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Tobacco exposure results in increased E6 and E7 oncogene expression, DNA

damage and mutation rates in cells maintaining episomal human
papillomavirus 16 genomes‡

Article  in  Carcinogenesis · July 2014

DOI: 10.1093/carcin/bgu156 · Source: PubMed

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 Carcinogenesis vol.35 no.10 pp.2373–2381, 2014
doi:10.1093/carcin/bgu156
Advance Access publication July 26, 2014

Tobacco exposure results in increased E6 and E7 oncogene expression, DNA damage

and mutation rates in cells maintaining episomal human papillomavirus 16 genomes

Lanlan Wei1,2, Anastacia M.Griego2, Ming Chu3 and tobacco smoking as a cause of cervical cancer (7). It is estimated that
Michelle A.Ozbun2,* 11.8% of cervical cancer deaths are attributable to smoking. Smoking
has been consistently linked with the progression of cervical neopla-
1
Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang sia, and female smokers have up to two times higher risk of developing
150081, China, 2Department of Molecular Genetics and Microbiology, The cervical cancer than non-smokers (8). Previous studies have focused
University of New Mexico School of Medicine, Albuquerque, NM 87131, USA on the impact of tobacco smoke on the prevalence (9,10), incidence
and 3The First Affiliated Hospital of Harbin Medical University, Department of
Neurosurgery, Harbin, Heilongjiang 150001, China
(11–13) and persistence of HPV infections (14–18). Tobacco smoke
contact includes mainstream tobacco smoke (MSTS) and side-stream
*To whom correspondence should be addressed. Tel: +1 5505 272 4950; tobacco smoke. MSTS refers to the exposure gained when a smoker
Fax: +1 505 272 6029; inhales directly from the tobacco source, whereas side-stream tobacco
Email: [email protected] smoke is that inhaled from the distal lit end of a cigarette, cigar or
High-risk human papillomavirus (HR-HPV) infections are neces- pipe. Both MSTS and side-stream tobacco smoke are heterogeneous
sary but insufficient agents of cervical and other epithelial cancers. mixtures of ~5000 chemical compounds, with several dozen carcin-
Epidemiological studies support a causal, but ill-defined, relation- ogens, cocarcinogens, mutagens and tumor promoters (5). Tobacco
ship between tobacco smoking and cervical malignancies. In this smoke has been shown to cause a variety of types of DNA damage
study, we used mainstream tobacco smoke condensate (MSTS-C) (19–21), including double-strand breaks (DSBs) (22,23). Yet, the
treatments of cervical cell lines that maintain either episomal or mechanisms by which tobacco smoke cooperates with HR-HPV
integrated HPV16 or HPV31 genomes to model tobacco smoke infection to enhance cancer progression are not clear.
exposure to the cervical epithelium of the smoker. MSTS-C expo- Few investigations have considered the effects of cigarette smoking
sure caused a dose-dependent increase in viral genome replication on HPV activities directly. Xi et al. (14,24) showed current but not
and correspondingly higher early gene transcription in cells with prior smoking is associated with higher baseline HPV16 and HPV18
episomal HPV genomes. However, MSTS-C exposure in cells with DNA load; however, there was no observed dose–response relationship
integrated HR-HPV genomes had no effect on genome copy number between cigarette smoking and HPV DNA load. Other studies showed
or early gene transcription. In cells with episomal HPV genomes, no association of smoking status and HPV viral load for women sin-
the MSTS-C-induced increases in E6 oncogene transcription led gly infected by any HR-HPV genotype, or specifically by HPV31 or
to decreased p53 protein levels and activity. As expected from loss HPV16 (25). Experimentally, benzo[a]pyrene (BaP), a major carcino-
of p53 activity in tobacco-exposed cells, DNA strand breaks were gen in cigarette smoke, was shown to cause DNA adducts and damage
significantly higher but apoptosis was minimal compared with cells and induce a p53 response in HPV-immortalized cells (26–28). Alam
