Chronic Exposure to Particulate Chromate Induces Spindle Assembly

Oct 6, 2006 - Health, UniVersity of Southern Maine, 96 Falmouth St., Portland, Maine 04104-9300, and Department of. Applied Medical Science, UniVersit...
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Chem. Res. Toxicol. 2006, 19, 1492-1498

Chronic Exposure to Particulate Chromate Induces Spindle Assembly Checkpoint Bypass in Human Lung Cells Sandra S. Wise,†,‡ Amie L. Holmes,†,‡ Hong Xie,†,‡ W. Douglas Thompson,‡,§ and John Pierce Wise, Sr.*,†,‡,§ Wise Laboratory of EnVironmental and Genetic Toxicology, Maine Center for Toxicology and EnVironmental Health, UniVersity of Southern Maine, 96 Falmouth St., Portland, Maine 04104-9300, and Department of Applied Medical Science, UniVersity of Southern Maine, Portland, Maine 04104-9300 ReceiVed June 23, 2006

One of the hallmarks of lung cancer is chromosome instability (CIN), particularly a tetraploid phenotype, which is normally prevented by the spindle assembly checkpoint. Hexavalent chromium [Cr(VI)] is an established human lung carcinogen, and Cr(VI) induces tumors at lung bifurcation sites where Cr(VI) particles impact and persist. However, the effects of Cr(VI) on the spindle assembly checkpoint are unknown and little is known about prolonged exposure to particulate Cr(VI). Accordingly, we investigated particulate Cr(VI)-induced bypass of the spindle assembly checkpoint after several days of exposure in WHTBF-6 cells. We found that lead chromate indeed induces spindle assembly checkpoint bypass in human lung cells, as 72, 96, and 120 h treatments with 0.5 or 1 µg/cm2 lead chromate induced significant increases in the percentage of cells with aberrant mitotic figures. For example, treatment with 1 µg/cm2 lead chromate for 96 h induced 11, 12.3, and 14% of cells with premature anaphase, centromere spreading and premature centromere division, respectively. In addition, we found a disruption of mitosis with more cells accumulating in anaphase; cells treated for 96 h increased from 18% in controls to 31% in cells treated with lead chromate. To confirm involvement of the spindle assembly checkpoint, Mad2 expression was used as a marker. Mad2 expression was decreased in cells exposed to chronic treatments of lead chromate, consistent with disruption of the checkpoint. We also found concentration- and time-dependent increases in tetraploid cells, which continued to grow and form colonies. When cells were treated with chronic lead alone there was no increase in aberrant mitotic cells or polyploidy; however, chronic exposure to a soluble Cr(VI) showed an increase in aberrant mitotic cells and polyploidy. These data suggest that lead chromate does induce CIN and may be one mechanism in the development of Cr(VI)-induced lung cancer. Introduction Hexavalent chromium [Cr(VI)]1 is a well-established human lung carcinogen; however, its carcinogenic mechanisms are uncertain. The most potent carcinogenic form of Cr(VI) is the water-insoluble or particulate form. Cr(VI)-induced tumors and increased tissue levels of Cr occur at lung bifurcation sites where Cr(VI) particles are likely to impact and persist, which strongly implicates particulate exposure in the etiology of these tumors (1, 2). Epidemiologic studies report a higher cancer risk for particulate-Cr(VI)-exposed workers (3), and experimental studies show that only particulate Cr(VI) compounds induce tumors in animal models and neoplastic transformation of cultured mouse embryo cells (3-5). Lead chromate is the most commonly studied particulate form of Cr(VI). In human lung cells, lead chromate induces chromosome aberrations and DNA damage, including double- and single-strand breaks and Cr adducts (6-11). This genotoxicity * Corresponding author. Phone: (207) 228-8050. Fax: (207) 228-8057. E-mail: [email protected]. † Wise Laboratory of Environmental and Genetic Toxicology. ‡ Maine Center for Toxicology and Environmental Health. § Department of Applied Medical Science. 1 Abbreviations: Cr(VI), hexavalent chromium; Cr, chromium; CIN, chromosome instability; Pb, lead; APC/C, anaphase-promoting complex/ cyclosome; HRP, horseradish peroxidase; LC, lead chromate, SC; sodium chromate; LG, lead glutamate.

