Chronic Exposure to Particulate Chromate Induces Spindle Assembly Checkpoint

Bypass in Human Lung Cells

Sandra WiseAmie L. Holmes

Hong XieW. Douglas ThompsonJohn Pierce Wise, Sr.

Chem. Res. Toxicology. 2006, 19, 1492-1498

Background

PG&E dumped Hexavalent Chromium into unlined ponds and polluted the ground water

The real Erin Brokovich

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.

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

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

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

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

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

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

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

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

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 anaphaseSTOP MITOSIS

MAD2 levels are reduced for APC/C to receive a “go” signalALLOW MITOSIS

So MAD2 levels are a useful marker for the spindle assembly checkpoint

Can be detected with western blotting

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

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

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

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

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

Arrested cells in Metaphase

Cells were arrested with colchicineActivates the spindle assembly checkpointPrevents progression of cells from anaphase to

metaphaseMechanism

Binds to tubulin Inhibits microtubule

polymerization

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)

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

Examined Cells for CIN

Chromosome Instability1.Centromere spreading

Disassociation of chromatids at centromere but not at the rest of chromosome

2.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

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

Centromere spreadingDisassociation of chromatids at centromere but not at the rest of chromosome

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

Premature AnaphaseCells in which all of the sister chromatids were completely separated from each other

..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)

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

SDS-PAGE

Western Blotting

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.

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

CIN with Lead Chromate

• Centromere Spreading

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

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

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

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

Lane

234

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)

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)

Chronic Exposure to Lead Chromate Causes Increased CIN

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

Permanent Tetraploid State

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

Sodium Chromate

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

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)

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

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

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

Future Directions

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

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

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.

Questions?