containing integrated viral genomes. Furthermore, DNA mutation et al. (29,30) demonstrated that exposure of cervical cells to a spe-
frequencies were higher in surviving cells with HPV episomes. These cific level of BaP could stimulate either higher levels of viral genomes
findings provide increased understanding of tobacco smoke expo- or higher virion synthesis, but oddly not both, in HPV-infected cells
sure risk in HPV infection and indicate tobacco smoking acts more grown as organotypic tissues. This group also showed that increased
directly to alter HR-HPV oncogene expression in cells that maintain viral replication resulted from heightened signaling via the mitogen-
episomal viral genomes. This suggests a more prominent role for activated protein kinase (MAPK) pathway (31). However, they failed
tobacco smoke in earlier stages of HPV-related cancer progression. to show dose responsiveness upon BaP exposure, and the BaP levels
tested were of questionable physiologic relevance (25).
Herein we aimed to study physiologically germane effects of all the
chemicals present in MSTS-condensate (MSTS-C) on cervical cells
Introduction that maintain HPV16 or HPV31 genomes either in extrachromosomal
forms or in an integrated state in the host cell DNA. Results show that
Cervical cancer is one of the most common cancers in women world- MSTS-C exposure leads to increased viral genome replication and
wide, with >0.5 M new cases and nearly 275 000 deaths among females early gene transcription in cells with episomal HR-HPV, but not in
annually (1). The causal relationship between high-risk human papillo- cells with integrated HR-HPV genomes. Consistent with increased
mavirus (HR-HPV) infection and cervical cancer is well documented oncogene E6 transcription, we found decreased p53 protein levels and
in epidemiological and functional studies, with detection of HR-HPVs activity. As expected from the loss of p53 activity in tobacco-exposed
in up to 99.7% of cervical malignancies (2). The HR-HPV E6 and E7 HPV episomal cells, DSB levels were significantly higher, but apop-
oncoproteins are expressed during and after cancer progression and tosis was not activated compared with tobacco-exposed cells contain-
contribute to cervical carcinogenesis in part by inactivating the cellu- ing integrated HPV genomes. Furthermore, mutation frequencies
lar tumor suppressor proteins p53 and pRb, respectively (3). However, were higher in surviving cells with HPV episomal genomes. These
HPV infection alone is insufficient for cervical cancer development. data show that tobacco smoke as a cofactor in HPV-related cancers
An estimated 80% of women will acquire an HPV infection during alters viral oncoprotein activities more predominantly in cells with
their lifetime, but most infections are transient, with only a minority episomal HPV genomes, suggesting a stronger role for tobacco smoke
resulting in recognizable cervical cancer (4). Therefore, additional early in cancer progression prior to HPV genome integration.
cofactors are required for development of cervical cancer.
Tobacco smoking exposure is associated with multiple cancers
(5,6). The International Agency for Research on Cancer has classified Materials and methods
Preparation of MSTS-C
A MSTS-C stock was generated from type 2R1 research cigarettes (Tobacco
Abbreviations: BaP, benzo[a]pyrene; cDNA, complementary DNA; CIN, Health Research Institute, Lexington, KY) using a Type 1300 smoking
cervical intraepithelial neoplasia; DSB, double-strand break; HFK, human machine (AMESA Electronics, Geneva, Switzerland). The condensate was
foreskin keratinocyte; HR-HPV, high-risk human papillomavirus; LCR, long bubbled through and collected in phosphate-buffered saline. MSTS-C expo-
control region; MAPK, mitogen-activated protein kinase; mRNA, messenger sure dosages were normalized based on nicotine concentrations, as measured
RNA; MSTS-C, mainstream tobacco smoke condensate; ORF, open-reading by reverse-phase high-performance liquid chromatography. MSTS-C stocks
frame; qPCR, quantitative PCR. were stored in aliquots at −80°C, and a fresh vial used for each experiment.

© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 2373
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Cell culture and viability (Sigma). At 2 weeks post-seeding, colonies were fixed, stained and counted.
W12 and CIN612 cell lines were established from human cervical intraepi- The mutation frequency was calculated as [mutation frequency = number of
thelial neoplasia grade I  (CIN1) biopsies (32,33). W12 clone 20863 (W12- colonies/(number of seeded cells × PE) × 106 cells]. In parallel control experi-
E), W12 clone 201402 (W12-I), CIN612 clone 9E (CIN612-9E) and CIN612 ments, cellular mutation response to 4-nitroquinoline 1-oxide (140 ng/ml for
clone 6 (CIN612-6) cell lines were derived from their respective parental cell 1.5 h), a well-characterized mutagen, was assayed using OuaR as described
lines. W12-E cells harbor episomal HPV16, whereas W12-I cells contain type above.
I integration of the HPV16 genome with an intact long control region (LCR) Statistics
(34). CIN612-9E cells maintain episomal HPV31 genomes; CIN612-6 cells
contain wild-type p53 and an integrated HPV31 genome of unclear structure Each experiment was performed at least three times. The results are expressed
(35,36). W12-E and CIN612-9E cells express wild-type p53 genes (37). C-33A as mean ± standard error of the mean. Statistical analysis was performed using
is an HPV-negative human cervical carcinoma cell line that expresses mutant Student’s t-test, and P value of ≤ 0.05 was considered as significant.
p53 (38). Primary human foreskin keratinocytes (HFK) and NIKS cells, a
spontaneously immortalized HFK line, both harbor wild-type p53 (39). All
cell lines were authenticated. Each cell line was grown as described previously Results
and generally grown in the presence of J2 3T3 fibroblast feeder cells (37). In
most cases remaining feeder cells were removed by differential trypsiniza- Smoking-relevant concentrations of MSTS-C are non-toxic to cervical
tion. As W12 cells often detached with the feeders, W12-E and W12-I cells epithelial cells
were seeded without fibroblast feeder cells prior to the 24 h MSTS-C treat- Many previous studies have detected nicotine, its major metabolite,
ments. After attachment in normal growth media for 16–20 h, subconfluent cotinine and tobacco-specific carcinogens in the cervical mucus of
cells were incubated with fresh media containing MSTS-C. Cell survival was
measured by Trypan Blue exclusion staining. Toxicity was measured based
smokers (42–47). To study the proliferation and cytotoxicity effects
on lactate dehydogenase detection in cell-free supernatants with lactate dehy- of MSTS-C on HPV-infected cells, cell cultures were exposed to
dogenase-based assay kit (Sigma). Apoptosis was detected by flow cytometry MSTS-C at increasing nicotine concentrations. This physiologically
with Annexin V-PE kit (BD Biosciences) according to the manufacturer. By relevant exposure range for both lung and cervical epithelium resulted
fluorescence-activated cell sorting analysis, Annexin V-PE fluorescence was in a dose-dependent inhibition of cell proliferation for W12-E cells
recorded in FL-2 and 7-amino-actinomycin D in FL-3. Apoptotic cells were that maintain episomal HPV16 genomes compared with untreated
labeled with only annexin V, necrotic cells with both annexin V and 7-amino- cells (Figure  1A). Toxicity measured by lactate dehydogenase
actinomycin D, and living cells were negative for both. release was only apparent at the highest MSTS-C dose of 20 µg/ml
Nucleic acid extraction, reverse transcription and qPCR assay (Figure  1B). W12-I, CIN612-9E, CIN612-6, NIKS and C33A cells
Total cellular DNA or RNA was harvested separately as described previ- responded similarly to W12-E cells in cell proliferation assays at all
ously (37). Reverse transcription of total RNAs (0.2–0.5 µg) was performed doses, and 2.5–10 μg/ml MSTS-C treatment was non-toxic to these
as reported (40). Relative levels of HPV DNA or each complementary DNA cells as well (data not shown).
(cDNA) was analyzed by quantitative PCR (qPCR) with the SYBR Green 1 kit
on the iCycler (Bio-Rad) in triplicate with PCR primers as we reported (37). MSTS-C exposure causes increased HPV viral genome replication
Primer placement and amplicons are illustrated in Supplementary Figure  1, and early viral transcription in cells containing HPV episomal
available at Carcinogenesis Online. qPCR results from cDNA reactions were genomes
normalized by TATA-box binding protein cDNA detection, which was previ-
ously validated as a control for HPV-infected cells (37,41), and we ensured Cells maintaining HPV genomes in either episomal (W12-E,
