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Molecular Cell Biology

Hexavalent Chromium–Induced ChromosomeInstability Drives Permanent and HeritableNumerical and Structural Changes and a DNARepair–Deficient PhenotypeSandra S.Wise1, Abou El-Makarim Aboueissa2, Julieta Martino1, and John PierceWise, Sr.1

Abstract

A key hypothesis for how hexavalent chromium [Cr(VI)]causes cancer is that it drives chromosome instability (CIN),which leads to neoplastic transformation. Studies showchronic Cr(VI) can affect DNA repair and induce centrosomeamplification, which can lead to structural and numericalCIN. However, no studies have considered whether theseoutcomes are transient or permanent. In this study, weexposed human lung cells to particulate Cr(VI) for threesequential 24-hour periods, each separated by about amonth. After each treatment, cells were seeded at colony-forming density, cloned, expanded, and retreated, creatingthree generations of clonal cell lines. Each generation ofclones was tested for chromium sensitivity, chromosomecomplement, DNA repair capacity, centrosome amplifica-tion, and the ability to grow in soft agar. After thefirst treatment, Cr(VI)-treated clones exhibited a normalchromosome complement, but some clones showed a

repair-deficient phenotype and amplified centrosomes. Afterthe second exposure, more than half of the treated clonesacquired an abnormal karyotype including numerical andstructural alterations, with many exhibiting deficient DNAdouble-strand break repair and amplified centrosomes. Thethird treatment produced new abnormal clones, with pre-viously abnormal clones acquiring additional abnormalitiesand most clones exhibiting repair deficiency. CIN, repairdeficiency, and amplified centrosomes were all permanentand heritable phenotypes of repeated Cr(VI) exposure.These outcomes support the hypothesis that CIN is a keymechanism of Cr(VI)-induced carcinogenesis.

Significance: Chromium, a major public health concernand human lung carcinogen, causes fundamental changesin chromosomes and DNA repair in human lung cells.Cancer Res; 78(15); 4203–14. �2018 AACR.

IntroductionHexavalent chromium [Cr(VI)] is a known respiratory carcin-

ogen. The most carcinogenic forms of Cr(VI) are the particulates,such as lead chromate, which deposit and persist at lung bifur-cation sites in the respiratory tract. These deposited particlesdissolve slowly over time, resulting in a chronic exposure of lungcells to Cr(VI) and an accumulation of Cr in tissues (1).Cr-induced tumors are typically found at these bifurcations andare associated with higher tissue burdens of Cr (2, 3). Thus,reoccurring exposure to Cr(VI) is key to its toxic effects.

Lung tumors are characterized by genomic instability.Cr-induced tumors are no exception and are characterized bymicrosatellite instability (MIN) and chromosome instability

(CIN). Markers of MIN were detected in 79% of Cr(VI)-inducedtumors, whereas only 15% of nonexposed tumors had increasesin these markers (4). In addition, MIN increased as workerchromium exposure increased (4) and was correlated with adecrease in hMLH1 expression (5). However, MIN is consideredto occur in cells when they are deficient in mismatch repair. Thus,MINmay play a role in the development of tumors but only aftermismatch repair deficiency has developed. Most people exposedto Cr(VI) would be expected to have proficient mismatch repair;thus, it is currently unclear if MIN is a driving factor or aconsequence of other changes in the genome.

CIN includes both structural and numerical chromosomeabnormalities. Studies have shown Cr(VI)-induced lung tumorsexhibit CIN. LOH was observed at 6 different loci in 50% to75% of Cr(VI)-induced lung tumors; however, this outcomewas not significantly different from non-Cr(VI) tumors (4).These findings may implicate LOH as a general mechanism forlung carcinogenesis as most lung cancers exhibit significant CIN(6). In addition, studies of chromate workers have shownincreased chromosomal aberrations in cultured lymphocytes(7), as well as increases in binucleated cells (8). This outcome isconsistent with cell culture studies showing profound andconsistent effects on chromosome structure in cultured cellstreated with Cr(VI) (9). Numerical abnormalities have not beenassessed specifically in Cr(VI)-induced tumors, but multiplestudies show Cr(VI) dramatically alters chromosome numberin cultured cells treated with Cr(VI) (10–13). No studies have

1Wise Laboratory of Environmental and Genetic Toxicology, Department ofPharmacology and Toxicology, University of Louisville School of Medicine,Louisville, Kentucky. 2Department of Mathematics and Statistics, University ofSouthern Maine, Portland, Maine.

Current address for J. Martino: Department of Microbiology and MolecularGenetics, University of Pittsburgh, Pittsburgh, PA.

CorrespondingAuthor: JohnPierceWise, Sr., Department of Pharmacology andToxicology, University of Louisville School of Medicine, 505 S Hancock St. CTRB522, Louisville, KY 40292. Phone: 502-852-8524; Fax: 502-852-7868; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-0531

�2018 American Association for Cancer Research.

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addressed CIN directly in chromium-induced lung tumors, andno analyses of chromosome complement have been done onCr(VI)-induced lung tumors.

