Quercetin, E7 and p53 in papillomavirus oncogenic cell transformation

R.G. Beniston, I.M. Morgan, V. O'Brien,1 and M.S. Campo,2

Department of Veterinary Pathology, Glasgow University, Garscube Estate, Glasgow G61 1QH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bovine papillomavirus type 4 (BPV-4) infects the upper alimentary canal of cattle causing benign papillomas which can progress to squamous carcinomas in cattle grazing on bracken fern (BF). We have previously shown that quercetin, a well characterized and potent mutagen found in BF, causes cell cycle arrest of primary bovine cells (PalF), but that a single exposure to quercetin can cause full oncogenic transformation of PalF cells partially transformed by BPV-4. Here we show that cell cycle arrest correlates with an increase in p53 protein levels and transcriptional activity. However, in cells transformed but non-tumorigenic, p53 protein is elevated and transcriptionally activated in response to quercetin or other DNA damaging stimuli, but the cells bypass quercetin-induced G1 arrest likely due to E7 expression. In transformed tumorigenic cells, p53 is elevated in response to quercetin but its transcriptional activity is inhibited due to mutation, and the cells fail to stop in G1 in the presence of quercetin.

Abbreviations: BF, bracken fern; BPV-4, bovine papillomavirus type 4; GI, gastrointestinal; HPV, human papillomavirus; PalF, primary bovine cells.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Papillomaviruses are oncogenic viruses (1) which infect cutaneous and mucosal epithelia, giving rise to benign hyperproliferative lesions, commonly called warts. In the great majority of cases, these lesions are under host immunological surveillance and regress spontaneously. However, if other factors intervene, the lesions can undergo malignant transformation and develop into squamous cell carcinomas. While the co-factors that promote neoplastic progression of warts are ill-defined in humans, they have been identified in animal systems (2). One such system is the papilloma–carcinoma syndrome of cattle (3).

Cattle are infected in their upper gastrointestinal (GI) tract by bovine papillomavirus type 4 (BPV-4) which induces squamous epithelial papillomas (4). In healthy cattle, the papillomas develop and persist for approximately 1 year and are then rejected by a cell mediated immune response (5). However, in cattle grazing on bracken fern the papillomas can transform and progress to squamous cell cancer (4). Bracken fern contains immunosuppressants and mutagens. Bracken-eating cattle become chronically immunosuppressed and incapable of mounting an appropriate immune response against the virus or virus infected cells (3). The bracken mutagens are probably responsible for the observed activation of ras (6), mutation of p53 (7) and increase in epidermal growth factor receptors (8).

The progression of papillomas to carcinomas has been experimentally reproduced in bracken-fed cattle (9) thus confirming the epidemiological studies conducted in the field.

We have recreated in vitro the multistep nature of BPV-4 cell transformation by using primary bovine cells (PalFs) from a fetus palate. E7 and E8, the transforming proteins of BPV-4, are not capable of fully transforming these cells (10). [E8 has recently been renamed E5 (11) and will be referred to as E5 hereafter]. In co-operation with Ha-ras, E7 induces morphological transformation of PalF cells and E5 is required for their anchorage independent growth but, although the cells have an extended life-span and are capable of growing independently of adhesion to the substrate, they are neither immortal nor tumorigenic (10). The inability to immortalize PalFs is almost certainly due to the absence in the BPV-4 genome of the E6 gene (12). The E6 protein of other papillomaviruses binds to and inactivates the cellular protein p53 (13) thus leading to cell immortalization. Indeed the addition of the E6 gene of HPV-16 to the BPV-4 system achieves the immortalization of PalF cells (10). Tumorigenic transformation is achieved by the addition of an exogenous mutant p53 gene (14), showing that, as in vivo, the action of p53 must be fully abrogated for tumour induction.

Quercetin is a well known mutagenic flavonoid widespread in nature but present in particularly high concentrations in bracken fern (15,16). It binds DNA causing single-strand DNA breaks (17), DNA rearrangements (18) and chromosomal damage (19), and arrests normal proliferating cells in the G1 phase of the cell cycle (20). Moreover, quercetin interferes with the proper signalling of numerous kinases (see ref. 21 and references therein).