is not altered by MSTS-C exposure (data not shown). DNA amplifications CIN612-9E) or integrated (W12-I, CIN612-6) forms were exposed
were normalized to detection of the glyceraldehyde-3-phosphate dehydroge- to increasing amounts of MSTS-C. After 24 h of treatment with
nase gene. Final results are shown as the average of three independent experi- MSTS-C, total DNAs and RNAs were harvested for qPCR quan-
ments, whose Q test <0.94. The melting curves and amplification efficiencies tification of HPV genome copies and reverse transcription–qPCR
are shown in Supplementary Figures 2–4, available at Carcinogenesis Online. analysis of early viral transcripts. The glyceraldehyde 3-phosphate
Protein expression assays dehydrogenase gene and TATA-box binding protein cDNA were tar-
Cells were lysed in standard radioimmunoprecipitation assay buffer. Proteins geted as internal controls in qPCR and reverse transcription–qPCR
were quantified by Bradford assay and subjected to 10% sodium dodecyl sul- assays, respectively. Exposure to increasing MSTS-C resulted in
fate–polyacrylamide gel electrophoresis. Following transfer to polyvinylidene dose-dependent and statistically significant increases of viral genome
difluoride membranes (Millipore), immunoblot was performed using anti-p53 replication and similar increases in viral E6, E7 and E1^E4 messen-
antibody (Calbiochem OP43). To control for protein loading, membranes were ger RNAs (mRNAs) in W12-E or CIN612-9E cells (Figure 2A and
stripped (62.5 mM Tris–HCl pH 6.8; 2% sodium dodecyl sulfate; 100 mM C). MSTS-C treatment of W12-E cells resulted in a 2.4-fold increase
2-mercaptoethanol) at 50°C for 20 min and reprobed with anti-β-tubulin in HPV16 viral genome levels, a 2.8-fold rise in E6 transcripts, a 2.0-
antibody (Sigma TO198). Cells were transfected with p53-luc using the fold increase in E7 mRNAs and a 3.2-fold climb in E1^E4 transcripts
Keratinocyte Nucleofector kit as reported (37). p53-luc contains 13 copies of
the p53 consensus sequence driving expression of luciferase. Luciferase assays
(Figure 2A). MSTS-C treatment of CIN612-9E cells resulted in 2.7-
(Promega) were performed according to the manufacturer’s instructions. fold higher HPV31 viral genome levels, 3.3-fold higher E6 mRNA
numbers, 2.9-fold augmentation of E7 transcripts and 4.1-fold more
Neutral single-cell gel electrophoresis (comet assay) E1^E4 mRNAs (Figure 2C). In contrast, the MSTS-C treatment had
Neutral comet assays were performed using the Comet Assay kit (Trevigen) no effect on HPV16 or HPV31 viral genome levels or viral mRNA
according to the manufacturer’s instructions. Briefly, trypsinized and resus- transcription in W12-I or CIN612-6 cells harboring integrated viral
pended cells were embedded in agarose on a slide and subjected to solution genomes (Figure  2B and D, respectively). These data suggest that
lysis followed by electrophoresis in 1× Tris/borate/EDTA buffer for 10 min at exposure to MSTS-C results in the increase in HPV transcription that
1 V/cm. The slides were then stained with SYBR Green I, photographed using
a Zeiss fluorescence microscope and analyzed with Comet Assay Software
follows increased genome load in cells containing episomal HPV
Project. At least 80 cells on each slide were chosen at random for the quantifica- DNA. The data in Figure 2 are shown relative to the levels of nucleic
tion of DNA damage using Comet Assay Software Project. Olive tail moment acids in the untreated cells for ease of comparison. It should be noted
(OTM) is defined as the product of distance (in x direction) between the center that viral oncogene levels are an average of ≈6–8 times greater in
of gravity of comet head (CGH) and the center of gravity of the tail (CGT) cells containing integrated cells compared with those with episomal
and the percent tail DNA (DNAT) wherein OTM = (CGTx − CGHx)/%DNAT. viral DNA (Supplementary Figure  5, available at Carcinogenesis
Mutagenesis Online). Finding higher levels of RNAs containing the E7 open-read-
Cells exposed to MSTS-C at various doses were cultured with fibroblast feeder
ing frame (ORF) than transcripts containing the unspliced E6 ORF
cells for 10 days to allow phenotypic expression, then seeded without feeder is consistent with other reports (48) (see Supplementary Figure  1,
cells to determine the mutation frequency of the OuaR gene: (i) 400 cells available at Carcinogenesis Online). Further, these data concur with
per 150 mm dish, three dishes/dose, for plating efficiency (PE); and (ii) 106 the prevailing notion that HPV genome integration leads to enhanced
cells/150 mm dish, five dishes/dose, in medium containing 10 μg/ml ouabain E6 and E7 RNA levels.

