Human Papillomavirus E 6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p 53 Gene Transfer in HeLa Cells

p53 is inactivated and degraded by the E6 protein encoded by the integrated human papillomavirus (HPV) in the cervical cancers (Scheffner et al., 1990). The susceptibility of p53 to degradation by the E6 protein varies between the arginine and proline variants at codon 72 of p53 (Storey et al., 1998). In vivo studies demonstrate that the arginine p53 variant is seven times more susceptible to degradation by the E6 protein, compared with the proline variant (Storey et al., 1998). However, the results of clinical studies are controversial, with the majority of studies demonstrating no significant difference between individuals harboring the arginine or proline p53 variant (Andersson et al., 2001; Klug et al., 2009; Nunobiki et al., 2011; Habbous et al., 2012). We considered that hormonal factors may affect these results. The estrogen response element (ERE) is a DNA sequence (-GGTCAnnnTGACC-) that when bound by the estrogen receptor, leads to the transcriptional activation of target genes (Klinge et al., 2001). We found that the ERE


Introduction
p53 is inactivated and degraded by the E6 protein encoded by the integrated human papillomavirus (HPV) in the cervical cancers (Scheffner et al., 1990).The susceptibility of p53 to degradation by the E6 protein varies between the arginine and proline variants at codon 72 of p53 (Storey et al., 1998).In vivo studies demonstrate that the arginine p53 variant is seven times more susceptible to degradation by the E6 protein, compared with the proline variant (Storey et al., 1998).However, the results of clinical studies are controversial, with the majority of studies demonstrating no significant difference between individuals harboring the arginine or proline p53 variant (Andersson et al., 2001;Klug et al., 2009;Nunobiki et al., 2011;Habbous et al., 2012).We considered that hormonal factors may affect these results.
The estrogen response element (ERE) is a DNA sequence (-GGTCAnnnTGACC-) that when bound by the estrogen receptor, leads to the transcriptional activation of target genes (Klinge et al., 2001).We found that the ERE

Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells
Koji Kajitani 1 , Ken-Ichi Honda 2,3 *, Hiroyuki Terada 2 , Tomoyo Yasui 3 , Toshiyuki Sumi 3 , Masayasu Koyama 3 , Osamu Ishiko 3 p53 gene with proline at codon 72 promotes adenovirusmediated p53 gene delivery in cultured cells derived from endometrial cancer.This transduction was strong in estrogen receptor-positive HHUA cells and weak in estrogen receptor-negative KLE cells (Kajitani et al., 2012).We studied the effect of the ERE on adenovirusmediated p53 transduction in HeLa S3 cells, which are derived from a human cervical cancer and contain integrated DNA of HPV type 18.
However, the transduction efficiency of a p53containing adenovirus vector was low in HeLa S3 cells, even when an ERE was linked to the p53 gene.When E6 mRNA was silenced in HeLa S3 cells by incubation with siRNA for HPV type 18 E6, adenovirus-mediated transduction of the ERE-linked p53 gene (proline variant at codon 72) was restored and early-phase apoptosis rates were increased.These results suggested an unknown effect of ERE on trans-membrane and nuclear transport of the adenovirus-mediated p53 gene (proline variant at codon 72) and therapeutic potential for HPV-associated neoplasm.