Cell culture studies have shown prolonged exposure to Cr(VI)(i.e., greater than 24hours) has significant effects on chromosomestability, inducing increases in aneuploidy (10–12), spindleassembly checkpoint (SAC) bypass (14, 12, 15), centrosomeamplification (16), and defects in HR repair (17, 18). All of thesecan severely affect the stability of chromosome structure andnumber. Defects in HR repair increase CIN in Cr(VI)-treated cells(19) and predispose them to oncogenic transformation (20).However, no studies have directly addressed chromosome trans-locations as a result of Cr(VI) exposure. Here, we show particulateCr(VI) induces permanent chromosome translocations, whicharise as a result of defective double-strand break (DSB) repair.In addition, we show Cr(VI) induces permanent alterations inchromosome number in which centrosome amplification mayplay a role.

Materials and MethodsCell culture

WHTBF-6 cells are hTERT-expressing human lung fibro-blasts. The cells exhibit a normal diploid karyotype, normalgrowth parameters, and extended lifespan. They have the samegenotoxic and cytotoxic response to metals as their parentprimary human lung fibroblasts. WTHBF-6 cells were culturedin a 50:50 mix of DMEM/F12 medium plus 15% cosmiccalf serum, 1% L-glutamine, and 1% penicillin/streptomycin.Cells were maintained in a 37�C humidified incubator with5% CO2. All cells originated in our laboratory and screenedmonthly for mycoplasma contamination. Dr. Sandra Wise is acertified cytogenetic technologist and routinely authenticatedcells through karyotyping.

Metal preparationsLead chromate was used as a particulate compound and

administered as a suspension in water; sodium chromate wasused as a soluble chromate compound and dissolved in steriledistilled water as previously described (21).

Clonal expansionWTHBF-6 cells were exposed to 5 mg/cm2 lead chromate for

24 hours in three independent treatments, each separated byabout a month. Workplace concentrations are allowed to reach5 mg/m3 (22), thus given an average respiration rate and an8 hour workday, then an occupational exposure would be33 mg Cr(VI) in a 24-hour period. The cell culture dose usedequals 17 mg particulate Cr(VI) for a 24-hour period and, thus,is relevant to human exposure. After each treatment, cells wereseeded at colony-forming density, cloned, expanded into celllines, and then retreated (Fig. 1). Cell lines at each stage wereevaluated for chromosomal changes, centrosome number, andability to repair DNA DSBs. There were 91 control clones and63 treated clones.

Karyotype analysisCellswere arrested atmetaphase using colchicine andharvested

using standard methods for chromosomal analysis (23). Meta-phases were Giemsa-banded; at least 20 metaphases were karyo-typed for each clonal cell line.

Aneuploidy analysisCellswere arrested atmetaphase using colchicine andharvested

using standardmethods for chromosomal analysis (23). A total of100 metaphase cells were counted for each clonal cell line.

Centrosome amplificationCells were seeded on glass chamber slides coated with Fibro-

nectin Coating Solution-coating matrix. Cells were then washedwith a microtubule-stabilizing buffer, fixed with methanol, airdried and then permeabilized with 0.05% Triton X-100, blockedin centrosome-blocking buffer then incubated with a-tubulinantibody, and washed with PBS, followed by incubation withAlexa Fluor 555 secondary antibody. Finally, slides were incubat-ed with anti-alpha tubulin FITC-conjugated antibody, washedand air dried, and then counterstained with DAPI. Centrosomenumbers in 100 mitotic cells were analyzed with an OlympusBX51 fluorescent microscope.

SAC analysisCloneswere harvested from subconfluent T25 flasks. Cells were

collected by trypsinization, given a hypotonic treatment, fixedwith 3:1 methanol:acetic acid. Metaphases were dropped ontoclean wet slides and stained with Giemsa. Metaphases wereassessed for evidence of SAC bypass including centromere spread-ing, premature centromere separation, and premature anaphaseas defined in previous studies (14). A total of 100 cells wereanalyzed.

Coimmunofluorescence for g-H2A.X and 53BP1 foci formationClones were treated with 1 mmol/L sodium chromate for 24

hours or for 24 hours followed by a chemical-free recovery periodof 24 hours. After the given time periods, cells were fixed in 4%paraformaldehyde and permeabilized with 0.2% Triton X-100.

Figure 1.

Experimental design for clonal expansion study. This figure shows theexperimental scheme for clonal expansion of Cr(VI)-treated cells. Cells wereexposed to lead chromate for 24 hours in three separate treatments. After eachtreatment, cells were seeded at colony-forming density, cloned, expanded intocell lines, and then retreated. Cell lines at each stage were evaluated forchromosomal changes, centrosome number, ability to repair DNA DSBs, and forgrowth in soft agar.

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Cells were then coincubated with anti–g-H2A.X and 53BP1 pri-mary antibodies, and then coincubated with Alexa Fluor 488 goatanti-rabbit and Alexa Fluor 555 goat anti-mouse secondary anti-bodies. Nuclei were counterstained with Prolong Gold antifadereagent with DAPI. Slides were analyzed on an Olympus BX51fluorescent microscope. H2A.X, 53BP1, and colocalized foci wereanalyzed by eye, and 50 cells per treatment condition werecounted.

