DNA fragile site breakage as a measure of chemical exposure and predictor of individual susceptibility to form oncogenic rearrangements
DNA fragile site breakage as a measure of chemical exposure and predictor of individual susceptibility to form oncogenic rearrangements
Christine E.Lehman 1
Laura W.Dillo n 1
Yuri E.Nikiforov 0
Yuh-Hwa Wang 1
0 Department of Pathology and Laboratory Medicine, University of Pittsburgh , Pittsburgh, PA 15261 , USA
1 Department of Biochemistry and Molecular Genetics, University of Virginia , 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0733 , USA
Chromosomal rearrangements induced by non-radiation causes contribution to the majority of oncogenic fusions found in cancer. Treatment of human thyroid cells with fragile site-inducing laboratory chemicals can cause preferential DNA breakage at theRET gene and generate theRET/PTC1 rearrangement, a common driver mutation in papillary thyroid carcinomas (PTC). Here, we demonstrate that treatment with non-cytotoxic levels of environmental chemicals (benzene and diethylnitrosamine) or chemotherapeutic agents (etoposide and doxorubicin) generates significant DNA breakage withinRET at levels similar to those generated by fragile site-inducing laboratory chemicals. This suggests that chronic exposure to these chemicals plays a role in the formation of non-radiation associatReEdT/PTC rearrangements. We also investigated whether the sensitivity of the fragRilEeT region could predict the likelihood of rearrangement formation using normal thyroid tissues from patients with and withouRtET/PTC rearrangements. We found that normal cells of patients with thyroid cancer driven byRET/PTC rearrangements have significantly higher blunt-ended, double-stranded DNA breaks atRET than those of patients withouRtET/PTC rearrangements. This sensitivity of a cancer driver gene suggests for the first time that a DNA breakage test at thReET region could be utilized to evaluate susceptibilityRtEoT/PTC formation. Further, the significant increase of blunt-ended, double-stranded DNA breaks, but not other types of DNA breaks, in normal cells from patients withRET/PTC-driven tumors suggests that blunt-ended double-stranded DNA breaks are a preferred substrate for rearrangement formation, and implicate involvement of the non-homologous end joining pathway in the formation of RET/PTC rearrangements.
Chromosomal rearrangements are a common genetic abno-r (PTC) (
) accounting for approximately 20% of adult3)( and
mality involved in the initiation of cancer development. These approximately 45% of childhood sporadic PTC cases 4(). PTC
rearrangements result in the disruption of genetic material, is responsible for the rising incidence of thyroid cancer in the
which can lead to the expression of oncogenic fusion proteins USA (
), where thyroid cancer is now the most rapidly incre-as
or the disruption of processes involved in tumor suppression1(). ing cancer type 5(,6). Although increases in thyroid cancer have
DNA strand breaks must occur in participating gene regions to been well documented following exposure to high doses of ra-di
initiate all chromosomal rearrangements. We have shown that ation (
), many tumors are sporadic in nature 4(
suggestfragile site-inducing conditions can create DNA breaks within ing other factors are involved in initiating DNA breaksRiEnT/
RET/PTC participating genes and ultimately lead to the for-ma PTC genes. RET, an oncogene involved in recurrent chromosomal
tion ofRET/PTC rearrangements2(). RET/PTC rearrangements are rearrangements found in thyroid3(
) and lungs (11,12) is
a common mutation observed in papillary thyroid carcinoma located within fragile site FRA10G. Our previous study offers
Abbreviations we found that patients with thyroid cancer driven bRyET/PTC
rearrangements have significantly more blunt-ended
doubleDEN diethylnitrosamine stranded breaks atRET intron 11 compared to patients without
PTC papillary thyroid carcinoma the rearrangements, suggesting that this increased breakage leads
NHEJ non-homologous end joining to the formation ofRET/PTC rearrangements. The results support
the hypothesis that chronic low-dose exposure to environmental
direct evidence for the role of fragile sites in cancer-specific fragile site-inducing chemicals or residual chemotherapy agents
rearrangements 2(). contributes to the formation of non-radiation induceRdET/PTC
DNA fragile sites are sensitive to a variety of chemicals and rearrangements, which are more common than radiation-induced
have been identified in regions with deletions and chromosomal RET/PTC in thyroid cancer. Also, the significant increase in
). Environmental agents such as benzene ended DNA breaks withinRET in normal cells ofRET/PTC patients
(in cigarette smoke and automobile exhaust) and diethylnit-ro implicate blunt-ended DNA breaks in the formation oRfET/PTC
samine (DEN) (in cigarette smoke, pesticides and cured meat), rearrangements. To our knowledge, this is the first study to d-em
and chemotherapeutic drugs, can significantly increase fragile onstrate the sensitivity of a cancer driver gene in normal cells,
site breakage 1(
) and are positively associated with the risk suggesting that a test to detect DNA breakageREaTtcould provide
of thyroid cancer1(
). Benzene has been associated with an an early indication of susceptibility to PTC.
increased risk of thyroid cancer after exposure to volcanic ac-tiv
ity at Mount Etna1(9), and an increased risk of thyroid cancer Materials and methods
was observed in female textile workers with 10 or more years
of benzene exposure (25). Specific nitrosamines, including DEN, Cell line and culture conditions
have been shown to cause thyroid tumors in animal studies2 7().
