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[Cancer Research 64, 6530-6536, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The Duration of Nuclear Extracellular Signal-Regulated Kinase 1 and 2 Signaling during Cell Cycle Reentry Distinguishes Proliferation from Apoptosis in Response to Asbestos

Ziqiang Yuan, Douglas J. Taatjes, Brooke T. Mossman and Nicholas H. Heintz

Department of Pathology, Environmental Pathology Program, and Vermont Cancer Center, University of Vermont College of Medicine, Burlington, Vermont


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asbestos exposure causes activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in lung epithelial cells, the targets of asbestos-associated lung carcinomas. The functional significance of ERK1/2 activation in pulmonary epithelial and mesothelial cells is unclear. Using serum-stimulated mouse alveolar type II epithelial cells as a model for cell cycle reentry, we show that the duration of phospho-ERK1/2 in the nucleus determines cell fate in response to crocidolite asbestos. In response to 10% serum, a proliferative stimulus, phosphorylated ERK1/2 initially accumulated in the nucleus, and reduction of nuclear phospho-ERK1/2 after 2 to 4 hours was followed by expression of cyclin D1 and S-phase entry. Low levels of asbestos (<0.5 µg/cm2) promoted S-phase entry in low (2%) serum through an epidermal growth factor receptor-dependent pathway but did not promote cell cycle progression or induce apoptosis in the presence of high (10%) serum-containing medium. Higher levels of asbestos (1.0 to 5.0 µg/cm2) prolonged the localization of phospho-ERK1/2 in the nucleus in the presence of high serum, impeded S-phase entry, and induced apoptosis in a dose-dependent manner. Immunofluorescence microscopy indicated that the duration of signaling by phospho-ERK1/2 in the nucleus was predictive of cell fate at any concentration of asbestos. After 8 hours of exposure, cells with nuclear phospho-ERK1/2 also were positive for nuclear localization of apoptosis-inducing factor (AIF), an early event in apoptosis. In contrast, asbestos-exposed cells that displayed cytoplasmic phospho-ERK1/2 at 8 hours expressed cyclin D1 and proceeded to S phase. Our studies show that prolonged localization of phospho-ERK1/2 in the nucleus is incompatible with expression of cyclin D1 and is predictive of asbestos-associated cell death by AIF, thereby providing an approach for determining cell fate in asbestos-induced tumorigenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Crocidolite asbestos is an iron-rich mineral fiber that has been linked to inflammation, fibrosis, and carcinogenesis in the lung and pleura (1 , 2) . The precise mechanism by which asbestos elicits pathogenic effects is not known, but chemical and physical properties of crocidolite fibers have been implicated in cellular responses to asbestos. After inhalation, asbestos fibers are deposited primarily at alveolar duct bifurcations, where they induce inflammatory and proliferative responses (3) . The lesions that develop at these sites show increased immunoreactivity for markers of cell proliferation, including incorporation of bromodeoxyuridine (BrdUrd) and nuclear proliferating cell nuclear antigen (3) . Immunostaining also shows that asbestos induces phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) at sites of proliferation in developing fibrotic lesions, suggesting that activation of ERK1/2 plays an important role in the injury and repair responses elicited by asbestos in the lung (4) .

Studies in cell culture models show asbestos is able to activate ERK1/2 for prolonged periods in a dose-dependent manner, whereas the related c-Jun NH2-terminal kinase (JNK) is minimally affected (5) . The activation of ERK1/2 by asbestos is linked to tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and activation of downstream kinase pathways that include MKK1 (6 , 7) , events that eventually lead to activation of the activator protein (AP-1) family of transcription factors (8) . Paradoxically, asbestos can elicit cell proliferation and cell death through activation of the EGFR, ERK1/2, and AP-1 (9 , 10) . At low doses of asbestos (<0.5 µg/cm2), asbestos acts in concert with low levels of growth factors to promote mitogenesis, whereas at higher doses it causes activation of p53 (11) , cell cycle arrest, and apoptosis. The balance between proliferation and cell death may be critical to outcomes of carcinogenesis in lung cancers and mesothelioma (12) .