results from particle dissolution outside of the cell releasing both Cr and Pb ions (12, 13); however, the Pb ions are not genotoxic and thus the damaging effects of lead chromate are due to exposure to soluble Cr(VI) ions (12). These findings indicate that soluble and particulate Cr(VI) operate through similar mechanisms but do not explain the difference in their potency as a carcinogen. Lung tumors are typically characterized by chromosome instability (CIN) consisting of alterations of both chromosome number and structure (14). These tumors frequently contain a triploid or tetraploid chromosome complement (14). The spindle assembly checkpoint protects cells from acquiring an aneuploid state. This checkpoint serves to regulate the progression from metaphase to anaphase, and only after all of the chromosomes have attached to the mitotic spindle are cells allowed to progress to cytokinesis (15). The ability of particulate Cr(VI) to induce spindle assembly checkpoint bypass is currently unknown, but arsenic, another human lung carcinogen, does induce spindle assembly checkpoint bypass, resulting in persistent aneuploid cells (16, 17). More specifically, chronic exposure of cells to arsenic induced centromere spreading and premature anaphase through an apparent disruption of the MAD2 protein. Mad2 is a key protein in the spindle assembly checkpoint and a reduction in MAD2 levels is known to cause spindle assembly checkpoint bypass (14). Accordingly, in this study we investigated the hypothesis that chronic exposure to particulate Cr(VI) induces

10.1021/tx0601410 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/06/2006

PbCrO4 Induces Spindle Assembly Checkpoint Bypass

spindle assembly checkpoint bypass manifested as centromere spreading, premature anaphase, and a decrease in MAD2 protein levels.

Materials and Methods Chemicals and Reagents. Lead chromate, sodium chromate, lead nitrate, L-glutamic acid, colcemid, potassium chloride, and sodium chloride were purchased from Sigma/Aldrich (St. Louis, MO). Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Trypsin/EDTA, sodium pyruvate, penicillin/ streptomycin, L-glutamine, and Gurr’s buffer were purchased from Invitrogen Corp. (Grand Island, NY). Methanol, acetone, and acetic acid were purchased from J.T. Baker (Phillipsburg, NJ). Dulbecco’s minimum essential medium and Ham’s F-12 50:50 mixture (DMEM/F-12) was purchased from Mediatech Inc. (Herndon, VA). Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Tissue culture dishes, flasks, and plasticware were purchased from Corning Inc. (Acton, MA). Cells and Cell Culture. WTHBF-6 cells, a clonal cell line derived from normal human bronchial fibroblasts that ectopically express human telomerase, were used in all experiments. These cells are diploid and have similar clastogenic and cytotoxic responses to metals compared to their parent cells (8). We chose to study lung fibroblasts because they are a target of chromium toxicity and most epithelial lung cell lines are aneuploid and thus not suitable for these studies (18). Furthermore, growing evidence indicates that damaged fibroblasts contribute to carcinogenesis in epithelial cells by producing an unhealthy microenvironment (1923). Cells were maintained as subconfluent monolayers in DMEM/ F-12 supplemented with 15% CCS, 2 mM L-glutamine, 100 U/mL penicillin/100 µg/mL streptomycin, and 0.1 mM sodium pyruvate and incubated in a 5% CO2 humidified environment at 37 °C. They were fed three times a week and subcultured at least once a week using 0.25% trypsin/1 mM EDTA solution. All experiments were performed on logarithmically growing cells. Preparation of Chemicals. Lead chromate was administered as a suspension in acetone, and sodium chromate, a model soluble Cr(VI) compound, was administered as a solution in water, as previously described (7). Sodium chromate treatment was refreshed every 24 h. Lead nitrate (CAS #10099-74-8, ACS reagent, 99+%) is insoluble in tissue culture medium; thus, a soluble form of Pb was created by mixing lead nitrate and glutamic acid (CAS# 5686-0) as previously described (24). Briefly, lead nitrate and glutamic acid were prepared by dissolving in distilled water and then filtersterilized through a 0.2 um filter. Lead glutamate (LG) was prepared by mixing the lead nitrate and glutamic acid in equimolar amounts. Chromosome Damage. Cells were prepared for chromosomal analysis as previously described (7). Cells were analyzed for centromere spreading, premature anaphase, and premature centromere division as defined by Yih et al. (16, 17). At least 100 metaphases per data point were analyzed in each experiment. Each experiment was repeated at least three times. In some experiments, to rule out an effect of colchicine, cells were incubated either with nocodazole or with no arresting agent and analyzed for premature anaphase. Clonogenic Aneuploidy. The fate of aneuploid cells was determined by seeding a monolayer of cells and treating them with 0 and 0.5 µg/cm2 lead chromate for 96 and 120 h. Cells were then replated at a colony-forming density onto glass coverslips and allowed to form colonies. When colonies contained 25-50 cells they were harvested for chromosome analysis. Media was aspirated, and cells were treated with 0.8% sodium chloride solution to swell the cells and then fixed with 3:1 methanol:acetic acid. Coverslips were then stained with 5% Giemsa. Colonies were analyzed for aneuploidy. Mitotic Stage Analysis. A monolayer of cells was seeded onto chamber slides and treated with 0 and 0.5 µg/cm2 lead chromate for 96 and 120 h. Cells were fixed in situ with 20:1 methanol: acetic acid, aged overnight, and stained with 5% Giemsa for 5 min.

