Ultrafine Airborne Particles Cause Increases in Protooncogene Expression and Proliferation in Alveolar Epithelial Cells

Toxicology and Applied Pharmacology 179, 98 –104 (2002) doi:10.1006/taap.2001.9343, available online at http://www.idealibrary.com on

Ultrafine Airborne Particles Cause Increases in Protooncogene Expression and Proliferation in Alveolar Epithelial Cells 1 Cynthia R. Timblin,* Arti Shukla,* Ingrid Berlanger,* Kelly A. BeruBe,† Andrew Churg,‡ and Brooke T. Mossman* *Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405; †Cardiff School of Biosciences, Cardiff University, Cardiff CF1 3US, Wales, United Kingdom; and ‡Department of Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada Received August 31, 2001; accepted November 25, 2001

aerodynamic diameters ⬍ 0.1 ␮m) are more pathogenic in comparison to fine (0.1–2.5 ␮m diameters) or coarse (2.5– 10-␮m diameters) particles after inhalation at equivalent airborne concentrations. However, whether finer particles are more bioreactive in lung due to their intrinsic toxicity is unclear. In work here, we hypothesized that ultrafine particle components of PM stimulate gene expression associated with the development of proliferation and/or cell injury. Based upon recent findings showing that interaction of pulmonary epithelial cells with PM in vitro causes activation of the c-jun kinase/stress-activated protein kinase cascade and transcriptional activation of activator protein-1 (AP-1)-dependent gene expression (Timblin et al., 1998), we examined whether PM would induce increases in expression of jun and fos family members, early response protooncogenes that comprise the AP-1 transcription factor. Results here show dose-related development of proliferation and apoptosis by PM and ultrafine particles in pulmonary epithelial cells. Moreover, our findings demonstrate unique patterns of early response protooncogene and apoptosis-related gene expression at concentrations of PM inducing mitogenesis vs apoptosis.

Ultrafine Airborne Particles Cause Increases in Protooncogene Expression and Proliferation in Alveolar Epithelial Cells. Timblin, C. R., Shukla, A., Berlanger, I., BeruBe, K. A., Churg, A., and Mossman, B. T. (2002). Toxicol. Appl. Pharmacol. 179, 98 –104. Exposure to ambient particulate matter (PM) is linked to increases in respiratory morbidity and exacerbation of cardiopulmonary diseases. However, the important components of PM and their mechanisms of action in lung disease are unclear. We demonstrate the development of dose-related proliferation and apoptosis after exposure of an alveolar epithelial cell line (C10) to PM or to ultrafine carbon black (ufCB), a component of PM. Ribonuclease protection assays demonstrated that increases in mRNA levels of the early response protooncogenes c-jun, junB, fra-1, and fra-2 accompanied cell proliferation at low concentrations of PM whereas apoptotic concentrations of PM caused transient increases in expression of fos and jun family members and dose responsive increases in mRNA levels of receptor-interacting protein, Fas-associated death domain, and caspase-8. Significant increases in steady-state mRNA levels of protooncogenes and apoptosis-associated genes, TNFR-associated death domain, and Fas were also observed after exposure of epithelial cells to ufCB, but not fine carbon black or glass beads, respectively, suggesting that the ultrafine particulate component of PM is critical to its biological activity. © 2002 Elsevier Science (USA) Key Words: particles; lung cancer; cardiopulmonary disease.

MATERIALS AND METHODS Cell cultures. Because the majority of ultrafine and respirable particles are deposited in the alveolar duct region and deep lung after inhalation, C10 cells, a contact-inhibited, murine pulmonary epithelial cell line characterized previously (Malkinson et al., 1997), were used for mechanistic studies. C10 cells were maintained and passaged in CMRL medium containing 10% fetal calf serum, 2 mM glutamine, and antibiotics (GIBCO BRL, Grand Island, NY). For most assays, cells between passages 88 and 98 were grown to confluence, and complete medium was removed before addition of 0.5% serum-containing medium 24 h prior to addition of particles.

