 0.2SSC for 2 min at curve analysis of products was performed routinely after room temperature. The slides were centrifuged at 3000 finishing amplification by a high-resolution data collec- rpm for 20 s to dry (Kim et al. 2009). tion during an incremental temperature increase from 608Cto958C with a ramp rate of 0.218C/s. We then converted real-time PCR cycle numbers to gene Analysis of microarray expression data amounts (ng) on the basis of the equation. Primer All the experiments were conducted using three sepa- sequences were as follows (forward and reverse, respec- rate array sets for control and the two concentrations tively) GMFB: ACA AGG ATA AAC GCC TGG TG, of chrysotiles used. Twelve replicates of the Roche AAT GAA GCG AGG TTG TCG TT; MAPK9: CCC NimbleGen Human whole genome were included per ATG TCC AGG TCT CAG TT, TGC TCT CCT GCT chip. An average of three different 60-base oligonucleo- CTC ACA GA; NFRKB: GGT CTG TCC TGG CTG tides (60-mer probes) represented each gene in the AGA AG, AGA CCT TGG AAC CCT GGA GT; genome. A quality control check was performed for OSP2: CCT AGG GAG CCT CTT CGA CT, TTT GTG each array, which contained on-chip control oligonu- GCA AGA AAG CAC AC; USP18: CCG CTC GAG cleotides. The arrays were analyzed using an Axon ACC CAT GAG CA, CGC GGA TCC GCA CTC CAT GenePix 4000B scanner with associated software CT; SMC3: GAG TAG AAG AAC TGG ACA GA, (Molecular Devices Corp., Sunnyvale, CA). Gene GAT TGT ACC TCA GTT TGC TG. expression levels were calculated with NimbeScan Version 2.4 (Roche NimbleGen, Inc., WI). Relative signal intensities for each gene were generated using the Results Robust Multi-Array Average algorithm. The data were processed based on the median polish normalization Cytotoxicity by chrysotile in human bronchial epithelial method using the NimbeScan Version 2.4 (Roche cell NimbleGen, Inc., WI). The normalized and log trans- The viability of BEAS-2B cells was determined by the formed intensity values were then analyzed using trypan blue dye exclusion test to determine the number GeneSpring GX 7.3.1 (Agilent technologies, CA). of viable cells present in a cell suspension. It is based on Fold-change filters included the requirement that the the principle that live cells possess intact cell mem- genes be present in at least 150% of controls for up- branes that exclude trypan blue dye, following exposure regulated genes and less than 67% of controls for to various concentrations of chrysotile for 48 hr down-regulated genes. For each gene, expression fold- (El-Said et al. 2009). Chrysotile exposure reduced cell changes between groups were derived from the mean viability in a concentration-dependent manner as expression level in three separate replicates. P-values shown in Figure 1. The exposure of BEAS-2B cell to are derived from student t-tests using log2 intensity 5, 10 and 20 mg/cm2 chrysotile resulted in an approxi- data assuming equal variance. Differentially expressed mately 20, 45 and 50% decrease in cell viability, genes in each point were selected using a volcano plot respectively. The concentrations of 5 and 20 mg/cm2 method. chrysotile, which showed approximately 20% and 50% Animal Cells and Systems 97

Figure 1. Effect of chrysotile on the viability of human bronchial epithelial cells. inhibition of cell proliferation, respectively, were used in the subsequent genomic analyses.