We are interested in the mechanisms by which environmental oxidants and carcinogenic fibers influence cell cycle progression and cell death. We recently showed that reactive oxygen and nitrogen species (ROS/RNS) alter the dynamics of cell cycle reentry in serum-stimulated lung epithelial cells (13) . Other studies showed that in lung epithelial cells, termination of nuclear ERK1/2 signaling is required for cells to exit G0 and express cyclin D1, a marker for entry into the G1 phase of the cell cycle (14) . Here we used laser scanning cytometry (LSC) and multifluorescence microscopic approaches to examine the responses of individual cells to various doses of asbestos. Our objective was to identify cellular markers that predict cell fate, either entry into the S phase or cell cycle arrest and death, in response to asbestos. These markers then could be used to predict the outcome of activating specific cell-signaling pathways in response to asbestos in various experimental settings. The studies here indicate that expression of cyclin D1 and migration of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus distinguish between proliferation and death in response to asbestos and that expression of these markers is linked to the duration of signaling by ERK1/2 in the cell nucleus.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Synchronization.
Murine type II alveolar C10 cells were propagated in Connaught Medical Research Laboratories medium 1066 with 10% fetal bovine serum (FBS) with 100 units penicillin/100 µg streptomycin/mL (17) . Cells were arrested in G0/G1 by incubation in medium containing 0.2% FBS for 72 hours and induced to reenter the cell cycle by adding DMEM containing 10% FBS, with or without the indicated concentration of crocidolite asbestos (NIEHS reference sample, Research Triangle Park, NC). Asbestos concentrations were based on those used to elicit activation of ERK1/2 in inhalation studies in mice (4) . Cell cycle progression and cells with sub-G1 DNA content were assessed by staining cells with propidium iodide (PI) and evaluating DNA content by flow cytometry (13) . AG1478 was used at concentrations that inhibit phosphorylation of the EGFR as described previously (6) .

Immunoblotting.
Immunoblotting was performed with total cell lysates as described previously (15) , using the ECL system (Amersham, Piscataway, NJ) for signal detection. The following rabbit polyclonal antibodies were obtained from Cell Signaling Technology (Beverly, MA): phospho-ERK1/2, #9102; total ERK1/2, #9101; phospho-JNK, #9251; total JNK, #9252; phospho-p38, #9211; and total p38, #9212. Mouse monoclonal antibodies were used to detect cyclin D1 (sc-450; Santa Cruz Biotechnology, Santa Cruz, CA) and retinoblastoma protein (14001A; Pharmingen, Hauppauge, NY).

Indirect Immunofluorescence Microscopy.
C10 cells were plated on glass coverslips (Corning, Corning, NY), synchronized by serum deprivation for 72 hours, and then treated with complete medium with 10% or 2% FBS, with or without the indicated concentration of asbestos. Coverslips were fixed and stained with specific antibodies using procedures described previously (16, 17, 18) . Confocal immunofluorescence and reflection image microscopy were performed with a Bio-Rad MRC 1024ES confocal scanning laser microscope system (Bio-Rad, Hercules, CA). Cells were stained for phospho-ERK1/2 using a rabbit polyclonal antibody (#9102; Cell Signaling Technologies) with an antirabbit conjugated to Alexa 488 (Molecular Probes, Eugene, OR) as secondary antibody and PI as a nuclear counterstain. For double-staining for phospho-ERK1/2 and cyclin D1, cells first were stained for phospho-ERK1/2 as described previously and then for cyclin D1 (mouse monoclonal sc-450; Santa Cruz Biotechnology) using an antimouse secondary antibody conjugated to Alexa 647 (Molecular Probes) with PI as a nuclear counterstain. For double-staining of phospho-ERK1/2 and AIF, cells were stained first for phospho-ERK1/2 (mouse monoclonal antibody #9106; Cell Signaling Technologies) using a goat antimouse secondary antibody conjugated to Alexa 568 (Molecular Probes) and then AIF (goat polyclonal sc-9416; Santa Cruz Biotechnology) using an rabbit antigoat secondary antibody conjugated to Alexa 488 (Molecular Probes) with TOTO-3 iodide as a nuclear counterstain. Staining for single-stranded DNA (Apostain; Alexis, San Diego, CA) was performed with mouse monoclonal antibody F7–26 with an antimouse secondary antibody conjugated to Alexa 568 (Molecular Probes).