Epidemiologic and clinical studies have associated acute and chronic exposures to ambient airborne particulates (PM) with a number of cardiopulmonary disorders and diseases (Committee of EOHA/ATS, 1996; Abbey et al., 1999). PM is a complex mixture of organic and inorganic chemicals, including metals and particulates. The particulate fraction of PM, especially smaller particles, has received recent attention (Kaiser, 2000), since insoluble particles may persist in lung and be intrinsically more bioreactive due to their accumulation over time. In addition, a number of experiments (reviewed in Oberdorster et al., 1997) show that ultrafine particles (defined as having

Preparation and characterization of particulates for in vitro studies. PM 2.5 samples were collected on Teflon filters from the Burlington and Waterbury, Vermont monitoring stations using a Wedding collection apparatus. Within 24 h after collection, PM 2.5 was removed from filters using sonication (4 ⫻ 30 s in 1 ml of pyrogen-free water). Preparations were then aliquoted, lyophilized, and stored at ⫺80°C prior to use. Ultrafine carbon black (ufCB; Monarch 880) and fine carbon black (fCB; Monarch 120) particles (free of organic components such as polycyclic aromatic hydrocarbons) were obtained

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Supported by NIH Grants RO1 HL39469 and RO1 ES/HL09213 to BTM and grants from the Medical Research Council of Canada to A. C. 0041-008X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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AIRBORNE PARTICLES AND GENE EXPRESSION from Cabot Corp. (Billerica, MA). The physicochemical characterization of carbon black and VT PM 2.5 has been previously reported (Timblin et al., 1998; Murphy et al., 1999). Glass beads (1– 4-␮m diameter) were obtained from Particle Information Services, Inc. (Kingston, WA). Particles were baked in a dry oven overnight to eliminate possible endotoxin contamination. All particulates were suspended in Hank’s balanced salt solution at 1 mg/ml and sonicated to disperse aggregates before addition to cultures at final concentrations. For each experiment, duplicate or triplicate dishes of untreated or particulate-exposed cells from each of five PM samples were assayed comparatively, and all experiments were performed in duplicate. Flow cytometry. Flow cytometry was used to determine the cell cycle distributions and amount of apoptosis induced by particulates as described previously (BeruBe et al., 1996). At 24, 48, and 72 h after addition of particles, cells were harvested by trypsinization, resuspended at 1 ⫻ 10 6 cells/ml in a propidium iodide staining solution [50 ␮g/ml propidium iodide, 0.1% Triton X-100, and 32 ␮g/ml RNase A in phosphate buffered saline (PBS)], and incubated at 37°C for 30 min before analysis of 10,000 cells per treatment group and time point. The distribution of cells in the various cell cycle compartments, including cells with a hypodiploid DNA content indicative of apoptosis (sub-G 0/G 1), was determined using a Coulter Epics Elite flow cytometer and appropriate software. Apoptosis in PM 2.5-exposed cells on coverslips was confirmed using an antibody to single-strand DNA (Apostain; Alexis, San Diego, CA) (see below). Cell proliferation assay. Cells were plated at 100,000 cells per 35-mm culture dish in 10% serum-containing medium and allowed to attach overnight. Cells then were exposed to PM 2.5 at 1, 3, or 10 ␮g/cm 2 area of culture dish or to 10 ␮g/cm 2 ufCB. Cells, including sham control dishes, then were trypsinized for determination of total cell numbers at 24 and 48 h thereafter using a Coulter counter. Apostain technique. For detection of apoptosis, confluent cell monolayers grown on glass coverslips were exposed to PM 2.5 for 72 h and then fixed in 100% methanol at ⫺20°C for 24 h. To induce DNA denaturation in situ, cells were heated to 100°C in PBS containing 5 mM MgCl 2 for 5 min and then immersed in ice-cold water for 10 min. After incubation with 40% fetal bovine serum in PBS on ice for 15 min, cells were incubated with a monoclonal antibody to single-stranded DNA (10 ␮g/ml, Apostain [F7-26]; Alexis) for 30 min and then washed twice in PBS and incubated with a horseradish peroxidase-conjugated secondary antibody (15 ␮g/ml, goat anti-mouse IgM; Jackson Laboratories, West Grove, PA) for 30 min at RT. To visualize secondary antibody binding, the peroxidase substrate DAB (Sigma) was used. Cells were washed and mounted on slides in 90% glycerol in PBS for subsequent examination using bright-field light microscopy. To determine numbers of apoptotic cells and total cell numbers per field, four fields were evaluated at 400⫻ magnification on duplicate coverslips. Ribonuclease protection assay. Confluent cultures maintained in 0.5% serum-containing medium for 24 h were exposed to 10 or 50 ␮g/cm 2 PM 2.5, 10 ␮g/cm 2 carbon black, or 20 ␮g/cm 2 glass beads for 8 or 24 h. Because of limited amounts of PM 2.5 available on filters, we also performed an experiment at 2 h using 10 and 25 ␮g/cm 2 PM 2.5, a concentration inducing apoptosis. Total RNA was prepared and quantitated by absorbance at 260 nm. Steady-state mRNA levels of c-jun, junB, junD, c-fos, fra-1, fra-2, and fosB, the ribosomal probe, L32, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were examined using the RiboQuant multi probe ribonuclease protection assay system and the mFos/Jun multiprobe template set (Pharmingen, San Diego, CA) according to the manufacturer’s protocol. A second riboprobe template set, mAPO-3, was used to examine the expression of genes associated with apoptosis. These included caspase-8, FasL, Fas/CD95/ApoI (Fas), FADD (Fas-associated death domain), FAP (Fas-associated phosphatase), FAF (Fasactivating factor), TRAIL (TNF-related apoptosis-inducing ligand), TNFRp55 (tumor necrosis factor receptor p55), TRADD (TNFR-associated death domain), and RIP (receptor-interacting protein). Gels were quantitated using a Bio-Rad phosphoimager (Bio-Rad, Hercules, CA). Results were normalized to expression of the housekeeping gene L32.