Anchorage independence assayAnchorage independence is considered themost stringent assay

for detecting transformation of cultured cells. To determineanchorage-independent cell growth, control and treated clonalcell lines were suspended in 0.35% agar, plated onto a 0.6% agarbase layer in a 60-mm dish at a density of 50,000 cells per dish,and grown for 4 to 6 weeks (24). Cultures were examinedmicroscopically 24 hours after plating to confirm single-cellsuspension. Colonies were detected by 5% 4-Nitro 545 tetrazo-lium chloride staining and counted.

Statistical analysisMean, median, variance, and SD were calculated for all groups

and subgroups. Mean values were compared using t tests. Becausethe distributions of most values were right skewed, a normalizinglogarithmic transformation was used for statistical testing. For thesmall subgroups and the nonnormally distributed subgroups,the Mann–Whitney test was used to compare median values ofthose subgroups. Proportion values of growth in soft agar andrepair deficiency were compared using the x2 and/or Fisherexact tests. P values less than 0.05 were regarded as statisticallysignificant. The statistical analyses were all conducted in SAS 9.4(SAS Institute).

ResultsPrevious studies have shown particulate chromate can induce

CIN with prolonged treatment; however, it is unclear whetherthese changes are a permanent state or if these cells are unstableand removed from the cell population over time. To addresswhether particulate Cr(VI) can induce permanent chromosomalabnormalities, we conducted a clonal expansion experiment asillustrated in Fig. 1. Briefly, cells were treated with lead chromatefor 24 hours, allowed to form colonies, which were isolated andexpanded into cell lines. Once the first-generation clonal cell lineswere established, they were characterized and treated for anadditional 24 hours and then again allowed to form coloniesand expanded into cell lines forming a second generation ofclones. These clones were also characterized and treated for anadditional 24 hours to create a third generation of clones foranalysis and characterization. Untreated control cells were alsoexpanded and characterized in each generation to ensure thecloning process did not induce changes.

Chromosome analysisParticulate Cr(VI) induced structural and numerical CIN in

treated clones compared with untreated clones (Fig. 2A). Thisimpact was observed at both the whole cell level with 4-foldincrease in the percentage ofmetaphaseswithCIN for each treatedclone, and at the chromosomal level with almost a 6-fold increasein the total number of chromosomes affected (Fig. 2A). There wasno increase in either structural or numerical CIN in the control

clones above background (parental cells have a background rateof approximately 10% aneuploidy). Lead chromate inducedstructural alterations in 34% all metaphases across all treatedclones. There were a total of 51 structural abnormalities per 100metaphases, indicating many metaphases had more than onestructurally altered chromosome (Fig. 2B). Lead chromateinduced numerical CIN or aneuploidy in 43% of all metaphasesacross all treated clones (Fig. 2C). The most striking numericalchange observed was an increase in tetraploidy (defined as 92� 4chromosomes; Fig. 2D).We also considered aneuploidy based on100 metaphases counted for each clone (Fig. 2E). The averageaneuploidy in control clones was 19% compared with 48% in thetreated clones. Considered another way, 98%of the treated cloneswere greater than the average of the control clones (Fig. 2E).

Both the numerical and structural CIN were heritable at thecellular level (Fig. 2A). The first generation had only a slightincrease in overall CIN across treated clones. Twenty percent ofmetaphases had some form of CIN, and there were a total of 21abnormal events per 100metaphases. CIN increased in the secondgeneration to 55% of metaphases and a total of 88 abnormalevents per 100 metaphases, indicating cells were accumulatingmultiple structural and/or numerical abnormalities. The thirdgeneration had 53% of metaphases in treated cells with someformofCIN and a total of 67 abnormal events in 100metaphases.There was no increase in CIN across the untreated controlgenerations.

Considering structural and numerical CIN separately, 6% ofmetaphases from first-generation treated cells had structuralabnormalities, which increased to 36% in the second generationand 39% in the third generation. Total structural abnormalitiesalso increased in treated cells with 6, 69, and 52 total alterationsper 100 metaphases examined in the first, second, and thirdgenerations, respectively (Fig. 2B). There was no increase incontrol cells.

For numerical CIN, lead chromate induced persistent aneu-ploidy in all generations with 16%, 46%, and 47% of aneuploidmetaphases in thefirst, second, and third generations, respectively(Fig. 2C). Of these aneuploid cells, 2% of treated clones weretetraploid or near tetraploid; in the second and third generations,22% and 20%of treated clones were tetraploid or near tetraploid,respectively; whereas less than 1% of control clones exhibitedtetraploidy (Fig. 2D). Thus, we saw increases in CIN across allgenerations in the treated cells but no increase in control cells.