Incorporation of Bromodeoxyuridine.
C10 cells were plated on glass coverslips, synchronized, and then stimulated to reenter cell cycle by adding DMEM with 10% FBS with or without different doses of asbestos. At the same time, 10 µg/mL BrdUrd (BrdU Flow Kit; BD Biosciences, San Jose, CA) were added to the cell culture media. After 20 hours, cells were washed two times with PBS, fixed in cold 80% EtOH for 30 minutes on ice, washed two times with PBS, denatured in 2 mol/L HCl for 20 minutes at room temperature, and then treated with 0.1 mol/L sodium borate [Na2B4O7 (pH 8.5)] for 2 minutes at room temperature. The coverslips then were washed two times with PBS, blocked with 1% BSA/PBS for 30 minutes, stained with FITC-conjugated anti-BrdUrd antibody (#23614L; BD Biosciences) for 1 hour, washed two times with 0.1% BSA, and counterstained with 2.5 µg/mL PI for 30 minutes at 37°C or overnight at 4°C. Slides were examined by confocal microscopy and LSC.

Laser Scanning Cytometry.
LSC was performed with C10 cells on coverslips using a laser scanning cytometer with WinCyte 3.3 data analysis software (CompuCyte, Cambridge, MA) as described previously (19) . To evaluate cells for expression of BrdUrd and cyclin D1, the primary contouring parameter was set on orange-red fluorescence of PI-stained nuclei with detector gain voltages set for a maximum of 75% saturation for the brightest pixel event scanned. Threshold contouring was performed as described previously (10) . Nuclear BrdUrd was detected as green fluorescence (FITC), and nuclear cyclin D1 was detected as far-red fluorescence (goat antimouse Alexa 647). To assess the relationship between expression of nuclear phospho-ERK and nuclear AIF, the primary contouring parameter was set on far-red fluorescence of TOTO-stained nuclei, and nuclear AIF was detected as green fluorescence (antigoat conjugated to Alexa 488), and phospho-ERK was detected as red fluorescence (antimouse conjugated to Alexa 568). Eight thousand to 10,000 cells were scanned in five fields per coverslip, and the LSC instrument automatically did the plotting and scales. LSC confirmed that BrdUrd was incorporated only into S-phase cells and that cyclin D1 persists in the C10 cell nucleus into S phase (data not shown).

Statistical Methods.
Responses to differing doses of asbestos were assessed by ANOVA using orthogonal contrasts to test the significance of a linear trend with increasing dose. P < 0.05 was considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To establish a model for the effects of asbestos on cell cycle reentry, mouse type II lung epithelial cells (C10) cells were synchronized in G0 by incubation in medium containing 0.2% FBS for 72 hours (13) . C10 cells then were induced to reenter the cell cycle by the addition of fresh medium with 10% FBS with or without concentrations of crocidolite asbestos that ranged from 0.2 to 5.0 µg/cm2. To measure S-phase entry, BrdUrd was added to the cultures at the time of serum stimulation, and the number of cells that incorporated BrdUrd into nuclear DNA was evaluated by LSC 20 hours later (Fig. 1A and B)Citation . Cell cycle distributions obtained by LSC were verified by cell cycle analysis of replicate cultures using flow cytometry (data not shown). In 10% FBS, ~60 to 65% of the C10 cell culture entered the S phase by 20 hours, whereas <1.0% entered the S phase in response to 0.2% FBS (Fig. 1A)Citation . In 2% FBS, ~25% of the population entered the S phase, and low levels of asbestos (0.2 and 0.5 µg/cm2) acted in concert with 2% FBS to promote S-phase entry. No dose of asbestos increased the fraction of cells that incorporated BrdUrd in response to 0.2% FBS (data not shown). Doses of asbestos >2.5 µg/cm2 inhibited S-phase entry at any concentration of FBS (Fig. 1ACitation and data not shown).



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  		Fig. 1. Effects of asbestos on S-phase entry. A. C10 cells were arrested in G0 by serum deprivation and then stimulated to reenter the cell cycle in media with either 2% or 10% FBS with or without the indicated concentration of crocidolite asbestos. In cultures treated with 2% FBS, 0.2 and 0.5 µg/cm2 of asbestos promoted S-phase entry (*P < 0.1 for treated versus control). Higher doses of asbestos inhibited entry into the S phase. B. Serum-starved C10 cells were treated with 10% FBS or 10% FBS with asbestos at 1.0 and 5.0 µg/cm2, and expression of cyclin D1 and incorporation of BrdUrd were measured at 20 hours. LSC showed C10 cells that expressed cyclin D1 in the presence of either 1.0 or 5.0 µg/cm2 asbestos also incorporated BrdUrd, suggesting cells capable of expressing cyclin D1 in the presence of any dose of asbestos are able to traverse G1 and enter the S phase.