FIG. 1. PM 2.5 stimulates changes in the cell cycle distribution of pulmonary epithelial cells using flow cytometry. *p ⱕ 0.05 compared to untreated controls at each time point.

Statistical analyses. All data were examined by analysis of variance using the Student–Newman–Keuls procedure to adjust for multiple pairwise comparisons between groups.

RESULTS

Alterations in cell cycle distributions and proliferation of pulmonary epithelial cells are induced by PM 2.5 and ultrafine carbon black. We have shown previously that PM 2.5 and ultrafine particles are rapidly phagocytized by C10 cells (Shukla et al., 2000). To initially characterize the subsequent responses of pulmonary epithelial cells to PM 2.5, we examined the cell cycle distribution of C10 cells by flow cytometry. Figure 1 shows the cell cycle distributions of confluent sham and particle-exposed cells after 24 and 48 h of exposure to 10 or 50 ␮g/cm 2 PM 2.5 or 10 ␮g/cm 2 ufCB particles. In cells exposed for 24 and 48 h to low doses of PM 2.5 (10 ␮g/cm 2) or ufCB (10 ␮g/cm 2), we observed significant increases ( p ⱕ 0.05) in numbers of cells in the S phase of the cell cycle. In cells exposed to high doses of PM 2.5 (50 ␮g/cm 2), we observed a significant decrease in numbers of cells in the S phase of the cell cycle at 24 h, followed by a significant increase in percentage of cells in S phase at 48 h, suggesting early injury and subsequent unscheduled DNA synthesis that may represent compensatory cell proliferation. At 24 and 48 h, significant decreases in the percentage of cells in G 2/M were also observed in cells exposed to ufCB. In cells exposed for 48 h to