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1493 Mitotic figures were analyzed under light microscopy. Mitotic figures were scored by stage (prophase, metaphase, anaphase, and telophase). Mad2 Expression. Mad2 levels were measured by Western blotting according to our published methods (10). Briefly, cells were lysed in 50 mM Tris (pH 8.0), 200 mM NaCl, 1% Igepal CA-630 supplemented with 5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 mM NaF, 20 mM 2-glycerophosphate, 1 mM sodium vanadate, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were clarified by microcentrifugation and the supernatant fractions were saved prior to protein concentration determination. Equal samples were resolved by 14% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were probed with antiMad2 antibody (Abcam, Cambridge, MA) overnight. The membranes were then probed with HRP-conjugated secondary antibody for 1 h and the blots visualized by chemilluminescent substrate (Pierce, Rockford, IL). Equal protein loading was confirmed by immunblotting with an antibody to constitutively expressed B-actin (Abcam, Cambridge, MA). Determination of Intracellular Lead and Chromium Ion Levels. The intracellular concentration of the metals was determined as previously described (24). Briefly, a monolayer of cells was treated with 0.1, 0.5, and 1 µg/cm2 lead chromate for 0, 24, 48, 72, 96, and 120 h or 0.5, 1, and 2.5 µM sodium chromate for 24, 48, 72, 96, and 120 h. Cells were then harvested and placed in a hypotonic solution followed by 2% SDS to degrade the cell membrane. This solution was then sheered through a needle seven times and filtered in order to remove remaining undissolved lead chromate particles. Cr and/or Pb ion concentrations of the samples were then measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) as described previously (24). Zero-hour harvests were performed with lead chromate to account for the possibility that some undissolved lead chromate particles passed through the filter. The final concentrations were corrected for this possible confounding factor by subtracting 0 h values from the 24, 48, 72, 96, and 120 h values as previously described (24). Statistical Analysis. The Student’s t-test was used to calculate p-values to determine the statistical significance of the difference in means. No adjustment was made for multiple comparisons. Interval estimates of differences are 95% confidence intervals, based also on Student’s t distribution.