Human thyroid epithelial cells immortalized by SV-40 (HTori-3) were p-ur
Increasing dietary nitrate intake (which generates nitrosamine chased from the European Collection of Authenticated Cell Culture and
by interacting with aminesin vivo, or by cooking) is positively were grown in RPMI1640 medium (Gibco) supplemented with 10% fetal
associated with thyroid cancer risk in older wome2n4() and men bovine serum and penicillin/streptomycin.
(17). Chemotherapy regimens which contain the topoisomerase
II inhibitors etoposide or doxorubicin have been linked to s-ec Tissue sample procurement
ondary PTC following treatment for primary osteosarcom1a8(), Tissue samples were obtained from the University of Pittsburgh Health
rhabdomyosarcoma (22), Ewing’s sarcoma and Wilms tumor Sciences Tissue Bank using a protocol approved by the University of
(15). We have found that DNA topoisomerases I and II partic-i Pittsburgh Institutional Review Board. Two paired samples (tumor and
pate in initiating fragile site breakage at tRhEeT oncogene (28). normal surrounding tissue) were obtained from each of six patients with
Therefore, investigating the sensitivity of tRhEeT region to these an RET/PTC+ tumor. Seven samples of normal thyroid tissue were also
chemicals is critical for understanding whether these agents are obtained from patients with benign nodules but without PTC (PTC−). Prior to
likely to contribute to the formation of non-radiation induced procurement for this study, tissue samples were histologically analyzed to
RET/PTC rearrangements, which are more common than radi-a tdreatnersmcrinipetatsuem-poorlytimsesuraesaencdhsauinrrroeuancdtiinogn n(RoTr-mPaClR)maasrgdienssc. rFiubretdhperre,vrieovuesrlsye
tion-inducedRET/PTC in thyroid cancer. (2), was used to verify the presence ofRET/PTC rearrangements in each
While common fragile sites are found in all individuals, the tumor sample and to confirm lack ofRET/PTC rearrangements in the su-r
extent of DNA breaks at particular fragile sites varies among rounding normal tissue or in the normal tissue provided by PTC− patients.
individuals (29). Double-stranded DNA breaks are commonly
repaired through homologous recombination or non-homol-o Cell treatments and analyses
gous end joining (NHEJ) (30), but dysfunction in these pathways Cell viability was assessed as previously described28() using propidium
can contribute to increased DNA breakage. Alternatively, an iodide staining and quantification by flow cytometry. Based on known
accumulation of chemical exposures could increase the number fragile site-inducing conditions13(), HTori-3 cells were treated for 24 h
of DNA breaks and overwhelm cellular DNA repair pathways, with 0.4 µM aphidicolin (APH) and various concentrations of benzene
resulting in the formation of chromosomal rearrangement1s).( (0.5–1 mg/ml; Sigma-Aldrich), DEN (3.5–7 mg/ml; Sigma-Aldrich),
etopoTherefore, the DNA fragility at rearrangement-participating and side (0.3–0.5 µM; Sigma-Aldrich) or doxorubicin (5–10 nM; Sigma-Aldrich),
breakage-sensitive gene regions may be indicative of an ind-i to dTeotaernmalinyzeetahcetiovpetaimpoapltdoosissa, gHeTowrhi-i3cchedllids wnoerteatlrteeartceedlwlivtihabtihleitcyh. emicals
vidual’s exposure to environmental factors and/or an unfavo-ra described above for 24 h, harvested and resuspended in 1X Annexin V bin-d
ble genotype for DNA stability, and thus predict a potential for ing buffer containing Annexin V (BD Biosciences). Early apoptotic cells were
the formation of cancer-causing chromosomal rearrangements. then quantified using a FACSCalibur flow cytometer and FlowJo software.