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of PM 2.5 was used in this experiment as subconfluent C10 cells are more sensitive to the cytotoxic effects of PM 2.5. Cells exposed to 1 ␮g/cm 2 PM 2.5 or 10 ␮g/cm 2 ufCB showed significant increases ( p ⬍ 0.05) in total cell numbers in comparison to untreated controls at 48 h. These data indicate that PM 2.5 and ufCB stimulate increases in cell proliferation in pulmonary epithelial cells. In contrast, the addition of glass beads (10 ␮g/cm 2) failed to alter cell proliferation (data not shown). FIG. 2. PM 2.5 and ufCB induce cell proliferation. *p ⱕ 0.05 compared to untreated controls.

high doses of PM 2.5 (50 ␮g/cm 2), significant elevations ( p ⱕ 0.05) occurred in the percentage of cells in the sub-G 0/G 1 fraction, representing apoptotic or necrotic cells, accompanied by corresponding decreases in the number of cells in the G 0/G 1 phase of the cell cycle. These patterns were also observed after exposure of cells to 10 ␮g/cm 2 ufCB at both time points (Fig. 1). The addition of 10 ␮g/cm 2 glass beads caused no alterations in the percentages of cells in the sub-G 0/G 1 fraction or in S phase (data not shown). To confirm that PM caused cell proliferation, we examined total cell numbers of subconfluent C10 cells exposed to a dose range of PM 2.5 (1, 3, and 10 ␮g/cm 2) or 10 ␮g/cm 2 ufCB for 24 and 48 h (Fig. 2). A lower dose range

Patterns of fos and jun gene expression in pulmonary epithelial cells exposed to PM 2.5. Previously, we demonstrated that PM 2.5 increased AP-1 transcriptional activity in a rat alveolar type II epithelial cell line (Timblin et al., 1998). To determine whether upregulation of AP-1 family genes occurred in epithelial cells exposed to PM 2.5, we examined patterns of fos (c-fos, fosB, fra-1, and fra-2) and jun (c-jun, junB, and junD) gene expression in C10 cells exposed to low (proliferative) and high (apoptotic) doses of PM 2.5 using a ribonuclease protection assay (Fig. 3). In cells exposed to high doses of PM 2.5 (25 or 50 ␮g/cm 2), we observed early (2 h) but transient increases ( p ⱕ 0.05) in mRNA levels of all fos family genes that returned to control levels by 8 and 24 h (Fig. 3B). In contrast, in cells exposed to low doses of PM 2.5 (10 ␮g/cm 2), we observed significant increases ( p ⱕ 0.05) in fra-1 and

FIG. 3. Increases in fos/jun family members by PM 2.5 as demonstrated by ribonuclease protection assays (RPA). (A) Representative autoradiograph of a RPA used to examine changes in levels of specific mRNAs in C10 cells exposed to particulates. (B) Quantitation of autoradiograms from RPAs showing PM 2.5-induced dose-related changes in steady-state mRNA levels of fos family genes. (C) Quantitation of autoradiograms from RPAs demonstrating PM 2.5-induced dose-related changes in steady-state mRNA levels of jun family genes. Note that 2-, 8-, and 24-h data were evaluated on separate gels. *p ⱕ 0.05 compared to untreated controls at each time point.

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FIG. 4. Photomicrographs of C10 cells indicating Apostain-positive nuclei (arrows) in sham control and PM 2.5-exposed cells after 72 h. Note black granular particulates in area of Apostain-positive cells.

fra-2 mRNA levels after 24 h of exposure to PM 2.5. Significant increases in c-fos mRNA levels were not observed at any time point in cells exposed to low doses of PM 2.5. A similar pattern of jun family gene expression was observed in cells exposed to high vs low concentrations of PM 2.5 (Fig. 3C). High amounts of PM 2.5 (25 and 50 ␮g/cm 2) induced early (2 h) and transient increases ( p ⱕ 0.05) in c-jun, junD, and junB mRNA levels, which returned to levels seen in sham groups at 8 and 24 h. In cells exposed to PM 2.5 at 10 ␮g/cm 2, we also observed increases in junB mRNA levels at 2 and 24 h and a delayed (24 h) increase in c-jun mRNA levels ( p ⱕ 0.05). PM 2.5-induced expression of apoptosis and apoptosis-related genes. To confirm the endpoint of PM-induced apoptosis in C10 cells, we measured apoptosis using the Apostain technique in cells exposed to PM 2.5 for 72 h (Fig. 4, Table 1). These studies showed that PM 2.5 caused dose-related increases