 
LSC showed S-phase entry at 20 hours, as measured by BrdUrd incorporation, correlated with expression of cyclin D1 at any dose of asbestos (Fig. 1B)Citation . For example, at 5.0 µg/cm2 asbestos, a dose that inhibited 90% of the cells from entering the S phase, >80% of the cells that expressed nuclear cyclin D1 also incorporated BrdUrd (Fig. 1B)Citation , indicating that once cells expressed cyclin D1 they were committed to enter the S phase. These responses are identical to those elicited in a dose-dependent manner by ROS/RNS in C10 cells, indicating that the transition from G0 to G1 represents a major checkpoint for environmental stress during cell cycle reentry (13 , 14) .

The induction of S phase in C10 cells by FBS or low levels of asbestos in the presence of 2% FBS depended on activation of signaling through the EGFR because an inhibitor of EGFR phosphorylation (AG1478) markedly reduced the number of cells that incorporated BrdUrd by 20 hours in response to 2% FBS or 2% FBS and 0.2 µg/cm2 asbestos (Fig. 2A)Citation . The proliferative effects of asbestos on cell cycle progression in 2% FBS did not appear to be related to the generation of ROS from fibers because catalase increased rather than decreased the number of cells entering the S phase in response to low doses of asbestos (Fig. 2B)Citation . Moreover, catalase was not able to rescue cells from cell cycle arrest at higher doses of asbestos (Fig. 2B)Citation . These experiments showed that serum-stimulated C10 cells that complete mitogenic signaling through the EGFR and express cyclin D1 are destined to traverse G1 and enter the S phase. Hence, expression of cyclin D1 in G1 serves as a statistically significant marker (P < 0.05) for cells destined to enter the S phase.



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  		Fig. 2. Induction of cell proliferation by asbestos requires signaling through the EGFR. A. Serum-starved C10 cells were exposed to media containing 2.0% FBS or 2% FBS with either 0.2 or 5.0 µg/cm2 asbestos, and BrdUrd incorporation was measured at 20 hours. Treatment of replicate cultures with AG1478 indicated BrdUrd incorporation required signaling through the EGFR. B. Serum-starved C10 cells were treated with 2.0% FBS and the indicated concentration of asbestos with or without 1000 units/mL catalase. S-phase entry was determined by incorporation of BrdUrd after 20 hours by LSC.

 
We next evaluated the effects of asbestos on induction of apoptosis by staining for single-stranded DNA (Apostain) and determining the number of cells with sub-G1 DNA content by flow cytometry. By 20 hours after serum stimulation, with or without exposure to asbestos, apoptotic C10 cells were detected by staining for single-stranded DNA or flow cytometry (Fig. 3)Citation . In 10% FBS alone, <5% of the cell population was positive for these markers of apoptosis, whereas concentrations of asbestos ≥0.5 µg/cm2 induced apoptotic cell death in a dose-dependent manner (Fig. 3A and B)Citation .



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  		Fig. 3. Asbestos induces cell death in a dose-dependent manner. Serum-starved C10 cells were incubated in medium with 10% FBS with or without the indicated concentration of asbestos. After 20 hours, the fraction of the population that was positive for single-stranded DNA using Apostain (A) or displayed a sub-G1 content as assessed by flow cytometry (B) was determined. A significant linear association was observed between the dose of asbestos and increases in sub-G1 cells (P < 0.001).

