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xome-wide mutation profile in benzo[a]pyrene-derived post-stasisnd immortal human mammary epithelial cells
aul L. Seversona, Lukas Vrbab, Martha R. Stampferb,c, Bernard W. Futschera,b,∗
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, 85724, USAUniversity of Arizona Cancer Center, Tucson, AZ, 85724, USALife Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
r t i c l e i n f o
rticle history:eceived 25 August 2014eceived in revised form 24 October 2014ccepted 27 October 2014vailable online 4 November 2014
eywords:enzo[a]pyrene16MECarcinogenesis
a b s t r a c t
Genetic mutations are known to drive cancer progression and certain tumors have mutation signaturesthat reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signa-ture and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis humanmammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality.We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa,184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing.The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons.Seventy percent of the mutations were C:G > A:T transversions, consistent with the known mutationspectrum of BaP. Mutations predicted to impact protein function occurred in several known and puta-tive cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutationssuch as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification andDNA repair were found to have mutations predicted to impact function in each of the post-stasis strains.Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared
greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomalanomalies during immortalization that arose independent of BaP. The results of this study indicate thatacute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and caninduce mutations in a number of cancer driver genes.
Cancer is a disease of genes, with DNA sequencing data show-ng tens to thousands of somatic mutations per tumor [1,2]. Solidumors of the breast, prostate and colon have an average of 33–66omatic mutations that are expected to alter protein structure
hile tumors linked to known environmental mutagen exposures
uch as melanomas or lung cancers have averages of 135 and 163
Abbreviations: BAP, Benzo[a]pyrene; p16, p16INK4A; HMEC, human mammarypithelial cells.∗ Corresponding author at: Department of Pharmacology and Toxicology, Collegef Pharmacy, University of Arizona, Tucson, AZ, 85724, USA. Tel.: +1 520 626 4646;ax: +1 520 626 4979.
[3]. It is thought that only a small portion of these mutations iscancer drivers.
Somatic mutations are caused by many distinct processes, andsome tumors have signature mutation patterns which reflect themajor mutational process at work in the development of thatparticular tumor [4]. For instance, lung cancers of smokers havea signature mutation pattern where C:G > A:T transversions pre-dominate due to mutagenic polycyclic aromatic hydrocarbonssuch as benzo[a]pyrene (BaP). BaP is one of the most extensivelystudied mutagens, a potent carcinogen and is a cause of cancerdriving mutations [5,6]. The mutagenic effects of BaP are pref-erentially targeted to epithelial cells due to exposure routes andspecific metabolism pathways that convert BaP to DNA-reactive,
diol epoxide intermediates [7–9]. BaP-derived epoxide intermedi-ates primarily form covalent adducts at the N2 position of guanine,which can lead to C:G > A:T transversions if not appropriatelyrepaired. Thus, BaP is a prototype complete carcinogen with a
Fig. 1. Development of BaP-derived post-stasis and immortal HMEC cells. Untreated(NT) pre-stasis 184 HMEC display complete growth arrest known as stasis. Pre-stasis 184 HMEC were exposed to 1 �g/mL (4 �M) BaP for two or three 24 h periods.From independent exposures, clones emerged that bypassed the stasis barrier; forinstance 184Aa, 184Be and 184Ce. The post-stasis cells continued proliferatinganother 10–40 population doublings until the telomeres were depleted, inducinggenomic instability, the DNA damage response and a p53-induced growth arrest. In
P.L. Severson et al. / Mutation
nown mutation signature; however, the exome-wide mutationattern it produces along with any potential cancer drivers it mayenerate, have not been studied in an in vitro human model systemelevant to cancer [10].