We have shown that although E7 by itself can transform PalF cells only partially, exposure of the cells to a single dose of quercetin can cause anchorage-independent growth, achieve oncogenic transformation and the cells can induce tumours in nude mice (22,23). Thus quercetin substitutes for E5 in inducing independence from substrate, for E6 in conferring immortality and for mutant p53 in inducing oncogenicity. Moreover, quercetin trans-activates the transcriptional promoter/enhancer (LCR) of BPV-4 (20), thus most likely contributing to cell transformation by elevating the expression of the viral transforming proteins.

However, activation of the viral LCR is unlikely to be quercetin's sole mechanism for promoting transformation as the timing of quercetin exposure is critical for the full transformation of the cell (23).

Here we present evidence that mutation of p53 and abrogation of quercetin-induced G1 arrest are two more critical events in the attainment of tumorigenic cell status. These in vitro observations reflect the involvement of bracken chemicals in BPV-4 associated carcinogenesis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmids
pZipneoE7 and pBV4 have been described (10,24). pT24 contains the activated c-Ha-ras gene and has been described (25). pRGCfosLuc carries two p53-binding sites from the ribosomal gene cluster promoter upstream of the murine c-fos promoter linked to a luciferase reporter gene. This plasmid has been derived from pRGC{Delta}foslacZ (26). The control plasmid, pfosLuc, lacks the p53-binding sites. Both plasmids were kind gifts from Dr J.D.Zhu (Glasgow University). pCH110 carries the LacZ reporter gene driven by the SV40 early promoter (Pharmacia, Herefordshire, UK).

Generation and transfection of PalF cells
PalF cells were derived from the soft palate of bovine fetuses as described before (27). The cells were transfected using DOTAP (Boehringer, Mannheim, Germany) following the manufacturer's instructions, both for the generation of stable lines and for transient transformants. Typically, for the generation of stable cell lines, 5 µg pZipneoE7 was mixed with 5 µg pT24 and 10 µg sonicated salmon sperm DNA and the mixture used to transfect 5x105 PalF cells. Twenty-four hours after transfection, cells were treated with 20 or 50 µM quercetin (with EtOH as solvent) for 48 h. There is no difference in outcome between the two quercetin concentrations (22,23). Transfected cells were selected in G418 containing medium (500 µg/ml) over 3 weeks and G418 resistant colonies were either pooled or isolated by ring cloning. The cell lines thus generated are designated E7R, if transfected with E7 and ras genes and not treated with quercetin; E7Q if treated with quercetin and clonal; E7QP{alpha}, ß, {gamma} etc. if derived from independently derived populations of pooled cells; E7QT if derived from tumours induced by E7Q; E7QP{alpha}T if derived from tumours induced by E7QP{alpha} (Table IGo). E7R cells are not immortal and therefore were kept frozen in liquid nitrogen and a fresh batch was used for each experiment.


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Table I. p53 transcriptional activity is incompatible with tumorigenesis
 
Additionally, two previously derived cell lines were used, Q0D and Q2D. These lines have been described before (23). Briefly, Q0D are PaF cells transfected with the whole genome of BPV-4 and activated ras gene; these cells are neither immortal nor tumorigenic. Q2D are Q0D treated with 20 µM quercetin for 48 h; they are both immortal and tumorigenic (23).

Transient transfections were carried out with DOTAP, using 4 µg pRGCfosLuc or pfosLuc, with 4 µg pCH110 as a transfection efficiency control. Cells were treated with 50 µM quercetin, or with EtOH only, for 36 h, with a change of medium and quercetin every 12 h. Cells were harvested and assayed for luciferase activity with luciferase assay buffer (Promega, Madison, WI) in a Tropix TR717 luminometer, protein concentration was determined by BCA assay (Sigma-Aldisch, Dorset, UK) and ß-galactosidase activity was measured by colour conversion of ONPG (Sigma) monitored at 420 nm. Expression of ß-galactosidase from the SV40 early promoter in the control plasmid pCH110 was not affected by quercetin (data not shown).