in numbers of apoptotic cells that were localized to areas of particle deposition (Fig. 4). To identify changes in gene expression that may mediate the apoptotic responses of epithelial cells to PM 2.5, we examined steady-state mRNA levels of genes comprising the TNF receptor and Fas/FasL apoptotic pathways. No changes in apoptosis-associated genes at either concentration of PM were observed at 8 h (data not shown). As shown in Fig. 5, exposure of C10 cells to 10 and 50 ␮g/cm 2 PM 2.5 for 24 h induced significantly increased ( p ⱕ 0.05) dose–response expression of RIP, FADD, and caspase 8. These data indicate that apoptosis-related gene expression is induced in epithelial cells by PM 2.5 in a dose-related fashion.

TABLE 1 Apoptosis in C10 Cells Exposed to PM 2.5 for 72 h as Detected Using an Antibody to Single-Stranded DNA (Apostain) Groups

Percentage a

Control PM 10 b PM 50 GB 10 d

1 3 16 c 6

a Mean of five fields per slide per two slides. Numbers of positively staining cells per total cells per field were counted. b ␮g/cm 2 dish. c p ⱕ 0.05 compared to unexposed sham controls. d GB, glass beads.

FIG. 5. Quantitation of autoradiograms from ribonuclease protection assays showing PM 2.5-induced changes in steady-state mRNA levels of apoptosis-associated genes at 24 h. *p ⱕ 0.05, dose–response trend.

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FIG. 6. Quantitation of autoradiograms from ribonuclease protection assays showing particle-induced changes in steady-state mRNA levels of fos and jun family genes (A) and apoptosis-associated genes (B) at 8 h. *p ⱕ 0.05 compared to untreated controls.

Patterns of fos and jun gene expression in pulmonary epithelial cells exposed to ultrafine and fine carbon black or glass beads. To determine whether particle size is important in the induction of early response protooncogenes, we examined patterns of gene expression in C10 cells exposed to low (10 ␮g/cm 2) doses of each particle for 8 h (Fig. 6). In cells exposed to ufCB, we observed significant increases ( p ⱕ 0.05) in mRNA levels of all fos and jun genes. In contrast, in cells exposed to fCB, we observed less striking, but significant increases ( p ⱕ 0.05) in mRNA levels of fra-1 only. Glass beads (10 ␮g/cm 2) had no effect on protooncogene expression. As shown in Fig. 6B, exposure of C10 cells to 10 ␮g/cm 2 ufCB, but not glass beads, induced significantly increased ( p ⱕ 0.05) expression of TRADD and Fas. These data demonstrate that ultrafine particles stimulate changes in expression of genes linked to both proliferative and apoptotic pathways. DISCUSSION

Airborne particulate matter is a focus of health concern as acute exposures to high levels of ambient air pollution are associated with increased admissions to hospitals, aggravation of asthma, particularly in children, and respiratory morbidity (Committee of EOHA/ATS, 1996; Abbey et al., 1999; Kaiser, 2000). Moreover, elevated risks of lung cancer, chronic respiratory disease, and death rates have been predicted as consequences of chronic exposures to PM. A pivotal cell type affected in asthma, bronchitis, pulmonary fibrosis, and lung cancer is the epithelial cell of the respiratory tract. In a contactinhibited murine alveolar cell line (C10), we observed doserelated and significant increases in the proportions of cells in S phase and apoptosis, respectively, after exposures to PM 2.5. To confirm that increases in S phase reflected cell proliferation, we examined total cell numbers after exposure of subconfluent C10 cells to low concentrations, i.e., ⱕ10 ␮g/cm 2, of PM 2.5.