Both canonical NHEJ and microhomology-mediated end-joining To analyze cell growth, the number of viable HTori-3 cells was de-ter
(MMEJ or alt-NHEJ) are implicated in the formation of inter- mined by trypan blue exclusion using a hemocytometer. Cells were plated
and intra-chromosomal rearrangements 3(
). Studies by at 1 × 105 viable cells and treated with the indicated chemical con-cen
Ghezraoui et al. (36) demonstrated that chromosomal translo-ca trations. After 24 h of chemical treatment, viability was determined and
tions depend on canonical NHEJ in human cells, and the ends of the cells were washed with PBS and re-plated in chemical-free media to
DNA breaks, such as blunt or long staggered, can influence the recover for an additional 24 h before being quantified again.
characteristics of rearrangement fusion points. platFeosrabnrdeatkrpeoaintetddaetppercotxioimna,HteTloyri1-83 chellalste×(1r 1 0w5)itwhe0rµ.e4M plAaPtHed,0in.5 smixg-/wmelll
In this study, we first demonstrated thRaEtT intron 11, the major benzene, 3.5 mg/ml DEN, 0.3 µM etoposide or 5 nM doxorubicin for 24 h.
patient break point region found in PTC patients37(–40) is sensi- Genomic DNA was then isolated from treated and untreated cells in pa-ral
tive to DNA breakage when exposed to non-cytotoxic, low doses lel for DNA break analyses.
of environmental chemicals (benzene or DEN) or chemother-a
peutic agents (etoposide or doxorubicin). This sensitivity was also Detection and quantification of all DNA breaks by
detected in the form of blunt-ended DNA double-stranded breaks ligation-mediated PCR (LM-PCR)
generated by these exposures. Using normal human thyroid tissue The total number of DNA breaks including single-stranded nicks and
samples from patients with or withoutRET/PTC rearrangements, double-stranded DNA breaks was detected and quantified as described
previously (2,28) (Supplementary Figure 1A, left panel). Briefly, genomic as benzene and DEN are encountered daily and are associated
DNA was isolated from HTori-3 cells or normal thyroid tissues of patients, with an increased risk of thyroid cancer17(
). In order to
followed by primer extension using a ′5-biotinylated primer matched mimic daily, low level exposure to environmental chemicals, we
to the regions of interest. DNA breaks were ligated to the asymm-etri used a cell viability assay to determine optimal, non-cytotoxic
cAamlpldifuipclaetxioLnLo3f/LtPh2e lDinNkAerbraenakds twhaesnaicshoileavteeddthursoinugghstnreesptteadviPdCinR obfetahdes. dosages of benzene and DEN to ensure that significant levels
extension-ligation products for each region of interest. Linker and primer of cell death were not induced relative to the untreated control
sequences have been described previously 2(). PCR products were resolved (Figure 1A). Treatment of human thyroid epithelial cells
(HToriby gel electrophoresis and sequenced to verify their identity. Each band 3) with 0.5 mg/ml benzene or 3.5 mg/ml DEN for 24 h did not
visualized on the gel represents one break isolated within the gene region induce apoptosis, as measured by flow cytometry analysis of
of interest. DNA breaks were then quantified as the number of DNA breaks annexin V staining F(igure 1B). Cell growth, measured by trypan
per 100 cells. blue exclusion over a period of 48 h, was not perturbed following
the same chemical treatment conditionsF(igure 1C).
Detection and quantification of double-stranded Therefore, these conditions were used to determine whether
DNA breaks by LM-PCR RET intron 11 was sensitive to DNA breakage upon exposure
To detect only blunt-ended double-stranded DNA breaks, genomic DNA to non-cytotoxic levels of benzene or DEN. HTori-3 cells were
isolated from HTori-3 cells or normal thyroid tissues of patients was -sub treated with 0.5 mg/ml benzene, 3.5 mg/ml DEN or 0.4µ M APH
jected to a protocol described previously41() (Supplementary Figure 1A, (as a positive control) for 24 h before being harvested for LM-PCR
right panel). Genomic DNA was directly ligated to the asymmetrical analysis of all types of DNA breaks, including single-stranded
SdeupphleaxdexLLG3-/1L0P02 cloilnukmenr,s parniodr ltioganteisotnedmPiCxRt.uPrCeRs pwroedreuctpusrwifeiered retshorlovuegdh nicks and double-stranded DNA breaks with ′-5overhangs,
by gel electrophoresis, sequenced and quantified as described above. 3′-overhangs and blunt ends (Supplementary Figure 1A, available
To detect all double-stranded DNA breaks, genomic DNA was first i n-cu at Carcinogenesis Online, left panel2();). Both benzene and DEN
bated withE. coli DNA polymerase I large (Klenow) fragment (New England induced significantly higher DNA breakage aRtET compared to
Biolabs) followed by heat inactivation at 75°C. The Klenow fragment -con untreated cellsFi(gure 2A and Supplementary Figure 1B,
availaverts double-stranded DNA breaks with eithe′r- 5or 3′-overhangs to blunt ble atCarcinogenesis Online). Also, the frequencies of DNA brea-k
ends through its polymerase and ′3-5′-exonuclease activities. The blunt- age induced by either of these chemicals were similar to that
ended DNAs were then ligated directly to the LL3/LP2 linker. Detection induced by APH, which we have shown leads to the formation of
and quantification of the DNA breaks was performed as described above. RET/PTC rearrangements 2(). FHIT, encompassing the fragile site
To detect the combination of ′5-overhang and blunt-ended double- FRA3B (42), also showed a significant increase in breakage upon
sFtrraagnmdeendt D(3N′–A5′ berxeoa-k)sl,agcekninogmic3′-5D′-NeAxonwuacsleiansceubaactteidvitwyit(hNetwheEnKglelannodw treatment with either chemical, while the non-common fragile
Biolabs), which creates blunt-ended DNA only from′-5overhang DNA site, G6PD region (2), was insensitive to fragile site induction by
breaks. Detection and quantification of the DNA breaks was performed as these environmental chemicals as well as APHFi(gure 2A).