Anchorage independence assay
Cells (1.25x105) were added to 5 ml methocel medium (0.9% methyl cellulose MC4000, 30% fetal calf serum in DMEM) and transferred to a bacterial grade 60 mm Petri dish. The dishes were left for 7–10 days before counting colonies greater than 0.1 mm in diameter in three random fields of 16 mm2 each. The average efficiency of colony formation was calculated as (number of colonies)/(number of cells).

Tumorigenicity assay
This was carried out essentially as described (10). Typically five athymic nude mice were used per cell line. Tumours were sized and excised either for storage in liquid nitrogen or to explant cells back into tissue culture where appropriate. The experiments were done in full compliance of the regulations of the Home Office of Great Britain.

Cell cycle analysis
This was carried out as described before (20). Briefly 5x105 cells were seeded into 90 mm culture dishes and 16 h later quercetin treatment was initiated. Fresh medium containing 50 µM quercetin (or EtOH alone) was replaced every 12 h for up to 48 h or up to 144 h (6 days) for long-term cell cycle analyses. Control cells were fed with fresh medium without quercetin. Cells were harvested, fixed in ice-cold 70% EtOH and stained using 20 µg/ml propidium iodide (Sigma) and 200 µg/ml RNAase A (Kramel Biotech, Northumberland, UK). Cells were then sampled on a FACScan (Becton-Dickinson, Mountain View, CA) and evaluated using Modfit v2.0 or CELLQuest software.

Western blotting
Cells (1x106) were seeded into 140 mm culture dishes and 29 h later quercetin treatment was initiated, at various concentrations for 36 h. Cells were also exposed to UV light (120 J/m2) and harvested 24 h later. Cells were harvested and lysed by boiling for 5 min in 1 M Tris–HCl (pH 6.8), 10% SDS and 20% glycerol. The lysates were run on SDS–PAGE gels, proteins were blotted onto HyBond nitrocellulose membranes (Amersham, Buckinghamshire, UK), and probed with the required antibody: Bp53.12 (Santa Cruz Biotechnology, Santa Cruz, CA) for p53, AB-1 (Calbiochem, La Jolla, CA) for bovine actin and C-19 (Santa Cruz) for p21Cip1. Horseradish peroxidase-linked secondary antibodies (anti IgG from New England Biolabs or anti IgM from Calbiochem) were used and the reaction visualized with ECL reagents (Amersham).

RNA isolation and RT–PCR
These techniques were carried out essentially as described by the manufacturer. Total RNA was isolated using RNeasy kit (Qiagen, West Sussex, UK) and stored at –70°C until use. Reverse transcriptase reactions of p53 mRNA were carried out using Omniscript RT (Qiagen) with 1 µg RNA and Oligo dT as primer. Typically 50% of the RT reaction was carried forward to PCR reactions. PCR was performed with HotStarTaq (Qiagen), using the 5'-TCGAAAGCTTATGGAAGAATCACAGGCA and 3'-TCGATCTAGATCAGTCTGAGTCAGGCCC primers (28) which include an HindIII site and XbaI site, respectively. Reactions were assayed by electrophoresis on 1% TBE agarose gel.

p53 cDNA cloning and sequencing
p53 RT–PCR products were purified on a PCR purification column (Qiagen) before being cloned into pcDNA3+ via HindIII and XbaI restriction sites. Clones were sequenced in a 373A DNA Sequencer (ABI Applied Biosystems) using a DNA Sequencing kit (Perkin-Elmer, Foster City, CA) with the above primers followed by internal primers: F2, TTCCGTCTAGGGTTCCTG; R2, AAGGCAGATCCCAAGGAC; F3, TATGAGTCCCCCGAGATC; R3, ATACTCAGGGGGCTCTAG; F4, CCTAGGAGCACTAAGCGA; R4, GGATCCTCGTGATTCGCT.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phenotypic analysis
We have analysed three types of cells as emblematic of oncogenic progression: normal primary PalF cells, cells transformed by E7 and Ras but not tumorigenic (E7R) and quercetin-treated tumorigenic E7R cells (E7Q). Although data will be presented for these three lines only, essentially identical results have been obtained with four independently derived transformed non-tumorigenic lines, three independent tumorigenic lines (including two previously described lines, Q0D and Q2D; ref. 23) and two lines explanted from nude mouse tumours. The cell lines and their characteristics are listed in Table IGo.