 
Quantification of the effects on cell proliferation and apoptosis in serum-starved C10 cells exposed to asbestos in 10% FBS showed that asbestos induced a dose-dependent and linear increase in apoptosis (Fig. 4)Citation but promoted cell proliferation at low doses only in the presence of 2% FBS (Fig. 1A)Citation . These results suggest that asbestos is able to act on cellular pathways that regulate proliferation and apoptosis in C10 cells. Given the ability of asbestos to activate ERK1/2 (6) , we then examined the effects of asbestos on levels of phospho-ERK1/2 in C10 cells at 6 and 12 hours after serum stimulation by immunoblot analysis (Fig. 5)Citation . We previously have shown that the activity of ERK1/2 in C10 cells measured by in vitro kinase assays mimics the level of phospho-ERK1/2 detected by immunoblot analysis (data not shown). The levels of phospho-JNK and p38 also were determined. At 6 and 12 hours, asbestos increased the levels of phospho-ERK1/2 in a dose-dependent manner (Fig. 5Citation , Lanes 2 through 7 and Lanes 8 through 13, respectively). At 6 hours, increased levels of phospho-ERK1/2 correlated with reduced expression of cyclin D1 (Lanes 2 through 7), but at this time reductions in cyclin D1 levels were not reflected in reduced phosphorylation of pRB because cells had not yet reached the G1 restriction point. After 12 hours of asbestos treatment, however, dose-dependent increases in the levels of phospho-ERK1/2 and corresponding inhibition of cyclin D1 expression by asbestos were reflected in reduced levels of phospho-pRB (Lanes 8 through 13), a result that correlated with inhibition of BrdUrd incorporation. Phosphorylation of JNK was not observed until 12 hours and then only at higher doses of asbestos (Lanes 12 and 13). p38 was phosphorylated in response to serum alone, and phospho-p38 was increased approximately twofold at higher doses of asbestos at 6 and 12 hours (Fig. 5Citation , Lanes 2 through 7 and Lanes 8 through 13).



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  		Fig. 4. Inhibition of S-phase entry correlates with cell death by asbestos. Serum-starved C10 cells were incubated in medium with 10% FBS with or without the indicated concentration of asbestos as before, and the number of cells incorporating BrdUrd was compared with the number of cells displaying a sub-G1 DNA content (see Fig. 3Citation ). As for the sub-G1 population, a significant linear association was observed between the dose of asbestos and inhibition of BrdUrd incorporation (P < 0.001).

 


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  		Fig. 5. Prolonged activation of ERK1/2 and inhibition of cyclin D1 expression by asbestos. Serum-starved C10 cells were incubated in medium with 10% FBS with or without the indicated concentration of asbestos as described previously, and at 6 and 12 hours, total cell lysates were prepared and examined for expression of total and phosphorylated mitogen-activated protein kinases, cyclin D1, and pRB by immunoblot analysis. Prolonged expression of phospho-ERK1/2 showed a significant linear association with inhibition of cyclin D1 expression (P < 0.05).

 
The subcellular location of phospho-ERK1/2 in serum-stimulated C10 cells changes as cells progress through the cell cycle, and this change in localization is perturbed by ROS/RNS (14) . To assess the effects of asbestos on the localization of phospho-ERK1/2, serum-starved C10 cells were exposed to media with either 10% FBS or 10% FBS and 5.0 µg/cm2 asbestos for various periods and then stained with an antibody specific for phospho-ERK1/2. By 15 minutes after addition of 10% FBS, increased levels of nuclear phospho-ERK1/2 were observed (Fig. 6Citation , top row). By 2 hours, nearly all of the phospho-ERK1/2 in serum-stimulated C10 cells was located in the cell cytoplasm or plasma membrane, and by 6 hours the levels of phospho-ERK1/2 in these compartments also had decreased but not to the level of serum-starved cells (Fig. 6)Citation . We previously showed that activation of ERK1/2 and its localization in the nucleus for 1 to 3 hours after serum stimulation is required for S-phase entry in C10 cells but that expression of cyclin D1 does not commence until signaling by ERK1/2 in the nucleus is terminated (14) . The immunostaining here shows that the phospho-ERK1/2 detected by immunoblot analysis at 6 and 12 hours in cells treated with 10% FBS alone (Fig. 5Citation , Lanes 2 and 8) was located in the periphery of the cell rather than within the cell nucleus (Fig. 6Citation , top). In cells exposed to asbestos, the subcellular localization of ERK1/2 did not differ from that of cells exposed to 10% FBS alone at 15 and 30 minutes (Fig. 6Citation , bottom row). At later time points, however, phospho-ERK1/2 was observed to persist in the nuclei of a fraction of the cell population for at least 6 hours (Fig. 6Citation , bottom right).