Gene expression profiles BEAS-2B cells were treated with 5 and 20 mg/cm2 chrysotile for 48 hr, and total RNA was subjected to Figure 2. Hierarchical cluster image showing the differential microarray analysis. Genes achieving a significant gene expression profiles in human bronchial epithelial cells by difference (PB0.05) in expression compared to the 5 and 20 mg/cm2 of chrysotile treatment. Rows represent control in three separate experiments were analyzed. genes and columns represent samples. Red and green indicate An arbitrary cutoff of 1.5-fold up- or down-regulated up-regulation and down-regulation, relative to the control sample, respectively, and black indicates equal expression in was used to further filter the data. In this analysis, 245 control. genes were up-regulated and 180 genes were down- regulated after 5 mg/cm2 of chrysotile exposure, and 191 Validation of differentially expressed genes by genes were up-regulated and 151 genes were down- quantitative real-time PCR regulated after 20 mg/cm2 chrysotile exposure. A hierarchical clustering was implemented for the visua- Six differentially expressed genes were selected from the lization and biological interpretation of the gene list of differentially expressed genes obtained by expression pattern (Figure 2). The cluster image microarray analysis. Real-time PCR was conducted revealed that two groups were well classified with for GMFB (glia maturation factor, beta), MAPK9 respect to chrysotile exposure. (mitogen-activated kinase 9), NFRKB (nuclear These up- and down-regulated genes revealed that factor related to kappaB binding protein integrin, several genes involved in various functional categories alpha 1), OSBP2 (oxysterol binding protein 2), USP were coordinately induced in the experimental groups 18 (ubiquitin specific peptidase 18) and SMC3 (struc- when compared with those in the control (Table 1). tural maintenance of 3). The GAPDH These genes were involved in processes including gene was used as an endogenous control for all genes. metabolism, signal transduction, transport, transcrip- The results from real-time PCR were consistent with tion, development, immune response, cell differentia- those from the DNA microarray (Figure 4). tion, cell cycling, apoptosis and other functions. Genes showing highly altered expression levels were aligned according to the magnitude of the altered expression. Discussion The differentially expressed genes that were grouped The central questions in this study concern the classes into functional categories and metabolic pathways and magnitude of gene expressions changed in BEAS- based on the KEGG databases are listed in Figure 3, 2B cells following chrysotile exposure. Genes with which shows a comparison of the expression levels expressions altered by at least 1.5-fold up or down between the experimental group and the control. with a significance of PB0.05 were examined. Notable 98 Y.-N. Seo et al.

Table 1. Up- and down-regulated genes based on comparison of gene expression between chrysotile- treated and non- treated human bronchial epithelial cells.

Fold change

5mg/ p- 20mg/ p- Description Gene symbol cm2 value cm2 value

BMetabolism Ubiquitin specific peptidase 18 USP18 2.16 0.039 3.25 0.028 Inositol polyphosphate-4-phosphatase, type I, 107kDa INPP4A 1.49 0.026 2.48 0.049 T-complex 1 TCP1 1.53 0.039 2.47 0.041 Prostaglandin E synthase PTGES 0.85 0.049 2.44 0.033 Zinc finger homeodomain 4 ZFHX4 1.46 0.016 2.29 0.032 Lactate dehydrogenase A LDHA 2.32 0.0007 1.93 0.005 Oxidative-stress responsive 1 OXSR1 1.94 0.020 1.55 0.049 Hydroxyacyl-CoA dehydrogenase HADH 1.40 0.049 1.22 0.043 H1 family, member X H1FX 0.56 0.008 0.57 0.007 Tetraspanin 4 TSPAN4 0.49 0.025 0.43 0.043 Mastermind-like 1 (Drosophila) MAML1 0.52 0.038 0.36 0.032 BSignal transduction Glia maturation factor, beta GMFB 3.02 0.003 2.74 0.049 G protein-coupled receptor 18 GPR18 1.30 0.029 2.73 0.025 START domain containing 8 STARD8 0.91 0.037 2.58 0.011 Serum/glucocorticoid regulated kinase 2 SGK2 1.87 0.016 1.82 0.023 Olfactory receptor, family 3, subfamily A, member 4 OR3A4 2.03 0.048 1.78 0.049 Mitogen-activated protein kinase kinase kinase 7 interacting protein 1 MAP3Kip1 1.89 0.025 2.68 0.014 G protein-coupled receptor 135 GPR135 0.51 0.011 0.78 0.019 Olfactory receptor, family 5, subfamily B, member 17 OR5B17 0.61 0.022 0.41 0.027 BTransport Oxysterol binding protein 2 OSBP2 1.35 0.044 3.21 0.049 Solute carrier family 39 (metal ion transporter), member 11 Slc39a11 1.30 0.049 2.75 0.032 Tubulin, alpha 1b TUBA1B 1.67 0.018 2.25 0.017 Solute carrier family 25, member 35 SLC25A35 1.14 0.012 2.25 0.032 Cell division cycle 37 homolog (S. cerevisiae) CDC37 1.94 0.003 2.20 0.019 SNAP-associated protein SNAPIN 2.05 0.043 2.07 0.049 RAB36, member RAS oncogene family RAB36 1.30 0.049 1.57 0.049 P0M121 membrane glycoprotein (rat) POM121 0.94 0.019 0.62 0.029 Collagen and calcium binding EGF domains 1 CCBE1 0.47 0.004 0.49 0.045 BDevelopment Small proline-rich protein 3 Sprr3 1.38 0.031 2.41 0.039 Mesenchyme homeobox 1 MEOX1 0.54 0.021 0.64 0.012 Transqelin TAGLN 0.57 0.005 0.62 0.012 5-azacytidine induced 1 AZI1 0.48 0.022 0.042 0.025 BTranscription Structural maintenance of 3 SMC3 2.18 0.049 3.02 0.033 Hypothetical LOC389300 2.43 0.016 2.74 0.021 Zinc finger and BTB domain containing 41 homolog ZBTB41 1.15 0.028 2.51 0.012 Ubiquitin-conjugating E2N (UBC13 homolog, yeast) UBE2N 1.52 0.049 2.23 0.042 Forkhead box D4 FOXD4 2.15 0.029 1.67 0.030 PC4 and SFRS1 interacting protein 1 PSIP1 0.44 0.026 0.98 0.049 -binding Rho activating protein ABRA 0.53 0.014 0.49 0.015 BImmune response UL16 binding protein 3 ULBP3 2.01 0.049 2.26 0.032 Twinfilin, actin-binding protein, homolog 2 (Drosophila) TWF2 1.89 0.040 2.13 0.029 Aquaporin 9 AQP9 0.48 0.049 0.42 0.026 Animal Cells and Systems 99