We have used an in vitro human mammary epithelial cellHMEC) model system that has proven useful for identifying andeflecting the genetic and epigenetic events involved in earlyuman breast carcinogenesis [11–16]. In this model system, theransformation of normal finite lifespan HMEC to malignancyequires overcoming three distinct senescence-associated barri-rs to immortality [11,17]. The first barrier, termed stasis ortress-induced senescence, is characterized by elevated levels ofhe cyclin-dependent kinase inhibitor p16INK4A (gene CDKN2A)
aintaining the retinoblastoma protein (RB) in an active state11,13,18]. The stasis barrier has been overcome or bypassed inultured normal HMEC by various means, such as exposure toaP [12–14,17,19]. The resultant post-stasis cells frequently exhibit
nactivation of p16 by gene mutation or by promoter hypermeth-lation [13,20]. HMEC that get past stasis proliferate further beforencountering a second more stringent proliferation barrier, replica-ive senescence, resulting from critically shortened telomeres11,14]. When approaching replicative senescence, HMEC exhibitncreased chromosomal instability and a DNA damage response.are cells that gain telomerase expression may escape this bar-ier and acquire immortal potential; additional perturbations canonfer malignant properties on the telomerase-expressing immor-ally transformed cells, which are no longer vulnerable to thehird barrier, oncogene-induced senescence [21–24]. To broadenur understanding of BaP-induced mutagenesis that helps renderMEC capable of bypassing stasis and progressing to immortal-
ty, we performed whole exome sequencing on normal pre-stasisMEC cells, and multiple isogenic BaP-induced post-stasis and
mmortal derivatives.In this study we show that three independent post-stasis HMEC
ultures generated by BaP exposure of normal primary HMEC, andheir two immortal derivatives, have hundreds of BaP-linked muta-ions. C:G > A:T transversions are the predominant type of mutationollowed by C:G < G:C and C:G > T:A in order of prevalence. The BaP-nduced mutations occur across a broad array of genes includingeveral known and putative cancer drivers. There is no overlapf putative driver gene mutations among the independent BaP-erived post-stasis cells, but the protein altering mutations doffect biological processes that typically harbor cancer drivers inach of the different post-stasis strains. The immortal lines havelmost identical mutation patterns as their post-stasis precursorsith 10 or fewer additional mutations. Our exome sequencing data
lso confirm that immortalized cells have gross copy number varia-ions not present in their post-stasis precursors, indicating that theenomic instability occurred after generation of the BaP post-stasistrains. This study provides new insights and confirmatory evi-ence into the exome-wide mutational effects of an environmental,omplete carcinogen in an established model system relevant touman carcinogenesis.
. Methods
.1. Cell culture
The HMEC cultures utilized were developed previously as described [12,19]. Inrief, HMEC were isolated from the reduction mammoplasty tissue of a 21-year-oldoman, specimen 184. In three separate experiments, primary cultures of pre-stasis
84 HMEC were exposed to 1 �g/mL (4 �M) BaP for two or three 24-h periods,eading to clonal post-stasis populations that maintained growth after control popu-
ations ceased growth at stasis. Final concentration of DMSO in the BaP-containingulture medium was 0.05%. The first experiment produced one post-stasis clone,hereas the second and third experiments gave rise to at least two post-stasis clones
ach [19]. One post-stasis clone from each experiment was examined. Rare immor-al cell lines emerged from post-stasis populations during the period of telomere
rare instances a cell was able to escape replicative senescence and acquire immor-tality. The immortal lines 184A1 and 184BE1 emerged from the post-stasis strains184Aa and 184Be respectively.
dysfunction. DNA was isolated from sub-confluent cultures using the DNeasy bloodand tissue kit (QIAgen).