Additional detail regarding specific chromosome changes isseen in Fig. 3. Control clones showed no significant structuralchanges. Chromosomes 1 (12%), 6 (6%), 7 (5%), 9 (15%), 10(10%), 13 (15%), and 21 (5%) were the most commonly alteredchromosomes in the treated clones (Fig. 3A). The most commonchromosome loss was the Y chromosome; 9% of all metaphasesanalyzed in the treated clones exhibited loss of chromosome Y,compared with 2% in the controls (Fig. 3B). Other chromosomescommonly lost in the treated clones included chromosomes 7(5%), 17 (5%), 21 (6%), and 22 (7%); chromosome loss incontrol clones was 3% or less for all chromosomes (Fig. 3B).Chromosome gain (other than ploidy changes) was much lesscommon; the most common chromosome gain for the treatedclones was chromosome 13 (Fig. 3C).

Pedigree analysisAlthough the analyses above show the overall and cumulative

effects of lead chromate on clonally expanded and treated cells, it

Cr Induces Persistent and Heritable CIN and Deficient DNA Repair

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does not take into consideration the fact that each generation ofclones is related to eachother. To demonstrate the relatednessof theclones, we assembled a pedigree for each family to show how they

are related and how they evolve over time and with additionaltreatment.Weused standard cytogeneticnomenclature according tothe International System for Cytogenetic Nomenclature (25);

Figure 2.

Repeated exposure to particulate Cr(VI)induces CIN. This figure shows the types ofCIN observed in all metaphases examined aswell as across generations. A, All CIN andall CIN across three generations. B, StructuralCIN shown as the percentage of structurallyabnormal metaphases and total abnormalchromosomes in 100 cells, accounting formetaphases with more than one damagedchromosome for all cells and across threegenerations. C, Numerical CIN in all cells andacross three generations. D, Tetraploid andnear-tetraploid cells in all cells and acrossthree generations (1,859 metaphases wereanalyzed for control clones and 1,343metaphases were analyzed for treated clones;144, 431, and 1,284 metaphases were analyzedin the G1, G2, and G3 generations in controlcells, respectively. 160, 388, and 795metaphases were analyzed in the G1, G2, andG3 generations in treated cells, respectively).� , statistically different from comparablecontrol cells (P <0.05). E,Aneuploidy found ineach treated clone. Analysis is based on 100metaphases for each clone. WTHBF-6 showsthe aneuploidy found in the parental cell line.C1–1 shows the highest level of aneuploidyfound in the comparable control clones.Overall aneuploidy in treated clones wasstatistically higher than the control clones (P <0.05).

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therefore, cells with a single cell missing a chromosome are notreflected here. In some instances, when a clonewas treated, the cellshad an increased sensitivity to lead chromate and no cells survivedtreatment; in other instances, only one or two clones survivedtreatment and successfully expanded into cell lines.

Most control clones had normal karyotypes (Fig. 4A). CloneC1–1hadabalanced translocationbetween chromosomes 13 and17 in half of the analyzed metaphases not seen in subsequentgenerations. Clone C51–1 had 12 normal cells and 8 cells withadditional material on chromosome 21. The remaining 89 cloneswere normal.

Within the treated pedigrees, we considered clones at threelevels of analysis: Individual clones, generation, andfamily. Figure 4B shows the pedigrees of the treated clones.At the level of individual clones, 44% of the clones wereabnormal. Specifically, 35 clones had normal karyotypes(T1, T2, T2-2, T21-1, T22-1, T22-3, T3, T3-1, T3-2, T31-2,T31-3, T4, T5, T5-1, T51-2, T51-3, T6, T6-1, T6-2, T6-3, T61-2, T61-3, T62-1, T62-2, T62-3, T63-2, T7, T7-2, T71-2, T71-3,

T72-1, T72-2, T72-3, T73-1, and T73-3), and 28 clones hadCIN (T1-1, T1-2, T2-1, T2-3, T21-2, T21-3, T22-2, T23-1, T23-2,T23-3, T3-3, T31-1, T32-1, T33-1, T33-2, T33-3, T4-1, T4-2, T41-1,T41-2, T41-3, T51-1, T61-1, T63-1, T7-1, T7-3,T71-1, and T73-2).

Based on generation analysis, all first-generation clones hadnormal karyotypes, 53% of clones in the second generationwere abnormal, and 49% of clones in the third generation wereabnormal. Clones that were abnormal in the second generationwere also abnormal in the third generation, demonstrating theheritability and persistence of the damage. The specific abnor-malities observed in the second generation persisted into thethird generation. For example, clone T2-3 had 20 cells with anunbalanced translocation between chromosomes 10 and 13,and 17 of those cells had an extra copy of chromosome 13; inthe daughter cells, the same abnormalities persisted but withdiffering frequencies (T23-1 had 20 cells with the translocationand 3 cells with the extra 13; T23-2 had 20 cells with thetranslocation and 11 cells with the extra 13; and T23-3 had 20cells with the translocation and 3 cells with an extra 13). Thispattern is also seen between clones T3-3 and T33-1 with bothlines being tetraploid, and between T2-1 and two of its daugh-ters T21-2 and T21-3.