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  		Fig. 6. Asbestos perturbs the intracellular localization of phospho-ERK1/2 during cell cycle reentry. Serum-starved C10 cells were incubated in medium with 10% FBS with or without 5.0 µg/cm2 asbestos as described previously. At the indicated times, cells were fixed and stained with an antibody specific for phospho-ERK1/2. Asbestos caused increased expression of nuclear and cytoplasmic phospho-ERK1/2 in a fraction of serum-stimulated C10 cells at later time points.

 
We next determined the fate of cells that displayed phospho-ERK1/2 in the nucleus for prolonged periods (>4 hours) in response to asbestos. After 20 hours of incubation with serum and any dose of asbestos, apoptotic cells detected by staining for single-stranded DNA had highly condensed nuclei and loss of cytoplasmic components, and phospho-ERK1/2 was not detected by immunostaining (data not shown). This result suggested that those cells that contained nuclear phospho-ERK1/2 at 6 hours represented the apoptotic fraction of the cell population detected at 20 hours by flow cytometry or staining for single-stranded DNA. To evaluate this possibility, cells were stained for phospho-ERK1/2 and AIF, a proapoptotic flavoprotein that migrates from the mitochondria to the nucleus during the initial phases of apoptotic cell death (20, 21, 22, 23) . At 8 hours, for cells stimulated with 10% FBS, AIF and phospho-ERK1/2 were located in the cell cytoplasm (Fig. 7A)Citation , and the morphology of C10 cells was normal as evaluated by phase microscopy (Fig. 7B)Citation . In contrast, C10 cultures treated with 10% FBS and asbestos showed a fraction of the population that was positive by immunostaining for nuclear phospho-ERK1/2 and nuclear AIF (Fig. 7C)Citation . No cells were detected that contained nuclear AIF without nuclear phospho-ERK1/2. Those cells that displayed nuclear phospho-ERK1/2 and nuclear AIF at 8 hours also displayed retraction of cell membranes, cell rounding, and nuclear blebbing (Fig. 7D)Citation , which are morphologic characteristics of apoptosis. Interestingly, we found no relationship between the pattern of deposition of asbestos fibers, persistence of nuclear phospho-ERK1/2, and migration of AIF from the cytoplasm into the nucleus (Fig. 7DCitation and data not shown). The induction of nuclear AIF by asbestos at 8 hours after serum stimulation was dose dependent (Fig. 7E)Citation and correlated with the levels of apoptotic cells at 20 hours, suggesting that those cells with nuclear phospho-ERK1/2 and nuclear AIF at 8 hours represented cells destined to die of apoptosis.



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  		Fig. 7. Prolonged localization of phospho-ERK1/2 in the nucleus correlates with the induction of apoptosis. Serum-starved C10 cells were incubated in medium with 10% FBS with or without 5.0 µg/cm2 asbestos as described previously. At 8 hours, cells were fixed and stained for phospho-ERK1/2 (red signal) and AIF (green signal), with TOTO as a nuclear counterstain (blue signal). In C10 cells stimulated with 10% FBS, AIF and phospho-ERK1/2 were located in the cytoplasm (A), and cell morphology was normal as assessed by phase microscopy (B). In C10 cultures exposed to 10% FBS and asbestos, a fraction of the population contained nuclear phospho-ERK1/2 and nuclear AIF (C, arrows), and phase microscopy (D) showed these cells had morphologic features consistent with the induction of apoptosis. Quantification of the number of AIF-positive cells by LSC at 8 hours showed a significant linear relationship between the dose of asbestos and the fraction of the population with nuclear AIF (P < 0.001).

 
Finally, we evaluated the relationship between cyclin D1 expression and nuclear phospho-ERK1/2 after 8 hours of serum stimulation, with or without exposure to asbestos. As shown in Fig. 8ACitation , C10 cells that expressed cyclin D1 by 8 hours in response to 10% FBS did not contain nuclear phospho-ERK1/2. Moreover, in serum-stimulated cultures treated with asbestos, those cells displaying nuclear phospho-ERK1/2 at 8 hours did not express cyclin D1 (Fig. 8B)Citation . Remarkably, cells adjacent to one another often showed different responses to asbestos. As shown in Fig. 8BCitation (arrows), one cell clearly expressed cyclin D1 and therefore would be predicted to proceed onto S phase. In contrast, a neighboring cell showed evidence of persistent nuclear phospho-ERK1/2 and no expression of cyclin D1 and therefore would be predicted to be positive for nuclear AIF and die of apoptosis. At 5.0 µg/cm2 of asbestos, every C10 cell was observed to be in contact with numerous asbestos fibers (Fig. 8C)Citation , precluding the conclusion that fiber deposition alone governs the duration of nuclear ERK1/2 signaling and therefore cell fate.