2.2. Exome sequencing
Exome sequencing was performed using the Life Technologies AmpliseqTM
Exome Kit (Cat. #4487084) according to the manufacturer’s protocols(MAN0008346). This kit uses a pool of 293,000 primer sets divided into 12multiplexed PCR reactions to selectively amplify exons. In brief, 100 ng of genomicDNA was used to seed 12 exon amplification reactions of 10 cycles. After completingPCR cycling the 12 reactions were combined prior to primer digestion and adapterligation. A unique barcoded adapter was used for each sample. After adapterligation, the libraries were purified and then quantified by qPCR. Library concen-trations were in the range of 150–600 pM. Pairs of barcoded libraries were dilutedin equimolar concentrations for template preparation using the Ion OneTouchinstrument and then sequenced on a single Ion Proton P1 chip. In total, exomesof 6 HMEC cultures were sequenced including pre-stasis 184, post-stasis 184Aa,post-stasis 184Be, post-stasis 184Ce, immortal 184A1 and immortal 184BE1. Thedata are publicly available through NCBI’s Sequence Read Archive (SRA) underproject code SRP045165 (www.ncbi.nlm.nih.gov/sra/).
2.3. Data processing and analysis
Reads were aligned to the hg19 reference genome including unassigned con-tigs but without alternative haplotypes using TMAP aligner, included in the torrentsuite software v4.0. An initial round of variant calling was performed using TorrentVariant Caller version 4.0-r76860 and hg19 reference using the parameters recom-mended by Life Technologies (Supplemental File 1). Variants from the first roundof calling were merged into a master VCF file using VCFtools v0.1.7. This masterVCF file was used in a second round of variant calling as a hotspots file. The secondround of variant calling was run with higher stringency parameters (SupplementalFile 2) and with the hotspots file, providing coverage statistics for all variants andall samples. Genomic positions that were unsuitable for variant calling due to lowcoverage or strand bias in any one of the samples were filtered out of the combinedvariant set, leaving a set of variants that exceeded the minimum coverage standardin each of the samples thus allowing even comparisons between samples. Variantswithin introns were filtered out of the variant files. Lists of BaP-induced mutationswere analyzed using IntOGen-mutations software to annotate and predict varianteffects [25]. Genes with mutations that were predicted to impact protein functionwere annotated to biological processes using DAVID [26]. IonReporter 4.0 softwarewas used to find copy number variations in the post-stasis and immortal cells rel-ative to pre-stasis, untreated 184 HMEC. Data from ten Affymetrix HT HG U133Agene expression arrays of pre-stasis 184 HMEC were downloaded from the GeneExpression Omnibus (Series GSE16058 and GSE37485) and normalized using thearoma.affymetrix package in R [18,27–29].
3. Results
Primary cultures of pre-stasis 184 HMEC were exposed to1 �g/mL (4 �M) BaP for two or three 24 hour periods, leading tothe outgrowth of post-stasis cells from three independent exper-iments (Fig. 1) [12,19]. The BaP post-stasis strains 184Aa, 184Be
50 P.L. Severson et al. / Mutation Research 775–776 (2014) 48–54
Fig. 2. Relative distribution of single base substitutions by type in the BaP-derivedpag
arltla
3
F1e1sth
stimBsCrcmgbHfr
gt1e
Fig. 3. Distribution of pre-stasis expression levels for all genes and genes that werefound mutated in post-stasis cell. The dashed curve shows the expression profile
ost-stasis and immortal HMEC. The 184 germline variants bar represents the devi-tion from the reference genome hg19. All other bars show the deviation from 184ermline which is the somatic mutations after subtracting 184 germline variants.
nd 184Ce have been cultured until they either ceased growth ateplicative senescence, or in rare instances, gave rise to immortalines, such as the post-stasis strains 184Aa and 184Be giving riseo the immortal lines 184A1 and 184BE1 respectively. Thus far, theess extensively cultured post-stasis strain 184Ce has not producedn immortal line.
.1. Exomic analysis of BaP-induced mutations
We performed exome sequencing on all the cells shown inig. 1: pre-stasis HMEC 184, its derivatives post-stasis HMEC 184Aa,84Be, 184Ce and the immortal HMEC 184A1 and 184BE1. Asxpected, the composition of variants in the untreated pre-stasis84 HMEC relative to the hg19 reference was predominately tran-itions, which is in agreement with the typical range of 2.8-3.0 forransition to transversion ratios of germline variants observed inuman exomes (Fig. 2) [30].