In some instances, these third-generation clones acquired addi-tional chromosome abnormalities. For example, clone T4-1 had91 chromosomes with an unbalanced translocation betweenchromosomes 1 and 9, and it had one daughter with the sametranslocation as well as some cells with an isochromosome 7 andanother unbalanced translocation between chromosomes 13 and14. This pattern was also seen between clone T3-3 and two of itsdaughters (T33-2 and T33-3) and between clone T7-3 and one ofits daughters (T73-2).

Analysis of the pedigrees by family showed CIN in all clonalfamilies. In addition, the family pedigrees show five clonal fam-ilies had clones that were more sensitive to additional Cr(VI)treatment and did not survive to form further generations. Inclonal families T1, T4, and T5, there was increased sensitivity inthe second generation; specifically, there were only two second-generation clones developed in the T1 family, two second-generation clones in the T4 family, and only one-second gener-ation clone in the T5 family.

Another pattern that becomes apparent with the pedigrees isthe induction of a tetraploid chromosome complement. Thereare four clonal families showing a tetraploid or near-tetraploidstate; specifically, families T2, T3, T4, and T7. In two of thesetetraploid clones, there is increased CIN in the subsequentgeneration. For example in the clonal family T3, tetraploidyis the only abnormality observed in one of the second-gener-ation clones. The next generation of this clone results in oneclone with the same karyotype; one clone with a tetraploidkaryotype and a structural abnormality with chromosome 7;and a clone with a near-tetraploid karyotype with additionalloss of either chromosome 5 or chromosome 7 or both. Thissuggests a tetraploid state may be an initiating event in Cr(VI)-induced CIN. This conclusion is also supported by other cloneswith a near-tetraploid chromosome complement that have oneof the four chromosomes altered as seen in clones T4-1 withdaughter clone T41-1 (Fig. 4C) and clones T7-1 with daughterclone T71-1. If the structural alteration had occurred prior tothe induction of tetraploidy, then two of the four chromosomeswould have the same affected chromosome; we did not observethis in any of the clones.

Figure 3.

Chromosome-specific effects observed after repeated particulate chromatetreatment. This figure shows the chromosome-specific effects in all metaphasesexamined. A, Percentage of metaphases with structural alterations tochromosomes. B, Percentage of metaphases with missing chromosomes.C, Percentage of metaphases with additional chromosomes.






Figure 4.

Karyotype pedigrees of untreated and treated clones. This figure shows the karyotype designation for each of the control (A) and treated (B) clone families.All karyotypes were derived from at least 20 analyzed cells. Clone C1–1 and C51–1 had mixed populations of half normal and half abnormal karyotypes, andthe remaining 89 control clones were normal. Some treated clones had mixed cell populations, and the number of cells in the brackets indicates the numberof cells for each type of abnormality; a cp in front of the number means the karyotype is a composite of several karyotypic lines with related karyotypes butnot all identical. Abnormal chromosomes are designated with an abbreviation (for example, der or t followed by the affected chromosome numbers; iso,isochromosome; t, balanced translocation; der, unbalanced translocation; add, additional material of unknown origin). Missing or additional chromosomes areindicated with a (–) or (þ) sign. C, Representative karyotype of treated clones. T4-1 has derivative chromosome made up of chromosomes 1 and 9, and thecomplementary chromosome 9 is missing. T41-1, the derivative chromosome persists into the third generation; in addition, the third-generation clone acquiredan isochromosome 7. T1-1 has 75 chromosomes, 22 ofwhich are structurally abnormal. Arrows, structurally abnormal chromosomes. Abnormal chromosomes includetranslocations, dicentric chromosomes, deletions, and additions. There are also numerical defects; for example, there are 6 copies of chromosome 1 (4 normal and 2abnormal), and there is only one copy of chromosomes 14 and 22. The analysis of clone T1-1 revealed every cell had a different chromosome complement and differentstructural abnormalities.

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We also observed clones with a heterogeneous population(not all 20 cells had the same karyotype). Six clonal familieshave at least one clone that is heterogeneous: T1, T2, T3, T4, T6,and T7. The only family without heterogeneous clones was T5,which consisted of only 5 clones; one was abnormal. Inaddition, there were two clones (T1-1 and T4-2) with severeCIN in which every cell examined had a different chromosomecomplement. Clone T4-2 had several cells with the samestructural aberrations. Clone T1-1 was highly unstable, withevery cell analyzed having different chromosome alterations(Fig. 4C).

Neoplastic transformationHaving determined lead chromate–induced permanent CIN,

we wanted to determine if this state was sufficient for neoplastictransformation. Normal human lung cells exhibit anchorage-dependent growth and require a surface that allows them toattach to, flatten out, and divide. Transformed cells do not requirea surface to attach to and have the ability to grow in suspension.Anchorage-independent growth strongly correlates with tumori-genicity and invasiveness in several cell types, including small-celllung carcinomas (20). Therefore, we assessed the ability of theclones to grow in soft agar. Figure 5A shows pictures of colonies inagar and microscopically. Eighteen percent of all treated clonesdeveloped the ability to grow in soft agar (Fig. 5B). Considered bygeneration, 14.4% of first-generation clones grew in agar, 12.5%

of second-generation clones grew in agar, and 20.5% of third-generation clones grew in soft agar (Fig. 5C). None of the controlclones grew in agar.