The degree of transformation of the newly generated lines was analysed by inspecting their gross morphology and by assessing their ability to grow in semi-solid medium and to induce tumours in nude mice. In agreement with previous results (22,23), the E7R and E7Q cells have a pronounced spindle-like `criss-cross' morphology (Figure 1AGo). In addition, while E7R cells would senesce after approximately 2 months, E7Q cells were immortal and could be passaged extensively without loss of viability.



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Fig. 1. Phenotype of cells at different stages of transformation. (A) Cell morphology under phase contrast. (B) Anchorage independent foci. Magnification 40x for all panels.

 
Neither PalF nor E7R cells were capable of growing independently of substrate and, among the quercetin-treated cells, only E7Q and E7QP{alpha}, and the previously reported Q2D (23), could form colonies in methocel (Figure 1BGo, Table IGo).

The transformed cell lines not treated with quercetin were unable to induce tumours (Table IGo). Of the four newly generated quercetin-treated lines (E7Q, E7QP{alpha}, E7QPß, E7QP{gamma}), only E7Q and E7QP{alpha} were capable of inducing tumours at a similar frequency as that previously reported for Q2D (Table IGo), indicating that quercetin treatment does not inevitably lead to a fully transformed phenotype, again in agreements with previous observations (22,23).

All cell lines expressed E7 and Ras, as assessed by RT–PCR of E7 RNA and by western blot analysis, respectively (data not shown).

Lack of apoptosis in E7R cells
The possibility that E7R cells are not tumorigenic because of predisposition to apoptosis due to E7 expression (29) could be discounted because of no evidence of a sub-G1 fraction, indicative of apoptosis, in asynchronously growing E7R cells (Figure 2Go) as assayed by both CELLQuest and Modfit software. The PalF and E7R cell cycle profiles were not significantly different; the profile of the E7Q cells showed a greater proportion of cells in S phase, indicative of a shorter doubling time. The small peak seen near the origin in all the CELLQuest profiles represented cell debris originated during sample preparation.



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Fig. 2. Lack of apoptosis in E7R cells. Representative FACS profiles of PalF, E7R and E7Q cells as assayed by (A) CELLQuest and (B) Modfit software. No sub-G1 fraction, indicative of apoptosis, is apparent in the asynchronous cultures.

 
It is known that activated Ras prevents apoptosis (30) and its expression in E7R cells is therefore likely to abrogate possible apoptotic signals.

p53 protein is elevated in response to quercetin
The tumour suppressor p53 has been found mutated in bovine cancers (7). To investigate if inhibition of p53 functions was at least partly responsible for quercetin-induced oncogenic cell transformation, we analysed the status of p53 in cells treated and untreated with quercetin.

Western blot analysis showed that, in agreement with previous results (31), in PalF cells the level of p53 was increased upon quercetin exposure (Figure 3Go; Table IGo). p53 was induced by UV light too, another DNA damaging stimulus (Figure 3Go). p53 was increased also in non-tumorigenic and tumorigenic lines (Table IGo and Figure 3Go), showing that these cells have a normal p53 response to DNA damaging agents, but was constitutively elevated in E7QT and Q2D lines (Table IGo). UV light induction of p53 appeared more marked than the induction due to quercetin; the reason for this was not investigated.



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Fig. 3. p53 and p21Cip1 proteins are elevated in cells exposed to DNA damaging stimuli. Cells were treated with quercetin at 50 µM or equivalent volume of EtOH for 36 h or with 120 J/m2 UV and harvested 24 h later. Membranes were probed for p53 (top row), p21Cip1 (middle row) and actin as a loading control (bottom row). An overexposure of the p21Cip1 western blot strip of PalF and E7Q cells confirmed the total absence of p21Cip1 in E7Q cells exposed to DNA damaging agents (data not shown).

 
p53 transcriptional activity in response to quercetin
To ascertain whether the increase in p53 protein level was accompanied by increased protein function, we investigated the expression of p21Cip1, which is transcriptionally controlled by p53 (ref. 32) by western blot analysis. p21Cip1 was elevated in PalF cells and in E7R and other non-tumorigenic lines in response to quercetin and UV light but was undetectable in E7Q and other tumorigenic lines (Figure 3Go, Table IGo). These results indicate that p53 is transcriptionally defective in the tumorigenic lines.

