1. Chronic Exposure to Particulate Chromate Induces Spindle Assembly Checkpoint Bypass in Human Lung Cells Sandra Wise Amie L. Holmes Hong Xie W. Douglas Thompson John Pierce Wise, Sr. Chem. Res. Toxicology. 2006, 19, 1492-1498

2. Background

3. PG&E dumped Hexavalent Chromium into unlined ponds and polluted the ground water The real Erin Brokovich

4. Hexavalent Chromium- Cr(VI) Established lung carcinogen An increased risk of lung cancer has been demonstrated in workers exposed to Cr(VI) compounds Occupational exposures to Cr(VI)  Occur during the production of stainless steel, chromate chemicals, and chromate pigments.  Also occur during other work activities such as stainless steel welding, thermal cutting, chrome plating, painting, and coating processes.  Occupational exposure to hexavalent chromium can occur from inhalation of dusts, mists, or fumes containing hexavalent chromium, or from eye or skin contact with hexavalent chromium The Occupational Safety and Health Administration says 380,000 U.S. workers are exposed to the chemical on the job each year.

6. Hexavalent Chromium- Cr(VI) continued Particulate form  Most potent carcinogenic form of Cr(VI) is water-insoluble or particulate form  Epidemiological studies report a higher risk of cancer for particulate Cr(VI) exposed workers  Only particulate Cr(VI) compounds induce tumors in animal models & neoplastic transformation of cultured mouse embryo cells Lead chromate  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 Ions  Genotoxicity results from particle dissolution outside of the cell releasing both Cr and Pb ions

7. Lung Cancer Cr(VI) induces tumors at lung bifurcation sites  This is where Cr(VI) particles impact and persist  Carcinogenic mechanisms are unknown Hallmark of lung cancer is chromosome instability (CIN)  Particularly tetraploid phenotype Normally prevented by spindle assembly checkpoint  Arsenic, another lung carcinogen induces spindle assembly checkpoint bypass Chronic exposure of cells to arsenic induced premature anaphase through an apparent disruption of the MAD2 protein MAD2 is a key protein in the checkpoint A reduction in MAD2 levels is known to cause spindle assembly checkpoint bypass

8. So.. Hypothesis is that chronic exposure to Cr (VI) also induces bypass of the spindle assembly checkpoint through a disruption of the MAD2 protein

10. Spindle Assembly Checkpoint Anaphase delayed until all of the chromosomes are correctly bioriented. Prevents cells from developing an aneuploid state Microtubules exist in shrinking/growing state  Probe 3D space around centromeres  When encounter kinetochore, they become stabilized

11. What is the switch for the checkpoint? 1. Checkpoint regulated by microtubule occupancy at the kinetochores & 2. Tension sensitive enzymes at kinetochores that send out negative regulators of anaphase  Before biorientation- unaligned chromosomes produce negative signals  After biorientation- tension from spindle fibers turns off negative signals

12. The spindle checkpoint detects the loss or impairment of functional connections between kinetochores and spindle microtubules during mitosis and disseminates signals that inhibit the APC/C The Anaphase Promoting Complex/cyclosome (APC/C) is a ubiquitin ligase complex that starts a cascade of events that lead to the separation of chromatids Cdc20 = Activator subunit

13. Genetic studies in yeast and mammals have implicated at least 7 genes in mitotic spindle checkpoint function :  BUB proteins: BUB1, BUBR1, BUB3  MAD proteins: MAD1, MAD2, MAD3  and CDC20 How these complexes work not fully understood Agreed that functions of one or more of these genes must be compromised for spindle assembly abrogation

14. Spindle Assembly Checkpoint genes and Cancer Differential expression of the BUB1, BUB3, BUBR1, MAD1 and MAD2 genes in various human tumors and cell lines (ex. ovarian cancer cells) Reduced expression of MAD1, MAD2, BUB1 and BUBR1 has been found in different human cancers (ex. Gastric cancer) Heterozygous MAD2, BUB3 or BUBR1 disruptions in mice result in partially downregulated checkpoint protein levels, an impaired spindle checkpoint and aneuploidy Overexpression of CDC20 has been observed in several oral squamous cell carcinoma cell lines and primary head and neck tumors