Table 1 (Continued )

Fold change

5mg/ p- 20mg/ p- Description Gene symbol cm2 value cm2 value

BCell cycte Cell division cycle 37 homolog (S. cerevisiae) CDC37 1.94 0.003 2.20 0.019 Tumor protein D52-like 1 TPD52L1 0.90 0.042 0.67 0.049 Baculoviral IAP repeat-containing 5 (survivin) BIRC5 1.20 0.004 0.61 0.049 Anaphase promoting complex subunit 1 ANAPC1 0.76 0.032 0.38 0.014 BApoptosis Beclin 1 (coiled-coil, myosin-like BCL2 interacting protein) BECN1 1.95 0.031 2.01 0.044 CD38 molecule CD38 1.91 0.02 1.95 0.019 Mitogen-activated protein kinase 9 MAPK9 1.72 0.040 1.93 0.049 Tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry TNFRSF14 1.69 0.003 1.33 0.040 mediator) Signal transducer and activator of transcription 1.91 kDa STAT1 0.69 0.021 0.83 0.039 Programmed cell death 10 PDCD10 0.45 0.008 0.56 0.049 Notch homolog 2 (Drosophila) Notch2 0.79 0.038 0.53 0.049 Blnflammatory response Nuclear factor related to kappaB binding protein NFRKB 1.21 0.049 2.70 0.027 Integrin, alpha 1 ITGA1 1.95 0.039 1.06 0.037 Olfactory receptor, family 7, subfamily A, member 17 OR7A17 0.73 0.049 0.61 0.012 Phosphomannomutase 2 PMM2 0.40 0.049 0.37 0.041 BCell proliferated Epithelial membrane protein 2 EMP2 1.89 0.029 2.35 0.033 Thy-1 cell surface antigen THY1 1.27 0.014 1.44 0.023 GLI-Kruppel family member GLI2 GLI2 0.74 0.049 0.42 0.043

Figure 3. classification of genes based on comparison of gene expression between chrysotile-treated and untreated human bronchial epithelial cells. 100 Y.-N. Seo et al.