Additional evidence for the transcriptional malfunction of p53 in the tumorigenic cells was provided by the transcriptional trans-activation of pRGCfosLuc, a p53-responsive promoter, by p53. As assessed by the response of the promoter, p53 transcriptional activity was elevated ~3-fold in quercetin-treated PalF cells and E7R cells (Figure 4Go). However, in E7Q not only was there no increase in p53 transcriptional activation in response to quercetin (Figure 4BGo), but there was very little or no p53 activity (Figure 4AGo), and there was no difference between pRGCfosLuc, the p53-responsive promoter, and pfosLuc, the control promoter (data not shown). Additional non-tumorigenic and tumorigenic lines showed the same p53 response (Table IGo). Similar results were obtained also with a construct containing the p53-responsive promoter of the p21Cip1 gene (data not shown).



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Fig. 4. Quercetin stimulates p53 transcriptional activity. Cells were transfected with 4 µg pRGCfosLuc together with 4 µg pCH110 as a control for transfection efficiency. Transfections were then treated with 50 µM quercetin over 36 h (50) or with an equivalent volume of EtOH (0). Luciferase activity was normalized to ß-galactosidase specific activity. In (A) the results are expressed as fold induction over activity in PalF cells and in (B) as fold induction over EtOH alone.

 
These results are in complete agreement with the p21Cip1 expression data and show that, although p53 is elevated in response to quercetin, its transcriptional activity is defective in the tumorigenic lines, and lead to the suggestion that loss of p53 transcriptional activity is a critical step for the attainment of tumorigenicity.

p53 gene mutations
To determine the reason for the loss of p53 transcriptional activity, we analysed possible mutations of p53 in the tumorigenic cell line E7Q (and in a derivative line, E7QT2, explanted from a tumour) by DNA sequencing. p53 cDNA produced in three separate RT–PCR reactions was cloned in pcDNA3+ and two plasmid clones for each reaction (six clones in total) were sequenced. Two point mutations were found at codon 278 in conserved region V (equivalent to codon 271 in human p53), GGA->GTT, which resulted in a change of amino acid residue from glycine to valine. The sequence of p53 cDNA was not analysed in transformed non-tumorigenic cells as p53 was wild type in its behaviour, but p53 cDNA from parental PalF cells was sequenced and no mutations were found.

Analysis of cell cycle in quercetin-treated cells
Quercetin induces reversible cell cycle arrest in normal PalF cells (20) and we reasoned that expression of BPV-4 E7 and/or malfunctioning p53 in the transformed cells might deregulate the cell cycle, and that cell proliferation in the presence of quercetin would lead to accumulated DNA damage and transformation.

Cells were exposed to 20 or 50 µM quercetin over 48 h with essentially the same results (20). PalF cells arrested in both G1 and G2/M phases of the cell cycle (Figure 5Go). There was a noticeable decrease in the percentage of cells in S phase accompanied by an increase in G2/M; the G1 population remained constant showing that the cells could not traverse G1 and move into S phase. The cell cycle arrest of PalF cells in G1 is in agreement with the quercetin-induced increased level and activity of the p53 protein (Figures 3 and 4GoGo) which is well known to be involved in G1 arrest following DNA damage (21,22).




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Fig. 5. Cell cycle profiles of normal and transformed PalF cells. Cells grown in 50 µM quercetin or equivalent volume of EtOH for 48 h were analysed by FACS. The histograms in (A) show the percentage of cells in different phases of cell cycle; they represent the average of at least three independent experiments and are shown with error bars. The histograms in (B) are derived from (A) and show differences in cell cycle phases between control cells grown in EtOH only and cells grown in quercetin.