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  		Fig. 8. Heterogeneity of cyclin D1 expression in response to crocidolite asbestos. Serum-starved C10 cells were incubated in medium with 10% FBS with or without 5.0 µg/cm2 asbestos as described previously. At 8 hours, cells were fixed and stained for phospho-ERK1/2 (green signal) and cyclin D1 (blue signal), with PI as a nuclear counterstain (red signal). In response to 10% FBS alone (A), cells containing nuclear cyclin D1 (purple signal) did not contain nuclear phospho-ERK (arrow). In the presence of asbestos (B), some cells showed a similar pattern of phospho-ERK expression as cells treated with serum alone (lower arrow), whereas adjacent cells (lower arrow) often were positive for nuclear phospho-ERK1/2 and negative for cyclin D1 (upper arrow). At 5.0 µg/cm2 of asbestos, phase microscopy showed all of the cells were in contact with numerous asbestos fibers (C), and no association between the pattern of fiber deposition and expression of cyclin D1 was noted.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Environmental and carcinogenic insults such as ROS, RNS, and crocidolite asbestos, a fiber capable of generating oxidants (24) , elicit a spectrum of responses that may ultimately govern cell fate. Understanding the mechanisms by which agents such as asbestos influence cell proliferation, cell survival, and cell death is fundamental to developing new approaches for management of debilitating diseases such as fibrosis and cancer. In an effort to understand how asbestos and other agents influence control of the cell cycle, we examined the sensitivity of C10 lung epithelial cells to asbestos at specific stages of the cell cycle. Two phases of the C10 cell cycle appear particularly sensitive to environmental insults: transition from G0 to G1 as assessed by expression of cyclin D1 (13 , 14) and progression through the S phase (data not shown). Here we examined the responses of serum-stimulated C10 cells to various doses of asbestos with the primary aim of identifying early response markers that distinguish between progression into the S phase and induction of cell death. Our results indicate expression of cyclin D1 represents a reliable marker for serum-stimulated cells that are destined to enter the S phase, whereas migration of AIF into the cell nucleus represents a marker for cells destined to die of apoptosis (Fig. 9)Citation . The expression of both of these markers correlated with the subcellular localization of phospho-ERK1/2 over time, supporting the notion that the duration of ERK1/2 signaling in the nucleus is intimately connected to cell fate.



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  		Fig. 9. A model for determining cell fate in response to asbestos during cell cycle reentry. In quiescent C10 cells treated with high serum or high serum plus asbestos, ERK1/2 is activated through an EGFR-dependent pathway and enters the nucleus. In cells treated with mitogens alone, signaling by ERK1/2 in the nucleus decays by 2 to 4 hours, and expression of cyclin D1 ensues, thereby driving cells through G1 and into the S phase. In a fraction of the cells treated with serum and asbestos, signaling by ERK1/2 in the nucleus is prolonged for at least 8 hours. In these cells, AIF relocates to the nucleus, and cell death ensues. At any concentration of asbestos, expression of cyclin D1 identifies those cells fated to enter the S phase, whereas those that express nuclear AIF are fated to undergo apoptosis.

 
Expression of cyclin D1 in response to mitogens in C10 cells requires activation of the ERK1/2 and phosphatidylinositol 3'-kinase/Akt pathways (14) . Activation of ERK1/2 by FBS in C10 cells occurs within 15 minutes and is likely a consequence of tyrosine phosphorylation of EGFR (Fig. 2)Citation . In C10 cells, phosphorylated ERK1/2 generated in response to mitogens relocates rapidly from the cytoplasm to the cell nucleus (Fig. 6)Citation , where the kinase phosphorylates substrates such as c-Fos (25 , 26) that are involved in gene expression and other aspects of cell cycle progression. After several hours, the levels of phospho-ERK1/2 in the nucleus decline, and the levels of the activated kinase in the cell cytoplasm and periphery increase. Phospho-ERK1/2 has been located in cell adhesion complexes (27) , suggesting it plays a role in monitoring adhesion to the extracellular matrix and the integrity of the cytoskeleton, both of which are critical for expression of cyclin D1. Therefore, our studies support the hypothesis that expression of cyclin D1 represents successful integration of signals from multiple sources and thereby represents a marker for quiescent cells that have traversed G0 and entered into the G1 phase of the cell cycle.