15. MAD2 (mitotic arrest deficient) MAD2 -- or a complex of checkpoint proteins -- inhibits the APC after it has sensed that the spindle attachments are defective. MAD2 levels are high:  In the presence of unrepaired DNA damage  Chromosomes not ready for anaphase STOP MITOSIS MAD2 levels are reduced for APC/C to receive a “go” signal ALLOW MITOSIS So MAD2 levels are a useful marker for the spindle assembly checkpoint Can be detected with western blotting

16. Cohesin A molecule that holds the sister chromatids together Located at the centromere during metaphase http://www.sciencemag.org/feature/data/prizes/ge/2004/haering.dtl Proteins: Smc1, Smc3, Scc1, Scc3 subunits of a "cohesin" complex holding sisters together

17. Separase Cleaves Cohesin Separase cleaves cohesin when the spindle assembly checkpoint is turned off 2 separase cleavage sites in the SCC1 subunit of cohesin cohesin

19. Materials and Methods  Hypothesis: Chronic exposure to particulate Cr (VI) induces bypass of the spindle assembly checkpoint manifested as:  Premature progression into anaphase  And a decrease in MAD2 protein levels

20. Cell line used: Chose to study fibroblasts (WHTBF-6 cells) :  Diploid cell line derived from normal human bronchial fibroblasts Have similar clastogenic and cytotoxic responses to metals compared to parent cells Ectopically express telomerase  Damaged fibroblasts contributes to unhealthy microenvironment Chose not to study epithelial lung cell lines  Derived from cancer cells  Most are aneuploid so not suitable for study

21. Chromate Carcinogenicity of Cr(VI) related to its solubility  Insoluble particulate Cr(VI) compounds are the most potent carcinogens  Reasons for difference remain unknown In this study:  Insoluble particulate Cr(VI) Lead chromate administered as suspension in acetone Main focus of the paper Most commonly studied particulate form of Cr(VI)  Soluble Cr(VI) Sodium chromate administered as a solution in water Used to determine lead was not causing deleterious effects

22. Arrested cells in Metaphase Cells were arrested with colchicine  Activates the spindle assembly checkpoint  Prevents progression of cells from anaphase to metaphase  Mechanism Binds to tubulin Inhibits microtubule polymerization

23. Cell Tissue Culture  Performed multiple cell tissue culture experiments Exposed a monolayer of cells to varying concentrations of lead chromate (0.1, 0.5 and 1.0 ug/cm^2) for Varying times (24, 48, 72, 96, and 120 hours)

24. Mitotic Stage Analysis Used to compare the number of cells in the stages of mitosis with or without lead chromate  Monolayer of cells treated with 0 and 0.5ug/cm^2 lead chromate for 96 and 120 h. Mitotic figures were stained with Giemsa and analyzed under light microscopy Scored by stage:  Prophase  Metaphase  Anaphase  Telophase

25. Examined Cells for CIN Chromosome Instability 1. Centromere spreading  Disassociation of chromatids at centromere but not at the rest of chromosome 2. Premature centromere division  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 3. Premature anaphase  Cells in which all of the sister chromatids were completely separated from each other

26. Centromere spreading Disassociation of chromatids at centromere but not at the rest of chromosome

27. Premature centromere division 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

28. Premature Anaphase Cells in which all of the sister chromatids were completely separated from each other

29. ..and they examined cells for spindle assembly checkpoint bypass MAD2 Protein  Used as a marker to confirm involvement of spindle assembly checkpoint bypass  MAD2 protein levels determined by Western Blotting Looked at cells treated with 0.5 ug/cm^2 lead chromate for 96 hours Compared to  Control cells just treated with acetone  And cells treated with 10 Gy Ionizing Radiation (used to induce spindle assembly checkpoint and raise levels of MAD2)