Results Chronic Exposure to Lead Chromate Causes Spindle Assembly Checkpoint Bypass. The spindle assembly checkpoint serves to ensure that cells do not progress to mitosis until all of the chromosomes are ready to separate properly (17). We found that longer exposures to lead chromate induced spindle assembly checkpoint bypass manifested as centromere spreading, premature centromere division, and premature anaphase (Figure 1). Centromere spreading was defined as the disassociation of chromatids at the centromere but not at the rest of the chromosome (Figure 1A). Premature centromere division was defined as a cell in which at least one chromosome was still attached to its sister chromatid and at least one chromosome was completely separated from its sister chromatid (an advanced state of centromere spreading) (Figure 1B). Premature anaphase was defined as cells in which all of the sister chromatids were completely separated from each other (Figure 1C). The number of cells in each of these stages increased with both time and concentration (Figure 2). For example, for cells in premature anaphase, no increases were observed after 24 or 48 h, but 72, 96, or 120 h of exposure to 0.5 µg/cm2 lead chromate induced 6, 9, and 18% of cells in premature anaphase. There was also an increase in premature anaphase at 72, 96, and 120 h as the concentration increased from 0.1 to 1.0 µg/ cm2.

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Figure 2. Chronic exposure to lead chromate causes increases in centromere spreading, premature centromere division, and premature anaphase. Centromere spreading, premature centromere division, and premature anaphase all represent forms of aberrant mitotic progression. After 72, 96, and 120 h of lead chromate treatment, the total number of disrupted metaphases increased. Controls each showed 1% of metaphases disrupted at all time points, while treatments of 0.5 µg/ cm2 lead chromate had 9% of metaphases disrupted after 72 h of treatment, 26% of metaphases disrupted after 96 h of treatment, and 54% of metaphases disrupted after 120 h of treatment. Statistics: exposure to 0.1 and 0.5 µg/cm2 LC for 96 h is statistically different from control (p < 0.04), exposure to 0.5 and 1 µg/cm2 LC for 120 h is statistically different from control (p < 0.05), exposure to 0.1 µg/ cm2 LC for 96 h is statistically different from 0.1 µg/cm2 LC for 24 h (p < 0.04), exposure to 0.5 µg/cm2 LC for 96 and 120 h is statistically different from 0.1 µg/cm2 LC for 24 h (p < 0.04), exposure to 0.5 µg/cm2 LC for 96 and 120 h is statistically different from 0.5 µg/cm2 LC for 48 h (p < 0.04), exposure to 0.5 µg/cm2 LC for 120 h is statistically different from 0.5 µg/cm2 LC for 72 h (p < 0.04), exposure to 1 µg/cm2 LC for 120 h is statistically different from 0.1 µg/cm2 LC for 24 h (p < 0.05), and exposure to 1 µg/cm2 LC for 120 h is statistically different from 1 µg/cm2 LC for 48 h (p < 0.05),

Figure 1. Representative examples of centromere spreading, premature centromere division and premature anaphase. (A) Centromere spreading consists of chromosomes in which the separation of the chromatids starts at the centromere, sometimes appearing as a hole in place of the typical centromere constriction. Arrows pointing to chromosomes show typical centromere spreading. (B) Premature centromere division is considered to be an advanced stage of centromere spreading in which at least one chromosome is still attached to its sister chromatid and at least one chromosome is completely separated from its sister chromatid. Circled chromatids are completely separated, and arrows show sister chromatids still in association with each other. (C) Premature anaphase is defined as all sister chromatids being completely separated. Circle shows separated sister chromatids.

The frequency of cells with centromere spreading and the total number of chromosomes with spread centromeres both increased. The tremendous increase in the total number of chromosomes exhibiting centromere spreading indicates that some cells have multiple chromosomes with this abnormality (Figures 2 and 3). Again, there was no increase at 24 or 48 h, but there was a significant concentration- and time-dependent increase at 72, 96, or 120 h exposure to 0.5 µg/cm2 lead chromate with 1, 8, and 20% of cells exhibiting spread centromeres, respectively (Figures 2 and 3A). We also found an increase in the total number of chromosomes exhibiting spread centromeres. Specifically, after a 72, 96, or 120 h exposure to 0.5 µg/cm2 lead chromate, there were 23, 50, and 292 chromosomes with spread centromeres, respectively (Figure 3B). Centromere spreading also increased at 72, 96, and 120 h as the concentration of lead chromate increased from 0.1 to 1.0 µg/cm2. The third indication of spindle assembly checkpoint bypass that we observed was premature centromere division (Figure 2). The frequency of this was 3, 9, and 17% for 72, 96, or 120 h of exposure to 0.5 µg/cm2 lead chromate, respectively. Premature centromere division also increased at 72, 96, and 120 h as the concentration increased from 0.1 to 1.0 µg/cm2. All of these events occurred despite the presence of colchicine, which activates the spindle assembly checkpoint and prevents the progression of cells from metaphase to anaphase. Thus, lead chromate was able to bypass the induction of the spindle assembly checkpoint and allow premature entry into anaphase.