Recombinant adenoviruses containing the human p53 gene
Total RNA was extracted from human chorionic tissue using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) from a woman who underwent elective abortion at 11 weeks of normal gestation.The woman provided written informed consent for this study, and the study was conducted with the consent of the ethical committee of Osaka City University.cDNAs were synthesized from the total RNA using the High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).The p53 gene (nucleotides 166-1143, including 21 non-coding nucleotides on the 3' side) was amplified using the following primers; forward primer 5'-GAAGACCCAGGTCCAGAT-3' and reverse primer 5'-TTTATGGCGGGAGGT-3'.To incorporate an EcoRI site together with the ERE and a start codon at the 5' terminus, the following primers were used to amplify the ERE-linked p53 gene; forward primer 5'-GAATTCGGTCATAGTGACCATATGGAAGACC CAGGTCCAGAT-3' and reverse primer 5'-AGTGTGA TGGATATCTGCAGAATTCTTTATGGCGGGAGGT-3'.To incorporate an EcoRI site together with the start codon, but not including the ERE at the 5' terminus, the following primers were used to amplify a non-ERE-linked p53 gene; forward primer 5'-AGGAATTCATGGAAGACCCAGGT C C A G A T -3 ' a n d r e v e r s e p r i m e r 5'-CAGAATTCTTTATGGCGGGAGGT-3'.
PCR products were agarose gel purified, ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA) and amplified in competent E. coli DH5α cells (Competent High, Toyobo, Osaka, Japan).For polymorphic variants of p53 at codon 72, the DNA isolated from chorionic tissue encoding the arginine variant (CGC) p53 gene was modified to the proline variant (CCC), using the QuikChange ІІ Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), and the following primers, 5'-GCCAGAGGCTGCTCCCCC-3' and 5'-CGTGCAAGTCACAGACTT-3'.The ERE-linked or non-ERE-linked p53 genes and their codon 72 arginine (EREp53R or p53R) or proline variants (EREp53P or p53P) were excised by digestion with EcoRV and XhoI, and inserted into the pShuttle-IREShrGFP-1 shuttle vector (Agilent Technologies) (Figure 1).After linearization by Pme I digestion and gel purification without dephosphorylation, the p53 gene constructs were electroporated into BJ5183-AD-1 cells pretransformed with E1-deficient adenovirus vector (Agilent Technologies).The adenoviruses were extracted from the supernatant of AD293 cells after four rounds of rapid freezing in methanol cooled with dry ice and thawing in a 37°C water bath and purified using a Virabind Adenovirus Miniprep kit (Cell Biolabs, San Diego, CA, USA) and stored with 10% glycerol at -80°C.

Cell culture and targeted silencing of HPV18 E6 by siRNA
The HHUA human endometrial cancer cell line, which expresses ERβ, was supplied by Riken Laboratories (Wako, Saitama, Japan) (Ishiwata et al., 1984;Zhi et al., 2007).The KLE human endometrial cancer cell line, which does not express ERα or ERβ, was supplied by DS Pharma Biomedical Co. (Osaka, Japan) ( Kumar et al., 1998).HeLa S3 cells, which express low levels of ERα in our study (data not shown), were a gift from Dr Akira Inoue (Laboratory of Immunology, Osaka City University Graduate School of Medicine, Japan) (Allgood et al., 1992).The presence of the HPV 18 E6 DNA sequence was confirmed in the nuclear DNA of these HeLa cells (data not shown).HeLa S3 cells were cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), in a humidified incubator with 5% CO 2 in air.One day prior to treatment of cells with siRNA, cells were detached by treatment with trypsin EDTA, washed, and cultured at 20% confluence in 24-mm diameter wells of 12-well, plastic tissue culture plates.
For targeted silencing of HPV 18 E6, a 21bp siRNA hairpin complementary to the HPV 18 E6 mRNA sequence at 755-773 nucleotides (5'-CCACAACGUCACACAAUGU-3') was constructed (Kuner et al., 2007).Cell culture medium was replaced with Opti-MEM medium (Invitrogen) containing the siRNA premixed with Lipofectamine RNAiMAX (Invitrogen) and incubated for 4 h.The medium was subsequently replaced with Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, and containing p53 gene-containing recombinant adenoviruses and incubated for 80 h until cell harvesting.Copy numbers of recombinant adenoviruses were quantified as described below.

Quantification of recombinant adenovirus containing the p53 gene by real-time polymerase chain reaction (PCR)
After cultured cells were lysed with 0.6% sodium dodecylsulfate (SDS) containing 10 mM EDTA, adenovirus DNA was precipitated with ice-cold ethanol and dissolved in water.For TaqMan real-time PCR of the cell extract or stock

GFP
vector-p53P vector-EREp53R vector-EREp53P DOI:http://dx.doi.org/10.7314/APJCP.2015.16.18.8239HPV E6 Knockdown Restores Adenovirus Mediated-Estrogen Response Element Linked p53 Gene Transfer in HeLa Cells solution of recombinant adenovirus, the forward primer 5'-GAATTGCTATTATTTGTCGTCATCA-3', reverse primer 5'-AGGTAGACTGACCCTTTTTGGACTT-3', and the probe (CCTTGTAGTCCTCGAGTTA) conjugated with FAM (6-carboxy fluorescein) at the 5' end and the minor groove binder (MGB) at the 3' end (Applied Biosystems), were used for detecting the junction region between the 3' end of the p53 gene insert and the XhoI site of the shuttle vector.The absolute copy number of shuttle vector or recombinant adenovirus was titrated in each well on a 7500 Fast real-time PCR system (Applied Biosystems).