Since all the samples used in this study were derived from aingle genotype, the BaP-induced mutations were isolated by sub-racting the germline variants from each of the post-stasis andmmortal samples. The acute BaP exposures resulted in roughly 3–9
utations per million base pairs of exome and almost 70% of theaP-induced single nucleotide mutations were C:G > A:T transver-ions while the remaining 30% of the mutations were mostly:G > G:C and C:G > T:A (Fig. 2, Supplemental Table). This mutationate and pattern is consistent with what has been described in lungancers of smokers [31–33]. Greater than 90% of the BaP-inducedutations occurred at C:G base pairs which is in agreement with
uanine as the major site of BaP-diol epoxide DNA adducts. Overall,oth the number and type of mutations detected in the BaP-derivedMEC samples match the somatic mutation pattern of lung tumors
rom smokers, supporting the BaP-HMEC model system as an accu-ate representation of BaP-induced carcinogenesis.
We next examined whether there was any correlation between a
ene’s expression level and its susceptibility to BaP-induced muta-ion. We analyzed previously published data from ten pre-stasis,84 HMEC gene expression arrays in order to estimate a genexpression profile for the untreated, parental cells. By combining
and the dashed vertical line the median of all genes on the Affymetrix HT HG U133Aarray (14732). The solid curve shows the genes that are mutated in post-stasis cells(348 with expression data).
the BaP-induced mutations with the pre-stasis gene expressionprofile we found that mutations of genes expressed in the tophalf prior to BaP exposure were significantly under-representedin post-stasis cells (hypergeometric test, p = 1.05 × 10−6) (Fig. 3).This under-representation of mutated genes amongst those thatare expressed could be due to transcription coupled nucleotideexcision repair. In support of this hypothesis, we found that of themutated genes, only 13 of the 78 (17%) G to T mutations in the tophalf of gene expression occurred on the transcribed strand. Con-versely, 58 of 139 (42%) G to T mutations in the bottom half of geneexpression occurred on the transcribed strand suggesting that theoverrepresentation of mutations amongst the weakly expressedgenes likely reflects less efficient repair in these genes. Anothercontributor to the uneven distribution of mutated genes is the pos-sibility that expressed genes, when mutated, are more likely tocause a decrease in cellular fitness than non-expressed genes. Thus,unaffected cells would overgrow the cells with mutations in essen-tial genes thereby preventing propagation and detection of thesemutations. Taken together, these data suggest that expressed genescould be partially protected from BaP-induced mutagenesis due totargeted DNA repair mechanisms (reviewed in [34]).
3.2. BaP-derived post-stasis cells harbor unique mutations thataffect cancer drivers and biological processes linked to cancer
HMEC undergo stasis through the induction of p16 expressionand can become post-stasis by defects in the RB pathway lead-ing to RB inactivation. Getting past stasis is often mediated byfunctional inactivation of p16, generally by deletion, mutation,or epigenetic silencing. Previous work identified a nonsense p16mutation (E88*) in the immortal line 184A1 suggesting that themutation was also present in the precursor cells [20]. In 184Aa wefound the expected nonsense mutation in the p16 coding region(E88*) which rationally drives the functional inactivation of oneallele while the other allele is silenced by DNA methylation [16].Although p16 expression is absent in each of the post-stasis strains
examined, it is not known how this silencing event was attainedin 184Be, 184BE1 and 184Ce. p16 inactivation could be achievedthrough distinct mutations to p16 in the other post-stasis strainsor other genes in this signaling pathway. In 184Be and 184BE1, we
ig. 4. Venn diagram of the BaP-induced mutations in post-stasis HMEC. Each post-tasis strain has a completely unique set of mutations.
ound a novel C to A transversion at the codon for amino acid 70 of16 (NM 000077), but this mutation is silent (P70=, Supplementalable). The alternate codon has comparable usage in the humanenome as the original and is therefore unlikely to affect proteinranslation. Therefore, in some instances such as 184Aa, BaPxposure contributes to the bypass of stasis by directly causing16 mutations, but in the other post-stasis strains there are likelyo be different defects that affect the RB pathway or cell cycle.