PbCrO4 Induces Spindle Assembly Checkpoint Bypass

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1495 Table 1. Lead Chromate Disrupts Mitotic Stage Distribution of Cells Treated for 96 or 120 h. lead chromate time concn (µg/cm2) (h) prophasea metaphase anaphase telophase 0b 0.5 0b 0.5

96 15 ( 2 96 7 ( 1.9c 120 15 ( 1.2 120 9 ( 1.2d

44 ( 3 50 ( 2.3 44 ( 1 36 ( 4.7

19 ( 2.1 31 ( 1.2e 17 ( 0.6 42 ( 3.9d

22 ( 1.8 12 ( 3.5 24 ( 1.2 14 ( 1f

total 100 ( 6 100 ( 0.3 100 ( 0.3 101 ( 0

a Data reflect an average of three independent experiments; 100 mitotic cells per experiment were analyzed. b Vehicle control: acetone. c Statistically different from control (p < 0.05). d Statistically different from control (p < 0.03). e Statistically different from control (p < 0.02). f Statistically different from control (p < 0.01).

Figure 3. Chronic exposure to lead chromate causes increases in the total number of chromosomes exhibiting centromere spreading. This figure shows increases in both the percent of cells with centromere spreading and total spread centromeres. Panel A shows these data based on percent of metaphases with spread centromeres. Panel B shows these data based on the total number of chromosomes with spread centromeres seen in 100 scored metaphases. Statistical analysis: (A) Exposure to 1 µg/cm2 LC for 72 and 120 h are statistically different from control (p < 0.05). (B) Exposure to 1 µg/cm2 LC for 72 h is statistically different from control (p < 0.05), exposure to 0.5 µg/cm2 LC for 96 h is statistically different from control (p < 0.04), exposure to 1 µg/cm2 LC for 120 h is statistically different from control (p < 0.04), and exposure to 0.5 µg/cm2 LC for 120 h is statistically different from 0.5 µg/cm2 LC for 72 h (p < 0.04).

Spindle Assembly Checkpoint Bypass Is Not Due to a CrColchicine Interaction. The chromosomal manifestation of spindle assembly checkpoint bypass (e.g., premature centromere division, centromere spreading, and premature anaphase) was performed in the presence of colchicine to allow for easy visualization of the chromosomes. To confirm that these events were due to lead chromate and not related to the presence of colchicine, we analyzed the effect of lead chromate on mitosis in situ and without colchicine to determine if there was an effect on the distribution of the phase of mitosis. We indeed found a significant increase in the number of mitotic figures in anaphase after lead chromate exposure, and no increase in the other mitotic stages. Specifically, we found an increase from 19% of cells in anaphase in the controls to 31% in those treated with 0.5 µg/ cm2 lead chromate for 96 h and an increase from 18% of cells in anaphase in the controls to 41% in those treated with 0.5 µg/cm2 lead chromate for 120 h (Table 1). Thus, lead chromate does induce spindle assembly checkpoint bypass, leading to premature entry into anaphase. To further rule out a Cr-colchicine interaction in the abnormal progression of mitotic cells, we used an alternative arresting agent, nocodazole, as well as no arresting agent. We found 40% of cells in premature anaphase and premature

Figure 4. Aberrant mitotic progression is not a result of a Crcolchicine interaction. This figure shows the percentage with premature centromere division and premature anaphase cells after a 96-h exposure to 0.5 µg/cm2 lead chromate using colchicine, nocodazole. or no arresting agent to block the cells in metaphase. Percentage of cells in anaphase is highest with no arresting agent and the lowest when treated with colchicine.