Analysis of p53 mRNA and CAR mRNA
Total RNA was extracted from HeLa S3 cells 80 h after adenovirus infection, using an RNeasy Mini kit (Qiagen), and cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).The relative expression of target mRNAs was measured by real-time PCR using TaqMan MGB probes (Hs00153349_m1 for p53, Hs00154661_m1 for CAR), and analyzed in comparison with 18S ribosomal RNA, by a multiplex threshold method on a 7500 Fast Real Time PCR System (Applied Biosystems).

Western blot analysis of CAR, p53, and β-tubulin
HeLa cells were lysed with 0.1% SDS 80 h after adenovirus infection, and the cell extracts were boiled for 2 min before storage at -20°C.The proteins in the cell extracts were treated with DNase Ι (New England BioLabs, Ipswich, MA, USA), separated by SDS polyacrylamide gel electrophoresis using Miniprotean TGX gel for any kD (BioRad, Hercules, CA, USA) and then transferred to PVDF Hybond P membrane (GE Healthcare).After blocking with 5% skimmed milk in PBS containing 0.2% Tween 20, the membrane was reacted with rabbit antibodies against human CAR (Bethyl Laboratories, Montgomery, TX, USA), human full-length p53 (Santa Cruz Biotechnology, Dallas, TX, USA), or human β-tubulin (Proteintech, Chicago, IL, USA), washed and then reacted with peroxidase-conjugated anti-rabbit immunoglobulins goat IgG.The chemiluminescent reaction was performed using ECL Prime Western Blotting Detection Reagent (GE Healthcare), and chemiluminescent intensity was quantified using ImageQuant TL software on an LAS4000mini image analyzer (GE Healthcare).

Statistical analysis
Data were analyzed by nonparametric methods using PASW statistics 18 Software (SPSS, Chicago, IL, USA), and results with p<0.05 were judged as significant.

Transduction efficiency of p53-containing adenovirus in HHUA or KLE cells
The efficiency of adenovirus-mediated p53 transduction was lower in KLE cells compared with HHUA cells.The transduction efficiency of ERE-linked p53 was, however, higher than that of non-ERE-linked p53 in either cell line (Figure 2).Although a higher transduction efficiency of ERE-linked p53P compared with ERE-linked p53R was observed in HHUA cells (Figure 2A), no significant difference between the transduction efficiencies of ERElinked p53R and ERE-linked p53P was observed in KLE cells (Figure 2B).

Transduction efficiency of p53-containing adenovirus in
HeLa S3 cells following pretreatment with HPV18 E6 siRNA When HeLa S3 cells were pretreated with HPV18 E6 siRNA, the number of cells positive for p53-containing adenovirus (as measured by the percentage of GFPpositive cells) was increased markedly 72 h after infection with ERE-linked p53P-containing adenovirus (Figure 3B).When HeLa S3 cells were not pretreated with HPV18 E6 siRNA, the number of cells positive for p53-containing adenovirus remained low 72 h after infection with ERElinked p53P-containing adenovirus (Figure 3A).
We observed no significant difference in the efficiency of HeLa S3 cell transduction between p53R-containing adenovirus and upstream ERE-linked p53R-containing adenovirus.Pretreatment of HeLa S3 cells with siRNA for HPV18 E6 significantly increased the transduction of ERE-linked p53P-containing adenovirus (Figure 4A) and non-ERE-linked p53P-containing adenovirus (Figure 4B).Furthermore, the transduction efficiency of ERE-  .2015.16.18.8239HPV E6 Knockdown Restores Adenovirus Mediated-Estrogen Response Element Linked p53 Gene Transfer in HeLa Cells linked p53P adenovirus was significantly higher than that of non-ERE-linked p53P adenovirus (Figure 4A).However, pretreatment of HeLa S3 cells with siRNA for HPV18 E6 did not change the transduction efficiency of ERE-linked p53R-containing adenovirus, and there was no difference between the transduction of ERE-linked p53R-containing adenovirus and non-ERE-linked p53Rcontaining adenovirus (Figure 4A).