 
In contrast, the quercetin-treated cells, with the exception of E7QP{gamma}, did not arrest in G1 whether tumorigenic or not (Table IGo). Figure 5Go shows the lack of G1 arrest in the tumorigenic E7Q line: the cells traversed G1, progressed through S phase and accumulated in G2/M. The ability of the tumorigenic lines to bypass quercetin-induced G1 arrest is consonant with a malfunctioning and, in the case of E7Q and E7QT2, mutated p53 (Figures 3 and 4GoGo; Table IGo). However, the observation that the non-tumorigenic E7QPß cells fail to arrest in G1, despite having a functional p53 (Table IGo), raises the possibility that expression of E7 and Ras is sufficient to overcome G1 arrest (see below).

The ability to overcome quercetin-induced G1 arrest could be due to the expression of the oncoproteins, to malfunctioning p53 or to other inheritable changes affecting cell cycle regulation originating from previous exposure to quercetin. Cell cycle analysis was undertaken for the E7R and Q0D cell lines which had had no previous exposure to quercetin. These cells too failed to arrest in G1 in the presence of quercetin (Figure 5Go, Table IGo), although their accumulation in G2 was not as dramatic as in E7Q cells during the same time interval. Thus previous exposure to quercetin is not required to overcome the G1 checkpoint. E7R and Q0D cells have a functional p53 response to quercetin, including induction of p21Cip1, so the abrogation of the G1 arrest is not due to malfunction of p53 as in the tumorigenic lines, but is probably due to expression of E7 and Ras, the oncoproteins common to E7R and Q0D.

To confirm that PalF cells were not merely cycling more slowly in the presence of quercetin, the cultures were grown in 50 µM quercetin for up to 6 days (Figure 6Go). Control PalF cells grown in normal medium became confluent and contact-inhibited after 3 days, and accumulated in G1; the arrest in G1 was accompanied by a decrease of the number of cells in S and in G2/M phases. In contrast, for the quercetin-exposed cells, the population of cells in G1 was stationary during the whole 7-day period; there was a decrease of ~50% in the number of cells in S phase after 12 h of treatment and at the same time the number of cells in G2/M started increasing; the S phase cells continued decreasing until they levelled off at approximately the level of the non-treated cells (which were growth-arrested due to contact inhibition), while the G2/M phase cells continued increasing and then levelled off after 36 h (Figure 6AGo).




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Fig. 6. Long-term cell cycle profiles of normal and transformed PalF cells. Cells grown in 50 µM quercetin or equivalent volume of EtOH for up to 144 h were analysed by FACS. The histograms show the percentage of cells in different phases of cell cycle. (A) Normal PalF cells; (B) tumorigenic E7Q cells.

 
A very different picture was obtained with the tumorigenic E7Q cells (Figure 6BGo). In the absence of quercetin the cells continued cycling, but in its presence they rapidly exited G1 and S, and accumulated almost in their entirety in G2/M.

The most likely explanation for these results is that PalF cells are arrested both in G1 and G2/M by quercetin, whereas transformed cells can traverse G1 and S even in the presence of quercetin, but are not capable of overcoming the G2/M block.

We have not investigated the G2/M arrest as this is common to all cell lines and is therefore less likely to contribute to the transformed phenotype, although at present this cannot be ruled out completely.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have reproduced in vitro the multi-step process of papillomavirus tumorigenesis, and assigned functions to the transforming proteins of BPV-4 (10) and roles to the cellular genes ras and p53 (14,27). We have shown that introduction of an exogenous mutated p53 in cells transformed by BPV-4, exogenous E6 and Ras leads to oncogenic transformation (14). We have also shown that in E7-transformed cells quercetin substitutes for E6 in cell immortalization, for E5 in conferring anchorage independence from substrate and for mutated p53 in inducing tumorigenicity (23). We show here that in tumorigenic cells the endogenous p53 is mutated and transcriptionally non-functional but that the abolition of p53 function is not necessary for the E7-transformed cells to bypass the quercetin-induced G1 cell cycle arrest.