centromere division in cells arrested with nocodazole as compared to 18% of cells arrested with colchicine and 45% of cells in premature anaphase in cells with no arresting agent added (Figure 4). Thus, the increase of cells in premature anaphase is not a result of Cr interacting with colchicine, as there are even higher levels of premature anaphase when no colchicine is added or and alternative arresting agent is used. Chronic Exposure to Lead Chromate Causes Decreased MAD2 Levels. To confirm that this was indeed spindle assembly checkpoint bypass, we examined the effects of lead chromate on the expression of the Mad2 protein. Mad2 is a key element of the spindle assembly checkpoint and can be a marker of spindle assembly checkpoint function (14). Its role is to bind to cdc20 to prevent the activation of APC/C and subsequent downstream cleavage of the sister chromatids (14). To receive a “go” signal for the spindle assembly checkpoint, Mad2 levels are reduced. Thus, in the presence of unrepaired DNA damage or chromosomes not ready for anaphase, Mad2 levels remain high. We examined Mad2 concentrations after chronic exposure to lead chromate. Figure 5A shows that lead chromate does indeed suppress the spindle assembly checkpoint as highly clastogenic concentrations of lead chromate significantly decrease Mad2 levels. This suppression was clearly the result of lead chromate, as colchicine was not present for these Mad2 studies. Figure 5B also shows that the spindle assembly checkpoint response is active in these cells as Mad2 levels were increased by high levels of X-rays. Chronic Exposure to Lead Chromate Causes Increased CIN. We also considered the ability of lead chromate to induce CIN. In particular, we focused on triploid and tetraploid cells, as one of the characteristics of lung tumors is a near triploid or tetraploid chromosome complement (14). We found no increase in triploid or near triploid cells (data not shown). By contrast, we did find an increase in tetraploid cells with increasing time

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Figure 6. Chronic exposure to lead chromate induces numerical chromosome instability. This figure shows that chronic exposure to lead chromate induces a permanent tetraploid state. No tetraploid colonies occurred in the controls. Data represent an average of three experiments ( standard error of the mean. Exposure to 0.5 µg/cm2 LC for 96 and 120 h is statistically different from control (p < 0.05 and p < 0.03 respectively). Figure 5. Lead chromate decreases the expression levels of Mad2. Mad2 levels are decreased in response to chronic exposure to lead chromate. (A) This figure shows a western blot of expression of Mad2. Lane one is the ladder, lane 2 is the control, lane 3 represents cells treated with 0.5 µg/cm2 lead chromate for 96 h, and lane 4 shows cells treated with 10 Gy IR as a positive control. β-actin was used as a loading control. (B) This table shows that Mad2 expression of cells treated with 0.5 µg/cm2 lead chromate for 96 h is decreased by more than 50% as compared to control cells. Table 2. Lead Chromate Induces Time- and Concentration-Dependent Increases in Tetraploidy lead chromate concn (µg/cm2)

24 h

48 h

72 h

0b 0.1 0.5 1

2.1 ( 0.3 1.5 ( 0.8 1.9 ( 0.9 1.9 ( 0.5

0.8 ( 0.3 1.0 ( 0.3 1.9 ( 1 2.5 ( 1.1

0.6 ( 0.2 1.9 ( 0.6 3.5 ( 2.7 4.8 ( 0.5e

% of tetraploid metaphasesa 96 h

120 h

0.8 ( 0.6 0.8 ( 0.3 5.4 ( 1.4 8.4 ( 2.5c 7.4 ( 1.7d 12 ( 1.7c,d 4.8 ( 0.5c,e 15.1 ( 2.5c,e,f

a Data reflect an average of three independent experiments. b Vehicle control: acetone. c Statistically different from control (p < 0.03). d Statistically different from 24 and 48 h (p < 0.05). e Statistically different from 24 h (p < 0.02). f Statistically different from 48 and 96 h (p < 0.02).