Comparing the karyotype results with the agar results raisesinteresting questions about the frequency with which cells withCIN grow in soft agar and the frequency with which cells thatgrow in soft agar exhibit abnormal karyotypes. First, consider-ing the clones that grew in agar, 50% were chromosomallyabnormal; none in the first generation, 100% in the secondgeneration. All of the clones in the third generation exhibitedsome degree of CIN, but only 50% had consistent CIN reflectedby the same chromosomes being affected in at least 85% of thecells analyzed (Table 1). Second, considering the percentage ofchromosomally abnormal clones that grew in agar, 14% of thechromosomally abnormal clones grew in agar; none in the firstgeneration, 25% in the second generation, and 15% in thethird generation (Table 1). Thus, CIN is a common phenotypein clones neoplastically transformed by particulate Cr(VI),but several with particulate Cr(VI)-induced CIN do not growin agar.

Underlying dysregulation leading to CINTo begin to understand the underlying causes of the observed

CIN, we investigated potential contributing factors to bothnumerical and structural CIN. We considered centrosomeamplification. Abnormal centrosome numbers are common in

Figure 5.

Cells treated with prolongedparticulate Cr(VI) grow in soft agar.This figure shows the results of thesoft-agar assay. A, Images of theclones in agar. The first row shows arepresentative clone (T73-3), with theagar colonies stained in the dish. Thesecond row of images shows the cellsunder 10�; clone T41-1 did not formcolonies in agar and only single cellsare seen, clone T73-1 grew smallcolonies in agar, and clone T23-2 grewlarge colonies in agar.B,Percentage ofall clones able to grow in agar. C,Percentage of clones able to grow inagar by generation. � , statisticallydifferent from control clones(P < 0.05).






tumors (26, 27). Incorrect centrosome number can lead tomultipolar segregation of chromosomes resulting in the incor-rect chromosome complement in daughter cells (28). In addi-tion, multipolar cells can abort cytokinesis and result in a singletetraploid cell (29, 30). Twenty-nine percent of all treatedclones exhibited centrosome amplification (Fig. 6A). Consid-ered by generation, 43% of first-generation clones had ampli-fied centrosomes, 44% of second-generation clones had ampli-fied centrosomes, and 21% of third-generation clones hadamplified centrosomes (Fig. 6B). None of the control clonesshowed amplified centrosomes. Broken down by generation,we saw 3 of 7 clones in the first generation, 7 of 16 clones in thesecond generation, and 21 of 39 clones in the third generationexhibited centrosome amplification.

Another aspect that may contribute to numerical CIN is defectsin the SAC. The SAC serves to ensure that cells do not progress toanaphase and cytokinesis until all of the chromosomes areproperly aligned. Thus, we analyzed metaphases for indicationof SAC bypass manifested as centromere spreading, prematurecentromere division, and premature anaphase. Forty-one percentof all treated clones exhibited SAC bypass (Fig. 6C). Consideredby generation, 29% of first-generation clones exhibitedSAC bypass, 44% of second-generation clones exhibited SACbypass, and 42% of third-generation clones exhibited SAC bypass(Fig. 6D).

DNA DSB repairIn order to better understand the structural alterations

observed, we sought to reveal the underlying contributingfactors leading to chromosome translocations. Chromosometranslocations require multiple DNADSBs and thus we chose tolook at the efficiency of DNA DSB repair. Previous studiesfrom our lab show Cr(VI) induces persistent DNA DSBs(17). We treated cells with soluble sodium chromate for24 hours and measured the induction of DNA DSBs usingcolocalized g-H2AX and 53BP1 foci. In complimentary dishes,we washed the treatment out and gave cells 24 hours to repairthe damage. Figure 6E shows a representative example of repairdeficiency in a control clone and a treated clone; after 24 hoursof treatment, the control and treated clones show similar levels(22 and 24, respectively) in the percentage of cells with morethan 5 foci, indicating DNA DSBs occurred. When the cells weregiven time to repair, we found the control clone showed areduced number of foci (10%) indicating repair, whereas thetreated clone failed to repair the DNA DSBs and actually hadmore damage (40%). Figure 6F shows 59% of all treated clonesdisplayed some level of repair deficiency. Considered by gen-eration, 29% of treated clones were repair deficient in the firstgeneration, 63% in the second generation, and 64% in the thirdgeneration (Fig. 6G). None of the control clones were repairdeficient.