These experiments showed that logrithmically growing cultures were exquisitely sensitive to low concentrations of PM 2.5, i.e., 1 ␮g/cm 2. At higher concentrations, PM 2.5 induced significant increases in numbers of apoptotic cells that were also accompanied by increases in proportions of cells in S phase. This may reflect accumulation of cells arrested in S phase prior to the development of PM-induced apoptosis. Alternatively, compensatory cell proliferation often follows epithelial cell damage, and a balance between proliferation and apoptosis is thought to be a key regulatory event in the development of a number of proliferative diseases, including pulmonary fibrosis and cancer (Mossman and Churg, 1998; Manning and Patierno, 1996). Previous work from our laboratory has demonstrated that exposure of pulmonary epithelial cells to PM 2.5 induces transcriptional activation of AP-1-dependent gene expression (Timblin et al., 1995). To further examine the mechanisms underlying PM 2.5-induced proliferation and apoptosis, we focused here on the time frame of expression of jun (c-jun, junB, and junD) and fos (c-fos, fra-1, fra-2, and fosB) family members that comprise the AP-1 transcription factor. These studies were further merited since different Jun/Jun homodimers and Jun/Fos heterodimers exhibit distinct transcriptional properties that are related causally to cell proliferation and apoptosis in a number of cell types (Angel and Karin, 1991; Ubeda et al., 1999). Our data show unique patterns of expression of jun and fos family members at concentrations of PM 2.5 eliciting epithelial cell proliferation vs apoptosis. At concentrations of PM 2.5 inducing cell proliferation, increases in steady-state mRNA levels of junB were early and transient (2 h) while increases in levels of c-jun, fra-1, and fra-2 mRNAs were more protracted (24 h). A causal link between expression of these genes, cell proliferation, and carcinogenesis has been established in sev-

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eral model systems. For example, we have demonstrated that overexpression of c-jun in tracheal epithelial cells leads to increased cell proliferation and morphological transformation (Timblin et al., 1995). Others have reported occasional immunocytochemical localization of cJun in basal cells of normal human bronchial epithelium, but dramatic increases in hyperplastic and metaplastic lesions in patients developing lung cancers (Szabo et al., 1996). Thus, aberrant expression of cJun appears to be important in proliferation and carcinogenesis in pulmonary cells. We have shown previously that increased phosphorylated cJun is observed in pulmonary epithelial cells exposed to PM 2.5 (Timblin et al., 1998), confirming that increased mRNA levels of jun/fos family members as reported here result in increased protein levels. In cells exposed to apoptotic doses of PM 2.5 (25 and 50 ␮g/cm 2), striking increases in steady-state mRNA levels of all jun and fos family genes were observed at 2 h, returning to levels comparable to those in sham cells by 8 h. To further examine the mechanisms underlying PM-induced cell injury, we next focused on the expression of genes important in TNF receptor and Fas pathways of apoptosis. PM 2.5 induced significant ( p ⱕ 0.05) dose-related increases in steady-state mRNA levels of RIP, FADD, and caspase 8. Less strikingly, elevated mRNA levels of TNFR p55, TRADD, Fas, and FasL were also seen. Others have shown in pulmonary epithelial and a variety of other cell types that signaling through the TNF receptor pathway results in either apoptosis or cell survival/proliferation (Stewart et al., 1995; Pryhuber et al., 2000; Beg and Baltimore, 1996). Moreover, stimulation of the Fas–Fas ligand pathway in pulmonary epithelium is causally involved in the development of apoptosis and bleomycin-induced pulmonary fibrosis (Kuwano et al., 1999). To determine whether the ultrafine component of PM 2.5 is uniquely responsible for the biological responses of epithelial cells to PM 2.5, we examined proliferation, apoptosis, and patterns of fos and jun family gene expression in cells exposed to ultrafine vs fine or coarse particles. Our data show both changes in cell cycle alterations and increases in fos and jun family gene expression by ufCB. The early induction (2 h) of protooncogenes was identical to the patterns of mRNA levels observed in cells exposed to high (25–50 ␮g/cm 2) concentrations of PM. In contrast, only elevated mRNA levels of fra-1 were observed in cells exposed to fCB, and glass beads, a coarse particle, had no effect on fos and jun gene expression. Since ufCB was more toxic than PM 2.5 preparations, we examined expression of apoptosis-related genes of 8 h after addition of particles, including glass beads as a negative control. In contrast to glass beads, significant increases ( p ⱕ 0.05) in TRADD and Fas were seen with ufCB exposures. Moreover, trends in elevated levels of other TNF- and Fasrelated death pathway members were observed. These observations suggest that the ultrafine fraction of PM 2.5 is critical to both protooncogene expression and apoptotic outcomes of exposure.