p53 malfunction in transformed cells
In normal PalF cells, quercetin treatment is accompanied by elevation and increase in transcriptional activity of p53, consonant with DNA damage induced by quercetin (17,18) and subsequent activation of p53 (ref. 33). These observations are in agreement with the results of Plaumann et al. (31) who found that quercetin activates wild-type p53. In transformed cells, the behaviour of p53 differs depending on whether or not the cells are tumorigenic. Thus, in transformed non-tumorigenic cells, p53 is elevated and transcriptionally activated by quercetin treatment with production of p21Cip1, whereas in quercetin-treated tumorigenic cells p53, despite being elevated, is transcriptionally dead and accordingly there is no production of p21Cip1. Indeed, although the number of tumorigenic cell lines analysed is perforce small given that the frequency of tumorigenic conversion is low, there is complete concordance between tumorigenic status and malfunctioning p53. Interestingly, in E7Q cells p53, although mutated, behaves in a wild-type manner with regard to protein level and inducibility by quercetin. On the contrary, p53 is constitutively elevated in E7QT and in Q2D cells (Table IGo). The reasons for these differences are not known but this observation is consistent with the progression of E7 cells toward increased transformation.

The lack of p53 transcriptional activity in E7Q can be ascribed to the mutation detected in the p53 sequence. The glycine to valine substitution has taken place in conserved region V, in the DNA binding domain (34) and the mutant p53 is incapable of trans-activating the p21Cip1 promoter (Scott VandePol, personal communication), supporting the conclusion that the mutation is the reason for the malfunction of p53 in these tumorigenic cells.

The mutation in the p53 gene may have resulted directly from the mutagenic action of quercetin, or from another unknown cause. Although we cannot at the moment distinguish between the two, we favour the first hypothesis as p53 mutations have been detected in two naturally occurring cancers in bracken fed cattle (7). In these cases the same mutation, CCC->ACC, was found at codon 243 in conserved region IV, leading to a proline to threonine substitution. Further analysis of p53 mutations in both naturally occurring cancers and in vitro transformed cells will be needed to determine whether the mutations detected so far are particular to bracken/quercetin-exposed cells.

Quercetin-induced G1 arrest is abrogated in E7 expressing cells
Contrary to normal PalF cells which reversibly arrest in G1 and G2/M in the presence of quercetin (20), transformed cells failed to arrest in G1 independent of whether they were tumorigenic or not, with one single exception (Table IGo). The transformed cells, however, maintained the G2/M check point. The G2/M check point is common to all cell lines and is therefore less likely to contribute to the transformed phenotype. It is to be remembered that, like the G1 check point, the G2/M check point is reversible when quercetin is withdrawn and this allows the outgrowth of transformed clones.

How can the abrogation of G1 arrest in non-tumorigenic cells be reconciled with an activated, transcriptionally functional p53? In other systems, E7 has been shown to bind and inhibit p105Rb and p21Cip1, thus promoting cell proliferation and preventing DNA damage-induced cell cycle arrest, respectively (35,36). p21Cip1 is an inhibitor of G1 cyclin-dependent kinases (37) and its expression is transcriptionally regulated by p53 (32). Accordingly, in our non-tumorigenic transformed cells, where p53 transcriptional activity is unaltered, p21Cip1 is elevated but fails to cause a G1 arrest. We hypothesize that this is due to a direct inhibition by E7. Alternatively, the activation of cyclin E by E7 (38) could be large enough to titrate out p21Cip1 (39). In either case, p21Cip1 would not be functional and this would explain how p53, apparently wild-type in its behaviour, fails to arrest the cells in G1.

The transformed cells also express activated Ha-ras. The activated Ras oncoprotein induces DNA synthesis and cell proliferation and prevents apoptosis via the PI3K/PKB pathway (30) and in cell lines with a functional Rho-GTPase suppresses p21Cip1 (40). Undoubtedly activated Ras contributes to PalF cell transformation, perhaps through further inhibition of p21Cip1 and abrogation of apoptosis, but it is worth stressing that neither activated Ras alone nor E7 alone are capable of PalF cell transformation, even partial.