Figure 4. Validation of the differential expression of genes on exposure to chrysotile by quantitative real-time PCR. The mRNA levels were compared between microarray analysis and real-time PCR. genes in this list were related to processes including (Robin et al. 2009), which include three pathways; metabolism, signal transduction, transport, transcrip- extracellular signal-related kinases (ERKs), p38 kinase tion, development, immune response, cell differentia- and c-Jun N-terminal kinases (JNKs) or stress-acti- tion, cell cycling, apoptosis and other functions. vated protein kinases (SAPK). ERK2, also known as Among these genes, a variety of genes involved in MAPK1, consists of 44- and 42-kDa Ser/Thr kinases, sensory perception of smell such as olfactory receptors and is a part of the Ras-Raf-ERK signal transduction 2 were found. At 5 (IC20) and 20 mg/cm chrysotile cascade often found downstream of growth factor (IC50), OR3A4 was up-regulated, while OR7A17 and receptor activation (Levchenko et al. 2000). ERK is OR5B17 were down-regulated. known to be involved in various cellular proliferation, Olfactory receptors (ORs) are known to constitute differentiation and cell cycle processes in response to a the first element in a biochemical cascade leading to the variety of extracellular signals. Asbestos fiber has been perception and recognition of smells (Fung et al. 1997; reported to selectively induce ERK phosphorylation Xu et al. 1999). The initial event in odor perception is and ERK activity in mesothelial cells, leading to the detection of smells by olfactory receptors, which are apoptosis and cell proliferation (Shukla et al. 2003). located on olfactory sensory neurons in the olfactory The initiation of the ERK cascade in mesothelial cells epithelium of the nose. ORs are seven transmembrane by asbestos was due to autophosphorylation of the domain G-protein-coupled receptors, which are en- EGF receptor. Notably, the magnitude of EGF recep- coded by a large multigene family. OR genes constitute tor expression depended on the class of the asbestos the largest mammalian gene family, with several used. Actually the EGF receptor was enhanced by hundred genes in the and up to 1550 crocidolite and erionite, whereas it was not by chryso- in the rat genome (Rimbault et al. 2009; Robin et al. tile (Faux et al. 2000). Moreover, the induction of AP-1 2009). They are expressed not only in the sensory activity by asbestos was thought to be mediated neurons of the olfactory epithelium, but also in various through the activation of MAPK family, ERK1 and other tissues where their potential functions are largely ERK2. The activation of ERK by proinflammatory unknown. It is supposed that the inhalation of stimuli was also thought to play important roles in chrysotile might affect the olfactory sensory neurons innate immune responses and autoimmunity (Ng and in the olfactory epithelium of the nose, as a variety of Bogoyevitch 2000; Shaul and Seger 2007). OR-related genes were differentially expressed by c-Jun N-terminal kinases (JNKs) (also called chrysotile. MAPK9, SAPK, stress activated protein kinase alpha The MAPK (mitogen-activated protein kinase) II, stress-activated protein kinase JNK2) are pathway-related genes, named MAPK9 and MAP3- exhibiting JUN kinase activity, mitogen-activated pro- K7IP1, were up-regulated 1.93- and 2.68-fold at 20 mg/ tein kinase binding, caspase activator activity and other cm2 chrysotile, respectively. The MAPK cascade is functions (Fusello et al. 2006). MAPK9 is known to be composed of sequential series of phosphorylation involved in positive regulation of cell morphogenesis, Animal Cells and Systems 101 differentiation, regulation of the JNK cascade, positive this study (Nymark et al. 2007). However, some of the regulation of apoptosis and other processes. Up- magnitudes of gene expression alteration by chrysotile regulation of the MAPK pathway by chrysotile here were not great, probably due to different molecular might produce the same result as growth factor responses of BEAS-2B cells to different kinds of stimulation by receptor tyrosine kinases, which were asbestos as reported in EGF receptor activation coupled to the activation of Ras G- and (Faux et al. 2000). Reported mesothelioma-associated malignant transformation, as reported earlier (Shapiro genes named TPD52L1 (tumor protein D52-like 1), 2002). Drugs that selectively down-regulate MAP BIRC5 (baculoviral IAP repeat-containing 5 (survi- kinase cascades were proven to be valuable as ther- vin)), CALU (calumenin) and TXN (thioredoxin) were apeutic agents to treat malignant diseases (Kortenjann detected in the chrysotile-exposed BEAS-2B cells in and Shaw 1995). this study. Other asbestos-related genes named chemo- Glia maturation factor beta (GMFB) is involved in kines, growth-regulated oncogene-alpha (Gro-alpha, nervous system development, angiogenesis and im- CXCL-1), interleukin-8 (IL-8) and