DiscussionCIN is thought to be a central component of the carcinogenic

mechanism for Cr(VI) as the model of clonal expansion of drivermutations does notfit well with the available data.Most studies ofthe carcinogenic mechanism of Cr(VI) involve data that considerimpacts in the immediate aftermath of exposure, i.e., assays aretreatedwithCr(VI) and the outcomesmeasured immediately afterexposure. Although this approach indicates impacts of exposure,it cannot informwhether those impacts are transient changes thatrequire the presence of Cr(VI) or if they are permanent changesthat persist long after the exposure has ended. The notableexception being assays for transformation, which occur weeksafter exposure ends. The cell lines we analyzed were all clonaloriginating from a single cell. Thus, the observed outcomesrepresent permanent changes. The second generation of cells wasderived from the first, and the third from the second allowingfor us to see changes heritable at a cellular level. Our data showall of our endpoints, CIN, DNA repair deficiency, centrosomeamplification, and growth in soft agar were permanent andheritable changes.

This article is the first to report Cr(VI)-induced transloca-tions. It is well established that Cr(VI) induces chromosomalaberrations including chromatid and chromosome lesions.Observations of dicentrics, triradial figures, and chromatidexchanges indicate Cr(VI) has the potential to induce translo-cations (23, 31, 32), but the only report of any chromosomespecificity is limited to observations that aberrations preferen-tially occur in euchromatic regions over heterochromaticregions (33). We found the amount of structural chromosomedamage was heritable and increased with generation, indicatingprogressively more CIN. This outcome is consistent with pre-vious observations of increasing chromosomal aberrations withincreasing length of Cr(VI) exposure in solid stained chromo-somes from human lung cells (10–12) and further supports thehypothesis that CIN is a significant part of Cr(VI)'s carcinogenicmechanism.

Chromosome translocations, deletions, and duplications arethe most frequent structural rearrangements observed in somat-ic tumors, but distinct and recurrent chromosome transloca-tions in tumors are rare (34). We found Cr(VI) caused recurrenttranslocations in chromosome numbers 1, 3, 6, 7, 9, 10, 13, 17,and 21. Moreover, of the recurring chromosomal translocationsobserved, 94% were nonreciprocal or unbalanced transloca-tions leading to either the loss or gain of partial chromosomes.Interestingly, all but one of the recurring translocationsobserved were whole-arm translocations resulting in loss ofchromosomes 1p, 3p, 9p, 10p, 16q, and 17p or gain ofchromosomes 9q and 13q. There are few reports of recurringwhole-arm translocations in any cancer. Whole-arm transloca-tions are frequent in head and neck squamous cell carcinomawith whole-arm losses seen at 3p, 8p, 9p, 17p, and 18q and

Table 1. Comparison of neoplastic and CIN phenotypes

Generation Number ofclones tested

Number ofclones with CIN

Number ofagarþ clones

Number ofagarþ with CIN

% Agarþclones with CIN

% CIN clonesalso agarþ

All 28 8 4 50 141 7 0 1 0 0 02 16 8 2 2 100 253 39 20 6 3 50 15

Wise et al.





gains at 3q, 5p, 7p, 8q, and 20q (35). Whole-arm translocationsleading to loss of 17p have been implicated in advancedhematologic malignancies with poor prognoses (36). Whole-arm translocations involving chromosomes 7, 10, 12, 14, and21 were report for cervical cancers (37). One study investigated

whole-arm chromosome translocations among adenocarcino-ma versus squamous cell carcinoma and found the whole-armtranslocations observed in squamous cell carcinomas werederived from centromere breakage and refusion (38). Chromi-um causes predominately squamous cell carcinoma in lungs

40

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G1 G2 G3

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G1 G2 G3

Control clones Control clones

Control clones

Treated clones

Control clones Treated clones

Control clones Treated clones Control clones Treated clones

Treated clones

Treated clones

Control clone 24 h treatment

Treated clone 24 h treatmentControl clone 24 h recovery

Treated clone 24 h recovery

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C11-1 T41-2

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Figure 6.

Repeated exposure to particulate Cr(VI) induces centrosome amplification, SAC bypass, and DNA repair deficiency. These data show some of the potentialunderlying causes for CIN. Overall centrosome amplification in treated clones was statistically higher than the control clones (P < 0.05). A, Percentage of all cloneswith centrosome amplification. B, Percentage of clones with centrosome amplification by generation. Analysis is based on 100 interphase cells for each clone.Experiments were done in triplicate. Overall SAC bypass in treated clones was statistically higher than the control clones (P < 0.05). C, Percentage of allclones with SAC bypass. D, Percentage of clones with SAC bypass by generation. Analysis is based on 100 metaphases for each clone. E, Representative exampleof a control clone and a treated clone (background levels have been subtracted). F, Percentage of clones with DNA repair deficiency. G, Percentage of cloneswith DNA repair deficiency by generation. � , statistically different from control clones (P < 0.05).






of exposed workers. Thus, Cr(VI) may induce centromericinstability, which is also supported by our previous observa-tions of premature centromere division in Cr(VI)-exposed cells(12, 14).

DNA DSBs have emerged as a critical step in the carcinogenicmechanism for Cr(VI) and underlies the structural chromo-somal changes (20). Remarkably, data show, in addition toinducing these breaks, Cr(VI) inhibits their repair, specificallytargeting the RAD51 step of homologous recombination (HR)repair (17, 18, 39). Loss of HR allows Cr(VI) to induce theneoplastic transformation of cells and increase levels of CIN. Inthis report, we find for the first time that Cr(VI) inhibition ofDNA repair persists long after exposure has ceased and isheritable at a cellular level. This outcome further strengthensthe importance of DSB repair inhibition in the carcinogenicmechanism of Cr(VI).