of exposure to lead chromate (Table 2). Specifically, after a 24-h treatment, there was no increase in the number of tetraploid cells above the control. However, by 72 h of lead chromate exposure there was a clear increase in the number of tetraploid cells as the number of tetraploid cells increased from 1% in the control to 5% at 1 µg/cm2 lead chromate. By 120 h of lead chromate treatment the percent of tetraploid cells increased from 1% in the controls to 8% at 0.1 µg/cm2 lead chromate, 12% at 0.5 µg/cm2 lead chromate, and 15% at 1 µg/cm2 lead chromate. Cells with Lead Chromate-Induced CIN Cause a Permanent Tetraploid State. Once we established that lead chromate induces numerical CIN, we wanted to determine the fate of these cells. We replated cells exposed to 0.5 µg/cm2 lead chromate for 96 or 120 h onto glass coverslips and allowed colonies to form. Once colonies formed, we stained the coverslips in situ and assessed colonies for the presence of tetraploid cells. We found that cells with a tetraploid chromosome complement were able to continue to grow and form colonies, and thus, the numerical CIN was a permanent state. We found that, after 96and 120-h treatment with 0.5 µg/cm2 lead chromate, 1.5 and 10% of the colonies exhibited a tetraploid chromosome complement, respectively, while control cells did not exhibit any tetraploid colonies (Figure 6).

Figure 7. Intracellular ion levels after chronic exposure to lead chromate, sodium chromate and lead glutamate. Figure 7 shows the comparison of intracellular ion levels between lead chromate and sodium chromate and lead glutamate. The data represent an average of at least three experiments ( standard error of the mean. (A) Exposure to 1 µM sodium chromate produces similar intracellular Cr ion concentration as 0.5 µg/cm2 LC. In order to ensure the cells were exposed to Cr(VI), the media and sodium chromate treatment was refreshed every 24 h. (B) Exposure to 50 µM lead glutamate produces similar intracellular Pb levels as 0.5 µg/ cm2 LC.

Spindle Assembly Checkpoint Bypass Is Due to Chromium Treatment and Is Not an Effect of Lead. Because lead often has its own deleterious effects on cells, we wanted to determine if the effects seen were due to chromium or lead. We found comparable intracellular concentrations of Cr and Pb ions when cells were treated with either particulate chromium or soluble chromium and lead, respectively. Exposure to 1 µM sodium chromate produces similar intracellular Cr ion concentrations as 0.5 µg/cm2 lead chromate (Figure 7A), while exposure to 50 µM lead glutamate produces similar intracellular Pb ion levels as 0.5 µg/cm2 lead chromate (Figure 7B). When

PbCrO4 Induces Spindle Assembly Checkpoint Bypass

Figure 8. Chronic exposure to Pb does not induce increases in centromere spreading, premature centromere division, and premature anaphase, but soluble Cr(VI) does. This figure shows that Cr ions induce centromere spreading, premature centromere division, and premature anaphase and that Pb does not. Cells treated with soluble Cr(VI) showed an increase in the percent of disrupted metaphases after 96 or 120 h of treatment; there was no increase at 24, 48, or 72 h over controls (data not shown).

Figure 9. Centromere spreading, premature centromere division and premature anaphase effects are a result of Cr(VI) dissolution and not a particle effect. This figure shows that cells do not need to be in contact with the particles to express these effects. Cells harvested from the bottom of a transwell plate with direct lead chromate exposure for 120 h showed 59% of disrupted metaphases. Cells harvests from the top of a transwell plate, separated from lead chromate particles by a membrane showed 57% of disrupted metaphases.

cells were treated with 50 µM lead glutamate for 120 h, we found no increase in disrupted metaphases (data not shown). By contrast, we found that sodium chromate did induce this effect (Figure 8). This shows that the disruption in mitosis is occurring due to the chromium ions and that the lead ions do not contribute to this effect. In addition, there is no increase in the number of polyploid cells when treated with chronic lead glutamate, while cells treated with chronic sodium chromate showed an increase of polyploid cells from 0.3% in the control to 8% in cells treated with 1 µM sodium chromate (data not shown). Spindle Assembly Checkpoint Bypass Is Not a Particle Effect. To determine the possible influence of the particulate, cells were seeded on the top and bottom layer of a transwell plate, cells on the bottom layer only were treated with lead chromate particles. We saw no difference in the percent of disrupted metaphases for either directly exposed cells or cells separated from lead chromate particles by a membrane, confirming this was a Cr ion effect and not a particle effect (Figure 9)