Localization of phospho-ERK1/2 in the nucleus and expression of cyclin D1 are mutually exclusive in C10 cells, suggesting that termination of nuclear ERK1/2 signaling is required to exit G0. However, once cells exit G0 and express cyclin D1, they are destined to continue through G1, phosphorylate pRB, and enter into the S phase. Flow cytometry experiments indicate that at any dose of ROS, RNS, or asbestos, the interval between expression of cyclin D1 and S-phase entry is fairly constant in C10 cells (data not shown), suggesting that delays in cell cycle progression elicited by environmental insults during cell cycle reentry are linked to lengthening of the G0 to G1 transition. For example, at a dose of asbestos that inhibited 90% of the cells from entering the S phase here (5.0 µg/cm2), >80% of the cells that expressed cyclin D1 proceeded to S phase by 20 hours (Fig. 1)Citation .

In some models of cell stress, inhibition of caspases does not prevent cell death (23) . AIF is a mitochondrial intermembrane flavoprotein that induces nuclear fragmentation when added directly to nuclei in vitro (28) and therefore may be responsible for some aspects of cell death that occur under conditions in which caspases have been inhibited. Necrosis and apoptosis also may share features in common when ATP levels are low (29) . Although we were able to detect apoptotic C10 cells by either flow cytometry or staining for single-stranded DNA, these methods detect cells in the late stages of apoptosis and therefore cannot be used to ascertain the relationship between activation of selected cell signaling pathways and the induction of apoptosis in individual cells. Here the migration of AIF from mitochondria to the nucleus provided a dose-dependent marker of cell fate in response to persistent nuclear phospho-ERK1/2. Nearly all of the cells that expressed nuclear phospho-ERK1/2 8 hours after serum stimulation in the presence of any dose of asbestos also stained positive for nuclear AIF. Previous work indicates that AIF is released from mitochondria slightly before cytochrome c, supporting the interpretation that it represents an early marker for the induction of apoptosis and, under some conditions, necrosis (29) . Therefore, analysis of those signaling events that correlate with migration of AIF into the nucleus represents a useful approach to examine the responses of individual cells to environmental agents.

Here cell fate in response to asbestos, either entry into the S phase or induction of apoptosis, was strongly correlated with the trafficking of ERK1/2 over time. In response to serum, phosphorylated ERK1/2 entered the nucleus by 15 minutes, and by 2 hours most of the phosphorylated kinase was observed in the cell periphery. It is not known whether nuclear ERK1/2 relocates to the cell periphery under these conditions or whether control of kinase activity in these locations occurs independently. However, sequestration of p42/p44 mitogen-activated protein kinase (i.e., ERK1/2) in the cytoplasm by expression of a catalytically inactive form of a cytoplasmic mitogen-activated protein kinase phosphatase does not prevent phosphorylation of cytoplasmic substrates (30) . Here cells expressing nuclear phospho-ERK1/2 at 8 hours in response to asbestos often showed high levels of phospho-ERK1/2 in the cytoplasm and the nucleus (Fig. 8)Citation , indicating that mechanisms that regulate the kinases in these two locations may be independent of one another. Our present efforts are directed toward understanding how cells in apposition to one another, and presumably staged at the same phase of the cell cycle, show such disparate patterns of ERK1/2 regulation. The ability to elicit proliferation and cell death in a cell autonomous fashion, as well as in adjacent cells that clearly are in contact with multiple fibers, may reflect an important property of asbestos in tumorigenesis.


   ACKNOWLEDGMENTS
 
We thank Jan Schwarz of the Microscopy Imaging Facility for assistance with LSC, Peter Burch for assistance with detection of cyclin D1, and Pamela Vacek for statistical analysis.


   FOOTNOTES
 
Grant support: NIEHS (grant ES09673) and NHLBI (grant PO1 HL67004).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Nicholas H. Heintz, Department of Pathology, HSRF 328, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington VT 05405. Phone: 802-656-0372; Fax: 802-656-8892; E-mail: Nicholas.Heintz{at}uvm.edu

Received 3/19/04. Revised 6/17/04. Accepted 7/13/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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