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 Tobacco smoke mutagenesis in HPV-infected cells

a slight increase in p53 transcriptional activity concordant with the

higher p53 protein levels (Figure 3B). Also consistent with the levels
of p53 induced by MSTS-C in HPV-negative, p53 wild-type NIKS
cells, MSTS-C treatment lead to a robust and dose-dependent increase
in p53 transcriptional activity (Figure 3B). Finally, in agreement with
the unchanged p53 protein levels in C33A cells, MSTS-C treatment
did not alter p53 transcriptional activity (Figure 3B). These data show
that p53 activity is directly related to p53 levels in each cell line,
and the levels of HPV E6 protein present in the HPV-infected cells
impacted both.

MSTS-C induces more DNA DSBs, but lower apoptosis, in cells

containing HPV episomes compared with cells with integrated
HPV genomes
Tobacco exposure causes DNA DSBs in mammalian cells (22,23), and
DSBs are the most cytotoxic of DNA lesions. As we found p53 activ-
ity to be lower upon MSTS-C exposure in cells maintaining episo-
mal HPV16 or HPV31 genomes, compared with cells with integrated
HPV genomes, we reasoned that DSBs would be more pronounced
in cells maintaining HPV episomes after MSTS-C exposure. To test
this idea, DNA DSBs were evaluated in W12-E and W12-I cells using
the neutral comet assay wherein cells sustaining DNA damage appear
as a comet with a specific bright head and tail. In contrast, cells with
undamaged DNA show an intact nucleus with no tail in this assay. As
predicted, exposure to increasing doses of MSTS-C resulted in signif-
icantly higher DNA DSBs in W12-E cells compared with W12-I cells
(Figure 4A and B). HPV-negative HFK cells showed little evidence of
DSBs after MSTS-C exposure.
In normal cells, DNA DSBs induce expression of p53 to trigger
either DNA repair or apoptotic cell death as a protective mechanism
(50). To determine whether apoptosis was induced or correlated with
lost wild-type p53 activity and MSTS-C-related DNA damage in
Fig. 1.  Effect of MSTS-C on cell proliferation and cytotoxicity. Subconfluent HPV-positive cells, annexin V and 7-amino-actinomycin D staining
W12-E cells were treated with the indicated concentrations of MSTS-C at was employed after 24 h of MSTS-C exposure. At 0 and 5 μg/ml
37°C. (A) Trypan blue exclusion staining during cell quantification was used
MSTS-C treatment there were similar, but not statistically significant
to determine the effect of MSTS-C on cell proliferation at 0, 24 and 48 h
posttreatment. Results are shown as the average of three separate experiments; increases of cell apoptosis in W12-E and W12-I cells (Figure  4C).
error bars represent standard error of the mean. (B) Cell cytotoxicity was However, when treated with 5 μg/ml MSTS-C, the HPV-negative
determined by detecting the release of lactate dehydogenase (LDH) in cell- NIKS cells showed a statistically significant increase of apoptosis
free culture supernatants 24 h after treatment with MSTS-C. Data represent (P < 0.05), which is consistent with the doubled levels of p53 pro-
an average from three separate experiments and error bars represent standard tein and activity induced in these cells under the same conditions
error of the mean. Statistical significance was achieved with P < 0.05(*). (Figure  3). Also consistent with induced levels of p53 activity in
the cells when treated with the higher MSTS-C dose (10 μg/ml),
Increased HPV E6 early viral transcription coincides with p53 both W12-I cells and NIKS cells displayed statistically significant
degradation in cells maintaining episomal HPV genomes increases in apoptosis (Figure  4C). However, W12-E cells failed to
The presence of E6 is considered a predisposing factor in the devel- mount a significant apoptotic response to MSTS-C at the higher dose
opment of HPV-associated cancers, allowing the accumulation of (Figure 4C) where significant DNA DSBs were detected (Figure 4B).
chance errors in host cell DNA to go unchecked (49). As relative lev- As expected, MSTS-C treatment did not induce apoptosis in C-33A
els of p53 protein can be used as an indirect readout for E6 activity, cells that express mutant p53 (Figure 4C). Thus, although MSTS-C
we detected p53 protein in cells by immunoblot. After 24 h treatment exposure led to more DSBs in cells with HPV16 episomal genomes,
with MSTS-C, both W12-E and CIN612-9E cells demonstrated a the increased E6 levels suppressed both the p53 activity and the pro-
dose-dependent decrease of wild-type p53 protein levels in a man- tective function of apoptosis in these cells.
ner consistent with increased E6 expression (Figure 3A). However, in
W12-I and CIN612-6 cells containing integrated HPV genomes, the MSTS-C causes accumulation of DNA mutations in cells containing
MSTS-C treatment lead to a mild increase in p53 protein levels con- HPV episomes
sistent with their unaffected E6 mRNA levels (Figure 3A). The HPV- DNA damage repair of DSBs is error prone and increases the likeli-
negative spontaneously immortalized HFK cell line, NIKS, responded hood of DNA mutations that can lead to cancer. As p53 is thought to
to MSTS-C treatment with substantially increased p53 levels as be involved in the repair process and data above show decreased p53
expected in cells with functional wild-type p53 genes (Figure  3A). activity in W12-E cells, but not in W12-I cells after MSTS-C treat-
Lastly, MSTS-C treatment did not readily change p53 levels in the ment, we quantified the accumulation of mutations in the cells after
HPV-negative C-33A human cervical carcinoma cell line harboring MSTS-C exposure. The data in Table I show the sensitivity of W12-E
mutant p53 (Figure 3A). and W12-I cells to ouabain. The spontaneous (0 μg/ml MSTS-C)
To functionally assess p53 transcriptional activity, a p53-responsive OuaR gene mutation frequencies were similar in W12-E (11.3 ± 2.9
luciferase reporter plasmid was transfected into the cell lines prior per 106 cells) and in W12-I (9.4 ± 2.1 per 106 cells). After 24 h treat-
to MSTS-C treatment. We found wild-type p53 activity was reduced ment with 5 or 10 μg/ml MSTS-C, we detected 1.5- or 2.2-fold
significantly in W12-E and CIN612-9E cells following MSTS-C increases (P < 0.05) of OuaR gene mutation frequencies in W12-E
treatment (Figure  3B) corresponding to the decreased p53 levels cells, but only 1.1- and 1.4-fold increases in W12-I cells as compared
(Figure  3A) and increased E6 RNA levels (Figure  2A and C). In with their untreated control groups. As a positive control, mutagen
the cells with integrated HPV genomes, MSTS-C treatment induced 4-nitroquinoline 1-oxide induced OuaR gene mutations at frequencies

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Fig. 2.  Quantification of viral genome copy numbers and early viral transcripts in HR-HPV-positive cell lines exposed to MSTS-C. Subconfluent plates of cells
were exposed to the concentrations of MSTS-C as indicated (A-D). After 24 h of treatment, the cells were harvested for total RNA and DNA. DNase-treated
total RNAs were reverse transcribed to cDNA and subjected to qPCR for E6, E7 or E1^E4 RNAs using TATA-box binding protein cDNA quantification for
normalization. Viral DNA levels were quantified by targeting amplification of the viral LCR in reference to levels of the cellular glyceraldehyde 3-phosphate
dehydrogenase gene. Results represent the mean normalized expressions from three separate experiments with error bars representing standard error of the mean.
Statistical significance was achieved with P < 0.05(*) and P < 0.01(**) as compared with the 0 mM control in each group.

