Chronic Exposure to Particulate Chromate Induces Spindle Assembly Checkpoint Bypass in Human Lung Cells

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• 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

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• Background

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The real Erin Brokovich

• PGE dumped Hexavalent Chromium into unlined
  ponds and polluted the ground water

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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.

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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

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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

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

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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

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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

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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

• 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

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• 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

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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

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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

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Cohesin

• A molecule that holds the sister chromatids
  together
• Located at the centromere during metaphase
• http//www.sciencemag.org/feature/data/prizes/ge/2
  004/haering.dtl

• Proteins Smc1, Smc3, Scc1, Scc3
• subunits of a "cohesin" complex holding sisters
  together

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Separase Cleaves Cohesin
cohesin

• Separase cleaves cohesin when the
• spindle assembly checkpoint is turned off
• 2 separase cleavage sites in the SCC1 subunit of
  cohesin

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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

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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

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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

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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

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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/cm2) for
• Varying times (24, 48, 72, 96, and 120 hours)

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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/cm2
  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

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Examined Cells for CIN

• Chromosome Instability
• Centromere spreading
• Disassociation of chromatids at centromere but
  not at the rest of chromosome
• 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
• Premature anaphase
• Cells in which all of the sister chromatids were
  completely separated from each other

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Centromere spreadingDisassociation of chromatids
at centromere but not at the rest of chromosome
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Premature centromere divisionA 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
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Premature AnaphaseCells in which all of the
sister chromatids were completely separated from
each other
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..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/cm2 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)

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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

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SDS-PAGE
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Western Blotting
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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.

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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/cm2 lead chromate
• But, increases were observed for 72, 96, or 120 h
  of exposure to 0.5 ug/cm2 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/cm2

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CIN with Lead Chromate
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• Centromere Spreading

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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/cm2 lead chromate
• Increase from 19 (controls) to 31 with No
  increase in the other mitotic stages
• 120 h 0.5 ug/cm2 lead chromate
• Increase from 18 (controls) to 41
• Reduction of number of cells in metaphase after
  chronic lead chromate exposure

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Lead chromate leads to premature entry into
anaphase Mitotic Stage Analysis
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Also, used alternative arresting agent,
Nocodazole and no arresting agent
Derivative of colchicine

• 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.

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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 4
Lane
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)
Lane 2
3
4
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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/cm2 lead chromate)
• By 120 h of exposure
• Increase in tetraploid cells form
• 1 in control to
• 8 (0.1 ug/cm2 lead chromate)
• 12 at (0.5 ug/cm2 lead chromate)
• And 15 at (1.0 ug/cm2 lead chromate)

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Chronic Exposure to Lead Chromate Causes
Increased CIN
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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/cm2 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

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Permanent Tetraploid State
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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/cm2 lead chromate
• Exposure to 50 uM lead glutamate
• produces similar intracellular Pb ion levels as
  0.5 ug/cm2 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

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Sodium Chromate
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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

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Conclusions

• Prolonged exposure to particulate Cr(VI) induces
  spindle assembly checkpoint bypass

Exposure to Cr(VI)
Checkpoint is bypassed Anaphase allowed to
progress MAD2 levels low Increased levels of CIN
Checkpoint is active Anaphase delayed MAD2 levels
high No CIN
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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

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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

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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

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Future Directions

• A look at some of the Papers that were published
  by the same group later in the summer of 2006

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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

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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.

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Questions?