In addition to p16 mutations, more than 200 other BaP-inducedutations were found in the post-stasis strains 184Aa and 184Behereas 184Ce had 93 (Fig. 4). When the specific nucleotide pos-
tions mutated by BaP were considered, each of the independentlybtained post-stasis strains had a completely unique set of muta-ions (Fig. 4). When the genes mutated by BaP were considered, fiveenes with mutations predicted to alter protein function were com-on in two of the three post-stasis strains; the mutations occurring
n different codons; no genes were common to all three post-stasistrains (Table 1).
Each of the post-stasis strains had at least two mutated geneslassified as high-confidence cancer drivers (Table 1). A previoustudy analyzed over 3000 tumors and combined several comple-entary methods to generate a list of 291 high-confidence cancer
able 1Top) Genes that have mutations predicted to alter protein function in more than oneost-stasis strain. (Bottom) Putative cancer drivers that have mutations predictedo alter protein function in post-stasis cells.
driver genes that mostly correspond to 5 broad biological modules[25]. Seven genes from this list had protein altering mutations in184Aa including p16, PLCG1, MED12, TAF1, MLL2, ARHGAP32 andKALRN (Table 1). 184Be had 4 cancer drivers with protein alteringmutations including PIK3CG, HSP90AB1 (one allele with two muta-tions), WHSC1L1 and LCP1. 184Ce had two cancer drivers withpredicted protein altering mutations, FANCA and LPP.
We chose six biological processes from the gene ontologydatabase to represent those processes typically affected by cancerdriver mutations and found that each of the post-stasis strains hasat least one mutation predicted to alter protein function in eachof these processes (Fig. 5). These results identify potentially impor-tant BaP-induced protein altering mutations in genes and pathwaysthat are known to be preferentially disrupted in human tumorsand suggest that defects in these processes can confer survival andproliferation advantages.
3.3. Characterization of the genetic alterations unique toimmortalized cells
Point mutations in the immortal cells have a high degree of over-lap with their post-stasis precursors (Fig. 6A). Overall 184Aa and184A1 share 226 BaP-induced mutations, many of which are pre-dicted to alter protein function (Fig. 6A). One of these overlappingmutations is the previously described nonsense mutation in p16(E88*) [20]; a known driver mutation. Our sequencing data con-firmed the presence of this mutation in 184A1 and here extendthe finding to the 184Aa post-stasis strain. Similarly, 184BE1 alsoshares the vast majority of its mutations with its post-stasis precur-sor 184Be. The immortal lines 184A1 and 184BE1 have additionalpoint mutations that appear to occur during or after immor-talization and are therefore not likely to be directly caused byBaP-induced DNA adducts (Fig. 6A). This interpretation is supportedby the fact that only 5 of the additional 15 mutations seen in 184A1and 184BE1 are the BaP signature C:G > A:T transversion. Of the 5unique mutations in 184A1, 3 are predicted to impact protein func-tion and only the A to G transition at the codon for amino acid 239of NKRF is predicted to have a medium impact (Table 2). Six of the10 mutations unique to 184BE1 are predicted to impact proteinfunction (Table 2). 184Aa has mutations that were not detectedin its immortal derivative 184A1, suggesting that they were lostduring immortalization, most likely due to chromosomal deletions(as described below). In summary, the majority of the mutationsdetected in the immortal lines were directly inherited from thepost-stasis precursors (>95%); however, a small portion of muta-tions that could possibly influence the immortal lines’ phenotype
appears to have been acquired during immortalization.