Discussion Particulate Cr(VI) is a known human lung carcinogen, but its carcinogenic mechanism is poorly understood; however,

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chronic exposure to particulate Cr(VI) after impaction at bronchial bifurcation sites is hypothesized as a key factor in its carcinogenicity (7-10, 12, 13). This study is the first to consider the effects of Cr(VI) on the spindle assembly checkpoint. The spindle assembly checkpoint operates between metaphase and anaphase and serves to prevent inappropriate separation of the sister chromatids and tetraploidy (15). A “go” signal, i.e., one that indicates it is time to go ahead and allow chromatids to separate and progress to anaphase, is thought to be signaled by microtubules occupying all of the empty kinetochores at the centromeres of the chromosomes and appropriate levels of tension in the microtubule fibers (15). Once a go signal is received, a number of proteins interact at the centromere, leading to the release of cdc20 from Mad2 (15). This release allows cdc20 to bind to APC/C, which activates it and allows the activated complex to ubiquinate securin, causing it to dissociate from separase (15). Separase then cleaves the cohesion molecules that hold the sister chromatids together, which in mammalian cells are located at the centromere, leading to sister chromatid separation and the onset of anaphase (15). Decreases in Mad2 levels are known to cause disruption of the checkpoint (25); thus, MAD2 levels have been used as a useful marker of spindle assembly checkpoint disruption (25). We find that prolonged exposure to particulate Cr(VI) induces spindle assembly checkpoint bypass manifested as centromere spreading, premature centromere division, premature anaphase, and decreased MAD2 levels. This effect is also caused by soluble Cr(VI) and not seen at all in cells treated with Pb, thus indicating that Pb is not involved in this effect. These events are considered to be manifestations of CIN (26) and to induce tetraploidy, which is common in lung cancers (14). Indeed, we also find that particulate chromate induces a persistent tetraploid state that is consistent with the observations that particulate chromate is a human lung carcinogen and human lung tumors exhibit a tetraploid phenotype. Disruption of the spindle assembly checkpoint is understudied for metals. Previously, effects on the spindle assembly checkpoint have only been considered after arsenic exposure. Arsenic induced centromere spreading, premature anaphase, and decreased expression of Mad2, resulting in cells with permanent CIN manifested as tetraploidy (15, 16). Our data are consistent with those results and suggest that spindle assembly checkpoint bypass may be an important general mechanism for chemical carcinogenesis. The mitotic stage analysis data is also consistent with bypass of the spindle assembly checkpoint and with results reported for arsenic (15, 16). There was a clear increase in the number of cells in anaphase and a reduction of the number of cells in metaphase after chronic lead chromate exposure, indicating that more cells are able to pass into anaphase. It is tempting to speculate that lead chromate may also be inducing anaphase arrest, as there was an increase in anaphase but not in telophase. This speculation may eventually prove to be a potential factor as in yeast, where there is an anaphase checkpoint, but currently no such checkpoint has been identified in mammalian cells (27). Currently, the only known checkpoint in mitosis is the spindle assembly checkpoint, which acts prior to the onset of anaphase. Thus, the mechanism for particulate chromate-induced carcinogenesis appears to begin with particle impaction at bronchial bifurcation sites followed by chronic extracellular dissolution releasing both the chromate oxyanion and the cation, which both enter the cell (13). Once inside the cell, the chromate ions are reduced to Cr(III) through a series of redox reactions releasing Cr(V), Cr(IV), and free radicals as intermediates (22). Cr(III),

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one of the intermediates, or some combination of them induce chromosomal aberrations, adducts, cross-links, and strand breaks (7, 11, 12, 28). We propose that either Cr(III) or one of the metabolites has a direct effect on the spindle assembly checkpoint or that CIN is a consequence of the damage itself, ultimately leading to carcinogenesis. Further work is aimed at elucidating the role of other genes in the spindle assembly checkpoint. Acknowledgment. We thank Geron Corp. for the use of the hTERT materials. This work was supported by NIEHS grant ES10838 (J.P.W.) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine.

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