We found Cr(VI)-induced aneuploidy, which is a consistenthallmark of cancer cells (40). We found Cr(VI) caused ampli-fication or loss of chromosome numbers 7, 13, 17, 21, 22, andthe Y chromosome, all have been observed in lung cancer (41).Loss of chromosome Y has been associated with increased riskof nonhematologic cancer in men (42). Loss of chromosome 7has been suggested as a marker for melanoma due to the lossof EGFR located at chromosome 7p12.3-p12.1 (43). Mono-somy of chromosome 17 has been implicated in breast cancerwith loss of p53, BRCA1, and TOP2A (44). LOH of chromo-some 22 has been associated with multiple cancers, includinglung cancer (45), and suggests a loss of tumor-suppressorgenes (46). Interestingly, we found a higher proportion ofsmall chromosomes were affected compared with largerchromosomes, which is consistent with a study considering43,205 human tumors with greater loss of small chromosomes(47). Gain of chromosome 13 has been implicated in colo-rectal cancer (40). These outcomes are also consistent withprevious observations showing Cr(VI) induces aneuploidy inhuman lung cells including hypodiploidy, hyperdiploidy,and tetraploidy (9, 12, 14, 23) and further supports thehypothesis that CIN is a significant part of Cr(VI)'s carcino-genic mechanism.

We found an increase in tetraploidy and near tetraploidy. Thisoutcome is consistent with observations in lung cancers withsevere aneuploidy (6). Several recent studies have shown tetra-ploidy is often found in precancerous lesions in a variety oftissue types (40, 48). Studies have also shown tetraploidy as anintermediate step in chemically induced aneuploidy and cellulartransformation (24, 49, 50).

Centrosome amplification and SAC bypass have emerged as afactor in the carcinogenic mechanism for Cr(VI) and underliethe numerical chromosomal changes (11, 12, 14). Cr(VI)induces centrosome amplification in human lung cells, whichis maintained in Cr(VI)-transformed cells (24). The targetappears to be the centrioles within the centrosomes withalterations of centriole numbers and an increase in centriolesplitting (16). In this report, we find for the first time Cr(VI)-

induced amplification persists long after exposure has ceasedand is heritable at a cellular level. In addition, we found SACbypass persisted and was heritable. These outcomes furtherstrengthen the importance of centrosome amplification andSAC bypass in the carcinogenic mechanism of Cr(VI).

Human pathology studies of workers with Cr(VI)-inducedlung cancer show Cr accumulates and tumors form at bronchialbifurcation sites where Cr(VI) particles affect, persist, anddissolve. This accumulation of Cr was noted as a key factor inthe incidence of tumors in these workers, more so than thedose. Our data are consistent with this conclusion as they showthe majority of effects do not fully manifest themselves untilafter two exposures to Cr(VI). Our previous data would alsoappear to implicate accumulation as the key factor in thosestudies and the major impacts were not seen until after at least48 hours of exposures.

In sum, we show for the first time Cr(VI) induces chromosometranslocations, and the chromosome translocations, aneuploidy,and polyploidy observed are permanent and heritable. Further,we show the underlying centrosome amplification and DNAdamage repair defects are also permanent and heritable. Thesechromosome imbalances likely lead to preferential selection andsurvival of cells in which oncogenes are activated and/or tumor-suppressor genes are lost providing a growth advantage forcancerous cells.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S.S. Wise, J.P. Wise, Sr.Development of methodology: S.S. Wise, J.P. Wise, Sr.Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.S. Wise, J. Martino, J.P. Wise, Sr.Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.S. Wise, A.E.-M. Aboueissa, J. Martino, J.P. Wise, Sr.Writing, review, and/or revision of the manuscript: S.S. Wise,A.E.-M. Aboueissa, J. Martino, J.P. Wise, Sr.Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.S. Wise, J.P. Wise, Sr.Study supervision: S.S. Wise, J.P. Wise, Sr.

AcknowledgmentsThis work was supported by a grant from the National Institute of Environ-

mental Health Sciences (ES016893 to J.P. Wise, Sr.). The authors would like tothank Rachel Speer, Greer Chapman, Kelsey Thompson, Therry The, Hong Xie,Christy Gianios, Jr., Kelly Holland, Aaron Howell, and Blair Cade for technicaland administrative assistance.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 19, 2018; revised May 3, 2018; accepted June 4, 2018;published first June 7, 2018.

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2018;78:4203-4214. Published OnlineFirst June 7, 2018.Cancer Res Sandra S. Wise, Abou El-Makarim Aboueissa, Julieta Martino, et al.

Deficient Phenotype−DNA Repair Permanent and Heritable Numerical and Structural Changes and a

Induced Chromosome Instability Drives−Hexavalent Chromium

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