Ongoing culture of the BaP post-stasis HMEC eventually leads toreplicative senescence resulting from telomere erosion [11,17,18].HMEC that have overcome this barrier, 184A1 and 184BE1,
Table 2Genes with mutations that are unique to the immortal lines and are predicted toalter protein function.
52 P.L. Severson et al. / Mutation Research 775–776 (2014) 48–54
F that a
e[(caftCSditccaufiilFs8FsMci
Fdman
ig. 5. Genes with mutations predicted to alter protein function in post-stasis cells
xpress telomerase activity and display genomic rearrangements14,35,36]. Using sequencing read depths, copy number variationsCNVs) were estimated in each of the post-stasis and immortalultures relative to untreated, pre-stasis HMEC (Fig. 6B). Over-ll, the post-stasis 184Aa, 184Be and 184Ce populations displayedew, very small CNVs (Supplemental Figs. S1–S3). In contrast,he immortal lines 184A1 and 184BE1 showed dramatically moreNVs than their post-stasis precursors (Fig. 6B, Supplemental Figs.1–S5). CNV analysis of the immortal 184A1 exome-sequencingata detected three copies of chromosome 20, previously observed
n late passage 184A1 as well as in several other cell lines cul-ured for long periods (Walen & Stampfer, 1989). Immortal 184A1ells also have previously reported large single copy deletions onhromosomes 3, 6 and 12 which account for all 7 mutations thatre present in 184Aa but not 184A1 (Fig. 6A, Supplemental Fig-re S4). These deletions also cause the loss of heterozygosity forve mutations that are predicted to alter protein function includ-
ng NMBR, KLHL32, COL12A1, SLC17A4 and HCAR2, associatingoss of the wild type alleles with immortalization (Supplementalig. S4). Immortal 184BE1 cells have an amplification on chromo-ome 2, approximately 4 copies of the long arm of chromosome
and a single copy deletion on chromosome 13 (Supplementalig. S5). Interestingly, amplification of the long arm of chromo-
ome 8 is common in immortal cell lines and encompasses theYC oncogene, a known genetic and etiologic event in breast can-
er. To summarize, the sequencing data showed that BaP-derivedmmortal cells have additional point mutations and substantially
ig. 6. (A) Venn diagrams showing the overlaps of point mutations between BaP-erived post-stasis and immortal HMEC. (B) Cumulative copy number variation aseasured by total base pairs covered. The genomic distance between consecutive
mplicons is included in the calculation even though those genomic regions wereot sequenced.
ffect biological processes known to be perturbed by cancer driver mutations.
more CNVs than their post-stasis precursor cells, providing sig-nificant evidence that the cells experienced a period of genomicinstability during their progression to immortality, but after andindependent of their exposure to BaP.
4. Discussion
The present work shows that acute exposures of normalfinite pre-stasis HMEC to the complete carcinogen BaP produceshundreds of mutations that reflect both the number and signatureof mutations observed in human lung cancers of smokers. Severalstudies have found p53 mutations in lung cancers of smokers witha BaP signature, but none of the post-stasis and immortal HMECexamined in this study had protein altering p53 mutations. Theabsence of p53 mutations in the post-stasis and immortal HMEC isconsistent with the absence of p53 mutations in most human breastcancers and the enforcement of stasis in cultured HMEC by p16 andnot p53-dependent p21 [5,6,17]. The 1 �g/mL (4 �M) dosage of BaPused for the original experiments produced 80% cell kill followingthe 24 h dosing periods [12]. Although this dose is a thousand timeshigher than what has been measured in serum from smokers, it isimportant to note that acute, high doses of BaP generate a similarmutation rate and pattern as are found in tumors [37]. This mightsuggest that for mutagenic carcinogens, the cumulative dose is adeterminant of cancer risk.