30. Western Blotting Can be used to detect the protein of interest from a mixture of a great number of proteins Can give information on protein expression when compared to a control such as an untreated sample. Steps  Obtain cell samples  Lyse the cells to release protein contents  Run these proteins on a gel that separates proteins by size In this study, an SDS-PAGE was used to separate proteins  Then transfer gel proteins onto a nitrocellulose membrane using electricity  The membrane can be used to probe for proteins of interest using a primary antibody In this study, membrane was probed with anti-MAD2 antibody  The membrane is then probed with a secondary antibody: HRP-conjugated secondary antibody The HRP converts a luminol substrate to a light releasing substance Light is detected as a spot on film Can determine how much protein is there relative to other spots

31. SDS-PAGE

32. Western Blotting

33. Reading a Western Blot Lane 1- Marker Ladder which shows different known sizes of proteins Lane 3- Cancer Sample Lane 5- Normal Sample In this example, protein has a higher expression in the cancer sample than the normal sample Compare protein spots in samples to the ladder in order to determine the protein size If the size of the spots matches the known size of the protein in question then you know the blot worked.

34. Results Longer exposure to lead chromate induced spindle assembly checkpoint bypass # of cells in each stage of CIN increased with both time and concentration of lead chromate For example (premature anaphase):  Time No increase observed for 24 or 48 h exposures to 0.5 ug/cm^2 lead chromate But, increases were observed for 72, 96, or 120 h of exposure to 0.5 ug/cm^2 lead chromate at 6, 9, and 18% respectively  Concentration Increase at 72, 96, or 120 h as concentration increased from 0.1 to 1.0 ug/cm^2

35. CIN with Lead Chromate

36. Centromere Spreading

37. Spindle Assembly Checkpoint Bypass is Not Due to a Cr-Colchicine Interaction Analyzed the effect of lead chromate on mitosis in situ and without colchicine Found  Significant increase in the number of mitotic figures in anaphase after lead chromate exposure 96 h 0.5 ug/cm^2 lead chromate  Increase from 19% (controls) to 31% with No increase in the other mitotic stages 120 h 0.5 ug/cm^2 lead chromate  Increase from 18% (controls) to 41%  Reduction of number of cells in metaphase after chronic lead chromate exposure

38. Lead chromate leads to premature entry into anaphase – Mitotic Stage Analysis

39. Also, used alternative arresting agent, Nocodazole and no arresting agent Increase in premature anaphase not a result of Cr interacting with colchicine Higher levels of premature anaphase when no colchicine added or an alternative arresting agent is used. Derivative of colchicine

40. Chronic exposure to Lead Chromate Causes Decreased MAD2 levels Examined MAD2 concentrations after chronic exposure to lead chromate  Highly clastogenic concentrations of lead chromate significantly decrease MAD2 levels 1 2 3 4Lane 2 3 4 Lane 1 Marker ladder which shows different known sizes of proteins B-Actin Used as a loading control MAD2 expression Lane 2-Control 100% (normal) Lane 3- Lead Chromate 44% (low) Lane 4- Ionizing Radiation 268% (high)

41. Chronic Exposure to Lead Chromate Causes Increased CIN Found no increase in triploid or near triploid cells Found an increase in tetraploid cells  Increased with time of exposure to lead chromate  By 72 h of exposure increase in tetraploid cells from  1% in control to  5% at (1 ug/cm^2 lead chromate)  By 120 h of exposure Increase in tetraploid cells form  1% in control to  8% (0.1 ug/cm^2 lead chromate)  12% at (0.5 ug/cm^2 lead chromate)  And 15 % at (1.0 ug/cm^2 lead chromate)

42. Chronic Exposure to Lead Chromate Causes Increased CIN

43. Cells with Lead Chromate-Induced CIN Cause a Permanent Tetraploid State Fate of aneuploid cells determined by:  Replating cells from the following groups exposed to 0.5 ug/cm^2 lead chromate for 96 or 120-h treatments  Replated cells at colony forming densities on coverslips  Allowed colonies to form When colonies contained 25-50 cells  harvested for chromosome analysis  Stained cells in situ  Assessed for presence of tetraploid cells Found that numerical tetraploid state was a permanent state