significantly higher in W12-E cells (P < 0.01) than in W12-I cells (P the p53 tumor suppressor protein and leaves HPV-infected cells at
< 0.05) when compared with their untreated control groups (Table increased susceptibility to tobacco smoke-induced DNA DSBs and
I). Therefore, MSTS-C exposure results in higher DNA mutation higher accumulated mutation rates. Interestingly, the MSTS-C actions
frequencies in W12-E cells where early HPV16 gene transcription on HR-HPV activities were seen in cells that maintain episomal viral
was induced, p53 levels decreased and apoptosis was suppressed in genomes, but not in isogenic cells with integrated viral genomes.
response to MSTS-C. This contrasted to W12-I cells where early Additionally, cells with episomal genomes had higher DNA strand
HPV16 gene transcription was unaltered. breaks and lower apoptosis than their integrated counterparts. These
observations suggest tobacco smoke exposure plays an important role
Discussion as a promoter of HPV-initiated carcinogenesis, with key effects on
HR-HPV activities occurring prior to, and perhaps promoting, viral
Nearly all cases of cervical cancer are linked to HR-HPV infections, genome integration.
with more than half attributable to HPV16 (3). Increasing numbers It appears that the primary effect of tobacco smoke on HR-HPV
of oropharyngeal squamous cell carcinomas also are due to HPV16 activities is that of augmented viral genome replication leading to
infections (51). However, HR-HPV infection is an insufficient car- increased early transcription, rather than genome replication driven
cinogen. Epidemiological studies demonstrate a causal relationship by a boost in HPV early transcription. Our reasoning is based on the
between tobacco smoking and cervical cancer development (52–56). findings that the fold change in viral genome levels and early gene
Chemical constituents of tobacco and their metabolites, including nic- transcription is similar in cells with episomal HR-HPV genomes com-
otine, nicotine-derived cotinine, carcinogens 4-(methyl nitrosamino)- bined with the fact that we do not observe increased viral early gene
1-(3-pyridyl)-1-butanone, BaP and others are present in the cervical transcription in cells with integrated HPV genomes. The structure of
mucus of active and passive smokers (42–47). Although these obser- HPV genomes when integrated in cancer cells includes maintenance
vations suggest that tobacco smoking acts as a cofactor with HR-HPV of the LCR that regulates gene expression (34,57–61). Many studies,
infection in cervical cancer progression, little is understood about the including our unpublished data (A.M.Griego and M.A.Ozbun), indi-
mechanisms by which the viral, cellular and environmental factors cate the LCR and early promoter are functional to drive expression
may cooperate in carcinogenesis. In this study, we found that expo- of E6 and E7 from integrated genomes (26,62–64). Therefore, it is
sure of HPV-infected cervical cells to physiologically relevant doses reasonable to expect integrated genomes would be responsive to tran-
of MSTS-C elicited increased viral genome replication and onco- scriptional stimulation if provided by the MSTS-C. It is interesting to
gene expression, which is known to be essential for HPV-mediated note that the MSTS-C constituent BaP was reported to activate both
cellular transformation. We showed that the MSTS-C-induced HPV MAPK and ERK1/2 signaling to increase viral titer in CIN612-9E
oncoprotein expression, in turn, neutralizes the protective effects of organotypic epithelial tissues, which maintain episomal HPV31

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 Tobacco smoke mutagenesis in HPV-infected cells

Fig. 3.  Detection of p53 expression and transcriptional activity after MSTS-C treatment in W12-E and W12-I cells. (A) Immunoblot analysis of p53 protein
expression after MSTS-C treatment. Subconfluent cells were exposed to different concentrations of MSTS-C for 24 h. Total protein (NIKS cells, 30 μg; C-33A
cells, 0.5 μg; other cells, 10 μg) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot for p53 (53 kDa); β-tubulin protein
(50 kDa) was detected as a loading control on the same blot after stripping the membrane. Images represent unaltered scans from films. Values directly above each
lane represent the relative p53 protein levels normalized to β-tubulin with the untreated control defined as 1.0; band intensities were performed on the original
autoradiographs. (B) p53 transcriptional activity assay in cells transfected with plasmid p53-luc. At 24 h posttransfection, cells were treated with MSTS-C at the
indicated concentration for 24 h, then lysed and analyzed for luciferase activity. The luciferase activity in cells control group (0 μg/ml) was defined as 1, and the
treated samples were normalized to this control. The graphs encompass averages from three independent experiments. Statistical significance was achieved with
P < 0.05(*) and P < 0.01 (**) as compared with 0 μg/ml control group.

genomes (31), as well as stimulate oncoprotein expression from inte- compared with those previous reports. This includes our finding no
grated HPV16 in CaSki cells (26). It is not surprising that BaP-induced enhanced viral gene expression from integrated HPV16 and HPV31
MAPK activation stimulated HPV oncoprotein expression from the genomes, in contrast to that seen in BaP-treated CaSki cells with
integrated HPV16 LCR (26), as the MAPK/ERK pathway activates integrated HPV16 genomes (26) and our observed MSTS-C induced
transcription factors known to bind in the LCR and promote viral gene increase in viral DNA levels, which were not found in response to
expression (65). Unfortunately, the effects of BaP on early viral tran- nitric oxide exposure (37).
scription were not assessed in CIN612-9E raft tissues (29–31); such The mechanism of episomal HPV genome replication in response
data might help to clarify confounding results in these reports indicat- to MSTS-C exposure is not clear. Extrachromosomal HPV genome
ing that genome amplification and virion production were not dose replication requires E1 and E2 proteins in addition to cellular replica-
responsive and were inversely related. In a previous study, we assayed tion factors, which can be influenced by E6 and E7 proteins (49). The
the response of the cervical cells maintaining episomal HPV16 and concentrations of MSTS-C we used slightly suppressed cellular pro-
HPV31 genomes (W12-E and CINC12-9E cells, respectively) to the liferation, suggesting cell proliferation was not a key factor in enhanc-
effects of nitric oxide, a free radical that is increased in the cervix ing viral genome replication. Interestingly, the interferon-inducible
in response to other factors that cooperate with HR-HPV in cervical protein p56 blunts HPV DNA replication via binding E1 (66), but
cancer progression, including tobacco smoke (37). In contrast to the tobacco smoke is known to suppress the interferon response. This
present work, physiologically relevant levels of nitric oxide promoted may in part explain increased HPV DNA levels following MSTS-C
oncogene transcription, but not increased viral genome replication. exposure. However, this is a complicated interplay of systems that
Nevertheless, the outcome of higher oncoprotein expression was sim- will take a great deal of experimentation to clarify.
ilar to what we found herein: increased DNA damage and survival The lack of reagents for quantifying E1 and E2 proteins is a detri-
of cells with higher mutational frequencies (37). We emphasize that ment to our understanding of how these proteins regulate genome
our current work, employing the complete and more tobacco smoke replication (67). As the viral RNAs that contain E1 and/or E2 ORFs
exposure-relevant complement of chemical constituents in MSTS, also include E6 (or E6*) and E7 sequences (see Supplementary
cannot be directly compared with the prior studies where cells were Figure  1, available at Carcinogenesis Online) (67), the levels of
exposed only to BaP or to nitric oxide. Our use of the more complex, E1 and/or E2 RNAs in undifferentiated cells are predicted to mir-
but physiologically relevant MSTS-C is the most likely explanation ror those of E6 and E7. Additionally, viral RNA levels may not
for the differences observed in overall effects to HR-HPV in this study be indicative of protein levels as it is unclear which ORFs in the