Analysis of p53 mRNA and CAR mRNA levels in HeLa S3 cells following transduction with p53-containing adenovirus
p53 mRNA levels were higher in siRNA-treated cells compared with non-siRNA-treated cells 80 h after infection with ERE-linked p53P-containing adenovirus (Figure 5).Furthermore, p53 mRNA levels in HeLa S3 cells pretreated with HPV18 E6 siRNA were higher in cells infected with ERE-linked p53P-containing adenovirus compared with non-ERE-linked p53P-containing adenovirus (Figure 5).However, p53 mRNA levels were not higher in siRNAtreated cells compared with non-siRNA-treated cells 80 h after infection with ERE-linked or non-ERE-linked p53Rcontaining adenovirus (Figure 5).Levels of CAR mRNA were significantly higher in siRNA-treated cells compared with non-siRNA-treated cells, 80 h after infection with ERE-linked p53P-containing adenovirus.CAR mRNA levels were also higher in HeLa S3 cells infected with ERE-linked p53P adenovirus compared with non-ERElinked p53P adenovirus (Figure 6A).CAR mRNA levels in HeLa S3 cells uninfected with either ERE-linked or non-ERE-linked p53-containing adenovirus, were higher in cells treated with HPV18 E6 siRNA compared with cells not treated with HPV18 E6 siRNA (Figure 6B).

Western blot analysis and quantification of CAR/β-tubulin and p53/β-tubulin ratios
The chemiluminescent intensity of western blot bands corresponding to p53, CAR, and β-tubulin (Figure B-D) was analyzed and is shown graphically in Figure 7E-G.Total protein content in each sample was slightly lower in siRNA-treated HeLa cells compared with non-siRNAtreated cells (Figure 7H), and β-tubulin content was lowest in siRNA-treated HeLa cells 80 h after infection with ERE-linked p53P-containing adenovirus (Figure 7E).The p53/β-tubulin and CAR/β-tubulin ratios were largest in siRNA-treated HeLa cells 80 h after infection with ERElinked p53P-containing adenovirus (Figure 7I-J).

Flow cytometric analysis of annexin V binding rates in HeLa S3 cells
Annexin V binding rates were significantly higher in siRNA-treated HeLa S3 cells compared with untreated HeLa S3 cells, even when cells were not exposed to p53containing adenovirus (Figure 8, no vector).Annexin V binding rates were also significantly higher in siRNAtreated HeLa S3 cells infected with ERE-linked p53containing adenovirus compared with cells not infected with p53-containing adenovirus (Figure 8).However, annexin V binding was not significantly higher in siRNAtreated HeLa S3 cells infected with non-ERE-linked p53containing adenovirus compared with cells not infected with p53-containing adenovirus.