activating transcrip- mune functions. The structures of mouse glia matura- tion factor 3 (ATF3), were also detected (Belitskaya- tion factors beta and gamma reveal homologies with Levy et al. 2007). ADF-H (actin depolymerization factor homology) Those genes that were reported to be common in domains and suggest a new function of this protein some epithelial and mesothelial cell lines after exposure family. GFMB was suggested to be involved in an to crocidolite were also detected in this study. Among inhibition process of IFN beta-1a on activation and them, NOTCH2 was about 2-fold down-regulated by apoptosis of LX-2 cells in liver, and as an independent 20 mg/cm2 chrysotile in this examination. Thioredoxin prognostic predictor for ovarian cancer (Li et al. 2010). (TXN) was reported to potentially contribute to GFMB was approximately 3-fold up-regulated by common responses to crocidolite in three epithelial chrysotile-exposed bronchial epithelial cells, and the and mesothelial lung cell lines (Nymark et al. 2007). over-expression was confirmed by both microarray and However, the magnitute of TXN alteration was not real-time PCR great in this study. Asbestos-induced cytotoxicity and Asbestos-induced inflammation and proliferation genotoxicity have been proposed to be associated with are thought to be associated with the development of enhanced oxidative stress via production of reactive asbestos-associated diseases, asbestos-induced redox oxygen species (Kakooei et al. 2007). The genes change, phosphorylation events and in vitro activated involved in redox potential, including peroxiredox- nuclear factor-kappa B (NFKB). Those genes involved in1(PRDX1), were also up-regulated by 20 mg/cm2 in NFKB/IKB pathways, including ubiquitin specific chrysotile. peptidase (USP18), tumor necrosis factor receptor The regulatory and sorting mechanisms involved in superfamily 14 (TNFRSF14) and inhibitor of kappa internalization of toxicants by endocytosis are still light polypeptide gene enhancer in B-cells, kinase beta poorly understood. However, the Rab family of small (IKBKB), were detected. Nuclear factors related to GTP-binding proteins plays crucial roles in regulating kappa B binding protein (NFRKB) were 2.70-times the trafficking pathways, and the Rab family is known up-regulated at 20 mg/cm2 chrysotile. to be directly implicated in regulating endocytic Notably, ubiquitin-specific peptidase 18 (USP18) recycling of cell surface receptors and junctional was enhanced approximately 3.25-fold at 20 mg/cm2 proteins, as well as controlling cytoskeletal rearrange- chrysotile. Mutation in ubiquitin-specific peptidase 18 ment (Chen et al. 2010). The RAB36, RAS oncogene (USP 18) caused hyperactivation of IFN-b signaling family and G-protein-coupled receptor 18 were notably and suppressed STAT4-induced IFN- production, up-regulated by both concentrations of chrysotile. resulting in enhanced susceptibility to bacterial infec- Oxysterol-binding protein 2, a membrane-bound tion (Malakhova et al. 2003). N-Ethyl-N-nitrosourea- protein encoded by OSBP2, contains a pleckstrin induced missense mutation in the USP18 gene resulted homology (PH) domain and an oxysterol-binding in an increased inflammatory response (IL-6, type 1 region. The proteins in different organisms indicate IFN) to salmonella and LPS challenge while para- roles of the gene family in diverse cellular processes doxically reducing IFN production during bacterial including control of lipid metabolism, regulation of infection. Thus, USP18 up-regulated by chrysotile vesicle transport, apoptosis and cell signaling events might be related to inflammation and cytokine-induced (Lehto and Olkkonen 2003). One member of this family death through reduced IFN-levels. in humans, HLM/OSBP2 protein, has recently been Previously identified mesothelioma-associated reported as a potential marker for solid tumor dissemi- genes, human homologs of asbestos- associated murine nation and worse prognosis in these cases (Henriques genes and other asbestos-related genes already present et al. 2003). Therefore, about 3.2-fold up-regulation of in crocidolite-treated lung cells, were also detected in OSBP by chrysotile might be involved in the tumor 102 Y.