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 L.Wei et al.

Fig. 4.  Effect of MSTS-C on DSBs and cell apoptosis during a 24 h time course of treatment. (A) Neutral comet assay for DNA DSBs is shown subsequent
to MSTS-C treatment with representative images of the nuclei depicted. (B) Quantification of the comet tail moments for each condition were calculated on
≥80 cells per group from A; data are shown as a box-and-whisker plot to illustrate the distribution and statistics of the datasets. The box bottom and top are
25th and 75th percentiles of each dataset, respectively; the central band is 50th percentile (median). Vertical lines extend to 10th and 90th percentiles. Outliers
are indicated as dots. (C) Apoptosis analysis on cells treated for 24 h using double staining with Annexin V-PE and 7-amino-actinomycin D (see Materials and
methods). Graphed are the averages of three separate experiments; statistical significance was achieved with P < 0.05(*) and P < 0.01 (**) as compared with
0 μg/ml control group.

polycistronic RNAs are actually translated. Although loss of E2

expression via HPV genome integration results in increased viral Table I.  MSTS-C induced OuaR gene mutation frequency after 24 h of
exposure (N = 3)
oncogene expression, viral integration would need to occur rapidly
and specifically in a majority of cells, which is highly improbable in Cell MSTS-C (μg/ml) OuaR mutants/106 cells
our 24 h period to assay for MSTS-C effects. Further, genome inte-
gration would preclude the increased viral DNA copies relative to W12-E 0 11.3 ± 2.9
genomic genes we observed. Thus, we do not feel viral integration 5 16.1 ± 5
and loss of E2 could account for our results. Nevertheless the height- 10 24.4 ± 4*
ened DNA strand breaks and lower apoptotic indexes are likely to 4-NQO, 140 ng/ml 28.1 ± 1.8**
W12-I 0 9.4 ± 2.1
support HPV integration at some point, whereby a subset of those
5 11.1 ± 3.5
events might lead to an eventual growth advantage, but this would 10 14.3 ± 3
require many cell generations to prevail. 4-NQO, 140 ng/ml 21.7 ± 6.3*
Although persistent HR-HPV infections are a clear prerequisite
for the development of cervical cancer (68–70), there is no con- 4-NQO, 4-nitroquinoline 1-oxide.
sistent association of HR-HPV DNA load (the levels of viral DNA *P < 0.05, compared with 0 μg/ml MSTS-C controls; **P < 0.01, compared
detected) with persistence (14,25). Increased viral load in the cervix with 0 μg/ml MSTS-C controls.

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 Tobacco smoke mutagenesis in HPV-infected cells

Fig. 5.  Model for HR-HPV and tobacco smoking interaction in cervical cancer progression. Infection with an HR-HPV is a necessary cause of cervical and other
epithelial cancers. Tobacco smoking is an epidemiologically defined cofactor for progression to cervical cancer and is known to increase nitric oxide levels and
induce DNA adducts and strand breaks (5), as well as suppress the immune system (71). Similar to many previous studies, we find in normal HPV-negative cells,
tobacco smoke exposure induces high levels of p53 leading to DNA repair or apoptosis wherein mutant cells generally fail to survive (right side). In HR-HPV-
infected cells that contain episomal viral genomes, tobacco smoke exposure causes an increase in HPV genome copies, higher HPV oncoprotein RNA expression
leading to a decrease in p53 activity and improved survival of cells with mutations (left side). Increased viral DNA load in the midst of DNA DSBs may promote
integration of viral genomes into the host cell chromosomes. As the cells with integrated HR-HPV genomes showed no further increase in oncoprotein activities
upon tobacco exposure, we conclude that the primary effects of tobacco smoke constituents are focused on cells with episomal genomes to promote early steps in
malignant conversion.