The overall pattern of increases in early response gene expression by PM 2.5, which can be attributed in studies here to the ultrafine particulate fraction, resembles that seen with the pathogenic particulates, crystalline silica, and asbestos (Shukla et al., 2001; Heintz et al., 1993). However, some differences exist. Elevations in JNK signaling are more striking and uniquely observed at proliferative concentrations of PM 2.5 or silica (Timblin et al., 1998; Shukla et al., 2001). Stimulation of the NF-␬B pathway by PM 2.5, silica, or asbestos has been observed in a number of models and may also be linked to increased cell survival (Shukla et al., 2001; Hubbard et al., 2001; Janssen et al., 1997). In contrast, the ERK activation pathway, associated with both injury and survival, is more striking and persistent in both C10 and RPM cells after exposure to asbestos (Zanella et al., 1996; Buder-Hoffmann et al., 2001; Shukla et al., 2001). Induction of fos and jun (AP-1) family members as a result of MAPK cascades may then lead to activation of fra-1, an AP-1-dependent gene that appears to be related directly to cell cycle changes by oxidant stress (Shukla et al., 2001). Future work in our laboratory is focusing on transfection approaches to determine the relative contribution of individual protooncogenes to AP-1 dimerization and phenotypic alterations by pathogenic particulates in pulmonary epithelial cells. REFERENCES Abbey, D. E., Nishino, N., McDonnell, W. F., Burchette, R. J., Knutsen, S. F., Beeson, W. L., and Yang, J. X. (1999). Long-term inhalable particles and other air pollutants related to mortality in nonsmokers. Am. J. Respir. Crit. Care Med. 159, 373–382. Angel, P., and Karin, M. (1991). The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim. Biophys. Acta 1072, 129 – 157. Beg, A. A., and Baltimore, D. (1996). An essential role for NF-␬B in preventing TNF-␣-induced cell death. Science 274, 782–784. BeruBe, K. A., Quinlan, T. R., Fung, H., Magae, J., Vacek, P., Taatjes, D. J., and Mossman, B. T. (1996). Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos. Am. J. Respir. Cell Mol. Biol. 15, 141–147. Buder-Hoffman, S., Palmer,, C., Vacek, P., Taatjes, D., and Mossman, B. (2001). Different accumulation of activated extracellular signal-regulated kinases (ERK1/2) and role in cell-cycle alterations by epidermal growth factor, hydrogen peroxide, or asbestos in pulmonary epithelial cells. Am. J. Respir. Cell Mol. Biol. 24, 405– 413. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society (1996). Health effects of outdoor air pollution. Am. J. Respir. Crit. Care Med. 153, 3–50. Heintz, N. H., Janssen, Y. M. W., and Mossman, B. T. (1993). Persistent induction of c-fos and c-jun expression by asbestos. Proc. Natl. Acad. Sci. USA 90, 3299 –3303. Hubbard, A. K., Timblin, C. R., Rincon, M., and Mossman, B. T. (2001). Use of transgenic luciferase reporter mice to determine activation of transcription factors and gene expression of fibrogenic particles. Chest 120, 24S–25S. Janssen, Y. M. W., Driscoll, K. E., Howard, B., Quinlan, T. R., Treadwell, M., Barchowsky, A., and Mossman, B. T. (1997). Asbestos causes translocation of p65 protein and increases NF-␬B DNA binding activity in rat lung epithelial and pleural mesothelial cells. Am. J. Pathol. 151, 389 – 401.

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