Since stasis in normal HMEC is a p16 mediated process actingon the RB pathway, we do not expect any of the mutated cancerdrivers reported here to be able to drive cells past stasis inde-pendent of p16/RB pathway inactivation. Although none of themutations predicted to alter protein function that we report hereare direct regulators of p16 or the RB pathway (with the exceptionof p16 E88*), it is possible that one or some of these defects indi-rectly contribute to p16 inactivation. In normal pre-stasis HMEC,p16 maintains an inducible bivalent epigenetic state thus allow-ing induction of p16 and consequently stasis. In post-stasis cellsand human tumors, p16 is commonly inactivated via aberrant DNAmethylation; therefore it would be interesting to test whether anycombination of these putative cancer driver mutations or theirexperimental surrogates can lead to epigenetic inactivation of p16in pre-stasis cells, thereby contributing to the bypass of stasis. Evenif these putative cancer driver mutations turn out to be ineffectivefor the bypass of stasis, they are likely to contribute to malignancyin other ways once the cells achieve immortality.
Replicative senescence occurs in post-stasis cells when telom-eres become critically short, leading to genomic instability, a DNAdamage response and growth arrest. In rare instances, this senes-
cence barrier can be overcome by the reactivation of telomerase.184BE1 contains an amplification of the long arm of chromosome 8,suggesting that MYC deregulation could have contributed to theirovercoming replicative senescence (Supplemental Fig. S5). On the
ther hand, 184A1 appears to have no chromosomal abnormalitiesnvolving MYC, indicating that other defects were important forts bypass of replicative senescence (Supplemental Fig. S4). Inhis study we identified 3 mutations predicted to alter proteinunction that occur in 184A1 but not in their post-stasis precursorells 184Aa (Table 2). One of these mutated genes, NKRF (I239T),eems an interesting candidate for further experimentation basedn its repressor function to NFKB. Other potentially importantandidates associated with the immortal phenotype are thoseutations that undergo loss of heterozygosity in 184A1, since loss
f heterozygosity is a typical identifier of a tumor suppressor gene.Inhalation of polycyclic aromatic hydrocarbons such as BaP is
learly a risk factor for lung cancer, but ingestion of BaP might be risk factor for breast cancer. BaP is a well-known mammary car-inogen in rodents. Several studies have shown that environmentalactors contribute to breast cancer risk [38–41]. Some studies haverovided evidence of aromatic DNA adducts in tissues of breastancer patients suggesting that BaP exposure could be involved in
portion of breast cancers which provides rationale for the studyf BaP in this HMEC model system [42,43].
In addition to this work, another recent study showed thatxposure of murine fibroblasts to BaP produced a mutation pat-ern that also resembled the mutation signature of lung cancers,upporting in vitro systems as models to study genome-wide muta-ional processes that drive cancer progression [44]. On the otherand, there are many critical differences relevant to carcinogenesisetween mouse fibroblasts and human epithelial cells that warrant
nvestigation of BaP-induced carcinogenesis in human epithelialells [8,18,45–47]. The model system used in this study is anccurate representation of normal epithelial tissue that progresseshrough defined stages of tumor development upon BaP exposure.n important feature of these cells is that they have not been virally
mmortalized, allowing the study of the normal protective mech-nisms that prevent tumor development, whereas most cell linesave these protective pathways artificially abrogated to facilitate
n vitro culture. This study provides additional evidence to theody of literature supporting the BaP driven mutagenic mechanismf carcinogenesis and highlights potentially important candidateriver gene mutations that are possible contributors to overcominghe normal proliferation barriers.
onflicts of interest
No competing interests.
cknowledgments
The authors would like to thank Gregory Metzger and theenomics Shared Services Laboratory at the University of Arizonaancer Center for their assistance with template preparation andequencing. This work was supported by the National Institutesf Health (P42 ES04940, ES006694, CA23074, 5T32ES16652-5)nd the Office of Science, Office of Biological and Environmentalesearch, of the U.S. Department of Energy under Contract No. DE-C02-05CH11231 (MRS).
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.mrgentox.2014.0.011.
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