described previously. Next, we investigated the induction of blunt-ended d-ou
To verify the ability of the assay to detect the specified type of DNA ble-stranded breaks withinRET intron 11 by these chemicals,
break, genomic DNA from HTori-3 cells was digested with various rest-ric since this type of break could be an immediate precursor in the
tion enzymes (New England Biolabs) to generate either single-stranded formation of rearrangements. Genomic DNA from treated or
nicks (by Nt.BstNBI digestion), ′5-overhangs (BbvI digestion), 3′-overhangs untreated cells was subjected to a modified LM-PCR procedure
(BanII digestion) or blunt-ended (HaeIII digestion) double-stranded DNA detecting only blunt-ended breaks (Supplementary Figure 1A,
Fbirgeuarkes ,1Aa,nadvasiulabbjelcetaetCdartcoinotgheenesfiosuOrnplrinoet.ocols listed in Supplementary available atCarcinogenesis Online, right panel). Treatment with
either benzene or DEN significantly increased the number of
Analysis of individual types of DNA breaks blunt-ended double-stranded DNA breaks in this region as
Normal thyroid tissue from patients withRET/PTC rearrangements or compared to untreated cellsFi(gure 2B). Further, blunt-ended
patients with benign nodules but without PTC, was evaluated indiv-idu double-stranded DNA breaks withinFHIT were also significantly
ally for each types of breaks as follows. To determine the amount of -sin increased compared to untreated cells, but with no difference
gle-stranded nicks only, the number of all double-stranded DNA breaks in the G6PD region. This result suggests that benzene and DEN
(from Klenow fragment-treated DNAs) was subtracted from the number induce fragile site-specific breakage within human thyroid e-pi
of all DNA breaks. To quantify ′3-overhang DNA breaks only, the num- thelial cells and demonstrate the sensitivity of human thyroid
ber of 5′-overhang and blunt-ended double-stranded DNA breaks (from epithelial cells to low-dose chemical exposures. This induction
3′ to 5′ exo-treated DNA) was subtracted from the number of all double- of DNA breakage atRET further suggests a role for these
longstranded DNA breaks (from Klenow fragment-treated DNAs). The amount term, low-dose chemical exposures in the formation of
nonof 5′-overhang DNA breaks was quantified by subtracting the number of radiation induced RET/PTC rearrangements, which are more
balnudnbtl-eunndte-ednddoeudbldeo-usbtlrea-nsdteradnDdNeAd bDrNeAakbsrweaitkhst(hfreonmu′3mtobe5r′ oe′xf-oo5v-terrehaatnegd common than radiation-inducedRET/PTC in thyroid cancer.
DNA). Treatment of primary tumors with chemotherapy regimens
including topoisomerase II inhibitors such as etoposide or dox-o
rubicin has been previously linked to secondary cancers incl-ud
Results ing PTC (
). Using previous pharmacokinetic studies to
mimic residual low doses following chemotherapy administr-a
Environmental chemicals and chemotherapeutic tion (43,44), we treated HTori-3 cells with various concentrations
drugs induce significant DNA breakage within of each drug for 24 hours and analyzed cell viability, apoptosis
fragile site genes including RET intron 11 and cell growthF.igure 1 shows that 0.3µ M etoposide or 5 nM
Intron 11 of theRET gene, the major patient break point region doxorubicin did not induce significant cell death or apoptosis,
(37–40), is sensitive to low doses of APH, a classic fragile site- and cell growth was not hindered.
inducing condition 2(). DNA breakage at this region, under fra-g Next, we examined the overall DNA breaks in HTori-3 cells
ile site-inducing conditions, leads to the formation RoEfT/PTC after 24 h treatment with 0.µ3M etoposide or 5 nM doxorubicin.
rearrangements 2(). Many fragile site-inducing chemicals such Both chemicals significantly increased breakage within intron
11 of RET as well as inFHIT (Figure 2A and Supplementary as a direct substrate to generate cancer-specific rearrangements.