-N. Seo et al. transformation following exposure to chrysotile, in rat pleural mesothelial cells correlates with carcino- although its precise function in the lung cells is not genicity of mineral fibres. Carcinogenesis 21:2275Á2280. Frank AL. 1993. Global problems from exposure to asbestos. defined yet. Environ Health Perspect 3:165Á167. Even more interesting was the observation that Fung H, Kow YW, Van Houten B, Mossman BT. 1997. SMC3 (structural maintenance ), which Patterns of 8-hydroxydeoxyguanosine formation in DNA is a key component of the nuclear multimeric protein and indications of oxidative stress in rat and human complex named cohesion, was up-regulated approxi- pleural mesothelial cells after exposure to crocidolite 2 asbestos. Carcinogenesis 18:825Á832. mately 3-fold at 20 mg/cm chrysotile. SMC3 was Fusello AM, Mandik-Nayak L, Shih F, Lewis RE, Allen PM, reported to be elevated in human carcinomas, and the Shaw AS. 2006. The MAPK scaffold kinase suppressor of up-regulation of SMC3 expression was also suggested Ras is involved in ERK activation by stress and proin- to be sufficient or permissive to trigger cell transforma- flammatory cytokines and induction of arthritis. J tion through ras-rho/GTPase and cAMP signaling Immunol 177:6152Á6158. Ghiselli G, Liu CG. 2005. Global gene expression profiling of pathway (Ghiselli and Liu 2005). The up-regulation cells overexpressing SMC3. Mol Cancer 4:1Á34. result for OSP2 and SMC3 in the microarray was well Henriques SN, Vasconcellos FM, Pimenta G, Pulcheri WA, consistent with that in real-time PCR. Spector N, Da Costa Carvalho Mda G. 2003. HLM/ In conclusion, the result provides chrysotile-specific OSBP2 is expressed in chronic myeloid leukemia. Int J gene expression patterns, and the biological and Mol Med 12:663Á666. Kakooei H, Sameti M, Kakooei AA. 2007. Asbestos metabolic processes of the genes might be implicated exposure during routine brake lining manufacture. Ind in the chrysotile toxicity. Also this study identifies Health 45:787Á792. several targets for further investigation in relation to Kim SJ, Park HW, Youn JP, HA JM, An YR, Lee CH, Oh asbestos-related diseases. In this regard, this profiling MJ, Oh JH, Yoon SJ, Hwang SY. 2009. Genomic alteration of bisphenol A treatment in the testis of mice. could confer insights into understanding the mechan- Mol Cell Toxicol. 5:216Á221. ism by which chrysotile affects the lung, and the genes Kortenjann M, Shaw PE. 1995. The growing family of modulated by chrysotile could serve as potential MAP kinases: regulation and specificity. Crit Rev Oncog biomarkers for the prediction and diagnosis of chry- 6:99Á115. sotile toxicity. Lehto M, Olkkonen VM. 2003. The OSBP-related protein: a novel protein family involved in vesicle transport, cellular lipid metabolism, and cell signaling. Biochim Biophys Acta 1631:1Á11. Acknowledgements Levchenko A, Bruck J, Sternberg PW. 2000. Scaffold proteins This project was supported by Korea Ministry of Environ- may biophasically affect the levels of mitogen-activated ment as SoonChunHyang Environmental Health Center for protein kinase signaling and reduce its threshold proper- asbestos related disease. ties. Proc Natl Acad Sci USA 97:5818Á5823. Li YL, Cheng XD, Hu Y, Zhou CY, Lu¨ WG, Xie X. 2010. Identification of glia maturation factor beta as an References independent prognostic predictor for serous ovarian Al-Wadei HM, Schuller HM. 2006. Cyclic AMP-dependent cancer. Eur J Cancer 46:2104Á2118. cell type-specific modulation of mitogenic signaling by Loomis D, Dement J, Richardson D, Wolf S. 2010. Asbestos retinoids in normal and neoplastic lung cells. Cancer fibre dimensions and lung cancer mortality among Detect Prev 30:403Á411. workers exposed to chrysotile. Occup Environ Med Belitskaya-Levy I, Hajjou M, Su WC, Yie TA, Tchou-Wong 67:580Á584. KM, Tang MS, Goldberg JD, Rom WN. 2007. Gene Malakhova OA, Yan M, Malakhov MP, Yuan Y, Ritchie KJ, profiling of normal human bronchial epithelial cells in Kim KI, Peterson LF, Shuai K, Zhang DE. 2003. Protein response to asbestos and benzo(a)pyrene diol epoxide ISGylation modulates the JAK-STAT signaling pathway. (BPDE). J Environ Pathol Toxicol Oncol 26:281Á294. Genes Dev 17:455Á460. Chen L, Hu J, Yun Y, Wang T. 2010. Rab36 regulates the Ng DC, Bogoyevitch MA. 2000. The mechanism of heat spatial distribution of late endosomes and lysosome shock activation of ERK mitogen-activated protein through a similar mechanism to Rab34. Mol Membr kinases in the interleukin 3-dependent ProB cell line Biol 27:24Á31. BaF3. J Biol Chem 275:40856Á40866. Dopp E, Yadav S, Ansari FA, Bhattacharya K, von Nymark P, Wikman H, Ruosaari S, Hollme´n J, Vanhala E, Recklinghausen U, Rauen U, Ro¨delsperger K, Shokouhi Karjalainen A, Anttila S, Knuutila S. 2006. Identification B, Geh S, Rahman Q. 2005. ROS-mediated genotoxicity of specific gene copy number changes in asbestos-related of asbestos-cement in mammalian lung cells in vitro. Part lung cancer. Cancer Res 66:5737Á5743. Fibre Toxicol 2:9. Nymark P, Lindholm PM, Korpela MV, Lahti L, Ruosaari S, El-Said WA, Yea CH, Kim H, Oh BK, Choi JW. 2009. Cell- Kaski S, Hollme´n J, Anttila S, Kinnula VL, Knuutila S. based chip for the detection of anticancer effect on HeLa 2007. Gene expression profiles in asbestos-exposed cells using cyclic voltammetry. Biosens Bioelectron epithelial and mesothelial lung cell lines. BMC Genomics 24:1259Á1265. 8:62. Faux SP, Houghton CE, Hubbard A, Patrick G. 2000. Park EK, Hannaford-Turner KM, Hyland RA, Johnson AR, Increased expression of epidermal growth factor receptor Yates DH. 2008. Asbestos-related occupational lung Animal Cells and Systems 103