Discussion
siRNA targeting the HPV oncogene E6 and/or E7 up-regulates expression of p53 and Rb in head and neck cancer cell lines (Adhim et al., 2013).Furthermore, co-expression of E6-specific siRNA and wild-type p53 suppresses the growth of human cervical cancer lines (Li et al., 2013).These findings indicate that intrinsic p53 expression is suppressed by HPV E6 and that knockdown of E6 is essential for p53 expression.However, additional treatments were required to induce apoptosis of cervical cancer cells (Hougardy et al., 2006;Tan et al., 2012).
In this study, treatment of HeLa cells with siRNA for HPV E6 permitted adenovirus-mediated transfer of the p53 gene (proline variant at codon 72).However, the transduction rate was large only when the p53 gene contained proline at codon 72 and was linked to an upstream ERE.These phenomena suggest unknown promoting effects of the ERE on adenovirus-mediated p53 gene transfer.
Expression of p53 and ERα is associated with the induction of leukemia inhibitory factor (LIF) during fertilized egg implantation into the endometrium and higher LIF induction was observed in mice transgenic for the human p53R gene compared with mice transgenic for the human p53P gene (Feng et al., 2011).A common set of genes has also been shown to be more strongly transcribed by p53R compared with p53P (Jeong et al., 2010).In our study, however, estrogen receptors binding with the ERE may work as a transcription factor and trigger expression of other genes involved in the transduction of p53P.
The proline-rich domain of p53 (encoded by codons 64-93) is required for transcriptional control of target genes (Liu et al., 2003;Bergamaschi et al., 2006).We constructed a p53P-containing adenovirus vector containing this domain and produced a high level of transduction of the p53P gene when linked to an upstream ERE in estrogen receptor-expressing HHUA cells.We also constructed ERE-and non-ERE-linked p53R genes, but found no significant difference between their efficiency of transduction in HHUA cells.Similar results were obtained in estrogen receptor-expressing SW48 cells, which are derived from colon cancer (Drewinko et al., 1984;Kajitani et al., 2012).Low transduction efficiency was observed in non-ER-expressing KLE cells, with relatively higher transduction rates for ERE-linked p53P and ERE-linked p53R gene-containing adenovirus, compared with non-ERE-linked p53 gene-containing adenovirus.However, no effect of upstream ERE-linkage was observed in HeLa S3 cells, and pretreatment of cells with HPV E6 siRNA restored the promoting effect of the upstream ERE for p53P transduction, but not for p53R transduction.
Pretreatment of HeLa S3 cells with siRNA for HPV type 18 E6 increased the efficiency of transduction of p53P, suggesting that the inhibitory effects of HPV E6 on adenovirus-mediated p53 gene delivery in these cells target the p53 gene with proline at codon 72.Furthermore, the effect of pretreatment of HeLa S3 cells with siRNA for HPV type 18 E6 was limited to transduction of ERElinked p53P.
Restoration of adenovirus-mediated p53 gene delivery to HeLa cells by HPV E6 siRNA suggests inhibitory effects of HPV E6 on the cellular responses to ERErelated gene expression.Increased levels of p53 and CAR mRNAs suggest that transcription of these genes is also ERE related.
The p53 codon 72 polymorphism appears to affect the transduction of the p53 gene; with transduction being more efficient for p53P than for p53R.These phenomena indicate that differences in the molecular structure of p53, which is synthesized from adenovirus-mediated transfer of the p53 gene, affect cellular responses.
p53 is rapidly degraded in normal cellular process but is stabilized in response to DNA damage, microtubule disruption, or other intracellular events.p53 is also stabilized by some oncoproteins (Blagosklonny MV., 1997).It is considered that siRNA treatment of the HPV E6 oncogene increased accumulation of p53 protein after adenovirus-mediated transfer of the p53 gene.
We constructed an adenovirus vector containing the human chorionic gonadotropin β chain gene (HCGβ) to study whether the effect of an upstream ERE on the transduction efficiency of linked genes is specific for p53, but we did not observe any difference in the transduction efficiency of ERE-and non-ERE-linked HCGβ adenovirus in HHUA cells (data not shown).These results suggest that the transferred p53-specific cellular response occurs against subsequently transferred exogenous p53 genes.These phenomena are, at least in part, dependent on transcriptional changes, including the induction of CAR mRNA.
Under cellular stress, the p53 gene is activated and leads to cell-cycle arrest, DNA repair, and/or apoptosis, via associated protein complexes, which control the transcription of target genes (Trigiante et al., 2006;Kruse et al., 2009).The ERE binds estrogen receptors in addition to other transcriptional factors, and may lead to activation of downstream target genes.Menendez et al. (2010) showed that an ERE-conjugated p53 response element could synergistically transactivate p53 target genes (Menendez et al., 2010).The effect of an upstream ERE on the transcription of p53 and CAR mRNA seems to depend on transcriptional complexes; however, p53 gene transduction may also be affected by other factors, as shown by the significantly higher transduction rates of adenovirus-mediated p53P in siRNA-treated cells compared with non-siRNA-treated cells.
There are many benefits of adenovirus-mediated p53 gene therapy for the treatment of human cancer.However, low rates of p53 transduction have limited the extent of these applications (Chen et al., 2014).We expect that transfer of adenovirus-mediated ERE-linked p53 into cancer cells or dysplastic cells of the cervix following pretreatment with HPV E6 siRNA may lead to apoptosis of these HPV integrating cells.

Figure 1 .
Figure 1.Upstream Estrogen Response Element (ERE)-Linked p53 genes with Codon 72 Polymorphisms Inserted into an Adenovirus Vector.Vectors containing arginine variant p53 (p53R), proline variant p53 (p53P), ERElinked p53 with arginine variant (EREp53R), and ERE-linked p53 with proline variant (EREp53P) are shown.A region of the p53 gene (166-1368 nt, indicated by orange lines) was inserted 5' to the green fluorescent protein (GFP) gene.The ERE and two additional nucleotides were linked to the 5' end of the start codon of p53

Figure 2 .Figure 3 .
Figure 2. Transduction of p53 genes with Codon 72 Polymorphisms into HHUA and KLE Cells.Copy number of recombinant adenoviruses (p53R, p53P, EREp53R, or EREp53P) in HHUA cells (A), or KLE cells (B) 72 h after infection.Cells were harvested 72 h after infection and recombinant adenovirus copy numbers were determined by real time PCR

Figure 8 .
Figure 8. Analysis of Annexin V Binding in HeLa S3 Cells Following Transduction of p53 gene-containing Adenoviruses.HeLa S3 cells pretreated with HPV 18 E6 siRNA (gray column) or no siRNA (white column), were infected with p53R, p53P, EREp53R, or EREp53P gene-containing adenoviruses or no adenovirus (no vecto), and 80 h later annexin V binding was assessed by flow cytometry