Figure 1B, available atCarcinogenesis Online) even at these low Therefore, DNA breakage at these sensitive fragile regions may
non-cytotoxic doses. Treatment with either drug, however, did be a useful metric for patient prognosis and the risk of devel-op
not induce significant breakage withinG6PD, again suggest- ing therapy-related secondary cancers.
ing the specific sensitivity of DNA fragile sites to these drugs.
Importantly, these low dose concentrations are approximately Frequency of RET breakage in RET/PTC patient
300–1000-fold lower than the concentrations found in the normal tissues is predictive of RET/PTC
plasma of patients immediately after chemotherapy treatment rearrangements
), demonstrating the deleterious consequences to cells The presence of RET/PTC rearrangements in patients withRET/
that are not killed by treatment with chemotherapeutic agents. PTC-driven thyroid cancer suggests prior chemical and en-vi
Further, we specifically examined blunt-ended double- ronmental agent exposures and/or unfavorable genotypes for
stranded DNA breakage induced after exposure to either che-mi DNA stability, which cause breakage in the intron 11 region
cal. The results show that these low-dose chemotherapy drugs of RET. Therefore, we hypothesize that the sensitivity oRfET
also induce significant blunt-ended DNA breakage within intron intron 11 signals the net balance of an individual’s ex-po
11 of RET and withinFHIT (Figure 2B). This suggests that cells are sure to environmental factors and unfavorable genotypes,
still susceptible to DNA damage in these regions when exposed and can predict the likelihood ofRET/PTC rearrangements.
to a residual dose of chemotherapy treatment, and the sign-ifi If indeed RET breakage can indicate a potential to form the
cant amount of blunt-ended double-stranded breaks could serve rearrangements, normal cells ofRET/PTC patients should
compared to PTC− patient tissues (Supplementary Figure 2,
available atCarcinogenesis Online). Also, no difference in DNA
breaks was observed between these two sample groups at the
FHIT/FRA3B region or theALK/FRA2N, indicating that individ-u
als susceptible toRET/PTC rearrangements might not have glo-b
ally susceptible fragile sites, but have fragile site breaks specific
to the individual’s translocation. These results suggest that DNA
breakage at specific fragile sites could be a valuable indicator
for the potential formation of chromosomal rearrangements at
Next, we chose to focus only on DNA breakage aRtET as it is
the common partner gene in 19 rearrangements known to cause
thyroid cancer9(). By analyzing all types of DNA breakage within
RET in each of the normal tissues from the two patient groups,
we found that there was no significant difference in the -fre
quency of breakage between these two groupsFi(gure 3B). There
was also no significant difference in total DNA breakage between
groups, when analyzingFHIT, 12p12.3 or G6PD. This indicates
that blunt-ended DNA breaks are increased specifically in the
normal cells of patients withRET/PTC-driven tumors, and
suggests that blunt-ended, double-stranded DNA breaks contribute
to the formation ofRET/PTC rearrangements.
Blunt-ended DNA breaks are the only type of
double-stranded DNA breaks increased in normal
cells of RET/PTC+ patients compared to RET/PTC−
Figure 2. Frequency of DNA breakage withinRET, FHIT and G6PD regions in
HTori-3 cells after 24 h treatment with APH, benzene, DEN, etoposide or do-xo
rubicin. (A) The breakage frequency is represented as all DNA breaks per 100
cells which includes single-stranded nicks′,-o5verhang, 3′ overhang and blunt- In addition to single-stranded DNA nicks, there are three types
ended double-stranded DNA breaks. B() The frequency of only blunt-ended
double-stranded DNA breaks per 100 cells after 24-h treatment. Both assays are of DNA double-stranded break ends: staggered ends with either
represented by the mean ± SEM of at least three replicated experiments. As-ter 5′- or 3′-overhangs and blunt ends. We observed that when co-m
isks indicateP < 0.05 as compared to the respective untreated sample following paring the amount of overall DNA breaks aRtET intron 11 to
analysis by one-way Anova and Dunnett’s multiple comparison test. that of blunt-ended double-stranded breaks, the vast majority of
DNA breaks in each patient tissue were not blunt-ended
doublehave higher DNA breakage atRET than patients without the stranded DNA breaks F(igure 3A compared to B). DNA
doublerearrangements. stranded breaks must occur for the formation of chromosomal
To test this, normal thyroid tissue surroundinRgET/PTC+ rearrangements, therefore, this prompted us to examine the d-is
tumors was compared with normal thyroid tissue from patients tribution of DNA breaks among the four types of break ends, and
with benign nodules. First, total RNA was isolated from each to shed light on the possible substrate required for the for-ma
tumor tissue to verify the presence ofRET/PTC rearrangement tion ofRET/PTC rearrangements.