Conclusions
In addition to its mutagenic action, quercetin interferes with several of the cell's signal transduction pathways (see ref. 21 and references therein) and this will have consequences for cell cycle regulation. Although the detailed role of quercetin in oncogenic cell transformation by E7 remains to be delineated, considering together all the observations listed above, we propose the following series of events as being likely to account for the synergism between virus and quercetin resulting in full tumorigenic cell transformation (Figure 7Go): quercetin induces DNA damage and in normal cells this causes G1 arrest through the activation of p53 and p21Cip1. In cells containing the viral genome, G1 arrest is abrogated as p21Cip1 is inhibited by E7, either through direct association or indirectly by increasing cyclin E; the traversing of G1 by damaged cells allows their expansion into transformed clones. During clonal expansion, p53 mutations contribute to the fully tumorigenic transformation of the cells. Mutations of p53 will further increase the genomic instability of the cell and thus lead to further damage. Indeed chromosomal analysis of quercetin-treated fully transformed cells has revealed numerous abnormalities (Stocco dos Santos, R.G.Beniston and M.S.Campo, in preparation), in agreement with previous results (19) and with the observed chromosomal instability in cells with non-functional p53 (33).



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Fig. 7. Proposed mechanism for the synergy between quercetin and papillomavirus E7 transforming protein. (A) In normal cells, quercetin induces DNA breaks, with consequent activation of p53 and p21Cip1, inhibition of cyclin/cdks and cell cycle arrest in G1. (B) In papillomavirus transformed cells, E7 inhibits p21Cip1, G1 cyclin/cdks are active and the cells are stimulated to cycle. (C) Exposure of cycling cells to quercetin results in DNA breaks; p53 is activated but p21Cip1 is inhibited by E7 and the cells overcome G1 arrest. (D) During the proliferation of these cells, p53 gene mutations take place and the cells become fully transformed and tumorigenic.

 
The requirement for activated Ras for BPV-4 in vitro cell transformation and for quercetin exposure and mutant p53 for cell progression to tumorigenicity parallels the activation of Ras and the mutations of p53 observed in naturally occurring cancers in bracken-feeding cattle (6,7). This supports the view that the interplay between E7 and quercetin observed in in vitro cell transformation may well play a vital role in carcinogenesis of the upper GI tract, although the contribution of other bracken chemicals cannot be excluded.

What is the relevance of the synergy between BPV-4 and bracken to human cancer? In humans too, exposure to bracken fern, whether in the diet or as spores, has been epidemiologically linked to cancer of the upper GI tract in several parts of the world (4145). All forms of culinary bracken have been shown to be carcinogenic in experimental animals (46,47) and DNA adducts have been found in the upper GI tissue of mice fed bracken extracts or bracken spores (48). Human papillomavirus (HPV) type 16 has been found in ~50% of cancers and pre-cancers of the oesophagus (4953), particularly in developing countries, and we ourselves have detected HPV-16 DNA in biopsies of oesophageal cancer from one area of Brazil (unpublished observations), where bracken fern is a common component of human diet and the relative risk for bracken fern exposure and oesophageal carcinoma has been estimated to be 5.47 (ref. 42). Additionally, a new study reports a relative risk of 3.64 for gastric cancer in Venezuela in areas where consumption of milk from bracken-fed cows is common (54). These findings suggest that some cases of cancer of the upper GI tract in humans may have the same aetiology as in cattle, i.e. papillomavirus and bracken.

Our unpublished observation that, like the LCR of BPV-4 (ref. 20), also the LCR of HPV-16 is trans-activated by quercetin, opens up the possibility that the molecular mechanisms we have started elucidating for cell transformation and cancer in cattle operate also in humans.


    Notes
 
1 Present address: The Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK Back

2 To whom correspondence should be addressed E-mail: s.campo{at}vet.gla.ac.uk Back


    Acknowledgments
 
This work was conducted while the authors were at The Beatson Institute for Cancer Research. Thanks are due to Prof. J.A.Wyke for his critical reading of the manuscript and helpful comments, to Prof. David Gillespie for his constructive interest in this work, and to Drs David Fergusson and Gary Sibbet for their invaluable technical help. The work was supported by the Medical Research Council. R.G.B. was the recipient of an MRC PhD Studentship. M.S.C. is a CRC Life Fellow.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 12, 2000; revised February 23, 2001; accepted March 14, 2001.