has also been inconsistently reported for current and/or ever smokers that our results can be generalized to HR-HPVs. Lastly, the MSTS-C
(15,16,24,25). Nevertheless, any observed association is proposed to dose-responsive results observed in cells containing both HPV16 and
favor HPV persistence in vivo (15,16), but this is yet inconclusive HPV31 episomes lends biological plausibility to our findings.
(25). Without a true quantitative assay for viral load in individual In summary, our demonstration of preferential upregulation of HPV
sampled cells, one cannot differentiate in a cell population between a oncoproteins in cells with episomal genomes, followed by increased
more homogeneous population with high DNA levels versus a hetero- DNA damage and higher mutation frequencies provides the bases for
geneous cell group where most have low HPV copies and a few cells a theory on the role for tobacco smoke exposure in the progression to
contain very high levels of HPV DNA. This limits the informative cancer of HR-HPV-positive infections (Figure 5). We propose that in
nature of the current assays in this regard. The general lack of a dose– HR-HPV-infected cells with episomal genomes, tobacco smoke not
response relationship observed between cigarette smoking and viral only causes DNA adducts and strand breaks (5), but independently
load could indicate a low threshold for the effect of smoking on HPV causes increased viral load within cells. This results in heightened
DNA load (25). Our data show cells with a ≤3-fold increase in viral HPV oncoprotein expression that further squelches cell cycle control,
DNA levels and similar increase in E6 RNAs have a significant deficit DNA damage repair and protective apoptosis. Increased viral genome
in ability to repair DNA or undergo apoptosis; this could mean that replication in the midst of DNA DSBs is a probable promoter of viral
small changes in the levels of HPV genomes in a minor population genome integration, wherein cells that maintain higher E6 and E7
of cells may have substantial functional effects in restraining tumor expression gain a growth advantage and can continue to accumulate
suppressor functions, thus leading to malignant progression of these mutations that permit malignant progression. The immune suppres-
few cells in vivo. sion that occurs in the context of tobacco exposure likely augments
A number of aspects differentiate our work from prior studies. The the survival of these cells (71). This experimental model provides
use of the tobacco smoke exposure-relevant chemical mixture and the impetus for expanded epidemiologic studies of the relationship
physiologically relevant levels of MSTS-C sets our work apart from between smoking-related cervical cancer and HPV integration sta-
many previous reports that have only used BaP to study the response tus, which may provide better understanding of tobacco smoke risk
of HPV-immortalized cells (26–31). This provides assessment that is to HPV infection and cancer risk. Finally, we expect this model to be
more similar to tobacco exposure in vivo. A limitation of our work, applicable to other cancers, especially those oropharyngeal malignan-
and that of the other studies with BaP, is that the local concentrations cies that are initiated by HR-HPV infections.
of the MSTS-C chemicals to the cells in the cervix is not known;
however, we have striven to mimic as closely as possible by normal- Supplementary material
izing our MSTS-C using nicotine levels that are found in the cervical
mucus (44). Prior studies on the effects of tobacco chemicals have Supplementary Figures 1–5 can be found at http://carcin.oxfordjour-
not compared isogenic cells and have typically analyzed only a single nals.org/.
HR-HPV genotype. Our use of two HR-HPV positive isogenically
matched sets of cervical cell lines with differing HPV structures allow Funding
us to rule out many aspects of cell line-to-cell line variation. Further,
we show consistent results comparing extrachromosomal HPV16 Primary support was by the American Cancer Society (RSG-05-
and HPV31 genome-carrying cells with their isogenic counterparts 149-01-MBC to M.A.O.); we also gratefully acknowledge the
that have integrated HPV genomes; this provides more confidence Oxnard Foundation (M.A.O.); the University of New Mexico Cancer

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 L.Wei et al.

Center (National Institutes of Health P30 CA118100); the National 20. Leanderson,P. et al. (1992) Cigarette smoke-induced DNA damage in cul-
Natural Science Foundation of China (NSFC 30902706 to L.W.); tured human lung cells: role of hydroxyl radicals and endonuclease activa-
the Heilongjiang Provincial Natural Science Foundation of China tion. Chem. Biol. Interact., 81, 197–208.
(ZD201020 to L.W.). 21. Nakayama,T. et  al. (1985) Cigarette smoke induces DNA single-strand
breaks in human cells. Nature, 314, 462–464.
22. Albino,A.P. et al. (2006) Induction of DNA double-strand breaks in A549
Acknowledgements and normal human pulmonary epithelial cells by cigarette smoke is medi-
ated by free radicals. Int. J. Oncol., 28, 1491–1505.
We thank Profs. P.Lambert and M.Stanley for W12-E and W12-I cells and 23. Moktar,A. et  al. (2009) Cigarette smoke-induced DNA damage and
L.Laimins for CIN612-9E and CIN612-6 cell lines. We are grateful to Dr repair detected by the comet assay in HPV-transformed cervical cells. Int.
J.-C.Seagrave at Lovelace Respiratory Research Institute for providing the J. Oncol., 35, 1297–1304.
MSTS-C, and to members of the Ozbun lab for critical comments on this 24. Xi,L.F. et al. (2009) Relationship between cigarette smoking and human
work. Dedication: This manuscript is dedicated to the fond memory of V.Fung, papilloma virus types 16 and 18 DNA load. Cancer Epidemiol. Biomarkers
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integrity, his love of sciences and the arts, his incredible culinary skills, and genesis? J. Virol., 82, 6084–5; author reply 6085.
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