in the tumor. To ensure a lack of significant tumor cell conta-mi We utilized the activity of the Klenow fragment Eo.f Coli
nation within the normal tissues, total RNA was isolated from DNA polymerase I to convert both ′5- and 3′-overhang to
bluntnormal tissue of six patients withRET/PTC rearrangements and ended double-stranded breaks, and the Klenow fragment
seven patients with benign nodules, and a lack oRfET/PTC rear- mutant (3′–5′ exo-) lacking 3′–5′ exonuclease activity to co-n
rangement in these thyroid tissues was verified by RT-PCR (data vert only 5′-overhang ends to blunt ends. The number of DNA
not shown). Genomic DNA was also isolated from each normal breaks detected from treatment of genomic DNA with these
tissue, and DNA breakage withinRET was evaluated. enzymes followed by the blunt-end double-stranded detection
Using the procedure to detect only blunt-ended, double- protocol (Supplementary Figure 1A, available aCtarcinogenesis
stranded DNA breakage, we found that normal cells surrou-nd Online, right panel) allows us to detect all four types of DNA
ing RET/PTC+ tumors have a significantly increased frequency breaks. To test the feasibility of our methods, genomic DNA from
of blunt-ended DNA breaks withinRET intron 11, as compared HTori-3 cells was digested with various restriction enzymes to
to the normal thyroid cells from patients withouRtET/PTC generate either single-stranded nicks (by Nt.BstNBI digestion),
(P < 0.001) (Figure 3A and Supplementary Figure 1C, available at 5′-overhang (BbvI digestion), 3′-overhang (BanII digestion) or
Carcinogenesis Online). This significant increase in DNA breakage blunt-ended (HaeIII digestion) double-stranded DNA breaks.
was specific toRET intron 11, as there were no significant diff-er The digested DNA was subjected to the four protocols listed
ences in the breakage frequency withinFHIT, ALK or two non- in Supplementary Figure 1A, available atCarcinogenesis Online.
CFS control regions, 12p12.3 orG6PD (45). ALK, located within In the RET intron 11 region, all three double-stranded breaks
the fragile site FRA2N, is involved in rearrangements found in up were detected as expected in Klenow fragment-treated DNA
to 10% of thyroid cancer cases4(6), therefore, the lack of a si-g (Supplementary Figure 3, available atCarcinogenesis Online, lane
nificant difference in theALK region between our patient groups 2), and only 5′ overhang and blunt-ended breaks were mea-s
demonstrates the specificity ofRET/PTC rearrangement forma- ured in Klenow (3′–5′exo-)-treated samples (lane 3). Lanes 1
tion following increased DNA breakage aRtET. Interestingly, and 4 showed the expected products for all four types of breaks
normal cells from patients withRET/PTC1 driven tumors have a and blunt-ended breaks, respectively, when using the all break
significant increase in DNA breakage at the partner genCeC,DC6, detection method and the blunt end detection method. All four
products were verified by Sanger sequencing to be located at the Discussion
expected restriction enzyme cut sites inRET intron 11. These
results demonstrate the specificity of our assay to detect single- In this study, we examined whether fragile site breakage could
stranded nicks, 3′-overhang, 5′-overhang and blunt-ended do-u be used to assess the consequence of long-term, low-dose
ble-stranded DNA breaks. chemical exposure and predict susceptibility to chromosomal
Applying these procedures to measure DNA breaks within rearrangement formation. We found that low-dose, non-cy-to
RET intron 11 from the normal thyroid cells surroundinRgET/ toxic exposure to two common environmental chemicals, be-n
PTC-driven tumors and from patients without PTC, no significant zene and DEN, generate significantly more DNA breaks within
difference between the two patient groups was observed in the RET intron 11 compared to untreated cells. We also found that
amount of all three types of double-stranded DNA breaks co-m both benzene and DEN induced significantly more blunt-ended
bined (Figure 4A, left panel). Similarly, the combination of′-5over- double-stranded DNA breaks withinRET intron 11 at a rate sim-i
hang and blunt-ended double-stranded DNA breaks showed no lar to that of APH treatment, which we have shown can lead
significant difference between the two groupsFi(gure 4A, right to RET/PTC rearrangements 2(). Another study demonstrated
panel). When individual types of DNA break were evaluated, that H2O2 can induce DNA double-stranded breaks and contr-ib
blunt-ended, double-stranded DNA breaks were the only type ute to RET/PTC rearrangements 4(7), also suggesting the role
of DNA break increased in normal thyroid cells of patients with of chemical exposures on DNA breakage atRET. Therefore, our
RET/PTC-driven tumors compared to PTC− patients, and no si-g study demonstrates the importance of investigating these and
nificant difference was observed when comparing single-strand other low-dose environmental chemicals and their role in the
DNA breaks, 3′-overhang or 5′-overhang DNA breaks between the formation of non-radiation associatedRET/PTC rearrangements
two patient groups F(igure 4B). This observation suggests that in thyroid cancer. While the HTori-3 cells used for this an-aly
blunt-ended double-stranded DNA breaks could be the preferred sis were immortalized by SV40 and therefore may have altered