diseases in NSW, Australia and potential exposure of the Shapiro P. 2002. Ras-MAP kinase signaling pathways and general population. Ind Health 46:535Á540. control of cell proliferation: relevance to cancer therapy. Rimbault M, Robin S, Vaysse A, Galibert F. 2009. RNA Crit Rev Clin Lab Sci 39:285Á330. profiles of rat olfactory epithelia: individual and age Shaul YD, Seger R. 2007. The MEK/ERK cascade: from related variations. BMC Genomics 10:572. signaling specificity to diverse functions. Biochim Bio- Riml S, Schmidt S, Ausserlechner MJ, Geley S, Kofler R. phys Acta 1773:1213Á1226. 2004. Glucocorticoid receptor heterozygosity combined Shukla A, Ramos-Nino M, Mossman B. 2003. Cell signaling with lack of receptor auto-induction causes glucocorti- and transcription factor activation by asbestos in lung coid resistance in Jurkat acute lymphoblastic leukemia injury and disease. Int J Biochem Cell Biol 35:1198Á1209. cells. Cell Death Differ Suppl 1:S65ÁS72. Xu A, Wu LJ, Santella RM, Hei TK. 1999. Role of Robin S, Tacher S, Rimbault M, Vaysse A, Dre´ano S, Andre´ oxyradicals in mutagenicity and DNA damage induced C, Hitte C, Galibert F. 2009. Genetic diversity of canine by crocidolite asbestos in mammalian cells. Cancer Res olfactory receptors. BMC Genomics 10:21. 59:5922Á5926. Rossana B, Carlo C, Paola M, Giovanna Z. 2009. Rocks with asbestos: risk evaluation by means of an abrasion test. Am J Environ Sci 5:500Á506.