44. Permanent Tetraploid State

45. Spindle Assembly Checkpoint Bypass is Due to Chromium Treatment – Not Lead Determined intracellular levels of Cr and Pb ions from exposure to lead chromate  Then found similar levels of Cr ions using sodium chromate &  Similar levels of Pb ions using lead glutamate Exposure to 1 uM sodium chromate  produces similar intracellular Cr ion concentrations as 0.5 ug/cm^2 lead chromate Exposure to 50 uM lead glutamate  produces similar intracellular Pb ion levels as 0.5 ug/cm^2 lead chromate Results:  Lead glutamate for 120 h Did not increase disrupted metaphases No increase in number of polyploid cells  Sodium chromate for 120 h Did induce disrupted metaphases Increase of polyploid cells from 0.3% in control to 8% with sodium chromate

46. Sodium Chromate

47. Spindle Assembly Checkpoint Bypass is Not a Particle Effect To determine possible influence of the particle  Cells seeded on top and bottom layer of transwell plate  Cells on bottom layer only treated with lead chromate particles Saw no difference in percent of disrupted metaphases for either  directly exposed cells (bottom layer) or  cells separated from lead chromate by membrane (top layer) Confirmed:  This was a Cr ion effect  Not a particle effect

48. Conclusions Prolonged exposure to particulate Cr(VI) induces spindle assembly checkpoint bypass Checkpoint is bypassed Anaphase allowed to progress MAD2 levels low Increased levels of CIN Checkpoint is active Anaphase delayed MAD2 levels high No CIN Exposure to Cr(VI)

49. Conclusions continued Caused by Cr ions  Not by Colchicine Pb ions Particle Mitotic stage analysis  Consistent with bypass of the spindle assembly checkpoint  Consistent with results reported for arsenic  Indicates that Chromium causes more cells to move into anaphase

50. Proposed mechanism Begins with particle impaction at bronchial bifurcation sites Followed by chronic extracellular dissolution releasing chromate oxyanion and Pb cation Once inside cell, chromate ions reduced to:  Cr(III) through redox reactions  Releasing Cr(V), Cr(IV), and free radicals as intermediates  Propose that Cr(III) has direct effect on the spindle assembly checkpoint or that CIN is a consequence of the damage itself, ultimately leading to carcinogenesis

51. Questions with the proposed mechanism Cr(VI) is reduced by the pulmonary epithelial lining fluid, alveolar macrophages, and peripheral lung parenchyma cells to the essentially nontoxic and noncarcinogenic Cr(III)  Why propose Cr(III) is Respiratory cancer can be induced only at airborne concentrations of Cr(VI) that overwhelm the reductive capacity of these extracellular components

52. Future Directions A look at some of the Papers that were published by the same group later in the summer of 2006

53. Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to human lung epithelial cells Studied the toxic effects of particulate and soluble Cr(VI) in immortalized human bronchial epithelial cells Important because fibroblasts provide mechanistic insight and contribute to carcinogenesis but Epithelial bronchial cells are the ones that transform and become tumors  Epithelial cell line was immortalized using HPV  Instead of looking at spindle assembly checkpoint looked for cell death and chromosomal aberrations: Chromatid lesions Isochromatid lesions Chromatid exchanges Dicentric chromosomes Results  Soluble Chromium Produced same toxicity in epithelial cells as in fibroblasts  Insoluble Chromium Produced less toxicity in epithelial cells than in fibroblasts This may be a result of the different modes of immortalization Also, dissolution of Cr(VI) ions may be an intracellular event for epithelial cells while it is an extracellular event for fibroblasts

54. The clastogenic effects of chronic exposure to particulate and soluble Cr(VI) in human lung cells Elucidate the reason for the different potencies between soluble and insoluble Cr(VI)  Hypothesis: The difference in potency between particulate and soluble Cr(VI) is due to more chronic exposures with particulate chromate because it can deposit and persist in the lungs while soluble chromate is rapidly cleared Used fibroblast cell line Results:  Chronic exposure to lead chromate induced a persistent amount of chromosome damage over time  Chronic exposure to sodium chromate induced a decrease in the amount of damage over the same time period  Produced similar trends in Cr ion uptake inducing both concentration- and time- dependent increases in intracellular Cr ion concentrations  Explanation: Pb cation in lead chromate may be causing the differences in toxicity over longer time periods.

55. Questions?