substrate for rearrangement formation, and that a test for blunt-DNA damage checkpoint and repair, we previously showed 4(
ended double-stranded DNA breakage aRtET could offer an early that in normal human hematopoetic stem and progenitor CD3+4
indication of susceptibility to PTC. cells, similar, non-cytotoxic levels of benzene, DEN, etoposide
biomarkers to predict cancer susceptibility. Our results dem-on
strate that normal thyroid cells of patients wRitEhT/PTC-driven
tumors have significantly increased blunt-ended,
doublestranded DNA breaks withinRET, and suggest that this diffe-r
ence could be utilized to predict the propensity to forRmET/
PTC rearrangements. This is the first study to show significantly
higher double-stranded DNA breakage at a cancer driver gene
in normal cells of patients with cancer driven by the respective
gene. A test to detect DNA breakage aRtET could offer an early
indication of susceptibility to PTC, and facilitate prompt prev-en
tion and timely treatments. Our data suggests that an ind-ivid
ual’s susceptibility to fragile site breakage and rearrangement
formation is due to the combined effects of both chemical exp-o
sures and their susceptibility to these exposures. Therefore, this
test could also be used to evaluate individuals at high risk for
PTC, either due to environmental exposures and/or unfavorable
genotypes. A diagnostic tool to measure individual susceptib-il
ity to PTC could improve early detection and possible long-term
clinical outcomes. Further, more than half of cancer-specific
recurrent chromosomal rearrangements possess DNA brea-k
points located within at least one fragile si4t9e).(Therefore, the
use of a test for DNA breakage within fragile regions could be
expanded to other breakage-sensitive regions in the genome
to predict the propensity to form a variety of cancer-specific
rearrangements involving these genes. This susceptibility test
would be widely helpful to tailor chemotherapy regimens, to
monitor high-risk populations and patients in remission, and
Figure 4. Detection of single-stranded,′-3overhang, 5′-overhang and blunt-ended to improve screening tests for other environmental toxins and
double-stranded DNA breaks at thReET intron 11 region in normal thyroid cells stress factors.
from RET/PTC+ or PTC− patients. A() The breakage frequency is represented for all Various studies have demonstrated that NHEJ and MMEJ are
double-stranded DNA breaks or the combination of′-5overhang and blunt-ended the DNA repair pathways required to generate chromosomal
bDlNuAntb-reenadkesd. ADlNlAdboruebalkes-s.Bt(r)aTnhdeebdrebarkeaagkse finrecqluudenes′c-yo3voefrehaacnhgD,5N′-Aovberrehaakntgyapendis translocations in human cancers 31(,32,34–36). The types
shown and represented as DNA breaks/100 cells. Diamonds represent the average of DNA break ends can influence the characteristics of r-ear
DNA breaks from at least three experimental replicates of each theRE6T/PTC+ rangement fusion points (36). Sequences at the fusion points
patient tissues and 7 PTC− tissues analyzed. The horizontal lines represent the of RET/PTC rearrangements often appear as short deletions
mean of each patient group. Asterisk representsP < 0.001 as compared to PTC−. or duplications 3(7,38,40). The significant amount of
bluntended double-stranded DNA breaks found in normal cells of
and doxorubicin can induce significant DNA breakage at fr-ag patients with RET/PTC rearrangements could represent the
ile site genes includingRET in the presence of optimal DNA immediate substrates for the NHEJ pathway to form rearran-ge
repair, indicating the relevance of DNA fragility induced by these ments. Ghezraoui et al. (36) demonstrated the formation of
chemicals. Further, our results suggest the need for more se-nsi chromosomal translocations following TALEN-induced bre-ak
tive tests to screen environmental chemicals, as genes within age (with 5′-overhang ends); however, the frequency of these
fragile sites are particularly susceptible to damage. translocations was 3-fold lower than that of Cas9-induced
Secondary cancers following chemotherapy and radiation breakage (with blunt ends), which further suggests the imp-or
treatment are a known risk4(
) and the ability to better mo-ni tant role of blunt-ended double-stranded DNA breaks in the
tor and predict deleterious consequences following treatment of formation of chromosomal translocations. Knockdown of co-m
primary cancer is needed. Here, we found that low dose etop-o ponents within the NHEJ pathway decreased the formation
side and doxorubicin, at levels 300–1000-fold lower than those of TMPRSS2 fusion transcripts in human prostate cells, which
found in the plasma of patients following chemotherapy4(
), demonstrates NHEJ as the major repair mechanism required to
can cause a significant increase in DNA breakage at genes in generate fusion genes 3(
fragile sites such asRET and FHIT (Figure 2). The low concentr-a Our study provides important insight into the potential for
tions used in our study mimic the residual conditions in which low-dose exposure to environmental chemicals to induce fr-ag
cells survive chemotherapeutic treatment but are still exposed ile sites and promote the formation of DNA breaks at genes
to these drugs. These results indicate that DNA breakage at involved in cancer specific chromosomal rearrangements.
genes within fragile sites can occur in surviving cells, with the Further, it provides a foundation for using DNA fragile site
potential to form cancer-causing gene rearrangements found in breakage to predict susceptibility to chromosomal rearran-ge
secondary cancers. Therefore, the capability of detecting DNA ments, which would be valuable to monitor high-risk patient
breakage at cancer-specific rearrangement-participating gene groups, tailor chemotherapy regimens, and increase the se-n
regions will be important for patients about to undergo ch-em sitivity of screening for carcinogenic chemicals. These results
otherapy. Identifying those at high risk before administering also further implicate NHEJ as the major repair mechanism
chemotherapy would help guide treatment decisions toward required to generate chromosomal translocations founds in
those that are less likely to affect DNA breakage. cancers such at PTC. To recapitulate these results in per-iph
While diagnostic techniques for detecting cancer formation eral blood mononuclear cells of patients witRhET/PTC-driven
have greatly improved, there is still an urgent need for additional tumors compared to healthy individuals would advance these
results to the development of a DNA diagnostic test . Paz-Elizur 16 . de Vathaire , F. et al. ( 1999 ) Second malignant neoplasms after a first et al. (50) has demonstrated that the activity of DNA repair cancer in childhood: temporal pattern of risk according to type of enzymes such as OGG in PBMCs is a sufficient surrogate for treatment . Br. J. Cancer , 79 , 1884 - 1893 .
DNA repair activity in lung tissues. As fragile site breakage is 17 . Kilfoy , B.A. et al. ( 2011 ) Dietary nitrate and nitrite and the risk of- thy the result of the combination of detrimental exposures and roid cancer in the NIH-AARP diet and health study . Int. J. Cancer , 129 , 160 - 172 .
insufficient DNA repair processes, this suggests that PBMCs 18 . Kim , M.S. et al. ( 2008 ) Secondary thyroid papillary carcinoma in ost-eo could serve as a sufficient surrogate for the analysis of fragile sarcoma patients: report of two cases . J. Korean Med . Sci., 23 , 149 - 152 .
site breakage in a variety of tissues. To expand on these results 19 . Pellegriti , G. et al. ( 2009 ) Papillary thyroid cancer incidence in the- vol and use DNA fragile site breakage as a tool to predict chro-mo canic area of Sicily . J. Natl. Cancer Inst. , 101 , 1575 - 1583 .
somal rearrangements in a variety of solid tumors would be 20 . Vane , D. et al. ( 1984 ) Secondary thyroid neoplasms in pediatric ca-n immensely valuable in advancing the field of clinical oncology cer patients: increased risk with improved survival . J. Pediatr. Surg. , 19 , diagnostics. 855 - 860 .
21. Veiga , L.H. et al. ( 2012 ) Chemotherapy and thyroid cancer risk: a report from the childhood cancer survivor study . Cancer Epidemiol . Biomar-k Supplementary material ers Prev ., 21 , 92 - 101 .
Supplementary data are available aCtarcinogenesis online . 22. Venkitaraman , R. et al. ( 2008 ) Childhood papillary thyroid cancer as second malignancy after successful treatment of rhabdomyosarcoma .
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Funding 23 . Verneris , M. et al. ( 2001 ) Thyroid carcinoma after successful treatment National Institute of Health (RO1GM101192) and National Cancer of osteosarcoma: a report of three patients . J. Pediatr. Hematol . Oncol., Institute (RO1CA113863) . 23 , 312 - 315 .
24. Ward , M.H. et al. ( 2010 ) Nitrate intake and the risk of thyroid cancer and thyroid disease . Epidemiology , 21 , 389 - 395 .
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analysis. In addition, we thank members of the Wang laboratory 26 . Yen , B.C. et al. ( 1993 ) Multiple hamartoma syndrome with osteos-ar for helpful discussions . coma. Arch. Pathol. Lab. Med ., 117 , 1252 - 1254 .
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